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University of Arkansas, FayettevilleScholarWorks@UARK
Theses and Dissertations
12-2013
Phenotypic, Physiological and Growth Interactionsamong Salmonella SerovarsJuliany Rivera CaloUniversity of Arkansas, Fayetteville
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Phenotypic, Physiological and Growth Interactions among Salmonella Serovars
Phenotypic, Physiological and Growth Interactions among Salmonella Serovars
A thesis submitted in partial fulfillment
of the requirements for the degree of
Master of Science in Food Science
by
Juliany Rivera Calo
University of Puerto Rico at Mayagüez
Bachelors of Science in Industrial Microbiology, 2005
December 2013
University of Arkansas
This thesis is approved for recommendation to the Graduate Council.
___________________________________
Dr. Steven C. Ricke
Thesis Director
___________________________________ ___________________________________
Dr. Young Min Kwon Dr. Steven L. Foley
Committee Member Committee Member
ABSTRACT
This thesis consists of four research parts: a literature review that covers Salmonella spp.,
one of the more prominent foodborne pathogens that represents a major risk to humans (chapter
1). Understanding the growth of Salmonella serovars and strains is an important basis for more in
depth research. In this case we studied a) the aerobic and anaerobic growth responses of multiple
strains from six different serovars, b) how the spent media from different serovars, more
importantly S. Heidelberg, affect the growth of S. Typhimurium, and c) determined whether or
not two different serovars undergo competitive interactions when they were grown together
(chapter 2). Growth responses of four Salmonella serovars, Heidelberg, Kentucky,
Typhimurium, and Enteritidis in enrichment and non-enrichment media were compared to
determine the effectiveness of a non-selective enrichment media, such as Luria Bertani broth
over selective enrichment methods (chapter 3). Since antimicrobial resistance continues to be
one of the major concerns about Salmonella, and consumers are demanding more natural
antimicrobials we studied the effectiveness of five essential oils against Salmonella Heidelberg.
Besides being one of the top five Salmonella enterica serovars associated with human infections,
S. Heidelberg is the primary focus for our research (chapter 4). Given that Salmonella enterica
are important facultative intracellular pathogens that are widely known to be isolated from
poultry and to cause infections in humans, understanding the ability of this pathogen to attach
and invade different cell types is of great importance. We studied the adhesion and invasion of
four different strains to chicken macrophage (HD11), and human epithelial (Caco-2) cells
(chapter 5).
ACKNOWLEDGMENTS
With deep gratitude and sincerity, I would like to thank my advisor, Dr. Steven C. Ricke,
for his guidance, support, mentorship, and expertise during my masters program in the
University of Arkansas. He provided me with constant motivation to do research with great
understanding. The scientific discussions with him were very fruitful. I also thank my committee
members, Dr. Steven L. Foley and Dr. Young Min Kwon for their contributions and support on
my thesis and research.
Special thanks to former and current members of the Center for Food Safety, Dr. Philip
G. Crandall, Dr. Corliss O’Bryan, Candy, specially Michelle, Dr. Si Hong Park, Nathan, Peter,
and Adam for their friendship, insights, support and all their help. I am very thankful to Dr. Ok
Kyung Koo for her mentorship, experimental advice and her infinite help during this process. I
also thank the Department of Food Science for providing graduate stipend support. Thanks to the
Food Science and Poultry Science staff and graduate students for their kindness, friendship and
all their help.
I thank all my family and friends, especially Mika, Jonah, Sofia, and Rafael for their
company, love, support, words of encouragement and constant motivation to keep going.
DEDICATION
I dedicate this thesis to my parents, Julie and Rafy, and my brother Rafael. Without their
infinite support, encouragement, words of advice and love this would not have been possible. I
thank them for their unconditional love and constant motivation in every step of the way and
everything I have ever wanted to accomplish. I sincerely dedicate this thesis to them, for being
instrumental in inspiring me at every step throughout my life on both personal and professional
fronts.
TABLE OF CONTENTS
INTRODUCTION..........................................................................................................................1
CHAPTER 1: Salmonella spp. - Literature Review .......................................................................3
1. Abstract ...............................................................................................................................4
2. Introduction ..........................................................................................................................5
3. Foodborne Pathogen, Salmonella enterica ..........................................................................9
3.1. Epidemiology of Salmonella .......................................................................................11
3.2. Salmonella and Food Contamination ..........................................................................12
3.3. Pathogenesis of Salmonella ........................................................................................13
3.4. Competitive Interactions among Serovars and Spent Media ......................................16
3.5. Salmonella in Poultry ..................................................................................................17
3.6. Salmonella Drug-resistant Strains ...............................................................................17
3.7. Mechanisms of Antimicrobial Action .........................................................................20
4. Natural Antimicrobials against Salmonella spp. ...............................................................22
4.1. Antimicrobials from Plant Sources ............................................................................23
5. Essential Oils ....................................................................................................................26
5.1. Properties ...................................................................................................................26
5.2. Mode of Action of Essential Oils ...............................................................................28
5.3. Essential Oils as a Pre-harvest Intervention ...............................................................32
5.4. Essential Oils as a Post-harvest Intervention .............................................................33
6. Hurdle Technology ............................................................................................................35
7. Conclusions ........................................................................................................................36
8. References ..........................................................................................................................39
9. Authorship Statement for Chapter 1 ..................................................................................66
CHAPTER 2: Growth Responses, Spent Media, and Competitive Interactions among
Salmonella Serovars ......................................................................................................67
1. Abstract .............................................................................................................................68
2. Introduction ........................................................................................................................69
3. Materials and Methods .......................................................................................................71
3.1. Salmonella spp. growth responses after aerobic and anaerobic incubation from
poultry Salmonella isolates ........................................................................................71
3.1.1. Bacterial strains ................................................................................................71
3.1.2. Aerobic and anaerobic growth responses ........................................................71
3.2. Effect of Salmonella spent media on the growth of S. Typhimurium .....................72
3.2.1. Aerobic and anaerobic spent media growth responses ....................................72
3.2.2. Salmonella Heidelberg and S. Typhimurium spent media ...............................72
3.2.3. Salmonella Heidelberg spent media .................................................................73
3.2.4. Salmonella Heidelberg spent media comparison between two different media
and an alternate temperature ....................................................................................73
3.3. Competitive growth between Salmonella Typhimurium ATCC 14028 and
Salmonella Heidelberg ARI-14 ..................................................................................74
3.3.1. Growth responses from mixed cultures ...........................................................74
3.3.2. DNA extraction ................................................................................................74
3.3.3. Bacterial quantification by real time quantitative PCR (qPCR) ......................75
3.4. Competitive growth between Salmonella Heidelberg ARI-14 and Salmonella
Enteritidis ATCC 13076 ............................................................................................75
3.4.1. Growth responses .............................................................................................75
3.5. Statistical analysis ....................................................................................................76
4. Results ................................................................................................................................76
4.1. Salmonella spp. growth responses after aerobic and anaerobic incubation from
poultry Salmonella isolates ........................................................................................76
4.2. Effect of Salmonella spent media on the growth of S. Typhimurium .....................77
4.2.1. Aerobic and anaerobic spent media growth responses ...................................77
4.2.2. Salmonella Typhimurium growth in S. Heidelberg spent media ....................77
4.3. Competitive growth between (1) S. Typhimurium and S. Heidelberg, and (2) S.
Heidelberg and S. Enteritidis .....................................................................................79
5. Discussion ..........................................................................................................................80
6. Acknowledgments ..............................................................................................................83
7. References ..........................................................................................................................84
8. Authorship Statement for Chapter 2 ................................................................................113
9. Appendix ..........................................................................................................................114
CHAPTER 3: Salmonella spp. Growth Rate Comparisons in Selective and Non-selective
Enrichment Media Methods ........................................................................................130
1. Abstract ...........................................................................................................................131
2. Introduction ......................................................................................................................132
3. Materials and Methods .....................................................................................................133
3.1. Bacterial cultures for non-selective enrichment media method ................................133
3.2. Bacterial cultures selective enrichment media method .............................................134
4. Results and Discussion ....................................................................................................134
5. Acknowledgments ............................................................................................................138
6. References ........................................................................................................................139
7. Authorship Statement for Chapter 3 ................................................................................151
8. Appendix ..........................................................................................................................152
CHAPTER 4: Antimicrobial Activity of Essential Oils against Salmonella Heidelberg Strains
from Multiple Sources.................................................................................................163
1. Abstract ...........................................................................................................................164
2. Introduction ......................................................................................................................165
3. Materials and Methods .....................................................................................................166
3.1. DNA extraction .........................................................................................................166
3.2. Confirmatory PCR for Salmonella Heidelberg strains .............................................167
3.3. Essential oils and cultures .........................................................................................167
3.4. Modified tube dilution assay .....................................................................................168
3.5. Statistical analysis .....................................................................................................168
4. Results and Discussion ....................................................................................................168
5. Acknowledgments ............................................................................................................171
6. References ........................................................................................................................172
7. Authorship Statement for Chapter 4 ................................................................................185
8. Appendix ..........................................................................................................................186
CHAPTER 5: Salmonella spp. Adhesion and Invasion Comparisons to Caco-2 and HD11 Cell
Lines ............................................................................................................................197
1. Abstract ...........................................................................................................................198
2. Introduction ......................................................................................................................199
3. Materials and Methods .....................................................................................................200
3.1. Bacterial strains for antibiotic susceptibility testing .................................................200
3.2. Antibiotic susceptibility testing ................................................................................200
3.3. Cell cultures ..............................................................................................................201
3.4. Bacterial cultures for adhesion and invasion assays .................................................202
3.5. Adhesion assays ........................................................................................................202
3.6. Invasion assays ..........................................................................................................203
3.7. Statistical analysis .....................................................................................................204
4. Results and Discussion ....................................................................................................204
5. Acknowledgments ............................................................................................................207
6. References ........................................................................................................................208
7. Authorship Statement for Chapter 5 ................................................................................222
8. Appendix ..........................................................................................................................223
CONCLUSIONS ........................................................................................................................234
LIST OF FIGURES
CHAPTER 2
Figure 2.1. Salmonella Typhimurium average from a) aerobic growth response, b) anaerobic
growth response ............................................................................................................91
Figure 2.2. Salmonella Heidelberg average from a) aerobic growth response, b) anaerobic
growth response ............................................................................................................92
Figure 2.3. Salmonella Kentucky average from a) aerobic growth response, b) anaerobic growth
response .........................................................................................................................93
Figure 2.4. Salmonella Newport average from a) aerobic growth response, b) anaerobic growth
response .........................................................................................................................94
Figure 2.5. Salmonella Enteritidis average from a) aerobic growth response, b) anaerobic growth
response .........................................................................................................................95
Figure 2.6. Salmonella Montevideo average from a) aerobic growth response, b) anaerobic
growth response ............................................................................................................96
Figure 2.7. Average aerobic spent media growth response ..........................................................99
Figure 2.8. Average anaerobic spent media growth response.....................................................100
Figure 2.9. Combinations of S. Heidelberg and S. Typhimurium spent media and inoculum ...101
Figure 2.10. Salmonella Typhimurium ATCC 14028 in S. Heidelberg spent media from
multiple sources ..........................................................................................................103
Figure 2.11. Confirmation that the drop showed during growth response only occurs when
growing S. Typhimurium on S. Heidelberg spent media ............................................104
Figure 2.12. Growth kinetic comparison for S. Typhimurium on S. Heidelberg spent media
growing at 42°C on LB and BPW ...............................................................................105
Figure 2.13. Growth kinetic comparison for S. Typhimurium on S. Heidelberg spent media
growing at 37°C on LB and BPW ...............................................................................106
Figure 2.14. Salmonella Typhimurium and Salmonella Heidelberg growth kinetic from a mixed
culture ..........................................................................................................................108
Figure 2.15. Salmonella Typhimurium and S. Heidelberg competitive interaction ...................109
Figure 2.16. Log CFU/mL for S. Typhimurium and S. Heidelberg from mixed culture ............110
Figure 2.17. Log CFU/mL from qPCR and plate counts from mixed culture ............................111
Figure 2.18. Growth of S. Heidelberg ARI-14 and S. Enteritidis ATCC 13076 individually and
as a mixed culture........................................................................................................112
CHAPTER 3
Figure 3.1. Optical density at 600 nm for each strain from overnight cultures (16 h) on LB and
BPW broth prior to enrichment ...................................................................................146
Figure 3.2. Optical density at 600 nm from each strain inoculated into 10 mL LB broth starting
on the first detectable time point .................................................................................147
CHAPTER 4
Figure 4.1. Gel electrophoresis of PCR products to confirm Salmonella Heidelberg strains .....178
Figure 4.2. Modified tube dilution assay for trans-cinnamaldehyde ..........................................180
Figure 4.3. Modified tube dilution assay for carvacrol ...............................................................181
Figure 4.4. Modified tube dilution assay for orange terpenes.....................................................182
Figure 4.5. Modified tube dilution assay for R-(+)-limonene .....................................................183
Figure 4.6. Modified tube dilution assay for cold compressed orange oil ..................................184
CHAPTER 5
Figure 5.1. Antibacterial activity of gentamicin against strains of S. Typhimurium, S. Heidelberg
and E. coli....................................................................................................................214
Figure 5.2. Gentamicin minimum inhibitory concentration against Salmonella Typhimurium
ATCC14028 ................................................................................................................216
Figure 5.3. Gentamicin minimum inhibitory concentration against S. Heidelberg ARI-14 .......217
Figure 5.4. Gentamicin minimum inhibitory concentration against E. coli 25922 .....................218
Figure 5.5. MIC of gentamicin based on CFU/mL for each strain .............................................219
LIST OF TABLES
CHAPTER 1
Table 1.1. The 10 most frequently reported Salmonella serovars from human infections reported
to CDC, 1998-2011 .......................................................................................................64
Table 1.2. The 10 most frequent serovars from analyzed PR/HACCP verification samples for
young chicken (broilers), 1998–2011 ...........................................................................65
CHAPTER 2
Table 2.1. Bacterial strains used in this study ...............................................................................90
Table 2.2. Optical density at 600 nm for Salmonella serovars under aerobic conditions .............97
Table 2.3. Optical density at 600 nm for Salmonella serovars under anaerobic conditions .........98
Table 2.4. Salmonella Heidelberg strains used in this study .......................................................102
Table 2.5. Primer pairs used in competitive growth studies .......................................................107
CHAPTER 3
Table 3.1. List of components for each media broth used for non-selective and selective
enrichment studies .......................................................................................................145
Table 3.2. Average CFU/mL for each Salmonella serovar inoculated on Luria Bertani broth ..148
Table 3.3. Average CFU/mL for each Salmonella serovar inoculated on Rappaport –Vassiliadis
broth ............................................................................................................................149
Table 3.4. Average CFU/mL for each Salmonella serovar inoculated on tetrathionate broth ....150
CHAPTER 4
Table 4.1. Salmonella Heidelberg specific primer pair used in this study ..................................176
Table 4.2. Salmonella Heidelberg strains used in this study .......................................................177
Table 4.3. Minimum inhibitory concentrations (in μg/mL) of five different EOs against
Salmonella Heidelberg strains .....................................................................................179
CHAPTER 5
Table 5.1. Salmonella strains used in this study .........................................................................212
Table 5.2. Zones of inhibition (in millimeters) of gentamicin against S. Heidelberg and S.
Typhimurium ..............................................................................................................213
Table 5.3. MIC of gentamicin against strains of S. Heidelberg, S. Typhimurium, and E. coli
examined .....................................................................................................................215
Table 5.4. Salmonella Typhimurium and S. Heidelberg adhesion and invasion to human
epithelial, Caco-2, cells ...............................................................................................220
Table 5.5. Adhesion and invasion of four Salmonella strains from different serovars to chicken
macrophage, HD11, cells ............................................................................................221
1
INTRODUCTION
Foodborne illnesses continue to be one of the primary public health concerns in the
United States. Annually, it is estimated that over 1 million Americans contract Salmonella
infections, resulting in approximately 35% of hospitalizations and 28% of deaths attributed to
foodborne pathogens (Scallan et al., 2011). Salmonella infections are typically contracted
through the consumption of contaminated food, water, or by direct contact with an infected host
(Alcaine et al., 2007). Eggs, poultry, poultry products, meat and meat products have all been
classified as common food vehicles of salmonellosis to humans (Ricke, 2003b; Patrick et al.,
2004; Braden, 2006; Dunkley et al., 2009; Foley et al., 2011; Finstad et al., 2012; Howard et al.,
2012; Koo et al., 2012; Alali et al., 2013). Salmonella are also associated with fresh fruits and
vegetables (Pui et al., 2011; Ganesh et al., 2010; Hanning et al., 2009). Almost all of the
Salmonella serovars that have been identified are of major concern to most sectors of the food
industry (Bell and Kyriakides, 2002).
The emergence and prevalence of Salmonella serovars in chickens has been studied, but
the predominant serovars have varied somewhat over the years. Understanding the basic growth
kinetics of Salmonella serovars and their respective strains is of great importance and serves as a
basis for in depth research. In this research, we studied a) the aerobic and anaerobic growth
responses of multiple strains from six different serovars, b) how the spent media from different
serovars, particularly S. Heidelberg, affect the growth of S. Typhimurium, and c) determined
whether or not two different serovars experience a competitive relationship when they are grown
together. Growth responses of four Salmonella serovars, Heidelberg, Kentucky, Typhimurium,
and Enteritidis in selective enrichment (Rappaport-Vassiliadis and Tetrathionate broth) and non-
2
selective enrichment (Luria Bertani broth) media were compared to determine the relative
effectiveness of the methods.
Historically, a number of antibiotics have been used to control foodborne pathogens but
the emergence of multidrug resistance pathogens has increased concerns over the consequences
and the impact in human health due to antibiotic resistance (Jones and Ricke, 2003; Kim et al.,
2005; Boerlin, 2010; Muthaiyan et al., 2011; Solórzano-Santos and Miranda-Novales, 2012).
Salmonella enterica serovar Heidelberg has become an important public health concern, since it
has been identified as one of the primary Salmonella serovars responsible for human outbreaks.
Because of this, we studied the effectiveness of five essential oils, orange terpenes, orange oil,
carvacrol, cinnamaldehyde, and R-(+)-limonene, against Salmonella Heidelberg strains from
multiple sources.
Adhesion of Salmonella to the intestinal epithelial surface is an important first step in
pathogenesis and is central to its colonization of the intestine. Understanding the ability of this
pathogen to attach and invade different cell types is of great importance. As a result, since
Salmonella is commonly known to be isolated from poultry and to cause human infections, we
studied the adhesion and invasion of four different strains to chicken macrophage (HD11), and
human epithelial (Caco-2) cells.
3
CHAPTER 1
Salmonella spp.
- Literature Review
Juliany Rivera Calo1,2
and Steven C. Ricke1,2
1Department of Food Science, University of Arkansas, Fayetteville, AR
2Center for Food Safety and Department of Food Science, University of Arkansas,
Fayetteville, AR
4
1. Abstract
Salmonella spp. are one of the more prominent foodborne pathogens that represent a
major health risk to humans, given they are facultative intracellular pathogens that cause
gastroenteritis. Many Salmonella serovars have been associated with the consumption of poultry
products, these being one of the main reservoirs of Salmonella (CDC, 2009). The emergence and
prevalence of Salmonella serovars in chickens has been studied but the predominant serovars
have varied somewhat over the years. Over time Salmonella spp. have developed resistance to
conventional and widely used antibiotics leading to considerable public health concerns.
Consequently, interest in more natural, non-synthesized, antimicrobials as potential alternatives
to conventional antibiotics to treat Salmonella infections has heightened. Aromatic plants and
their components have been examined as potential growth and health promoters and most of their
properties have been linked to essential oils (EOs) and other secondary plant metabolites.
Historically, EOs from different sources have been widely promoted for their potential
antimicrobial capabilities against microorganisms. In this review, multi-drug resistant
Salmonella strains, mechanisms of antimicrobial action, and the antimicrobial properties of plant
EOs, especially from citrus, are discussed, including their mode of action, effectiveness,
synergistic effects, major components and benefits.
5
2. Introduction
Foodborne illnesses continue to be one of the primary public health concerns in the
United States. Each year, it is estimated that over 1 million Americans contract Salmonella
infections (Scallan et al., 2011). Annual costs for Salmonella control efforts are estimated to be
up to $14.6 billion (Scharff, 2010; Heithoff, et al., 2012). Usually Salmonella infections are self-
limited in healthy individuals, only causing mild gastroenteritis with recovery occurring
anywhere from 4 to 7 days after initial exposure. Symptoms of salmonellosis include diarrhea,
fever, and abdominal cramps occurring 12 to 72 h after consumption of food containing the
pathogen (Finstad et al., 2012). Salmonella infections are typically contracted through the
consumption of contaminated food, water, or through direct contact with an infected host
(Alcaine et al., 2007). Eggs, poultry, poultry products, meat and meat products have all been
classified as common food vehicles of salmonellosis in humans (Ricke, 2003b; Patrick et al.,
2004; Braden, 2006; Dunkley et al., 2009; Hanning et al., 2009; Foley et al., 2011; Finstad et al.,
2012; Howard et al., 2012; Koo et al., 2012). Salmonella are also associated with contamination
of fresh fruits and vegetables (Pui et al., 2011; Ganesh et al., 2010; Hanning et al., 2009). Almost
all of the Salmonella serovars that have been identified are of major concern to most sectors of
the food industry (Bell and Kyriakides, 2002).
Historically, a number of antibiotics have been used to control foodborne pathogens but
the emergence of multidrug resistant pathogens has increased concerns over the consequences
and the impact in human health due to antibiotic resistance (Jones and Ricke, 2003; Kim et al.,
2005; Boerlin, 2010; Muthaiyan et al., 2011; Solórzano-Santos and Miranda-Novales, 2012).
Antibiotic resistance essentially confers the capacity of bacteria to inactivate or exclude
antibiotics or a mechanism that blocks the inhibitory or lethal effects of antibiotics (FDA, 2002).
6
Based on the definitions set by the FDA, antibiotics are drugs produced by a microorganism that
has the capacity, in dilute solutions, to inhibit the growth of or to kill other microorganisms;
while an antimicrobial compound is any substance that kills bacteria (bactericidal) or suppresses
(bacteriostatic) their multiplication or growth, including antibiotics and synthetic agents (FDA,
2002). Antimicrobials are probably one of the most successful forms of chemotherapy in the
history of medicine (Aminov, 2010).
Salmonella is not only a public health concern due to the significant number of cases per
year, but also because many strains are becoming more resistant to antimicrobial agents (Kim et
al., 2005; Alcaine et al., 2007; Foley and Lynne, 2008; Bajpai et al., 2012; Kollanoor-Johny et
al., 2012) due to the previous relatively unrestricted use of antimicrobials in feeds and increased
therapeutic use (Sørum and L’Abée-Lundm 2002; Su et al., 2004; Kim et al., 2005). Since their
discovery, antimicrobials have been extensively used in poultry and livestock for the treatment,
prevention and/or control of animal diseases, as well as for production purposes, for example to
improve feed efficiency and growth enhancement (Hur et al., 2012). The two mechanisms known
to be critical for the spread of antimicrobial resistance in Salmonella are 1) horizontal transfer of
antibiotic resistance genes and 2) clonal spread of antimicrobial drug-resistant Salmonella
isolates (Mølbak 2005; Alcaine et al., 2007). A major concern has been the acquired resistance to
cephalosporins (ceftriaxone and ceftiofur), since ceftriaxone is required to treat severe
salmonellosis in children and ceftiofur is the only antibiotic approved for veterinary use in the
U.S. (Bradford et al., 1999; Rabsch et al., 2001). Because of this, the development of strategies
that focus less on specific antibiotic administration to control pathogens is of major interest from
both a food safety and a medical perspective (Burt, 2004; Bajpai et al., 2012).
In recent years, aromatic plants and their extracts have been examined for their
7
effectiveness for food safety and preservation applications (Fisher and Phillips, 2008) and have
received attention as growth and health promoters (Brenes and Roura, 2010). Most of their
properties are due to their EOs and other secondary plant metabolite components (Brenes and
Roura, 2010). Phytochemicals, such as EOs, are naturally occurring antimicrobials found in
many plants that have shown to be effective in a variety of applications by decreasing growth
and survival of microorganisms (Callaway et al., 2011a). EOs exhibit antimicrobial properties
that may make them suitable alternatives to antibiotics (Chaves et al., 2008). These potential
attributes and an increasing demand for natural medicinal treatment options have led to an
interest in the use of EOs as potential alternative antimicrobials (Fisher and Phillips, 2008;
Solórzano-Santos and Miranda-Novales, 2012). Essential oils derived as by-products of the
citrus industry have been screened for antimicrobial properties against common foodborne
pathogens and several have been shown to possess antimicrobial properties (Dabbah et al.,
1970).
Historically, citrus oils have been incorporated into human diets (Dabbah et al., 1970;
Kim et al, 1995) because of their flavor and beneficial health properties and are approved as
generally recognized as safe (GRAS) compounds by the Food and Drug Administration (FDA),
due to their natural role as flavoring agents in citrus juices (Chalova et al., 2010; Hardin et al.,
2010; Callaway et al., 2011a). Their use as alternatives to chemical-based antimicrobials is not a
new concept as they have been used for medicinal purposes from ancient times into present day
applications and considerable research has been conducted to demonstrate their bioactivity
against bacteria, yeasts and molds (Fisher and Phillips, 2008). Approximately 400 compounds of
citrus oils have been identified from citrus and their content depends on the specific citrus
cultivar, separation and extraction methods (Caccioni et al., 1998; Robinson, 1999; Tao et al.,
8
2009). The most well known and characterized of the EOs from citrus products include citrullene
and limonene (Dabbah et al., 1970; Caccioni et al., 1998), which can exert potent, broad-
spectrum antimicrobial activity (Di Pasqua et al., 2006).
Citrus fruits, including oranges, contain a number of compounds, such as EOs, that are
toxic to bacteria, can exert antimicrobial activity and can alter the microbial ecology of the
gastrointestinal tract (Fisher and Phillips, 2006; Kim et al., 1995; Callaway et al., 2011a). In their
research, Callaway et al., (2008) reported that Salmonella populations were reduced by more
than 2 log colony forming units (CFU) per gram by the addition of up to 2% orange peel and
pulp in in vitro mixed ruminal microorganism fermentations. In addition to possessing
antimicrobial activity, citrus EOs can serve as optimal sources of antioxidants (Chalova et al.,
2010; Callaway et al., 2011a). Due to the antioxidant effect, the antimicrobial activity and other
health benefits of citrus consumption, there has been increased interest in these compounds for
additional applications (Callaway et al., 2011a). Little research has been done on how these
compounds affect the microbial ecosystem of the human gut or food animals, and the subsequent
impact in human health (Callaway et al., 2011a). In this review, multi-drug resistant Salmonella
strains, mechanisms of antimicrobial action, and the antimicrobial properties of plant EOs,
especially from citrus, are discussed, including their mode of action, effectiveness, synergistic
effects, major components and benefits. Since there have been numerous studies in terms of
Salmonella genetics and physiology, the stress and virulence characteristics of this organism
under antimicrobial treatment conditions has been well documented (Foley et al., 2011; 2013; Li
et al., 2013) and is beyond the scope of this review.
9
3. Foodborne Pathogen, Salmonella spp.
The genus name Salmonella was first suggested by Lignieres in 1900 in recognition of
the work carried out by the American bacteriologist, D.E. Salmon, who, with T. Smith in 1886,
described the hog cholera bacillus causing ‘swine plague’ (Topley and Wilson, 1929; D’Aoust,
1989). Salmonella species are Gram-negative, facultative anaerobic, flagellated, rod-shaped,
motile bacteria of the enterobacteria group, of approximately 2-3 x 0.4-0.6μm in size (Pui et al.
2011; Li et al., 2013). Salmonella can multiply under various environmental conditions outside
their respective living host (Park et al., 2008; Pui et al., 2011). Most Salmonella serovars grow at
a temperature range of 5 to 47°C with an optimum temperature of 35 to 37°C, but some can grow
at temperatures as low as 2 to 4°C and as high as 54°C (Gray and Fedorka-Cray, 2002). Given
the ability of Salmonella spp. to grow under a variety of different growth conditions and their
well-characterized genetics, they are a fairly ideal foodborne pathogen model for developing an
understanding of the mechanism(s) involved in the efficacy of antimicrobial compounds.
Classification of Salmonella is based on their susceptibility to different bacteriophages, as
well as grouping based on their somatic (O), flagellar (H), and capsular (Vi) antigenic patterns
(Dunkley et al., 2009; Pui et al., 2011). Salmonella H and O surface antigens can serve as typing
characteristics for potential reservoirs for each serovar (Hur et al., 2012). Currently the
Salmonella genus is composed of three species: S. enterica, S. bongori and S. subterranea
(Brenner, 1998; Su and Chiu, 2007; Foley et al., 2013). The species S. enterica is divided into six
subspecies: I (enterica), II (salamae), IIIa (arizonae), IIIb (diarizonae), IV (houtenae), and VI
(indica) based on biochemical and genetic properties (Le Minor and Popoff, 1987; Brenner,
1998; 2000). Salmonella bongori contains the members of subspecies V, and S. enterica consists
10
of members of the remaining six subgenera (Brenner, 1998; Foley et al., 2013). Salmonella
subterranea was described as a species in 2005 (Su and Chiu, 2007; Foley et al., 2013).
The Salmonella genus is widely heterogeneous, currently comprised of over 2,500
serovars (Popoff et al., 2004). The majority of the serovars that are pathogenic to human belong
to S. enterica, which contains over 99% of the serovars (Brenner et al., 2000; Tindall et al., 2005;
Hur et al., 2012). Members of this species have usually been named based on the isolation
geographical location of the serovar (Alakomi, 2007). Salmonella enterica subspecies I is mainly
isolated from warm-blooded animals and more than 99% of clinical isolated pertain to this
group, whereas the remaining subspecies are mostly isolated from cold-blooded animals and
account for less than 1% of clinical isolates (Pui et al., 2011; Bell and Kyriakides, 2002; Brenner
et al., 2000). Almost all of the Salmonella serovars that have been identified are of major
concern to nearly all sectors of the food industry (Bell and Kyriakides, 2002).
Salmonella cause gastroenteritis commonly referred to as salmonellosis, which can result
in a much more systemic infection, depending on several factors including susceptibility of the
host (Owens and Warren, 2012). Salmonella enterica serovars Typhimurium and Enteritidis
cause gastroenteritis and these two are the most common serovars responsible for human
infections (Olsen et al., 2001a; CDC, 2011; Dunkley et al., 2009; Foley et al., 2011; Finstad et
al., 2012; Howard et al., 2012). S. Enteritidis cases have often been associated with consumption
of contaminated eggs (Guard-Petter, 2001; Cogan et al., 2004; Howard et al., 2012). S.
Typhimurium cases are usually linked to the consumption of contaminated poultry, swine and
bovine meat (Jorgensen et al., 2000). Salmonella enterica serovar Typhi is the most invasive,
causing typhoid fever in humans. Some Salmonella strains may exhibit low infectious doses (500
or less) while others require 100,000 or more organisms to cause infection depending on the
11
virulence of the strain (Finstad et al., 2012). At least 106 to 10
9 cells are required to cause
salmonellosis in healthy human adults (Nester et al., 2009). Antibiotic therapy is not
recommended for simple cases of gastroenteritis (Hohmann, 2001). However for more systemic
infections, treatment with chloramphenicol may be needed (Li et al., 2013). Even though
chloramphenicol is used as treatment for systemic infections, a large number of cultures taken
from the bone marrow over the first week of treatment remained positive for Salmonella (Gasem
et al., 1995; Hussein Gasem et al., 2003). This persistence of Salmonella, potentially surviving
inside macrophages, could be responsible for a relapse of the infection (Sanders, 1965; Hussein
Gasem et al., 2003). In invasive life-threatening infections, the use of antimicrobial drugs is
required, but the efficacy of these drugs is decreasing due to the emergence of antimicrobial
resistant Salmonella strains (Shaw et al., 1993; Angulo et al., 2000; Winokur et al., 2000; Varma
et al, 2005a; Chen et al., 2007). Administration of antimicrobial agents such as ampicillin,
chloramphenicol, and tetracycline has become limited due to the increased resistance of
Salmonella to these agents. Fluoroquinolones, such as ciprofloxacin, or extended-spectrum
cephalosporins are commonly used to treat adult patients infected with Salmonella enterica or to
treat a severe case of gastroenteritis (Mølbak, 2005; Chen et al., 2007; Hur et al., 2012; Schmidt
et al., 2012). The emerging resistance to fluoroquinolones, the main treatment of severe
salmonellosis, has become a greater public health concern more recently (Olsen et al., 2001b;
Mølbak, 2005; Alcaine et al., 2007; Chen et al., 2007; Brichta-Harhay et al., 2011).
3.1. Epidemiology of Salmonella
Salmonella is a foodborne pathogen that causes gastroenteritis known as salmonellosis,
often resulting in systemic infection. Typhoid cases are stable with low numbers in developed
12
countries, but nontyphoidal salmonellosis has increased worldwide (Pui, et al., 2011). Annually,
it is estimated that over 1 million Americans contract Salmonella (Scallan, et al., 2011).
Epidemiologically Salmonella is classified into three groups based on the host
preferences that may include, 1) host-restricted, which are the serotypes capable of causing a
typhoid-like disease in a single host species (for example Salmonella Typhi in humans), 2) host-
adapted, which are serotypes associated with one host species, but also able to cause disease in
other hosts; some of these are human pathogens and may be contracted from foods (for example
S. Choleraesuis in swine and S. Gallinarum in poultry) and 3) unadapted serovars (no host
preference), which are pathogenic for humans and other animals, and they include most
foodborne serovars, for example S. Typhimurium (Boyen et al., 2008; Pui et al., 2011; Li et al.,
2013). Depending on the host and serotype, Salmonella can cause diseases ranging from mild
gastroenteritis to typhoid fever (Chiu et al., 2004; Humphrey, 2004). Antibiotic therapy is not
recommended for simple cases of gastroenteritis (Alcaine et al., 2007). In case of a systemic
infection, treatment with chloramphenicol may be needed (Shaw et al., 1993; Alcaine et al.,
2007).
3.2. Salmonella and Food Contamination
The primary sources of Salmonella are the gastrointestinal tract of humans, domestic and
wild animals, birds and rodents (Bell and Kyriakides, 2002). Because of this, they are
widespread in the natural environment including waters and soil, where they do not usually
multiply in a significant way but may survive for long periods (Bell and Kyriakides, 2002).
Salmonella enterica species are normally orally acquired pathogens, which cause one of four
major syndromes: enteric fever (typhoid), enterocolitis (gastroenteritis) or diarrhea, bacteremia
13
and chronic asymptomatic carriage (Coburn et al. 2007). Although the primary habitat of
Salmonella is the intestinal tract, it may also be found in other parts of the body and can also be
found in water. The organisms are excreted through the feces from which they can then be
transmitted by insects and others to a wide range of places (CDC, 2012).
Salmonella is among the most commonly isolated foodborne pathogens that are
associated with fresh fruits and vegetables (Pui et al., 2011). Eggs, poultry, meat and meat
products are the most common food vehicles of salmonellosis to humans. From 1996 to 1997,
four of the top-ranked Salmonella serovars were Typhimurium, Enteritidis, Heidelberg, and
Newport respectively (Olsen et al., 2001a).
After ingestion with contaminated food, symptoms usually develop in 12 to 14 hours, but
this time has been reported to be shorter or longer, up to 72 hours. Prevention of Salmonella can
be achieved by proper food handling, avoiding cross-contamination, personal hygiene and
sanitation and education to the public. Proper cooking following the recommended minimum
internal temperatures followed by prompt cooling to 3 to 4°C or freezing within 2 h would
eliminate Salmonella from foods (USDA/FSIS, 2013a).
3.3. Pathogenesis of Salmonella
Salmonella growth and virulence responds to a series of factors such as pH, oxygen
availability and osmolarity. When there is a lack of nutrients, such as carbon sources, this can
trigger global stress responses in Salmonella and a change in gene expression patterns can be
seen toward a more increased virulence and survivability (Altier, 2005; Kundinger et al., 2007).
Some of the genes necessary for invasion of intestinal epithelial cells and induction of
intestinal secretory and inflammatory responses are encoded in Salmonella Pathogenicity Island
14
1 (SPI-1) (Watson et al., 1995; Hur et al., 2012). Salmonella Pathogenicity Island 2 (SPI-2) is
key for the establishment of systemic infection beyond the intestinal epithelium; it also encodes
essential genes for intracellular replication (Cirillo et al., 1998; Ohl and Miller, 2001; Hur et al.,
2012). Adhesion of Salmonella to the intestinal epithelial surface is an important first step in
pathogenesis and is central to its colonization of the intestine. Once Salmonella is attached to the
intestinal epithelium, normally it expresses a multiprotein complex, known as T3SS, that
facilitates endothelial uptake and invasion (Foley et al., 2008, 2013; Winnen et al., 2008). This
type III secretion system (T3SS) is associated with SPI-1, which contains virulence genes
associated in Salmonella adhesion, invasion and toxicity (Foley et al., 2013). The human
intestinal Caco-2 cell line has been extensively used over the last twenty years as a model of the
intestinal barrier. The parental cell line, originally obtained from a human colon
adenocarcinoma, goes through a spontaneous differentiation process that leads to the formation
of a monolayer of cells, expressing several morphological and functional characteristics of the
mature enterocyte (Sambuy et al., 2005). The underlying mechanisms used by intracellular
pathogens such as Salmonella to penetrate the host epithelium are not well understood. As a
result, researchers have used cultured mammalian cells as in vitro models to study interaction
and internalization of Salmonella (Gianella et al., 1973; Finlay et al., 1988; Durant et al., 1999).
Invasion in cultured epithelial cells is commonly used to measure the pathogenicity of
Salmonella (van Asten et al., 2000, 2004; Shah et al., 2011). Previous studies have reported that
Salmonella invasion into cultured mammalian cells can be influenced by several environmental
stimuli such as osmolarity (Galán and Curtis, 1990; Tartera and Metcalf, 1993) carbohydrate
availability (Schiemann, 1995), and oxygen availability (Ernst et al., 1990; Lee and Falkow,
1990; Francis et al., 1992). In their research, Durant et al., (1999) reported that Salmonella can
15
encounter high concentrations of short-chain fatty acids (SCFA) in the lower regions of the
gastrointestinal tract, this in addition to changes in pH, oxygen tension, and osmolarity. Since a
combination of environmental conditions are what regulate virulence gene expression, SCFA
may contribute to the environmental stimuli that regulate cell-association and invasion of S.
Typhimurium epithelial cells (Durant et al., 1999). Shah et al., (2011) reported that in cultured
Caco-2 cells, isolates with high invasiveness were able to invade and/or survive in significantly
higher numbers within chicken macrophage cells than other isolates with low invasiveness.
HD11 cells are a macrophage-like immortalized cell line derived from chicken bone
marrow and transformed with the avian myelocytomatosis type MC29 virus (Beug et al., 1979).
In their research, He et al., (2012) compared Salmonella cell invasion and intracellular survival
of five different poultry-associated serovars (S. Heidelberg, S. Enteritidis, S. Typhimurium, S.
Senftenberg, and S. Kentucky) to HD11 cells. Their results showed that S. Enteritidis was more
resistant to intracellular killing, leading them to believe that the intracellular survival ability of
this serovar may be related with systemic invasion in chickens (He et al., 2012). Shah et al.,
(2011) suggested that not all isolates of S. Enteritidis recovered from poultry might be equally
pathogenic or have similar potential to invade cells; and that S. Enteritidis pathogenicity is
related to both the secretion of T3SS effector proteins and motility. Saeed et al., (2006) reported
that compared to isolates of S. Enteritidis recovered from chicken ceca, isolates recovered from
eggs or from human clinical cases exhibited a greater adherence and invasiveness of chicken
ovarian granulosa cells. Several pathogens and host factors may be an important key to
determine the mechanisms for the differences in Salmonella responses within different cell types
(Bueno et al., 2012; Foley et al., 2013). Understanding the ability and mechanisms of this
16
pathogen to attach and invade different cell lines could be helpful for the reduction of Salmonella
infections.
3.4. Competitive Interactions among Serovars and Spent Media
A significant shift in the predominant Salmonella serovars associated with poultry and
human infections had been seen over the last several decades. S. Enteritidis and S. Heidelberg are
among the top five serovars associated with human infections and some of the most commonly
detected serovars in chickens over the last 25 years. S. Enteritidis remains as a significant
problem in commercial poultry, but its prevalence has declined in chickens in the U.S. since the
mid 1990s. From 1997 to 2006 S. Heidelberg supplanted S. Enteritidis as the predominant
serovar, but for 2007 S. Kentucky became the most commonly isolated serovar (Foley et al.,
2008). This serovar is less commonly identified as a source of human salmonellosis and it is
unclear why it has become a predominant colonizer in chicken ceca but has not been a significant
threat to humans as are S. Typhimurium, S. Enteritidis or S. Heidelberg. Although S. Kentucky is
not among the most common serovars causing human diseases, it has a significant prevalence of
multidrug resistance isolates from poultry and humans in other parts of the world (Foley et al.,
2011).
Spent media is a growth medium exhausted of nutrients due to extensive growth of
bacteria species and is known to influence the gene expression patterns of S. Typhimurium. The
filtered supernatant of this media has the potential to serve as an in vitro screen of ecosystems
after growth of either specific microorganisms or a microbial consortium from a specific in vivo
site such as the gastrointestinal tract (Nutt et al., 2002; Kundinger et al., 2007). The use of spent
17
media has been previously applied to simulate chicken gastrointestinal tract conditions after feed
withdrawal (Nutt et al., 2002).
3.5. Salmonella in Poultry
Numerous Salmonella serovars have been associated with the consumption of poultry
products, these being one of the main reservoirs of Salmonella (Shah et al., 2011; He et al.,
2012). Contaminated poultry, meat, and eggs are important vehicles of Salmonella infections,
especially when the organism is in the egg contents (Foley et al., 2011). Kuehn (2010) reported
this contamination problem when a salmonellosis outbreak caused by S. Enteritidis was traced
back to contaminated eggs from Iowa. Some of the factors that can affect the colonization of
Salmonella in poultry include the age and genetic susceptibility of the birds, bird stress due to
overcrowding or underlying illness, level of exposure to the pathogen, competition with gut
microflora for the colonization sites, infecting Salmonella serovar, and whether or not the strains
carry genetic factors that may facilitate the attachment to the gastrointestinal tract of the bird or
evade host defenses (Bailey, 1988). In the past few years, the emergence and prevalence of
Salmonella serovars in chickens has been studied but the predominant serovars have varied
somewhat over the years (Foley et al., 2008; 2011; 2013).
3.6. Salmonella Drug-resistant Strains
A microbial strain is considered resistant to a particular antimicrobial when the
antimicrobial is no longer efficacious for treatment of a clinical disease caused by a particular
bacterial pathogen (Alcaine et al., 2007). Extensive research with the purpose of characterizing
antimicrobial resistance strains of Salmonella enterica from different food and animal sources
18
has revealed that numerous serotypes may have multiple antimicrobial resistance determinants
(White et al., 2001; Zhao et al., 2007; Brichta-Harhay et al., 2011). Multi-drug resistant
Salmonella (MDR) strains are defined as those strains which are resistant to two or more
antimicrobial agents. These may carry their resistance determinants on chromosomal sites,
resistance gene carrying plasmids or on both (Chen et al., 2004; Alcaine et al., 2007; Lindsey et
al., 2009). Resistance to antimicrobial compounds can result from one or more target gene
mutations (Condell et al., 2012b). Overall the human clinical Salmonella isolates resistant to one
or more drug classes declined from 1996 to 2003 in the United States (CDC, 2004b). Moreover,
in 2011 the CDC reported that resistance to one or more drug classes was lower than during the
period of 2003 to 2007 (CDC, 2013).
However, infections by MDR Salmonella remain a concern due to potential treatment
difficulties resulting in human infections with these strains being more severe and patients in
turn, are at greater risk of bacteremia, hospitalization and death compared to patients infected
with antibiotic susceptible strains (Helms et al., 2002; CDC, 2004b; Mølbak, 2005; Talbot et al.,
2006). Eight of the ten serotypes most commonly reported by the CDC, include at least some
isolates that displayed resistance to at least five or more antimicrobial drugs (CDC, 2004a;
2004b). Salmonella serotypes reported with the highest multidrug resistance were Typhimurium,
Heidelberg, and Newport (CDC, 2004a). The frequency of MDR in Typhimurium and Newport
is reportedly increasing (Zhao et al., 2008; Hur et al., 2012). Additionally, serovars such as
Agona, Anatum, Cholerasuis, Derby, Dublin, Kentucky, Pullorum, Schwarzengrund, Seftenberg,
and Uganda also contain MDR Salmonella strains (Chen et al., 2004; Zhao et al., 2008; Hur et
al., 2012).
19
There are two common resistance patterns for MDR Salmonella Typhimurium isolates:
1) resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline or 2)
resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline, the
resistance profile most commonly associated with S. Typhimurium DT104 (CDC, 2004a).
Salmonella Heidelberg isolates were typically characterized by resistance to ampicillin,
amoxicillin-clavulanic acid, ceftiofur, and cephalothin (CDC, 2004a).
The difference between these two resistance patterns for MDR S. Typhimurium is that
one has kanamycin and the other one has chloramphenicol. These antibiotics pertain to different
families of antibiotics, and each of them possesses a specific mechanism of action. Streptomycin
and kanamycin belong to the aminoglycosides family, which bind to conserved sequences within
the 16S rRNA of the 30S ribosomal subunit, leading to a codon misreading and translation
inhibition (Alcaine et al., 2007). Aminoglycoside resistance in Salmonella has been associated
with the expression of aminoglycoside-modifying enzymes. Ampicillin belongs to the beta-
lactam family, where the ability to interfere with the penicillin-binding proteins (involved in the
synthesis of peptidoglycan) confer antimicrobial effects on the target microorganism (Alcaine et
al., 2007). In Salmonella, the mechanism of resistance to beta-lactams that has been most
commonly described involves the secretion of beta-lactamases (Alcaine et al., 2007).
Chloramphenicol belongs to the phenicols family and its mode of action is via the
prevention of peptide bond formation. There are two resistance mechanisms in Salmonella from
this antibiotic: 1) enzymatic inactivation of the antibiotic and 2) removal of the antibiotic via an
efflux pump. The mechanism of action for tetracyclines is by inhibiting protein synthesis; and its
resistance to Salmonella is conferred by production of an energy-dependent efflux pump, which
excretes the antibiotic from the bacterial cell (Alcaine et al., 2007). Sulfamethoxazole belongs to
20
the sulfonamides family of antibiotics, which act by inhibiting enzymes that are involved in the
synthesis of tetrahydrofolic acid. Resistance in Salmonella is due to the presence of an extra sul
gene, which expresses an insensitive form of dihyrdropteroate synthetase (DHPS) (Alcaine et al.,
2007).
Varma et al., (2005b) reported that antimicrobial resistance in nontyphoidal Salmonella is
characterized by an increased frequency of bloodstream infections and hospitalizations of the
corresponding patients. Bloodstream infections have been reported to occur more frequently
among people infected with a nontyphoidal Salmonella isolate resistant to more than one
antimicrobial agent (CDC, 2003a; CDC, 2003b). Human-health consequences of increasing
resistance may be substantial if antimicrobial-resistant infection increases the risk of bloodstream
infection (Varma et al., 2005b). Previous research has revealed a strong association between
resistance, bloodstream infection, and hospitalization (Holmberg et al., 1984; 1987; Lee et al.,
1994; Helms et al., 2002; WHO, 2005; Varma et al., 2005b). Patients who become infected with
resistant Salmonella isolates are more prone to experience failure when administered
antimicrobial therapy, resulting in a more invasive illness.
3.7. Mechanisms of Antimicrobial Action
Antimicrobial agents are not essential to treat most cases of Salmonella infections, but
they are more likely to be essential at times of severe or systemic infection (Chen, et al., 2007).
In bacteria, resistance to antimicrobial agents may be mediated by a variety of mechanisms such
as (1) energy-dependent removal of antimicrobials via membrane-bound efflux pumps, (2)
changes in bacterial cell permeability, which restricts the access of antibiotics to target sites
(Ravishankar et al., 2010) (3) modifications of the site targeted by drug action, and (4)
21
inactivation or destruction of antimicrobials (Chen et al., 2004). It has been suggested that the
high resistance level of Salmonella to fluoroquinolones is due to the combination of two major
resistance mechanisms, active efflux mediated by AcrAB-Tolc and multiple target gene
mutations (Velge et al., 2005; Hur et al., 2012). In the case of tetracyclines, bacteria normally
acquire resistance to these antibiotics through an efflux mechanism by which the drug is pumped
out from the cell (Kim et al., 2005). In their research, Hur et al., 2012 described the resistance to
tetracycline and chloramphenicol to be highly associated with the acquisition and expression of
efflux pumps, which are responsible of reducing toxic levels of the drug in the bacterial cells.
To inhibit microbial growth, antimicrobials tend to act on several specific target sites
such as inhibition of synthesis of specific proteins, DNA and RNA synthesis, interference with
cell wall synthesis, disruption of membrane permeability structure and inhibition of essential
metabolite synthesis (Hughes and Mellows, 1978; Reynolds, 1989; Falla et al., 1996; Hooper,
2001). For example, antibiotics such as penicillins and cephalosporins inhibit bacterial cell wall
synthesis by interference with the enzymes associated with peptidoglycan layer synthesis; while
tetracyclines and streptogramins work by disrupting protein synthesis (Tenover, 2006). Due to
their different biological structures and properties, target sites for each antimicrobial could vary
(Li et al., 2011) depending on the type of microorganism that is being exposed to the respective
antimicrobial agent. The potential target site for Gram-positive cocci involves the cell wall,
cytoplasmic membrane, functional proteins, DNA and RNA. In the case of Gram-negative
bacteria they share the same target sites, except that the target is the outer membrane instead of
the cell wall (Li et al., 2011).
The continuous and widespread use of antibiotics are considered two of the important
factors that have led to the rapid increase of antibiotic resistance; resulting in more demand to
22
either replace or at least reduce the use of antibiotics by using more natural, non synthesized,
alternatives (Bajpai et al., 2012; O’Bryan et al., 2008b; Li et al., 2011).
4. Natural Antimicrobials against Salmonella
Control of food spoilage and pathogenic bacteria is mainly achieved by chemical control
but the use of synthetic chemicals is limited due to undesirable aspects including carcinogenicity,
acute toxicity, teratogenicity and slow degradation periods, which could lead to environmental
problems, such as pollution (Faleiro, 2011). The negative public perception of of industrially
synthesized food antimicrobials has generated interest in the use of more naturally occurring
compounds (Sofos et al., 1998; Faleiro, 2011). There has been an extensive search for potential
natural food additive candidates that retain a broad spectrum of antioxidant and antimicrobial
activities while possessing the ability to improve the quality and shelf life of perishable foods
(Fratianni et al., 2010). The emergence of bacterial antibiotic resistance and the negative
consumer attitudes toward food preservatives have led to an increased interest in the use of plant
components that contain EOs and essences as alternative agents for the control of food spoilage
and harmful pathogens (Shelef, 1983; Smith-Palmer et al., 1998; 2001; Burt, 2004; Nostro et al.,
2004; Fisher and Phillips, 2008). There are certain limitations to the use of antimicrobial plant
oils in livestock waste, for example their cost effectiveness is not clear, there could be potential
environmental effects and/or there could be little to no degradation of the waste (Varel, 2002).
Despite these limitations, Varel (2002) described some potential advantages of using natural
antimicrobial chemicals such as EOs including: 1) inhibition of microbial fermentation of waste,
hence reducing the rate of odor emissions as well as the production of global warming gases, and
thus conserve nutrients in the waste for increased fertilizer value; 2) destruction of pathogenic
23
fecal coliforms; 3) the phenolic plant oils are stable under anaerobic conditions, although they
degrade under aerobic environments; 4) these products are classified as GRAS; and 5) these oils
can also be used as pesticides, which could play a significant role in controlling flies in livestock
waste.
Natural compounds with animal, plant or microbiological origins have been used in order
to kill or at least prevent the growth of pathogenic microorganisms (Roller and Lusengo, 1997;
Li et al., 2011, Muthaiyan et al., 2011; Juneja et al., 2012). A number of naturally occurring
antimicrobial agents are present in animal and plant tissues, where they probably evolved as part
of their hosts’ defense mechanisms against microbiological invasion and exist as natural
ingredients in foods (Sofos et al., 1998).
4.1. Antimicrobials from Plant Sources
Substances that are naturally occurring and directly derived from biological systems
without alteration or modification in a laboratory setting are recognized as natural antimicrobials
(Lopez-Malo et al., 2000; Sirsat et al., 2009; Li et al., 2011). An ideal antimicrobial would be
one that is available in large volumes as a co-product and one that has GRAS status because it
has already been part of the typical human diet for years (Friedly et al., 2009; Nannapaneni et al.,
2009b; Chalova et al., 2010; Callaway et al., 2011a). There is great potential for new drug
discoveries based on collecting and characterizing traditional medicinal plants throughout the
world (Lewis and Elvin-Lewis, 1995). Some plant extracts such as Garcinia kola and Vernonia
amygdaina have previously been investigated for their phytochemical properties and
antibacterial effects on bacterial pathogens such as Escherichia coli, Klebsiella pneumonia,
Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus.
24
Chemicals deployed by plants against bacteria to obtain an advantage in their ecosystem include:
EOs, organic acids, and specific toxins (Friedman et al, 2002; Callaway et al., 2011a).
The use of pesticides in farming and the use of growth hormones and antibiotics in
livestock production (Magkos et al., 2003; Van Loo et al., 2010), lower environmental impact
and animal welfare practices (Magnusson et al., 2003; Van Loo et al., 2010); and quality and
safety of organic foods are some of the factors that meet consumers concerns and increase the
demand of organic foods (Magkos et al., 2006; Van Loo et al., 2010). Most of the research on
organic foods has concluded that there is no evidence that organic food is safer, healthier, or
more nutritious (Magkos et al. 2003; Van Loo et al., 2012). Organic foods are required to meet
the same food safety standards as foods that are conventional; there are no stricter food safety
requirements (Van Loo et al., 2012).
The United States Department of Agriculture (USDA) defines organic food as: “Organic
food is produced by farmers who emphasize the use of renewable resources and the conservation
of soil and water to enhance environmental quality for future generations. Organic meat, poultry,
eggs, and dairy products originate from animals that are given no antibiotics or growth
hormones. Organic food is produced without using most conventional pesticides; fertilizers made
with synthetic ingredients or sewage sludge; bioengineering; or ionizing radiation. Before a
product can be labeled ‘organic,’ a government-approved certification agent inspects the farm
where the food is grown to confirm that the farmer is following all the rules necessary to meet
USDA organic standards. Companies that handle or process organic food before it gets to your
local supermarket or restaurant must be certified, too” (USDA, 2013).
In their survey, Van Loo et al., (2010) reported that consumers leaned towards buying
organic chicken due to their perception that organic chicken is safer, healthier and has fewer
25
pesticides, antibiotics and hormones. Van Loo et al., (2011) assessed consumer’s willingness to
pay for organic chicken, and their results demonstrated that consumers were willing to pay 35%
premium for a general organic labeled chicken breast and 104% for a USDA certified organic
labeled based on an overview of current published research reports. Van Loo et al., (2012)
concluded that consumers should not assume that chickens labeled as organic, all natural, or free
range are any less contaminated with Salmonella than conventionally raised chickens. Crandall
et al., (2011) surveyed a total of 305 consumers to determine their concerns and beliefs about the
safety of foods obtained at three Arkansas farmers’ markets. Results showed that 36% of the
consumers preference for organic foods was that they wanted food free of chemicals; 45% of the
consumers biggest safety concerns were pesticides; and only 2 to 6% were actually concerned
about foodborne pathogens. A total of 76% of the surveyed consumers believed that organic
foods were safer than conventional foods (Crandall et al., 2011).
Based on health, economic and environmental issues, natural plant extracts represent a
safer choice as an alternative for synthetic antimicrobials (Burt, 2004; Jo et al., 2004; Callaway
et al., 2011a; Muthaiyan et al., 2011; Bajpai et al., 2012). Citrus products and other EOs sources
can be used as a green alternative to reduce antibiotic use (Callaway et al., 2011a; Warnke et al.,
2009). Including essential citrus oils in animal feeds and human foods was reported to be a
means to improve human health, also to be a green solution and economically feasible (Callaway
et al., 2011a). Jo et al., (2004) reported that lyophilized citrus extracts might be useful as natural
antioxidants to benefit health and also have the potential to be environmentally friendly and cost
effective. These naturally occurring antimicrobials have extensive histories of their use in foods
and can be identified from various components of the plants leaves, barks, stems, roots, flowers
and fruits (Rahman and Gray, 2002; Erasto et al., 2004; Zhu et al., 2004).
26
A wide range of products derived from plant sources (e.g., fruit preparations, vegetable
preparations or extracts and spices) have been used throughout history for the preservation and
extension of the shelf life of foods (Billing and Sherman, 1998; Cutter, 2000; McCarthy et al.,
2001; Islam et al., 2002; Draughon, 2004; Kim et al., 2011). Plant EOs have been widely used
for a variety of medicinal and cosmetic purposes (Crandall et al., 2012). It has been recognized
that certain plant extracts, including their EOs and essences possess antimicrobial properties
(Smith-Palmer et al., 1998; 2001; Brenes et al., 2010; Kim et al., 2011; Shannon et al., 2011b;
Solórzano-Santos and Miranda Novales, 2012) against bacteria, yeast and moulds (Smith-Palmer
et al., 1998).
5. Essential Oils
5.1. Properties
Historically, EOs and their components isolated from aromatic and medicinal plants
possess antimicrobial properties when used for health and food preservation (Faleiro, 2011).
Essential oils are natural, complex, multi-component systems composed mainly of terpenes in
addition to other non-terpene components (Edris, 2007). Essential oils are concentrated oils
containing volatile aroma compounds such as terpenes of plant material that are obtained by
solvent extraction, or fermentation; but the most common method used is steam distillation
(Burt, 2004; Kelkar et al., 2006; Edris, 2007; Faleiro, 2011; Shannon et al., 2011a; Solórzano-
Santos and Miranda Novales, 2012). During the juice extraction from the oranges, cold pressed
oils are recovered from the orange peels. The essence oils are what are collected during the
process while the orange juice is being evaporated into concentrate (Crandall and Hendrix 2001).
27
Bakkali et al., (2008) defined EOs as liquid, volatile, limpid and rarely colored, lipid
soluble and soluble in organic solvents with a usually lower density than that of water. The
composition of EOs is influenced by the respective extraction methods and the antimicrobial
properties of these are determined by the components recovered in the particular extraction
process (Burt, 2004). They can also act as biopreservatives, reducing or eliminating pathogenic
bacteria and increasing the overall quality of animal and vegetable food products (Solórzano-
Santos and Miranda-Novales, 2012).
Essential oils are primarily used for fragrances, flavors as well as pharmaceuticals due to
their chemopreventive and chemotherapeutic activities, antioxidants activities against low
density lipoproteins, antiviral and antidiabetic activities and even potential as insect repellents
(Burt, 2004; Edris, 2007; Nannapaneni et al., 2008; Chalova et al., 2010; Callaway et al., 2011a;
Jacob and Pescatore, 2012). Essential oils also retain characteristic odor and flavor of the plant
they were extracted from (Jacob and Pescatore, 2012). Approximately 3000 EOs are known, of
which approximately 300 are commercially important and used for the flavours and fragrances
market (Burt, 2004). Essential oils have been documented to be effective antimicrobials against
several foodborne pathogens including Escherichia coli O157:H7, Salmonella Typhimurium,
Staphylococcus aureus, Listeria monocytogenes, Campylobacter and others (Friedman et al.,
2002; Moreira et al., 2005; Callaway et al., 2008; Nannapaneni et al., 2008; O’Bryan et al.,
2008a; Friedly et al., 2009; Nannapaneni et al., 2009a; 2009b; Soković et al., 2010; Pittman et
al., 2011b; Shannon et al., 2011a; 2011b; Callaway et al., 2011b; Callaway et al., 2011c;
Muthaiyan et al., 2012; Pendleton et al., 2012).
28
5.2. Mode of Action of Essential Oils
The antimicrobial effects of EOs have been constantly screened in a wide range of
microorganisms, but their mechanism(s) of action are still not completely understood. Several
mechanisms have been proposed to explain the chemical compounds contained in the EOs (Cox
et al., 2000; Burt, 2004). Because EOs contain several components, their antimicrobial activity
cannot be confirmed based on the action of only one compound (Bajpai et al., 2012). Several
researchers have proposed that the antimicrobial action of EOs may be attributed to their ability
to penetrate through bacterial membranes to the interior of the cell and exhibit inhibitory activity
on the functional properties of the cell, and to their lipophilic properties (Smith-Palmer et al.,
1998; Fisher and Phillips, 2009; Guinoiseau et al., 2010; Bajpai et al., 2012). The phenolic nature
of EOs also elicits an antimicrobial response against foodborne pathogen bacteria (Shapira and
Mimran, 2007; Bajpai et al., 2012). Phenolic compounds disrupt the cell membrane resulting in
the inhibition of the functional properties of the cell, and eventually cause leakage of the internal
contents materials of the cell (Bajpai et al., 2012). The mechanisms of action may be the ability
of phenolic compounds to alter microbial cell permeability, damage cytoplasmic membranes,
interfere with cellular energy (ATP) generation system, and disrupt the proton motive force
(Burt, 2004; Friedly et al., 2009; Li et al., 2011; Bajpai et al., 2012). The disrupted permeability
of the cytoplasmic membrane can result in cell death (Li et al., 2011). An important
characteristic of EOs and their components is hydrophobicity, allowing the EOs to separate the
lipids of the bacterial cell membrane and mitochondria and in the process causing the bacterial
cell to become more permeable (Burt, 2004; Friedly et al., 2009).
The interaction of EOs with microbial cell membranes results in the growth inhibition of
some Gram-positive and Gram-negative bacteria (Calsamiglia et al., 2007). Gram-positive
29
bacteria such as Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus are more
susceptible to EOs than Gram-negative bacteria such as E. coli and S. Enteritidis
(Chorianopoulos et al., 2004). It is generally believed that EOs mechanistically should be more
effective against Gram-positive bacteria due to the direct interaction of the cell membrane with
hydrophobic components of the EOs (Shelef, 1983; Smith-Palmer et al., 1998; Chao and Young,
2000; Cimanga et al., 2002; Soković et al., 2010). Conversely, based on this premise, Gram-
negative cells should be more resistant to plant oil because they possess a hydrophilic cell wall
(Kim et al., 2011). This outer layer helps to prevent the penetration of hydrophobic compounds
(Calsamiglia et al., 2007; Ravichandran et al., 2011). Deans and Ritchie (1987) concluded that
Gram-positive and Gram-negative bacteria were equally sensitive to citrus EOs and their
components.
In a study by Dorman and Deans (2000) carvacrol and thymol acted differently against
Gram-positive and Gram-negative bacteria. Carvacrol and thymol were able to cause
disintegration of the outer membrane of Gram-negative bacteria, releasing lipopolysaccharides
(LPS) and increasing the permeability of the cytoplasmic membrane to ATP (Burt, 2004).
Previous studies by Ravishankar et al., (2010) suggested that the various S. enterica isolates may
not be equally susceptible to inactivation, and that the antimicrobials may act by similar
mechanisms against resistant and susceptible S. enterica isolates. In their research, Ravishankar
et al., (2010) stated that the use of cinnamaldehyde did not cause the bacterial outer membrane to
disintegrate and suggested that instead, cinnamaldehyde may interfere with the activity of some
enzymes. When comparing inactivation susceptibility of the bacteria because of the EOs,
Ravishankar et al., (2010) found that antibiotic-resistant isolates exhibited similar susceptibility
to inactivation as the susceptible isolates.
30
Factors present in complex food matrices such as fat content, proteins, water activity, pH,
and enzymes can potentially diminish the efficacy of EOs (Burt, 2004; Firouzi et al., 2007;
Friedly et al., 2009). According to Vigil (2001) low pH can increase the solubility and stability
of EOs, enhancing the antimicrobial activity. Additional methods to enhance EOs activity
include increasing salt content, and decreasing storage temperatures (Burt, 2004; Friedly et al.,
2009). Sivropoulou et al. (1996) reported that the antimicrobial effect of EOs is also
concentration dependent. Ravishankar et al., (2010) observed that at a 0.2% concentration,
antimicrobials, carvacrol and cinnamaldehyde completely inactivated the antibiotic-resistant and
-susceptible isolates of S. enterica. The antibacterial effects of organic acids increase as the
concentration increases (Dickson and Anderson, 1992). Organic acids, chlorine dioxide, and
trisodium phosphate are classified as safe (GRAS) and effective sanitizers (Cutter, 2000).
Besides being used as food additives and preservatives, organic acids and their salts also have
food processing applications (Ricke, 2003a; Ricke et al., 2005; Milillo et al., 2011). Organic
acids have several different antimicrobial mechanisms, such as disruption of intracellular pH,
osmotic stress, and membrane perturbation (Russell, 1992; Hirshfield et al., 2003; Ricke, 2003a).
Friedly et al., (2009) observed significant synergistic antimicrobial properties against Listeria by
combining citrus EOs with four different organic acids, citric, malic, ascorbic and tartaric acid,
making citrus EOs a more attractive antimicrobial control measure. Their study revealed that low
concentrations of citrus EOs in combination with organic acids could be effective to control
Gram-positive microbial growth, in this case Listeria. By combining organic acids and EOs in
Listeria, Friedly et al., (2009) concluded that their findings would allow for a reduction of the
concentration of EOs by more than 10-fold. Zhou et al., (2007b) reported that the combination of
different antimicrobials such as thymol or carvacrol with EDTA, thymol or carvacrol with acetic
31
acid, and thymol or carvacrol with citric acid all resulted in significantly reduced populations of
S. Typhimurium. In samples treated with combinations, these antimicrobials exhibited
synergistic effects compared with samples treated with thymol, carvacrol, EDTA, acetic acid, or
citric acid alone (Zhou et al., 2007b).
The production of off-flavor or strong odor limits the use of EOs as food preservatives to
increase the safety and shelf life of food products (Friedly et al., 2009; Tiwari et al., 2009;
Soković et al., 2010; Bajpai et al., 2012; Solórzano-Santos and Miranda-Novales, 2012). As a
result of this, an inhibitory dose (minimum concentration at which no bacterial growth will be
observed) instead of a bactericidal dose (which will kill the bacteria) is usually applied
(Chorianopoulos et al., 2006, Li et al., 2011). McMahon et al., (2007) reported that the increased
use of bacteriostatic (sublethal), rather that bactericidal (lethal), food preservation systems may
contribute to the development and dissemination of antibiotic resistance in food related
pathogens. High doses of EOs (0.05% v/v) have been shown to have a cytotoxic effect on Caco-2
cells and actually increase the damage to the tissue cell population caused by E. coli, although
medium to low doses (≤ 0.01%) of thyme, oregano and carvacrol over a short period cause no
detectable damage, which emphasizes the necessity to find a balance between the risk of food
poisoning and/or food spoilage and the required dose of EOs to have an effect (Fisher and
Phillips, 2008). In their research, Firouzi et al., (2007) reported that although in vitro work with
EOs and their components indicated that compounds such as oregano and nutmeg possessed
substantial antimicrobial activity, when used in food systems the amounts required were
approximately 1 to 3% higher, often higher than what would normally be organoleptically
acceptable (Firouzi et al., 2007).
32
Tassou and Nychas (1996) found that it required 10 times as much mastic gum to achieve
the same results in pork sausages as compared to in vitro. Mastic gum is a white,
semitransparent, natural resin obtained as a trunk exudate from mastic trees Pistacia lentiscus
(Koutsoudaki et al., 2005). Mastic gum possesses numerous qualities, such as antimicrobial,
antioxidant, anti-inflammatory, anticancer; and used as a health promoter, used in cosmetic
industry, culinary, toothpaste and lotions, among others (Koutsoudaki et al., 2005; Giaginis and
Theocharis, 2011; Dimas et al., 2012). The in vitro antimicrobial activity of P. lentiscus extracts
has also been tested on bacteria and fungi (Koutsoudaki et al., 2005). Huwez et al., (1998)
reported that 1 mg of mastic gum per day could cure peptic ulcers and that mastic gum could kill
Helicobacter pylori, which was in turn correlated with their effect on peptic ulcers. Al-Said et
al., (1986) reported that an oral dose of 500 mg/kg significantly reduced gastric secretions,
protected cells and the intensity of gastric mucosal damage. Mastic gum can also act as an oral
antiseptic, tighten gums and prevent or reduce plaque formation (Topitsoglou-Themeli et al.,
1984). Moreover, mastic gum has also been applied for culinary uses, for example, in biscuits,
ice cream, and mastic “sweets of the spoon” (Koutsoudaki et al., 2005). Smith-Palmer et al.
(1998) found that foods with higher lipid content required more EOs to inhibit bacterial growth.
Further studies will be needed to confirm the effects of these orange EOs in vivo as well as
incorporating them into multiple-hurdle approaches to improve food safety for meat products
(Ricke et al., 2005; Ganesh et al., 2010; Sirsat et al., 2011).
5.3. Essential Oils as a Pre-harvest Intervention
The use of antimicrobials as a pre-harvest intervention, before crops or livestock products
are sold, is an important part of animal production and food safety. These interventions have
33
been examined as feed additives to improve feed characteristics, increase digestion and
performance if the animal is being fed these compounds, or to enhance desirable characteristics
of the meat produced (Jacob and Pescatore, 2012). Alali et al., (2013) determined the effect of
non-pharmaceuticals (a blend of organic acids, a blend of EOs, lactic acids, and a combination of
levulinic acid and sodium dodecyl sulfate) on weight gain, feed conversion ratio, and mortality
of broilers and their ability to reduce colonization and fecal shedding of Salmonella Heidelberg
following S. Heidelberg challenge and feed withdrawal. Their results showed that the broilers
that received the EOs had significantly increased weight gain and mortality was lower compared
to the other treatments. Salmonella Heidelberg contamination in crops was significantly lower in
challenged and unchallenged broilers that received EOs and lactic acids in drinking water, than
any of the other treatments (Alali et al., 2013).
Although orange peel and pulp feeding will not prevent all human foodborne illnesses,
including citrus oils and products in the diet can be utilized as a part of a multiple-hurdle system
with the objective of reducing the passage of foodborne pathogens from farm to human
consumers (Callaway et al., 2011b). In their research, Callaway et al., (2011b) showed that
including orange peel and pulp as dietary components reduced populations of Salmonella
Typhimurium in the gut of experimentally inoculated sheep.
5.4. Essential Oils as a Post-harvest Intervention
The antimicrobial activity of several EOs against foodborne pathogens have been studied
in the laboratory. Orange and lemon oil are known to have in vitro antibacterial effects on
Salmonella and other foodborne microorganisms (Subba et al., 1967). Components of citrus EOs
such as carvacrol, citral, and geraniol exhibit antimicrobial activity against S. Typhimurium and
34
its rifampicin resistant strain (Kim et al., 1995). In this research they inoculated S. Typhimurium
onto fish cubes and reported that carvacrol at 3% w/v completely killed the inoculated bacteria.
Pittman et al., (2011a) studied the use of naturally occurring compounds citrus EOs
extracted from orange peel to reduce or kill Salmonella spp. and E. coli. They found that 3%
cold-pressed terpeneless Valencia orange oil could be used as an additional intervention against
these two foodborne pathogens at refrigerated storage stages of processing. Dabbah et al. (1970)
assessed the antibacterial properties of orange, lemon, grapefruit, and mandarine citrus oils and
their derivatives, lime terpeneless oil, orange terpeneless oil, lemon terpeneless oil, d-limonene,
terpineol, and geraniol in vitro against S. Seftenberg, E. coli, Staphylococcus aureus, and
Pseudomonas spp. In their findings they observed the fractions of citrus oil were more active
against all bacteria than the composite oils themselves. The citrus oils used in this research did
not reduce the populations of S. Seftenberg, but lime terpeneless, terpineol, and geraniol reduced
corresponding populations by 100%.
Some studies have reported that it would require a much higher concentration of EOs in
actual food systems to inhibit bacterial growth (O’Bryan et al., 2008a). In their research,
O’Bryan et al., (2008a) reported that differences were observed between serotypes of Salmonella
in response to the antibacterial activity of various fraction orange EOs, with S. Enteritidis having
a minimal inhibitory concentration of 0.13% v/v for terpenes from orange juice essence as
compared to 0.5% v/v for S. Typhimurium. Ravishankar et al., (2010) reported that S. Newport
was found to be resistant to six of the seven EOs tested, exhibiting different susceptibilities to
carvacrol and cinnamaldehyde than the other strains demonstrated. Carvacrol and
cinnamaldehyde antimicrobial activity against antibiotic-resistant and antibiotic-susceptible
Salmonella strains on PBS, and S. Newport on celery and oysters were tested. Results from
35
inoculation of S. enterica in PBS buffer showed that at low concentrations (0.1 to 0.2% v/v)
carvacrol exhibited stronger antimicrobial activity than cinnamaldehyde (Ravishankar et al.,
2010). Carvacrol also showed to have greater effectiveness against antibiotic-resistant
Salmonella Newport on celery; while carvacrol and cinnamaldehyde were equally effective
against antibiotic-resistant S. Newport on oysters (Ravishankar et al., 2010).
6. Hurdle Technology
The hurdle technology developed by Leistner and Gorris (1995) sought to prevent the
survival and regrowth of pathogens in food by using a series of combinations of preservation
techniques. A hurdle intervention is any treatment, physical, chemical or biological that is used
to reduce or eliminate the presence of pathogens on a food surface (Leistner, 1985; Leistner and
Gorris, 1995).
When used in combination of more than one component, EOs could work differently than
when they are used as independent compounds. Combinations of more than one antimicrobial
intervention treatment in lower doses have been often found to increase bactericidal activity
more than any single treatment, working synergistically (Leistner and Gorris 1995; Leistner,
2000; Ricke et al., 2005; Tiwari et al., 2009; Li et al., 2011). Zhou et al., (2007a) investigated the
antimicrobial activity of cinnamaldehyde, thymol and carvacrol alone or in combinations against
S. Typhimurium, and reported the lowest concentrations of cinnamaldehyde, thymol and
carvacrol inhibiting the growth of S. Typhimurium significantly were 200, 400 and 400 mg/L,
respectively. Results from the combinations, cinnamaldehyde/thymol, cinnamaldehyde/carvacrol
and thymol/carvacrol, demonstrated that the concentration of cinnamaldehyde, thymol and
36
carvacrol could be decreased from 200, 400 and 400 mg/L to 100, 100 and 100 mg/L,
respectively (Zhou et al., 2007a).
Barnhart et al., (1999) studied the potential to eliminate Salmonella in the presence of
organic matter and reported a synergistic combination of d-limonene (DL) and citric acid (CA).
Individually combinations of less than 10% DL or less than 2% CA did not reduce Salmonella
recovery, but when used in combination, 2% CA with 0.5% DL, completely eliminated
detectable Salmonella (Barnhart et al., 1999). Their observations suggested that, in nature, the
unknown anti-Salmonella action of DL/CA in combination might be bactericidal, rather than
bacteriostatic.
The application of more than one hurdle treatment at a time is advantageous because it
may decrease the requirement for a higher dose of the specific hurdle (Leistner, 2000). Multiple
hurdle technologies could be more exploited in order to control contaminant bacteria. Instead of
using high doses of a single antimicrobial compound, combinations of more than one
antimicrobial at lower concentrations will effectively inhibit the contaminants to the same
degree, and due to synergism, result in minimal development of antimicrobial resistance (Sirsat
et al., 2009; Ricke, 2010; Milillo et al., 2011; Muthaiyan et al., 2011).
7. Conclusions
Through the years, the pharmaceutical industry has heavily invested in the research and
discovery of natural products with the end goal of producing therapeutic antibiotic formulations.
Today more than two-thirds of the antibiotics in clinical use are natural products or a result of
their semisynthetic derivatives (Benowitz et al., 2010). The constant problem of increasing drug
resistance has created a more urgent demand to develop new and improved antimicrobials
37
against resistant microorganisms (Spellberg et al., 2004; Berghman et al., 2005). Unfortunately
at the moment the status of potential novel antibacterial drugs indicates that there are only a few
compounds in development by the large pharmaceutical companies (Overybye et al., 2005).
There are approximately 10,000 plants in the world that have documented medicinal use.
In western medicine it is reported that only around 150 to 200 of such agents are in use
(McChesney et al., 2007). Understanding the mechanisms involved in bacterial response, could
facilitate the refining and optimization of antimicrobial formulations or find new ways to
overcome potential tolerance mechanisms, leading to an improvement in biosecurity measures
and to enhance the protection of farm-animal and as a result, public health (Condell et al.,
2012a).
Foodborne illnesses continue to be one of the primary public health concerns in the U. S.
Salmonella is one of the major foodborne pathogens responsible for foodborne illnesses. The
potential use of EOs as natural antimicrobial agents is less exploited than their uses as flavoring
and antioxidant compounds (Chorianopoulos et al., 2006). Previous research confirmed that EOs
from citrus could be used as effective antimicrobial agents against Salmonella (O’Bryan et al.,
2008a). For an optimally targeted use of EOs and organic acids against Salmonella, more
research is needed for a better understanding of the synergestic antimicrobial mechanisms and
effects when natural antimicrobials, such as citrus EOs are used in combinations with multiple
hurdle technologies.
As we previously discussed, one of the previous concerns about Salmonella is its
antimicrobial resistance. In this research we studied the effectiveness of five essential oils against
Salmonella Heidelberg, the primary focus for our research. Understanding the growth of
Salmonella serovars and strains serves as an important basis for our research. In this case we
38
studied a) the aerobic and anaerobic growth responses of multiple strains from six different
serovars, b) how the spent media from different serovars, more importantly S. Heidelberg, affect
the growth of S. Typhimurium, and c) determined whether or not two different serovars undergo
competitive exclusion when are grown together. The growth rates of four Salmonella serovars,
Heidelberg, Kentucky, Typhimurium, and Enteritidis in enrichment and non-enrichment media
were compared to determine the effectiveness of a non-enrichment media, such as Luria Bertani
broth over enrichment methods. Given that Salmonella enterica are important facultative
intracellular pathogens that are widely known to be isolated from poultry and to cause infections
in humans, understanding the ability of this pathogen to attach and invade different cell types is
of great importance. We studied the adhesion and invasion of four different strains to chicken
macrophage (HD11), and human epithelial (Caco-2) cells.
39
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Table 1.1. The 10 most frequently reported Salmonella serovars from human infections reported to CDC, 1998-2011. (CDC, 2011)
Rank 1998 2001 2006 2011
1 Typhimurium Typhimurium Typhimurium Enteritidis
2 Enteritidis Enteritidis Enteritidis Typhimurium
3 Newport Newport Newport Newport
4 Heidelberg Heidelberg Heidelberg Javiana
5 Javiana Javiana Javiana I 4,[5],12:1:-
6 Agona Montevideo I 4,[5],12:1:- Montevideo
7 Montevideo Oranienburg Montevideo Heidelberg
8 Oranienburg Muenchen Muenchen Muenchen
9 Muenchen Thompson Oranienburg Infantis
10 Infantis Saintpaul Mississippi Braenderup
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Table 1.2. The 10 most frequent serovars from analyzed PR/HACCP verification samples for young chicken (broilers), 1998–2011.
(USDA/FSIS, 2013b).
* Typhimuriu
Rank 1998 2001 2006 2011
1 Kentucky Kentucky Kentucky Kentucky
2 Heidelberg Heidelberg Enteritidis Enteritidis
3 Typhim* var. Copenhagen Typhimurium Heidelberg Typhim* var. 5-
4 Typhimurium Typhim* var. Copenhagen 4,[5],12:I:- Infantis
5 Hadar Montevideo Typhim* var. Copenhagen Heidelberg
6 Schwarzengrund Schwarzengrund Typhimurium Typhimurium
7 Montevideo Hadar Montevideo Johannesburg
8 Enteritidis Thompson Schwarzengrund 4,[5],12:1:-
9 Thompson Enteritidis Infantis 8,20:-:z6
10 Infantis Berta Mbandaka Mbandaka
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9. Authorship Statement for Chapter 1
Juliany Rivera Calo is the first author of the paper and completed at least 51% of the studies
among coauthors, which the title is “Salmonella spp. – Literature Review ” in chapter 1.
Major Advisor: Dr. Steven C. Ricke
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CHAPTER 2
Growth Responses, Spent Media, and Competitive Interactions among Salmonella Serovars
Juliany Rivera Calo1,2
Ok Kyung Koo3 and Steven C. Ricke
1,2
1Department of Food Science, University of Arkansas, Fayetteville, AR
2Center for Food Safety and Department of Food Science, University of Arkansas,
Fayetteville, AR
3Food Safety Research Group, Korea Food Research Institute, Seongnam-si, Gyeonggi-do,
Republic of Korea
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1. Abstract
Salmonella enterica is one of the most prevalent pathogens responsible for foodborne
illness worldwide. Many of its serovars have been associated with the consumption of poultry
products, these being one of the main reservoirs of Salmonella. The emergence and prevalence of
Salmonella serovars in chickens has been studied but the predominant serovars have varied
somewhat over the years. The purpose of this research was to a) understand the aerobic and
anaerobic growth responses of predominant Salmonella serovars b) study how spent media from
different serovars affect the growth of S. Typhimurium, and c) determine if there is serovar
interactions as a result of growing two Salmonella strains from independent serovars on the same
culture. Results showed that among all six serovars, the growth under aerobic conditions was
comparable; while under anaerobic conditions the growth in LB broth was slower. For spent
media, results were similar for most strains, except from S. Heidelberg ARI-14, where a decrease
in the growth of S. Typhimurium ATCC 14028 was observed. Competitive growth studies were
conducted to determine if there was any inhibition from one serovar against the other. These
studies will provide a better understanding of the differences in growth among Salmonella
serovars versus S. Typhimurium in chickens.
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2. Introduction
Salmonella enterica is one of the most prevalent pathogens responsible for foodborne
illness worldwide. Annually, an estimated 1,027,561 Americans contract Salmonella (Scallan, et
al., 2011). Yearly costs for Salmonella control efforts are estimated to be up to $14.6 billion
(Scharff, 2010; Heithoff, et al., 2012). Salmonella can multiply under various environmental
conditions outside their respective living host (Park et al., 2008; Pui et al., 2011). Most
Salmonella serovars grow at a temperature range of 5 to 47°C, with an optimum temperature of
35 to 37°C, although some can grow at temperatures as low as 2 to 4°C and as high as 54°C
(Gray and Fedorka-Cray, 2002).
More than 99% of Salmonella strains causing human infections belong to Salmonella
enterica. These are mainly isolated from warm-blooded animals and account for more than 99%
of clinical isolates (Pui, et al., 2011). Many of its serovars have been associated with the
consumption of poultry products, these being one of the main reservoirs of Salmonella (CDC,
2009). The emergence and prevalence of Salmonella serovars in chickens has been studied but
the predominant serovars have varied somewhat over the years. Salmonella enterica serovars
Typhimurium, Enteritidis, and Heidelberg cause gastroenteritis and these are the most common
serovars responsible for human infections (Olsen et al., 2001; Dunkley et al., 2009; CDC, 2011;
Foley et al., 2008; 2011; 2013; Finstad et al., 2012; Howard et al., 2012). These three serovars
along with S. Kentucky are the most common serovars found in poultry.
Salmonella Heidelberg foodborne illnesses have been found to be associated with several
foods such as chicken (Snoeyenbos et al., 1969; Mahony et al., 1990; Layton et al., 1997;
Hennessy et al., 2004), pork (O’Mahoney et al., 1983; Di Guardo et al., 1992), as well as eggs
and eggshells (Jones et al., 1995; Schonei et al., 1995). Salmonella Enteritidis cases have often
70
been associated with consumption of contaminated eggs (Guard-Petter, 2001; Cogan et al., 2004;
Howard et al., 2012). Salmonella Typhimurium cases are usually linked to the consumption of
contaminated poultry, swine and bovine meat (Jorgensen et al., 2000). S. Typhimurium is an
invasive enteric pathogen that is remarkably adaptable to diverse hosts including humans,
poultry, rodents, cattle, sheep and horses (Luo et al., 2012).
Spent media is defined as a bacterial growth medium that has been exhausted of nutrients
due to extensive growth of the microorganism and is then filter sterilized (Nutt et al., 2002). One
of the benefits from spent media is that it has the potential to serve as an in vitro screen of
ecosystems after growth of the microorganism (Nutt, et al., 2002). Competitive interaction
phenomena have been studied with homologous as well as heterologous bacteria, and we
hypothesize whether competition exists between Salmonella serovars. Therefore, the objectives
of this research were to a) understand the aerobic and anaerobic growth responses of several
Salmonella serovars, b) study how spent media from different serovars affect the growth of S.
Typhimurium, and c) determine if there is serovar interaction as a result of growing two
Salmonella strains from independent serovars on the same culture. These studies will provide a
better understanding of the effect of spent media from multiple Salmonella serovars and the
competitive growth between different Salmonella serovars versus S. Typhimurium in chickens.
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3. Materials and Methods
3.1. Salmonella spp. growth responses after aerobic and anaerobic incubation from poultry
Salmonella isolates.
3.1.1. Bacterial strains
A total of 21 strains from six of the most predominant Salmonella serovars, S.
Typhimurium, S. Kentucky, S. Heidelberg, S. Newport, S. Enteritidis and S. Montevideo isolated
from poultry were studied and are listed in Table 2.1. Growth responses were generated under
aerobic and anaerobic conditions.
3.1.2. Aerobic and anaerobic growth responses
One colony of each strain was inoculated in 5 mL of Luria Bertani (LB) broth and
incubated for 16 h at 37°C, 190 rpm shaking using a C76 Water Bath Shaker (New Brunswick
Scientific, Edison, NJ, USA). Using a Nunclon 96-well plate, 200 μL of fresh LB broth and 2 μL
(1%) of each Salmonella overnight culture were added to each well. Optical density (OD) was
measured at 600 nm every hour over 24 h using a TECAN Infinite M200 (Tecan Systems Inc.,
San Jose, CA, USA). For the anaerobic growth response, the 96-well plate was stored in an
anaerobic box (BD Biosciences, Franklin Lakes, NJ, USA), argon gas was used to provide
anaerobic conditions, and incubated at 37°C. The OD values were measured at 600 nm at 0, 1, 2,
4, 8, 12, 16 and 24 h using the TECAN Infinite M200.
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3.2. Effect of Salmonella spent media on the growth of S. Typhimurium
3.2.1. Aerobic and anaerobic spent media growth responses
One colony from 11 Salmonella strains from six different serovars was inoculated into 16
mL of LB broth and incubated for 16 h at 37°C. Tubes were centrifuged at 7000 rpm for 10 min
using the Centrifuge 5804 R (Eppendorf, Westbury, NY, USA), and supernatant was filter
sterilized using 0.2 μm sterile syringe filters (VWR, Radnor, PA, USA) in 15 mL tubes. Eight
milliliters of each spent media were used to generate growth curves; the other 8 mL were saved
for further analysis. One percent (80 μL) of S. Typhimurium ATCC 14028 culture was
inoculated into each spent media sample. Optical density (OD) at 600 nm was measured at 0, 1,
2, 3, 4, 5, 6, 7, 8, 12, 14 and 24 h using a Spectronic 20D+ Spectrophotometer (Thermo
Scientific, Rockford, IL, USA). For the anaerobic conditions, the tubes were stored in an
anaerobic jar (BD Biosciences, Franklin Lakes, NJ, USA) and GasPak (BD Biosciences) were
used to provide anaerobic conditions. Optical density at 600 nm was measured at 0, 24, 32, 48,
60, 72, 84 and 96 h using Spectronic 20D+ Spectrophotometer (Thermo Scientific).
3.2.2. Salmonella Heidelberg and S. Typhimurium spent media
Salmonella Heidelberg and S. Typhimurium were selected to conduct further spent media
studies. One colony of S. Heidelberg ARI-14 and S. Typhimurium ATCC 14028 was inoculated,
per duplicate, into 8 mL of LB broth and incubated for 16 h at 37°C. One tube from each strain
was centrifuged at 7000 rpm for 10 min and supernatant was filter sterilized using 0.2 μm sterile
syringe filter (VWR). One percent (80 μL) of S. Typhimurium ATCC 14028 culture was
inoculated into S. Typhimurium and S. Heidelberg spent media and incubated for 24 h at 37°C.
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Optical density (OD) at 600 nm was measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 14 and 24 h using a
Spectronic 20D+ Spectrophotometer (Thermo Scientific).
3.2.3. Salmonella Heidelberg spent media
Based on the results from growing S. Typhimurium on multiple Salmonella spent media
from different serovars, several S. Heidelberg strains were selected for spent media growth
curves with S. Typhimurium ATCC 14028. One colony of 15 different S. Heidelberg strains and
one colony of S. Typhimurium ATCC 14028 was inoculated into 8 mL of LB broth and
incubated for 16 h at 37°C. All S. Heidelberg cultures were centrifuged at 7000 rpm for 10 min
and supernatant was filter sterilized using 0.2 μm sterile syringe filter (VWR). One percent (80
μL) of S. Typhimurium ATCC 14028 culture was inoculated into all 15 S. Heidelberg spent
media and incubated for 24 h at 37°C, 190 rpm shaking using a C76 Water Bath Shaker (New
Brunswick Scientific). Optical density (OD) at 600 nm was measured at 0, 2, 4, 6, 7:45, 8, 12
and 24 h using Spectronic 20D+ Spectrophotometer (Thermo Scientific).
3.2.4. Salmonella Heidelberg spent media comparison between two different media and an
alternate temperature
One colony of S. Heidelberg ARI-14 and S. Typhimurium ATCC 14028 was inoculated
into 8 mL of LB broth and 8 mL of BPW and incubated for 16 h at 37°C, 190 rpm shaking using
a C76 Water Bath Shaker (New Brunswick Scientific). S. Heidelberg overnight cultures were
centrifuged at 7000 rpm for 10 min and supernatant was filter sterilized using 0.2 μm sterile
syringe filter (VWR). One percent (80 μL) of S. Typhimurium ATCC 14028 culture was
inoculated into both S. Heidelberg spent media and incubated for 24 h at 42°C, 190 rpm shaking
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using a C76 Water Bath Shaker (New Brunswick Scientific). The same was repeated on BPW
and incubated at 37°C. Optical density (OD) at 600 nm was measured at 0, 2, 4, 6, 7, 8, 8:30; 12
and 24 h using Spectronic 20D+ Spectrophotometer (Thermo Scientific).
3.3. Competitive growth between S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14.
3.3.1. Growth response from mixed cultures
One colony of S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14 respectively,
were inoculated into 8 mL of LB broth, individually, and incubated for 16 h at 37°C, 190 rpm
shaking using a C76 Water Bath Shaker (New Brunswick Scientific). From each strain, 500 μL
(1% total) were inoculated into 100 mL of LB broth, individually, and as a mix culture, and
incubated at 37°C for 24 h, 190 rpm shaking. Optical density values at 600 nm were measured at
0, 2, 4, 6, 8, 12, 14 and 24 h. At each time point, 1 mL of culture was collected for genomic
DNA extraction and qPCR; and 1 mL collected for serial dilutions and plating on LB agar plates
to determine colony forming units per mL (CFU/mL).
3.3.2. DNA extraction
Samples were centrifuged at 14,000 x g for 10 min and supernatant was discarded.
Genomic DNA was extracted using the Qiagen DNeasy Blood Tissue kit (Qiagen, Valencia, CA,
USA) according to the manufacturer’s instruction. The isolated genomic DNA concentration and
purity were measured using a NanoDrop ND-1000 (Thermo Scientific) and DNA samples were
stored at -20°C until used.
75
3.3.3. Bacterial quantification by real time quantitative PCR (qPCR)
Quantification of bacteria was performed using qPCR and strain-specific primers,
STM4497M2-f and STM4497M2-r (310 bp) for S. Typhimurium; and SH_SHP-2f and SH_SHP-
1r (180 bp) for S. Heidelberg (Table 2.5). The qPCR was conducted using an Eppendorf
Masterplex thermocycler ep (Eppendorf, Westbury, NY, USA). Real Time PCR assays were
conducted as three independent experiments and triplicate samples in each experiment. A 20 μL
total reaction volume composed of 1 μL of template DNA, 1 μL of each primer (IDT, Coralville,
IA, USA), 10 μL of SYBR Green SYBR Green Premix Ex Taq (Takara, Clontech, Mountain
View, CA, USA), and 7 μL of DNase-RNase free water. The PCR conditions consisted of pre-
denaturation at 94°C for 5 min, then 35 cycles of denaturation at 94°C for 30 s, annealing at
65°C for 30 s, extension at 72°C for 30 s, with fluorescence being measured during the extension
phase. Because SYBR green was being used, a subsequent melting curve was required and
consisted of 94°C for 30 s, 65°C for 20 min increasing in 0.5°C increments to 95°C.
3.4. Competitive growth between Salmonella Heidelberg ARI-14 and Salmonella Enteritdis
ATCC 13076.
3.4.1. Growth responses
One colony of S. Heidelberg ARI-14 and S. Enteritidis ATCC 13076 was inoculated in 8
mL of LB broth, individually, and incubated for 16 h at 37°C, 190 rpm shaking using a C76
Water Bath Shaker (New Brunswick Scientific). From each strain, 500 μL were inoculated
individually into 100 mL of LB broth, and 500 μL of each strain into 100 mL LB broth as a mix
culture; flasks were incubated at 37°C for 24 h, 190 rpm shaking. Optical density values at 600
76
nm were measured at 0, 2, 4, 6, 8, 12 and 24 h. At each time point, 1 mL collected for serial
dilutions and plating on LB agar plates to determine CFU/mL.
3.5. Statistical analysis
All experiments were repeated at least three times. Statistical comparisons were carried
out using the Student t-test comparison of means at P < 0.05 to show significant differences. All
statistical analyses were conducted using JMP Pro Software Version 11.0 (SAS Institute Inc.,
Cary, NC).
4. Results
4.1. Salmonella spp. growth responses after aerobic and anaerobic incubation from poultry
Salmonella isolates.
Among all six serovars, the growth under aerobic conditions was comparable, while
under anaerobic conditions the growth in LB broth was slower (Figures 2.1 to 2.6). Optical
density values at different time points are listed on tables 2.2 (aerobic) and 2.3 (anaerobic). In
general, under aerobic conditions the fastest growing serovars were S. Montevideo followed by
S. Kentucky; while under anaerobic conditions the fastest growing was S. Heidelberg followed
by S. Montevideo. Under aerobic conditions, the range in optical density (600 nm) at 24 h for S.
Typhimurium was between 0.913 to 0.964, for S. Heidelberg 0.732 to 0.864, 0.831 to 1.003 for
S. Kentucky, S. Newport 0.770 to 0.968, S. Enteritidis 0.564 and 0.869, and S. Montevideo
between 0.915 and 0.994. Compared to the anaerobic conditions the ranges in OD values at 600
nm were different. S. Typhimurium was between 0.807 and 0.833, S. Heidelberg 0.677 and
0.928, S. Kentucky 0.719 and 0.904, S. Newport 0.751 and 0.919, S. Enteritidis 0.483 and 0.747
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and S. Montevideo ranged between 0.761 and 0.871. Among all serovars, strain differences were
observed based on the optical density. Based on these results, 11 strains from the six different
serovars were selected for further studies using spent media from these strains.
4.2. Effect of Salmonella spent media on the growth of S. Typhimurium
4.2.1. Aerobic and anaerobic spent media growth responses
In general, under anaerobic conditions, after 96 h no growth was observed (Figure 2.8).
As a control on this experiment we included S. Typhimurium ATCC 14028 growing on LB broth
and growth under anaerobic conditions was observed. Under aerobic conditions the growth of S.
Typhimurium ATCC 14028 in different Salmonella strains spent media was slower (Figure 2.7).
When grown in LB, these strains reach exponential phase faster. Results were similar for most of
the spent media from different strains, except from S. Heidelberg ARI-14. A decrease in the
growth of S. Typhimurium ATCC 14028 was observed between 6 to 12 h after inoculating it on
S. Heidelberg ARI-14 spent media. Based on this effect we selected these two strains to conduct
more experiments using different combinations of spent media and inoculum to confirm and
better describe this effect.
4.2.2. Salmonella Typhimurium growth in S. Heidelberg spent media
In order to confirm the phenomena observed when S. Typhimurium ATCC 14028 was
grown on S. Heidelberg ARI-14 spent media, we repeated the experiment inoculating S.
Typhimurium in S. Typhimurium and S. Heidelberg spent media (Figure 2.9). As controls, we
grew (1) S. Typhimurium in LB broth and (2) both S. Typhimurium and S. Heidelberg in spent
media. Results showed that only when S. Typhimurium is grown on S. Heidelberg spent media a
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decrease in growth between 6 to 12 h is observed. No growth was observed on S. Typhimurium
and S. Heidelberg spent media used as a control.
To further confirm the previous results and determine if the effect being observed was
specific to S. Heidelberg ARI-14 spent media, we screened 14 additional S. Heidelberg strains
from multiple sources (Table 2.4). Results showed that the growth of S. Typhimurium on 12 of
the 15 S. Heidelberg strains spent media decreased at different points between hours 6 and 12
after inoculation (Figure 2.10). The three strains that did not show this effect are from different
sources, two from turkey (163 and 824) and one from cattle (114). This suggests there are strain
differences. We also inoculated S. Heidelberg strains on S. Heidelberg spent media (Figure 2.11)
to further confirm the effect is observed specifically for S. Typhimurium on S. Heidelberg spent
media. When inoculating the strains on BPW prior to spent media, the drop on the growth of S.
Typhimurium was observed.
The same experiment was conducted at 37°C and 42°C, and using two different media,
LB broth and BPW. Growth of S. Typhimurium was faster on LB broth than BPW, but there
appeared to be no effect caused by the spent media when incubated at 42°C (Figure 2.12). When
incubated at 37°C, the drop in the growth of S. Typhimurium was observed for both media used
(Figure 2.13). These results suggested that the previous effect caused by spent media could be
temperature dependent and media independent. Further studies are needed to have a better
understanding of the growth differences at different temperatures.
79
4.3. Competitive growth between (1) S. Typhimurium and S. Heidelberg, and (2) S.
Heidelberg and S. Enteritidis
The above spent media growth studies showed an interesting phenomenon when growing
S. Typhimurium ATCC 14028 on S. Heidelberg spent media. Competitive growth studies were
performed where the two Salmonella strains were grown together as a mixed culture to observe
any serovar interaction (Figure 2.14). Quantitative PCR was used to determine the cell number of
each strain at specific time points over 24 h. Growth responses showed similar initial
concentrations for the mixed culture containing S. Typhimurium ATCC 14028 and S. Heidelberg
ARI-14, of approximately 6 log CFU/mL (Figure 2.16). Salmonella Heidelberg appeared to
grow at a faster rate than S. Typhimurium. Positive controls for both strains showed an initial
cell concentration of 7 log CFU/mL for S. Typhimurium ATCC 14028 and 8 log CFU/mL for S.
Heidelberg ARI-14 (Figure 2.16). Growth of S. Heidelberg ARI-14 is not affected by the
presence of S. Typhimurium ATCC 14028, instead the growth of S. Typhimurium was inhibited
by S. Heidelberg, as the growth response was reduced after the 6 h time point. CFU/mL growth
determination on LB agar plates was chosen to verify the results from the qPCR experiments
(Figure 2.15).The qPCR results were compared to CFU/mL obtained from the mixed culture
(Figure 2.17). These results correlate with results of previous spent media results; the growth of
S. Typhimurium also decreases when it grows in a mixed culture with S. Heidelberg. This effect
is not limited just to spent media.
Preliminary studies were conducted to determine if there was any serovar exclusion
between S. Heidelberg ARI-14 and S. Enteritidis ATCC 13076. In a growth curve we compared
each strain grown by itself as well as both strains as a mixed culture. The growth for all three
cultures appears to be similar until they reached stationary phase (Figure 2.18). At 24 h the
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OD600 nm for individual S. Heidelberg ARI-14 was slightly higher than S. Enteritidis and the
mixed culture respectively. Further studies, such as qPCR of the mixed culture, should be
conducted to determine if the differences in OD600 nm observed in the growth curve are due to
serovar interactions, where S. Heidelberg is inhibiting the growth of S. Enteritidis as it does with
S. Typhimurium.
5. Discussion
Growth responses for 22 strains from 6 different Salmonella serovars, Typhimurium,
Heidelberg, Kentucky, Newport, Enteritidis, and Montevideo (Figures 2.1 to 2.6) were
conducted. Under aerobic conditions S. Montevideo and S. Kentucky appeared to not be
significantly different between each other and were the fastest growing strains. Growth of S.
Heidelberg strains was faster than S. Typhimurium, but slower than S. Kentucky strains. In
general, both S. Typhimurium strains showed slower growth. Under anaerobic conditions most
of the serovars were not significantly different. S. Enteritidis ARS-50 appeared to be the only
one significantly different from the rest of the strains and exhibited slower growth under both
conditions. Based on these results, 11 strains from these six serovars were selected for further
research using spent media, in order to study the effect on the growth of S. Typhimurium ATCC
14028 caused by spent media from multiple strains.
Spent media, which contains unspecified and autostimulatory compounds may be used to
initiate or promote the growth of uncultivable microorganisms (Weichart and Kell, 2001; Yang
et al., 2006; Brehm-Stecher et al., 2009). Our results showed that S. Typhimurium grows slower
on spent media than it does on Luria Bertani (LB) broth. Previous research have found that spent
culture supernatant of the probiotic Lactobacillus rhamnosus GG exert antimicrobial activity
81
against Salmonella Typhimurium and other intestinal pathogens (Silva et al., 1987; Hudault et
al., 1997; Lehto and Salminen, 1997; De Keersmaecker et al., 2005, 2006). In their research, De
Keersmaecker et al., (2006) reported that antimicrobial activity of spent culture supernatant of
Lactobacillus rhamnosus GG against Salmonella Typhimurium to be mediated by lactic acid. In
contrast, Silva et al., (1987) reported the produced antimicrobial substance to be different than
lactic acid, and to have inhibitory activity against other bacteria within a pH from 3 to 5. Stevens
et al., (1991) reported the inhibitory activity against S. Typhimurium was not due to the
production of bacteriocin. Alverdy and Stern, (1998) reported growth environment and the
composition of the growth media to be important factors in regulating constitutive expression of
several virulence factors. Salmonella expression of invasion genes is regulated by several factors,
such as the HilA regulator and environmental conditions. Durant et al., (2000) in an attempt to
mimic the effects of Lactobacillus on the crop environment, studied if the growth of a poultry
probiotic lactobacilli strain can influence S. Enteritidis hilA expression. This was conducted by
growing S. Enteritidis in spent media from a 24 h growth of a poultry Lactobacillus sp. strain.
Their data suggested that the pH of the medium could be a key factor for the expression of hilA
(Durant et al., 2000). Durant et al., (2000) discussed possible explanations for less hilA
expression in Lactobacillus spent media compared to Salmonella spent media, 1) the possibility
that Salmonella spent media encompass a completely depleted nutrient source than Lactobacillus
spent media; 2) the growth of Lactobacillus may produce soluble signals that are specifically
inhibitory to the virulence of S. Enteritidis; 3) compounds could be present in Salmonella spent
media which serve as a signal to enhance the virulence expression of Salmonella. Nutt et al.,
(2002) studied the ability of spent media from both a pure culture and a cecal mixed culture to
influence virulence expression in a Salmonella Typhimurium hilA:lacZY fusion strain. Results
82
showed a dramatic increase in the virulence expression of the Salmonella Typhimurium strain
when exposed to spent media recovered after 23 h growth, where nutrients are depleted, as
compared to 2 h of growth (Nutt et al., 2002). This data suggests that during the first hours of
exposing the bacteria to spent media, the organism uses all the nutrients for growth and
reproduction; once the nutrients are exhausted the virulence expression increases.
Competitive interactions have been studied with bacteria from the same species as well as
using different species, but the mechanisms causing the inhibition are poorly understood.
Inhibition of colonization has been demonstrated with E. coli and Salmonella in chickens (Linton
et al. 1978; Barrow et al., 1987; Berchieri and Barrow, 1990). Berchieri and Barrow (1990)
reported that an explanation for the inhibition occurring between Salmonella and E. coli could be
that one strain, which acts as a protector, is occupying the niche required by the challenge
organism. That same mechanism is the one that takes place for the inhibition of K88+
enterotoxigenic E. coli by K88+ non-enterotoxigenic E. coli, which involves the occupation of an
attachment site (Davidson et al., 1976). Crhanova et al., (2011) presented some possible effects
of competitive exclusion products, such as the ability of the bacteria to compete directly with
pathogens and also the possibility of stimulating the gut immune system of newly hatched
chickens to mature. Our results showed that the growth of S. Typhimurium decreases when it
grows in a mixed culture with S. Heidelberg. This effect is not limited just to spent media.
Results suggest that the inhibition is not dependent on live cells, but on some factor that is
filterable within the spent medium of S. Heidelberg. To the best of our knowledge this is the first
experiment that studies the effect of S. Heidelberg spent media on the growth of S. Typhimurium
and whether there is competitive interaction between these two serovars.
83
6. Acknowledgments
This research was funded by the National Integrated Food Safety Initiative (NIFSI)
(2008-51110-04339), the Institute of Food Science and Engineering (IFSE), and the U.S. Poultry
& Egg Association. We thank Dr. Si Hong Park, Center for Food Safety, University of Arkansas,
Fayetteville, AR, for providing Salmonella strain specific primers and for his training to conduct
the quantitative PCR.
84
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Table 2.1. Bacterial strains used in this study.
Salmonella strain Source
S. Typhimurium
ATCC 14028 tissue, animal
LT2 wild type from natural source
S. Heidelberg
ATCC 8326
ARI-14 poultry product in Arkansas
ARI-15 poultry product in Arkansas
ARI-16 poultry product in Arkansas
S. Kentucky turkey as a source
WVN-216 left over feed
WVN-222 turkey ceca
WVN-343 litters
WVN-352 drinkers
S. Newport
ARS-14 retail
ARA-2 retail
282 retail
S. Enteritidis
ARS-49 farm
ARS-50 farm
ARS-58 retail
ATCC 13076
S. Montevideo
G4639
ARS-11 farm
ARS-12 farm
ARS-13 farm
91
a)
b)
Figure 2.1. Salmonella Typhimurium average from a) aerobic growth response, b) anaerobic
growth response.
92
a)
b)
Figure 2.2. Salmonella Heidelberg average from a) aerobic growth response, b) anaerobic
growth response. Strains labeled as ARI were isolated from poultry products in Arkansas.
93
a)
b)
Figure 2.3. Salmonella Kentucky average from a) aerobic growth response, b) anaerobic growth
response. WVN strains have Turkey as a source.
94
a)
b)
Figure 2.4. Salmonella Newport average from a) aerobic growth response, b) anaerobic growth
response. Strains ARS-14 and ARA-2 are from retail.
95
a)
b)
Figure 2.5. Salmonella Enteritidis average from a) aerobic growth response, b) anaerobic growth
response. ARS-49 and ARS-50 are strains from the farm and ARS-58 from retail.
96
a)
b)
Figure 2.6. Salmonella Montevideo average from a) aerobic growth response, b) anaerobic
growth response. The source of ARS strain is the farm.
97
Table 2.2. Optical density at 600 nm for Salmonella serovars under aerobic conditions.
Isolate
2 h 8 h 12 h 24 h
S. Typhimurium
ATCC 14028 0.156 ± 0.01c,d,e,f
0.539 ± 0.01h,i
0.752 ± 0.02d,e,f
0.964 ± 0.02a,b,c
LT2 0.157 ± 0.02c,d,e
0.517 ± 0.02i,j
0.797 ± 0.04c,d
0.913 ± 0.03c,d,e
S. Heidelberg
ATCC 8326 0.131 ± 0.01e,f
0.750 ± 0.02a
0.891 ± 0.03a,b
0.801 ± 0.06f,g
ARI-14 0.158 ± 0.01c,d,e
0.558 ± 0.01g,h
0.692 ± 0.01f,g,h
0.864 ± 0.04d,e,f
ARI-15 0.120 ± 0.01f
0.461 ± 0.02k,l
0.554 ± 0.05i
0.732 ± 0.02h
ARI-16 0.184 ± 0.01b,c,d
0.560 ± 0.01g,h
0.702 ± 0.03f,g
0.847 ± 0.06e,f
S. Kentucky
WVN-216 0.185 ± 0.01b,c,d
0.776 ± 0.01a
0.940 ± 0.02a
1.003 ± 0.01a
WVN-222 0.166 ± 0.03b,c,d,e
0.571 ± 0.03e,f,g,h
0.733 ± 0.03e,f,g
0.924 ± 0.03b,c,d
WVN-343 0.170 ± 0.02b,c,d
0.571 ± 0.02e,f,g,h
0.717 ± 0.02e,f,g
0.834 ± 0.02f,g
WVN-352 0.169 ± 0.02b,c,d
0.561 ± 0.01f,g,h
0.709 ± 0.03f,g
0.831 ± 0.02f,g
S. Newport
ARS-14 0.151 ± 0.01d,e,f
0.529 ± 0.01h,i
0.641 ± 0.03h
0.770 ± 0.03g,h
ARA-2 0.157 ± 0.01c,d,e
0.648 ± 0.02b,c
0.837 ± 0.02b,c
0.968 ± 0.03a,b,c
282 0.186 ± 0.02b,c,d
0.589 ± 0.01d,e,f,g
0.720 ± 0.03e,f,g
0.850 ± 0.03e,f
S. Enteritidis
ARS-49 0.159 ± 0.02c,d,e
0.547 ± 0.01g,h,i
0.681 ± 0.05g,h
0.820 ± 0.04f,g
ARS-50 0.153 ± 0.02d,e,f
0.434 ± 0.05l 0.511 ± 0.07
i 0.564 ± 0.09
j
ARS-58 0.158 ± 0.01c,d,e
0.582 ± 0.01d,e,f,g
0.725 ± 0.03e,f,g
0.869 ± 0.04d,e,f
ATCC 13076 0.160 ± 0.02c,d,e
0.481 ± 0.04j,k
0.558 ± 0.04i
0.638 ± 0.05i
S. Montevideo
G4639 0.225 ± 0.06a
0.658 ± 0.02b
0.864 ± 0.02b
0.994 ± 0.01a,b
ARS-11 0.196 ± 0.01a,b,c
0.604 ± 0.02d,e,f
0.735 ± 0.02d,e,f,g
0.915 ± 0.02b,c,d,e
ARS-12 0.203 ± 0.02a,b
0.608 ± 0.01c,d,e
0.744 ± 0.01d,e,f,g
0.938 ± 0.02a,b,c,d
ARS-13 .0182 ± 0.03a,b,c,d
0.621 ± 0.03b,c,d
0.775 ± 0.04c,d,e
0.958 ± 0.03a,b,c
Values labeled with same letters are not significantly different between each time point.
98
Table 2.3. Optical density at 600 nm for Salmonella serovars under anaerobic conditions.
Isolate
2 h 8 h 12 h 24 h
S. Typhimurium
ATCC 14028 0.162 ± 0.01a,b,c,d
0.607 ± 0.07c,d
0.658 ± 0.03d,e,f
0.807 ± 0.07b,c,d,e
LT2 0.137 ± 0.01c,d
0.584 ± 0.04d,e
0.756 ± 0.03b,c
0.833 ± 0.05a,b,c,d
S. Heidelberg
ATCC 8326 0.160 ± 0.02a,b,c,d
0.735 ± 0.06a,b
0.812 ± 0.02b
0.928 ± 0.03a
ARI-14 0.152 ± 0.03a,b,c,d
0.587 ± 0.05d,e
0.611 ± 0.04e,f,g
0.681 ± 0.05f
ARI-15 0.127 ± 0.01d
0.495 ± 0.07e,f
0.564 ± 0.05g
0.677 ± 0.07f
ARI-16 0.176 ± 0.03a,b
0.591 ± 0.06d,e
0.629 ± 0.02d,e,f,g
0.741 ± 0.07d,e,f
S. Kentucky
WVN-216 0.171 ± 0.02a,b,c
0.784 ± 0.06a
0.882 ± 0.04a
0.904 ± 0.08a,b
WVN-222 0.183 ± 0.04a,b
0.632 ± 0.06b,c,d
0.638 ± 0.02d,e,f
0.819 ± 0.05b,c,d,e
WVN-343 0.170 ± 0.03a,b,c
0.635 ± 0.04b,c,d
0.676 ± 0.06d,e
0.751 ± 0.05d,e,f
WVN-352 0.185 ± 0.04a
0.649 ± 0.04b,c,d
0.691 ± 0.06c,d
0.719 ± 0.03e,f
S. Newport
ARS-14 0.146 ± 0.01b,c,d
0.612 ± 0.05c,d
0.614 ± 0.04e,f,g
0.768 ± 0.03d,e,f
ARA-2 0.161 ± 0.02a,b,c,d
0.702 ± 0.05a,b,c
0.771 ± 0.04b
0.919 ± 0.03a,b
282 0.169 ± 0.02a,b,c
0.660 ± 0.06b,c,d
0.686 ± 0.03d
0.751 ± 0.04d,e,f
S. Enteritidis
ARS-49 0.157 ± 0.02a,b,c,d
0.598 ± 0.07d,e
0.601 ± 0.03f,g
0.713 ± 0.04e,f
ARS-50 0.167 ± 0.03a,b,c
0.443 ± 0.12f 0.447 ± 0.08
h 0.673 ± 0.02
f
ARS-58 0.163 ± 0.02a,b,c,d
0.629 ± 0.07c,d
0.669 ± 0.03d,e
0.747 ± 0.04d,e,f
ATCC 13076 0.169 ± 0.02a,b,c
0.497 ± 0.06e,f
0.482 ± 0.04h
0.483 ± 0.06g
S. Montevideo
G4639 0.176 ± 0.03a,b
0.660 ± 0.05b,c,d
0.769 ± 0.02b
0.871 ± 0.05a,b,c
ARS-11 0.163 ± 0.02a,b,c,d
0.618 ± 0.06c,d
0.630 ± 0.03d,e,f,g
0.761 ± 0.06d,e,f
ARS-12 0.159 ± 0.02a,b,c,d
0.620 ± 0.06c,d
0.639 ± 0.03d,e,f
0.766 ± 0.06c,d,e,f
ARS-13 0.166 ± 0.01a,b,c
0.623 ± 0.05c,d
0.644 ± 0.03d,e,f
0.816 ± 0.05b,c,d,e
Values labeled with same letters are not significantly different between each time point.
99
Figure 2.7. Average aerobic spent media growth response.
100
Figure 2.8. Average anaerobic spent media growth response. No growth was observed when S.
Typhimurium ATCC 14028 was grown on spent media from multiple serovars and strains.
Growth observed represents the control, S. Typhimurium ATCC 14028 on LB broth.
101
Figure 2.9. Combinations of S. Heidelberg and S. Typhimurium spent media and inoculum.
Confirmation that the decrease in the growth of S. Typhimurium is observed only when this
strain is grown on S. Heidelberg ARI-14 spent media.
102
Table 2.4. Salmonella Heidelberg strains used in this study.
Strain Source Reference
ARI-14 poultry products In this study
SL 476 ground turkey Fricke et al., 2011
SL 486 human Fricke et al., 2011
692 chicken egg house Lynne et al., 2009
945 human Han et al., 2011
114 cattle Lynne et al., 2009
163 turkey Kaldhone et al., 2008
136 swine Lynne et al., 2009
1148 human Han et al., 2012
824 turkey Kaldhone et al., 2008
130 chicken Lynne et al., 2009
937 human Han et al., 2011
118 cattle Lynne et al., 2009
144 swine Lynne et al., 2009
146 swine Lynne et al., 2009
103
Figure 2.10. Salmonella Typhimurium ATCC 14028 in S. Heidelberg spent media from multiple sources. From all strains, thirteen of
them showed the drop in the growth.
104
Figure 2.11. Confirmation that the drop showed during growth response only occurs when
growing S. Typhimurium on S. Heidelberg spent media. This effect is not observed when
Salmonella Heidelberg strains are inoculated on any S. Heidelberg spent media.
105
Figure 2.12. Growth response for S. Typhimurium on S. Heidelberg spent media growing at
42°C on LB and BPW. When incubated at 42°C, S. Typhimurium ATCC 14028 growing on S.
Heidelberg spent media did not show the drop that was observed at 37°C.
106
Figure 2.13. Growth response for S. Typhimurium on S. Heidelberg spent media growing at
37°C on LB and BPW. The drop in the growth of S. Typhimurium ATCC 14028 growing on S.
Heidelberg spent media was observed when strains were incubated at 37°C.
107
Table 2.5. Primer pairs used in competitive growth studies.
Primer Target Sequences PCR product
size (bp)
Reference
STM4497M2-f Salmonella
Typhimurium
5’-AACAA CGGCT CCGGT
AATGA GATTG-3’
310 Park et al., 2009
STM4497M2-r 5’-ATGAC AAACT CTTGA
TTCTG AAGAT CG-3’
SH_SHP-2f Salmonella
Heidelberg
5’-GCATA GTTCC AAAGC
ACGTT-3’
180 In this study
SH_SHP-1r 5’-GCTCA ACATA AGGGA
AGCAA-3’
108
Figure 2.14. Salmonella Typhimurium and Salmonella Heidelberg growth responses from a
mixed culture. Growth response of S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14
growing together in LB broth for 24 h.
109
Figure 2.15. Salmonella Typhimurium and S. Heidelberg competitive interaction.
110
Figure 2.16. Log CFU/mL for S. Typhimurium and S. Heidelberg from mixed culture.
111
Figure 2.17. Log CFU/mL from qPCR and plate counts from mixed culture.
112
Figure 2.18. Growth of S. Heidelberg ARI-14 and S. Enteritidis ATCC 13076 growth as
individual and mixed cultures.
113
8. Authorship Statement for Chapter 2
Juliany Rivera Calo is the first author of the paper and completed at least 51% of the studies
among coauthors, which the title is “Growth Responses, Spent Media, and Competitive
Interactions among Salmonella Serovars” in chapter 2.
Major Advisor: Dr. Steven C. Ricke
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9. Appendix
9.1. Motility of S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14 on different agar
concentrations.
9.1.1 Materials and Methods
One colony of S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14 was inoculated
into 8 mL of Luria Bertani (LB) Broth (Alfa Aesar, Ward Hill, MA, USA) and incubated for 16
h at 37°C, 190 rpm shaking. A sterile cotton swab was dipped into the overnight bacterial culture
and gently squeezed against the tube to remove any excess fluid. The cotton swab subsequently
streaked on three different LB agar concentrations, 0.3, 0.5 and 1.5%, to observe the motility of
these strains and their ability to move through the filter papers, 4.25 cm (Whatman, Sigma
Aldrich, St. Louis, MO, USA), inserted on the center of the agar plate. For the plates where spent
media was used, 100 μL of spent media were inoculated on the center of the plate and streaked
evenly across the plate using a sterile cotton swab. One μL of bacteria was then inoculated on the
center of the plate.
115
a)
b)
c) (Photo taken by author)
Figure 1. Motility of S. Heidelberg and S. Typhimurium at different agar concentrations a) 1.5%,
b) 0.5%, and c) 0.3%. As the agar concentration decreases, S. Heidelberg and S. Typhimurium
were able to move across the plate and in some cases, cross through the filter paper. Plate F
reperesents S. H. inoculated on S. T. spent media; plate G is S. T. inoculated on S. H. spent
media.
116
(Photo taken by author)
Figure 2. When inoculating S. Heidelberg on S. Typhimurium spent media it was observed how
the growth of S.T. spread across the plate.
(Photo taken by author)
Figure 3. The plate on the left represents S. T. inoculated on S. H. spent media; and the plate on
the right is S. H. inoculated on S. T. spent media, at 0.3% agar concentration. We can observe
how the growth is different in both cases.
117
(Photo taken by author)
Figure 4. Filter papers were cut and used as paper disk. We dip the paper disk in 100 μL of
overnight bacterial strain and placed them on the corner of the plates. On the other side we used
a sterile cotton swab to streak bacteria on the plate. Without filter papers we can observe how the
two strains, S. T. and S. H. do not cross between each other. On the plate at the top it is shown a
a line dividing the two strains.
118
(Photo taken by author)
Figure 5. Multiple combinations of S. Typhimurium and S. Heidelberg, filter papers in the center
of the plate, paper disks on the corner of the plate, and spent media were conducted.
119
9.2. Institutional Biosafety Committee (IBC) Number
120
9.3. Institutional Biosafety Committee (IBC) Number
IBC#: 13008
Please check the boxes for each of the forms that are applicable to the research project you
are registering. The General Information Form - FORM 1 (this form) MUST be completed
on all submitted project registrations, regardless of the type of research.
Recombinant DNA (EVEN IF IT IS EXEMPT from the NIH Guidelines.) (FORM 2) Pathogens (human/animal/plant) (FORM 3) Biotoxins (FORM 4) Human materials/nonhuman primate materials (FORM 5) Animals or animal tissues and any of the above categories; transgenic animals or tissues; wild vertebrates or tissues (FORM 6)
Plants, plant tissues, or seed and any of the above categories; transgenic plants, plant tissues, or seeds (FORM 7)
CDC regulated select agents (FORM 8)
Notice to Pat Walker Health Center (FORM 9)
To initiate the review process, you must attach and send all completed registration forms
via email to [email protected]. All registration forms must be submitted electronically. To
complete the registration, print page 1 of this form, PI sign, date, and mail to: Compliance
Coordinator-IBC, 210 Admin. Building, Fayetteville, AR 72701, or FAX it to 479-575-3846.
As Principal Investigator:
I attest that the information in the registration is accurate and complete and I will submit changes to the Institutional Biosafety Committee (IBC) in a timely manner.
I am familiar with and agree to abide by the current, applicable guidelines and regulations governing my research, including, but not limited to: the NIH Guidelines for Research Involving Recombinant DNA Molecules and the Biosafety in Microbiological and Biomedical Laboratories manual.
I agree to accept responsibility for training all laboratory and animal care personnel involved in this research on potential biohazards, relevant biosafety practices, techniques, and emergency procedures.
If applicable, I have carefully reviewed the NIH Guidelines and accept the responsibilities described therein for principal investigators (Section IV-B-7). I will submit a written report to the IBC and to the Office of Recombinant DNA Activities at NIH (if applicable) concerning: any research related accident, exposure incident, or release of rDNA materials to the environment; problems implementing biological and physical containment procedures; or violations of NIH Guidelines. I agree that no work will be initiated prior to project approval by the IBC.
I will submit my annual progress report to the IBC in a timely fashion.
121
Principal Investigator Typed/Printed Name: Steven C. Ricke
Signature (PI): ____________________________________ Date: ____________________
CONTACT INFORMATION:
Principal Investigator:
Name: Steven C. Ricke
Department: FDSC
Title: Professor
Campus Address: FDSC E 27
Telephone: 575-4678
*After Hours Phone:
Fax: 575-6936
E-Mail: [email protected]
Co-Principal Investigator:
Name: Philip G. Crandall
Department: FDSC
Title: Professor
Campus Address: FDSC N221
Telephone: 575-7686
*After Hours Phone:
Fax: 575-6936
E-Mail: [email protected]
*Required if research is at Biosafety Level 2 or higher
PROJECT INFORMATION:
Have you registered ANY project previously with the IBC? Yes
Is this a new project or a renewal?
Project Title: Production of a New Vaccine for Poultry to Prevent Salmonella
Project Start Date: 11/1/2012
Project End Date: 10/31/2013
Granting Agency: USDA/SBIR
Indicate the containment conditions you propose to use (check all that apply):
Biosafety Level 1 Ref: 1
2
Biosafety Level 1A Ref: 1
2
Biosafety Level 1P Ref: 1
2
Biosafety Level 2 Ref: 1
2
Biosafety Level 2A Ref: 1
2
Biosafety Level 2P Ref: 1
2
122
Biosafety Level 3 Ref: 2
Biosafety Level 3A Ref: 2
Biosafety Level 3P Ref: 2
References:
1: Biosafety in Microbiological and Biomedical Laboratories (BMBL) 4th Edition
2: NIH Guidelines for Research Involving Recombinant DNA Molecules
3: University of Arkansas Biological Safety Manual
If you are working at Biosafety Level 2 or higher, has your laboratory received an onsite
inspection by the Biosafety Officer or a member of the IBC?
If yes, enter date if known: 1/9/2012
If no, schedule an inspection with the Biological Safety Officer.
Please provide the following information on the research project (DO NOT attach or insert
entire grant proposals unless it is a Research Support & Sponsored Programs proposal).
Project Abstract:
Avirulent live Salmonella vaccines are considered to be more effective for preventing the spread
of Salmonella in poultry flocks. A live Salmonella vaccine should be completely genetically
stable and avirulent for both animals and humans. We have developed a double deletion mutant
that is deficient in its ability to synthesize lysine (lysA-) and is defective in its virulence
properties (hilA-).The goal of this project is to determine the survival and control of this vaccine
strain and differences in attachment or invasiveness in human Caco-2 cell model.
Specific Aims:
a. Determine motility of the vaccine strain.
b. Determine survival of vaccine strain under acid conditions.
c. Determine the ability of the vaccine strain to withstand thermal treatment.
d. Evaluate the attachment and invasiveness of the vaccine strain in human Caco-2 cell
model.
Relevant Materials and Methods (this information should be specific to the research
project being registered and should highlight any procedures that involve biohazardous or
recombinant materials):
Motility (Shah et al, 2011; Vikram et al., 2011; Wang et al. 2007): Grow Salmonella overnight
(~16 h). Stab-inoculate 1 µl of culture in the middle of semi-solid media (0.3% LB agar).
Incubate at 37 °C for 7-8 hr and measure the diameter of the halo of growth.
Survival under acid conditions (Malheiros et al., 2008): Prepare overnight Salmonella culture in
LB broth. Wash the cells with PBS three times and add the cells to PBS acidified to pH 3.5, 4.0,
123
4.5, and 5.0 with HCl. Incubate the cells in 30°C water bath and take 1 ml at each interval time
(20~30 min, up to 3 h) to plate on LB agar with appropriate dilutions for enumeration.
Survival under thermal condition (Malheiros et al., 2008; Alvarez-Ordonez et al., 2008): Prepare
overnight bacteria culture. Wash the cells with PBS three times and add the bacterial cells to
PBS. Incubate the cells in 52, 56, 60°C water bath and take 1 ml at each interval time (10 – 20
min up to 2h and 5 min up to 30 min for 60°C) to plate on LB agar with appropriate dilution for
enumeration.
Adhesion and Invasion:
Caco-2 cells preparation: Media: Dulbecco’s modified Eagle medium (DMEM) (Invitrogen)
supplemented with 10 % (v/v) fetal bovine serum (FBS; Invitrogen) in 95 % air/5 % CO2 at
37°C. Cell condition: Caco-2 human colon adenocarcinoma cells with passage of 20-35. When
confluence is achieved (~10 days), harvest the cells by trypsinization (add trypsin for 10-15min,
disrupt the cells) and resuspend in D10F. Plate the resuspeneded cells onto 12-well plates
(Cellstar) at 1x10E5 ~10E6 cells per well. Change the medium every other day until confluency
(10~12 or up to 14 days until differentiated). Cell differentiation can be monitored by estimation
of the production of intestinal alkaline phosphatase using SensoLyte pNPP alkaline phosphatase
assay kit (AnaSpec). (Intestinal alkaline phosphatase is a brush border enzyme expressed
exclusively in villus-associated enterocytes, and expression indicates the development of
digestive and absorptive function (Goldberg et al., 2008; Hara et al., 1993).)
Adhesion assay: Prepare overnight bacteria culture (~16 h, grown in LB broth). Wash the cells
with PBS three times and dilute the bacterial cells to MOI ration of 10: 1 (10E6 Salmonella:
10E5 Caco-2 cells) Add the bacteria to Caco-2 cells and incubate for 2 h. Wash the cells with
cell-PBS (137 mM NaCl, 5.4 mM KCl, 3.5 mM Na2HPO4, 4.4 mM NaH2PO4, 11 mM glucose,
pH 7.2) four times. Treat the adherent cells with 0.1% Triton-X 100 (Sigma, St. Louis, MO) in
cell-PBS for 10 min at 37°C and collect the disrupted cells. Plate the bacteria on LB agar with
appropriate dilution for enumeration.
Invasion assay: Prepare overnight bacteria culture (~16 h, grown in LB broth). Wash the cells
with PBS three times and dilute the bacterial cells to MOI ration of 10: 1 (Salmonella: Caco-2
cells). Add the bacteria to Caco-2 cells and incubate for 2 h. Wash the cells with cell-PBS for
four times. Add 100 µg gentamicin ml/1 in D10F in each well and incubate for 2 h to kill
extracellular bacteria. Wash the cells with cell-PBS four times, add 0.1% Triton-X 100 in cell-
PBS to the Caco-2 cells for 10 min at 37°C and collect the disrupted cells. Plate the bacteria on
LB agar with appropriate dilution for enumeration (Shah et al, 2011).
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EMERGENCY PROCEDURES:
128
In the event of personnel exposure (e.g. mucous membrane exposure or parenteral
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For a spill inside the biological safety cabinet, alert nearby people and inform laboratory
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130
CHAPTER 3
Salmonella spp. Growth Responses Comparisons in Selective and Non-selective
Enrichment Media Methods
Juliany Rivera Calo1,2
and Steven C. Ricke1,2
1Department of Food Science, University of Arkansas, Fayetteville, AR
2Center for Food Safety and Department of Food Science, University of Arkansas,
Fayetteville, AR
131
1. Abstract
The use of enrichment culture is one of the most common methods to recover Salmonella
from food samples. Growth rate and recovery of different Salmonella strains on Luria Bertani
broth versus buffered peptone water (BPW) followed by selective enrichment on Rappaport-
Vassiliadis (RV) broth and Tetrathionate (TT) broth were compared. One strain from Salmonella
serovars Typhimurium, Enteritidis, Heidelberg and Kentucky was tested. Strains were grown
overnight on LB broth and BPW at 35°C. Approximately 100 colony forming units per mL
(CFU/mL) of each strains was then sub cultured on LB, TT and RV broth and incubated at 42°C
for 24 hours. CFU/mL were determined by serial dilutions and plating on LB agar. Strains
inoculated on LB broth exhibited a faster growth rate than those on RV and TT broth, and were
detectable at an earlier time point. At 8 hours of incubation, approximately 30 to 60 CFU/mL
were recovered from samples on LB broth while no CFU/mL were recovered from samples on
RV or TT broth at that time point. The use of a non-selective enrichment media such as LB broth
could potentially be more effective for earlier quantification and recovery of different Salmonella
serovars present in samples.
132
2. Introduction
Salmonella enterica is one of the most prevalent pathogens responsible for foodborne
illness worldwide. Salmonella has been a leading cause of risk to humans with 1.0 million cases
occurring that represents 11% of all foodborne pathogens (Moore-Neibel et al., 2011;
Ravichandran et al., 2011). Annual costs for Salmonella control efforts are estimated to be up to
$14.6 billion (Scharff, 2010; Heithoff, et al., 2012). Many of its serovars have been associated
with the consumption of poultry products, these being one of the main reservoirs of Salmonella
(CDC, 2009). The emergence and prevalence of Salmonella serovars in chickens has been
studied but the predominant serovars have varied somewhat over the years. Salmonella
Enteritidis, S. Typhimurium and S. Heidelberg are the three most frequent serovars recovered
from humans. These three serovars along with S. Kentucky are the most common serovars found
in poultry.
Isolation and identification of Salmonella through conventional methods includes five
steps: pre-enrichment, selective enrichment, plating on selective media, biochemical screening,
and serotyping (Afflu and Gyles, 1997). The use of enrichment culture is one of the most
common methods to recover Salmonella from food samples, faeces, and other material (Banič,
1964; Afflu and Gyles, 1997). Theoretically, enrichment methods are used to detect very low
levels of Salmonella, while these may not be detectable without the use of enrichment broth.
Brehm-Stecher et al., (2009) reported that as generally used, enrichment culture effectively
erases valuable information about initial microbial numbers within a sample, downgrading a
potentially quantitative test into a qualitative one. Singer et al., (2009) reported that the
probability of detecting a specific Salmonella strain had little to do with its starting concentration
in the sample. The bias introduced by culture growth-based systems could be dramatically
133
biasing Salmonella surveillance systems (Singer et al., 2009). We hypothesized that the use of a
non-selective enrichment media such as Luria Bertani (LB) broth could potentially be more
effective and equally representative for earlier quantification and recovery of different
Salmonella serovars present in samples. In this project, the growth rate and recovery of four
Salmonella strains on LB broth versus Buffered Peptone water (BPW) followed by selective
enrichment on Rappaport-Vassiliadis (RV) broth and Tetrathionate (TT) broth were compared.
RV broth serves as a base for the selective enrichment of Salmonella; and TT broth is a base for
enrichment medium for isolation of Salmonella. The composition of each broth media is listed
on Table 1.
3. Materials and Methods
3.1. Bacterial cultures for non-selective enrichment media method
One colony of S. Typhimurium ATCC 14028, S. Heidelberg SL 476, S. Enteritidis ATCC
13076 and S. Kentucky WVN-222 was inoculated in 8 mL of LB broth and incubated for 16 h at
35°C, 190 rpm shaking using a C76 Water Bath Shaker (New Brunswick Scientific, Edison, NJ,
USA). Overnight cultures were diluted to 10-3
CFU/mL and 100 μL of each strain were
inoculated in 10 mL LB broth. Tubes were incubated for 24 h at 42°C, 190 rpm shaking. Optical
density at 600 nm was measured at 0, 2, 4, 6, 8, 12, and 24 h. From the 10-3
dilutions, 1 mL of
each strain was inoculated into 100 mL of LB broth. One mL of culture was collected at 0, 2, 4,
6, 8, 12, and 24 h for serial dilutions and plating, per duplicate, on LB agar plates for
enumeration, colony forming units per mL (CFU/mL).
134
3.2. Bacterial cultures for selective enrichment media method
One colony of S. Typhimurium ATCC 14028, S. Heidelberg SL 476, S. Enteritidis ATCC
13076 and S. Kentucky WVN-222 was inoculated in 8 mL of BPW broth and incubated for 16 h
at 35°C, 190 rpm shaking using a C76 Water Bath Shaker (New Brunswick Scientific). Optical
density at 600 nm was measured for all four overnight cultures. These were then diluted to 10-3
CFU/mL and 1 mL of each strain were inoculated in 100 mL RV broth, and 5 mL of each strain
inoculated in 100 mL TT broth. Flasks were incubated for 24 h at 42°C, 190 rpm shaking. One
milliliter of culture was collected at 0, 2, 4, 6, 8, 12, and 24 h for serial dilutions and plating, per
duplicate, on LB agar plates for enumeration, CFU/mL. This experiment was conducted
following the flow chart specific for FSIS laboratory Salmonella analyisis (USDA/FSIS, 2013).
4. Results and Discussion
Many researches have concluded that enrichment media are of great importance for the
isolation of Salmonella (Banič, 1964; Palumbo and Alford, 1970; Harvey et al., 1979; Harvey
and Price, 1980; 1981; Afflu and Gyles, 1997; AOAC, 1995; FDA, 1995; Hammack et al., 1999;
2001). Through history, the most commonly used enrichment media have been TT broth (Bohls,
1950; Palumbo and Aflord; 1970; Harvey et al., 1979; Harvey and Price, 1981; Beckers et al.,
1986; Hammack et al., 1999; 2001), selenite F broth (Leifson, 1936; Palumbo and Alford, 1970;
Harvey et al., 1979; Hammack et al., 1999) and RV broth (Beckers et al., 1986; Harvey et al.,
1979; Harvey and Price, 1980; 1981; Fricker et al., 1985; Hammack et al., 1999; 2001). These
media have been introduced with the purpose of inhibiting non-pathogenic enteric bacteria, while
allowing for Salmonella to grow (Iveson et al., 1964; Palumbo and Alford, 1970).
135
Tetrathionate broth was first used by Mueller (1923); later Kauffmann (1941) modified
the medium by the addition of bile and brilliant green. It is believed that most Salmonella and
Proteus reduce tetrathionate to thiosulfate in the media, while species such as E. coli and
Shigella do not, which may explain why Salmonella is able to flourish in the media (Knox et al.,
1942; 1945; Pollock et al., 1942; Knox et al., 1943; Pollock and Knox, 1943; Knox and Pollock,
1944; Jeffries, 1959; Palumbo and Alford, 1970).
In 1956, Rappaport et al., (1956) published a new medium for isolating Salmonella from
human faeces, claiming it to be better and more efficient than selenite and TT broth. In 1976,
Vassiliadis et al., (1979) modified the medium in order to make it more suitable for incubation at
43°C, this is why the media is known as Rappaport-Vassiliadis. The ability of the RV medium
for the isolation of Salmonella has been said to depend on the ability of the pathogen to multiply
at low pH-value, high osmotic pressure, and also on the resistance of Salmonella to malachite
green (Peterz et al., 1989). Previous researches have reported that RV media is more effective
than TT and selenite media when recovering Salmonella from different samples, including food
products (Harvey et al., 1979; Harvey and Price, 1980; 1981; Vassiliadis et al., 1981; Fricker et
al., 1983; Kalapothaki et al., 1983; van Schothorst and Renaud, 1983; Vassiliadis, 1983; Fricker,
1984; Tongpim et al., 1984; Vassiliadis et al., 1985; Rhodes and Quesnel, 1986; Allen et al.,
1991a; 1991b; June et al., 1995; 1996; Hammack et al., 1999; 2001). Brest Nielsen and Grunnet,
(1976) reported no difference when isolating Salmonella from meat and bone-meal, sewage or
receiving waters, while isolates were significantly less frequent from RV than from TT. In order
to be effective, the Rappaport medium should be very lightly inoculated; otherwise the media
loses its sensitivity and selectivity to isolate Salmonella (Rappaport et al., 1956; Collard and
136
Unwin, 1958; Hooper and Jenkins, 1965; Vassiliadis et al., 1985). Contrary to the case of
tetrathionate broth where the initial inoculation is required to be higher than RV broth.
We hypothesized that the use of a non-enrichment media such as LB broth could
potentially be more effective for earlier quantification and recovery of different Salmonella
serovars present in samples. To prove this, we compared the growth responses and recovery of
four Salmonella strains on LB broth versus BPW followed by enrichment on RV and TT broth.
Results showed that all four strains inoculated on LB broth exhibited a faster growth response
than those on RV and TT broth, and were detectable at an earlier time point. At 8 hours of
incubation, approximately 20 to 70 CFU/mL were recovered from samples on LB broth at a 10-5
dilution (Table 3.2), while no CFU/mL were recovered from samples on RV or TT broth at that
time point. Using enrichment methods, RV and TT broth, Salmonella numbers were lower at
every time point studied. Rappaport-Vassiliadis showed no detectable levels of Salmonella at
dilutions higher than 10-6
CFU/mL (Table 3.3); while using tetrathionate broth, no growth was
detected at dilutions higher than 10-4
(Table 3.4). For Salmonella strains inoculated on LB broth,
Salmonella was detected as early as 4 h at 10-2
and 10-3
dilutions, while the recovery was 0
CFU/mL for RV and TT broth at the same time point. At initial time points, the growth of
Salmonella on TT broth was higher than on RV, while at 24 h the growth on RV was
significantly higher. An explanation for this could be the fact that for enrichment on TT broth it
is required for the initial inoculum to be higher, 5 mL, while it was only 1 mL for RV broth.
Zhang et al., (2013) reported tryptic soy broth (TSB) to be significantly more efficient
than nutrient broth and BPW. Buffered peptone water is the most commonly used by food
industries before conducting enrichment, and was used for our experiment before inoculating on
RV and TT broth. After enrichment in RV broth, Zhang et al., (2013) observed S. Enteritidis
137
population to be 0.40 to 1.11 log CFU/mL lower than those in preenrichment cultures. Afflu and
Gyles, (1997) reported that enrichment in TT broth appears not to be necessary for enrichment.
Their findings showed the same results when inoculating directly from pre-enrichment or after
enrichment with TT broth (Afflu and Gyles, 1997). Because the BAM Method (FDA, 1995;
Hammack et al., 1999; 2001) recommends using selective enrichment pairs, TT at 35°C and RV
at 42°C are used from foods with low microbial load. June et al., (1995 1996) recommended
incubating TT broth at 42°C for the recovery of Salmonella with high microbial loads. In our
experiment, we incubated both enrichment media at 42°C.
In general, out of all four serovars studied, Salmonella Kentucky was the fastest growing
on every single media tested. A reason for this could be the hypothesis where S. Kentucky may
devote all metabolic efforts toward growth. González-Gil et al., (2012), studied virulence
expression of the hilA gene in S. enterica serovars in response to acid stress. Their results
showed no significant changes in hilA expression in S. Kentucky, which supports the idea of
focusing on growth rather than virulence (González-Gil et al., 2012). This could explain why S.
Kentucky is one of the most commonly isolated from poultry but so far is not of public health
concern in the United States. According to the CDC (2011), S. Kentucky is one of the most
common serovars isolated from broilers in the US and commonly found in dairy catle as well
(Foley et al., 2011; 2013). Several factors could contribute for the increase in isolating this
serovar from chickens, such as flock immunity, genetic changes in the organism, and
management practices (Foley et al., 2013). Several researchers have reported that it is most likely
for S. Kentucky to lack genes necessary to cause disease in humans, but that it does possess
characteristics that allow it to be a competitive colonizer in chicken cecum, such as having a
better ability to grow under moderately acidic conditions (pH 5.5) (Joerger et al., 2009;
138
González-Gil et al., 2012). It has been reported that S. Kentucky possess plasmids with factors
associated with antimicrobial and disinfectant resistance, bacteriocin production, iron
acquisition, and complement resistance, which could benefit and enhance their abilities to
survive in chickens (Han et al., 2012; Foley et al., 2013).
In conclusion, comparing the growth of different Salmonella serovars, Typhimurium,
Heidelberg, Enteritidis and Kentucky, it appears that the type of media used to recover
Salmonella serovars can influence the population levels detectable by culturing. Further
examination of additional serovars and more strains within serovars is needed to further
differentiate differences in recovery media. This may be critical for assessing the original
distribution of serovars in a sample prior to enrichment for detection and quantification.
5. Acknowledgments
This research was funded with a grant obtained from the U.S. Poultry & Egg Association.
We thank Dr. Corliss O’Bryan, Center for Food Safety, University of Arkansas, Fayetteville,
AR, for sharing her expertise about enrichment protocols of Salmonella and providing insights to
conduct this research.
139
6. References
Afflu, L., and C. L. Gyles. 1997. A comparison of procedures involving single step
Salmonella, 1-2 test, and modified semisolid rappaport-vassiliadis medium for detection of
Salmonella in ground beef. Int. J. Food Microbiol. 37:241-244.
Allen, G., V. R. Bruce, W. H. Andrews, F. B. Satchell, and P. Stephenson. 1991a.
Recovery of Salmonella from frozen shrimp: evaluation of short term selective enrichment,
selective media, post-enrichment, and a rapid immunodiffusion method. J. Food Prot. 54:22-27.
Allen, G., V. R. Bruce, P. Stephenson, F. B. Satchell, and W. H. Andrews. 1991b.
Recovery of Salmonella from high-moisture foods by abbreviated selective enrichment. J. Food
Prot. 54:492-495.
AOAC International. 1995. Official methods of analysis. 16th
ed., 967.25, 967.26, 967.28,
and 995.20. AOAC International, Gaithersburg, Md.
Banič, S. 1964. A new enrichment medium for salmonellae. J. Hyg. Camb. 62:25-28.
Beckers, H. J., F. M. Van Leusden, and R. Peters. 1986. Comparison of Muller-
Kauffmann’s tetrathionate broth and modified Rappaport’s medium for isolation of Salmonella.
J. Food Saf. 8:1-9
Bohls, S. W., and L. H. Mattman. 1950. Use of tetrathionate broth in isolation of
salmonella, shigella, and paracolon bacilli. J. Lab. Clin. Med. 35:654-657.
Brehm-Stecher B, C. Young, L. A. Jaykus, and M. L. Tortorello. 2009. Sample
preparation: the forgotten beginning. J. Food Prot. 72:1774-1789.
Brest Nielsen, B., and K. Grunnet. 1977. A comparison of tetrathionate broth and
rappaport’s medium as enrichment media for Salmonella. Nord. Vet.-Med. 29:141-143.
Centers for Disease Control and Prevention (CDC). 2009. Multistate outbreaks of
Salmonella infections associated with live poultry – United States, 2007. MMWR. 58:25-29.
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Centers for Disease Control and Prevention (CDC). 2011. Salmonella surveillance:
annual summary, 2009. Centers for Disease Control and Prevention, Atlanta, GA.
Collard, P., and M. Unwin. 1958. A trial of Rappaport’s medium. J. Clin. Pathol. 11:426-
427.
Foley, S. L., R. Nayak, I. B. Hanning, T. J. Johnson, J. Han, and S. C. Ricke. 2011.
Population dynamics of Salmonella enterica serotypes in commercial egg and poultry
production. Appl. Environ. Microbiol. 77:4273-4279.
Foley, S. L., T. J. Johnson, S. C. Ricke, R. Nayak, and J. Danzelsen. 2013. Salmonella
pathogenicity and host adaptation in chicken-associated serovars. Microbiol. Mol. Biol. Rev.
77:582-607.
Fricker, C. R., R. W. A. Girdwood, and D. Munro. 1983. A comparison of enrichment
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sewage sludge. Zentralbl Bakteriol. Mikrobiol. Hyg. B. 179:170-178.
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Hammack, T. S., R. M. Amaguaña, G. A. June, P. S. Sherrod, and W. H. Andrews. 1999.
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Harvey, R. W. S., T. H. Price, and E. Xirouchaki. 1979. Comparison of selenite F,
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Harvey, R. W. S., and T. H. Price. 1980. Salmonella isolation with Rappaport’s medium
after pre-enrichment in buffered peptone water using a series of inoculum ratios. J. Hyg. 85:125-
128.
Harvey, R. W. S., and T. H. Price. 1981. Comparison of selenite F, Muller-Kauffmann
tetrathionate and Rappaport’s medium for salmonella isolation from chicken giblets after pre-
enrichment in buffered peptone water. J. Hyg. Camb. 87:219-224.
Heithoff, D. M., W. R. Shimp, J. K. House, Y Xie, B. C. Weimer, R. L. Sinsheimer, and
M. J. Mahan. 2012. Intraspecies variation in the emergence of hyperinfectious bacterial strains in
nature. PLoS Pathog. 8:1-17.
Hooper, W. L., and H. P. Jenkins. 1965. An evaluation of Rappaport’s magnesium
chloride/malachite green medium in the routine examination of faeces. J. Hyg. (Camb). 63:491-
495.
Iveson, J. B., N. Kovacs, and W. M. Laurie. 1964. An improved method of isolating
salmonellae from contaminated desiccated coconut. J. Clin. Path. 17:75-78.
Jeffries, L. 1959. Novobicin-tetrathionate broth: a medium of improved selectivity for the
isolation of salmonellae from faeces. J. Clin. Path. 12:568-571.
Joerger, R. D., C. A. Sartori, and K. E. Kniel. 2009. Comparison of genetic and
physiological properties of Salmonella enterica isolates from chickens reveals one major
difference between serovar Kentucky and other serovars: response to acid. Foodborne Pathog.
Dis. 6:503-512.
June, G. A., P. S. Sherrod, T. S. Hammack, R. M. Amaguaña, and W. H. Andrews. 1995.
Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis
142
medium for the recovery of Salmonella from raw flesh and other highly contaminated foods:
Precollaborative study. J. AOAC Int. 78:375-380.
June, G. A., P. S. Sherrod, T. S. Hammack, R. M. Amaguaña, and W. H. Andrews. 1996.
Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis
medium for the recovery of Salmonella from raw flesh, highly contaminated foods, and poultry
feed: Collaborative study. J. AOAC Int. 79:1307-1323.
Kalapothaki, V., P. Vassiliadis, C. H. Mavrommati, and D. Trichopoulos. 1983.
Comparison of Rappaport-Vassiliadis enrichment medium and tetrathionate brilliant green broth
for isolation of salmonellae from meat products. J. Food Prot. 46:618-621.
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salmonella group. J. Path. Bact. 54:469-483.
Knox, R., P. G. H. Gell, and M. R. Pollock. 1943. The selective action of tetrathionate in
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26:146-150.
Leifson, E. 1936. New selenite enrichment media for isolation of typhoid and
paratyphoid (Salmonella) bacilli. Amer. J. Hyg. 24:423-432.
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Antimicrobial activity of lemongrass oil against Salmonella enterica on organic leafy greens. J.
Appl. Microbiol. 112:485-492.
Mueller, L. 1923. Un nouveau milieu d’enrichissement pour la recherche du bacille
typhique et des paratyphiques. C. R. Seances Soc. Biol. Fil. 89:434-437.
Palumbo, S. A., and J. A. Alford. 1970. Inhibitory action of tetrathionate enrichment
broth. Appl. Microbiol. 20:970-976.
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magnesium chloride concentration on growth of salmonella in home-made and in commercially
available dehydrated Rappaport-Vassiliadis broth. J. Appl. Bacteriol. 66:523-528.
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Nature (London). 150:94.
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37:476-481.
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salmonellae. J. Clin. Pathol. 9:261-266.
Ravichandran, M., N. S. Hettiarachchy, V. Ganesh, S. C. Ricke, and S. Singh. 2011.
Enhancement of antimicrobial activities of naturally occurring phenolic compounds by nanoscale
delivery against Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella
Typhimurium in broth and chicken meat system. J. Food Saf. 31:462-471.
Rhodes, P., and L. B. Quesnel. 1986. Comparison of Muller-Kauffmann tetrathionate
broh with Rappaport-Vassiliadis (RV) medium for the isolation of salmonellas from sewage
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Accessed at: http://publichealth.lacounty.gov.
Singer R. S., A. E. Mayer, T. E. Hanson, and R. E. Isaacson. 2009. Do microbial
interactions and cultivation media decrease the accuracy of Salmonella surveillance systems and
outbreak investigations? J. Food Prot. 72:707-713.
Tongpim, S., R. R. Beumer, S. K. Taminga, and E. H. Kampelmacher. 1984. Comparison
of modified Rappaport’s medium (RV) and Muller-Kauffmann medium (MK-ISO) for the
detection of Salmonella in meat products. Int. J. Food Microbiol. 1:33-42.
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Van Schothorst, M., and A. M. Renaud. 1983. Dynamics of Salmonella isolation with
modified Rappaport’s medium (R10). J. Appl. Bacteriol. 54:209-215.
Vassiliadis, P., D. Trichopoulos, G. Papoutsakis, and E. Pallandiou. 1979. A note on the
comparison of two modifications of Rappaport's medium with selenite broth in the isolation of
Salmonellas. J. Appl. Bacteriol. 46:567-569.
Vassiliadis, P., V. Kalapothaki, D. Trichopoulos, C. H. Mavrommati, and C. H. Serie.
1981. Improved isolation of salmonellae from naturally contaminated meat products by using the
Rappaport-Vassiliadis enrichment broth. Appl. Environ. Microbiol. 42:615-618.
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Salmonella with the use of 100 ml of the R10 modification of Rappaport’s enrichment medium.
J. Hyg. Camb. 87:35-41.
Vassiliadis, P. 1983. The Rappaport-Vassiliadis (RV) enrichment medium for the
isolation of salmonellas: an overview. J. Appl. Bacteriol. 54:69-76.
Vassiliadis, P., V. Kalapothaki, and D. Trichopoulos. 1985. Efficiency of Rappaport-
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Zhang, G., E. W. Brown, and T. S. Hammack. 2013. Comparison of different
preenrichment broths, egg: preenrichment broth ratios, and surface disinfection for the detection
of Salmonella enterica spp. enterica serovar Enteritidis in shell eggs. Poult. Sci. 92:3010-3016.
145
Table 3.1. List of components for each media broth used for non-enrichment and enrichment
studies.
Media Ingredient g/liter
Luria Bertani (LB) broth Tryptone 10.0
Yeast extract 5.0
Sodium chloride 10.0
Buffered peptone water
(BPW)
Peptone from casein 10.0
Sodium chloride 5.0
Disodium hydrogen phosphate dodecahydrate 9.0
Potassium dihydrogen phosphate 1.5
Tetrathionate (TT) Broth
Base
Yeast extract 2.0
Tryptose 18.0
Dextrose 0.5
D-Mannitol 2.5
Sodium desoxycholate 0.5
Sodium chloride 5.0
Sodium thiosulfate 38.0
Calcium carbonate 25.0
Brilliant green 0.01
Rappaport-Vassiliadis (RV)
R10 Broth
Pancreatic Digest of Casein 4.54
Sodium chloride 7.2
Monopotassium Phosphate 1.45
Magnesium chloride 13.4
Malachite green oxalate 0.036
146
Figure 3.1. Optical density at 600 nm for each strain from overnight cultures (16 h) on LB and
BPW broth prior to enrichment. When compared, OD values were higher for S. Typhimurium
and S. Kentucky when grown on LB broth; and values were similar for S. Enteritidis and S.
Heidelberg.
147
Figure 3.2. Optical density at 600 nm from each strain inoculated into 10 mL LB broth starting
on the first detectable time point. Salmonella growth is not detected until the 8 h.
148
Table 3.2. Average CFU/mL for each Salmonella serovar inoculated on Luria Bertani broth.
Time (h) Dilution S. Typhimurium S. Heidelberg S. Enteritidis S. Kentucky
0
Inoculum 2 2 1 1
10-1 BD
* BD* BD
* BD*
10-2 BD
* BD* BD
* BD*
2
Inoculum 24 27 27 45
10-1 3 2 3 2
10-2 BD
* BD* BD
* 1
10-3 BD
* BD* 1 BD
*
4
Inoculum TNTC+
TNTC+ TNTC
+ TNTC
+
10-1 104 101 55 TNTC
+
10-2 9 12 6 21
10-3 1 1 2 2
6
10-2
TNTC+ TNTC
+ TNTC
+ TNTC
+
10-3 85 41 84 160
10-4 10 3 6 10
10-5 2 BD
* BD* 2
8
10-4 TNTC
+ 64 239 TNTC
+
10-5 38 24 24 72
10-6 7 3 2 11
12
10-6 123 137 113 115
10-7 13 16 9 9
10-8 2 1 2 3
24 10
-7 25 23 27 22
10-8 2 2 1 3
TNTC+ (too numerous too count) - number of colonies over 250
BD* - below detection level. Detection level was estimated to be approximately 10 CFU/mL.
149
Table 3.3. Average CFU/mL for each Salmonella serovar inoculated on Rappaport –Vassiliadis
broth.
Time (h) Dilution S. Typhimurium S. Heidelberg S. Enteritidis S. Kentucky
0 Inoculum 1 1 1 2
10-1 BD
* BD* BD
* BD*
2
Inoculum 1 2 1 2
10-1 BD
* BD* BD
* BD*
10-2 BD
* BD* 1 BD
*
4
Inoculum 3 7 2 27
10-1 BD
* 4 BD* 4
10-2 BD
* 1 BD* 1
10-3 BD
* BD* BD
* BD*
6
10-2 BD
* 2 BD* 4
10-3 BD
* BD* BD
* 1
10-4 BD
* BD* BD
* BD*
8
10-3 BD
* 11 BD* 11
10-4 BD
* 1 BD* 1
10-5 BD
* BD* BD
* BD*
10-6 BD
* BD* BD
* BD*
12
10-3 BD
* TNTC+ BD
* TNTC+
10-4 BD
* TNTC+ BD
* TNTC+
10-5 BD
* TNTC+ BD
* TNTC+
10-6 BD
* 20 BD* 17
24
10-2 9 TNTC
+ BD
* TNTC+
10-4 1 TNTC
+ BD
* TNTC+
10-6 BD
* 62 BD
* 103
TNTC+ (too numerous too count) - number of colonies over 250
BD* - below detection level. Detection level was estimated to be approximately 10 CFU/mL.
150
Table 3.4. Average CFU/mL for each Salmonella serovar inoculated on Tetrathionate broth.
Time (h) Dilution S. Typhimurium S. Heidelberg S. Enteritidis S. Kentucky
0 Inoculum 6 2 4 5
10-1 BD
* BD* BD
* BD*
2
Inoculum 5 9 6 7
10-1 BD
* 2 2 BD*
10-2 BD
* BD* BD
* BD*
4
Inoculum 2 13 17 18
10-1 BD
* 1 1 BD*
10-2 BD
* BD* BD
* BD*
6 Inoculum 19 32 26 20
10-2 1 1 1 1
8
Inoculum 20 45 28 106
10-2 BD
* 3 2 4
10-3 BD
* BD* BD
* BD*
12 10
-2 60 15 2 4
10-4 BD
* BD* BD
* BD*
24
10-2 230 28 2 130
10-4 4 1 BD
* 4
10-6 BD
* BD* BD
* BD*
BD* - below detection level. Detection level was estimated to be approximately 10 CFU/mL.
151
7. Authorship Statement for Chapter 3
Juliany Rivera Calo is the first author of the paper and completed at least 51% of the studies among
coauthors, which the title is “Salmonella spp. Growth Responses Comparisons in Selective and
Non-selective Enrichment Media Methods” in chapter 3.
Major Advisor: Dr. Steven C. Ricke
152
8. Appendix
8.1. Institutional Biosafety Committee (IBC) Number
153
8.2. Institutional Biosafety Committee (IBC) Number
IBC#: 13008
Please check the boxes for each of the forms that are applicable to the research project you
are registering. The General Information Form - FORM 1 (this form) MUST be completed
on all submitted project registrations, regardless of the type of research.
Recombinant DNA (EVEN IF IT IS EXEMPT from the NIH Guidelines.) (FORM 2) Pathogens (human/animal/plant) (FORM 3) Biotoxins (FORM 4) Human materials/nonhuman primate materials (FORM 5) Animals or animal tissues and any of the above categories; transgenic animals or tissues; wild vertebrates or tissues (FORM 6)
Plants, plant tissues, or seed and any of the above categories; transgenic plants, plant tissues, or seeds (FORM 7)
CDC regulated select agents (FORM 8)
Notice to Pat Walker Health Center (FORM 9)
To initiate the review process, you must attach and send all completed registration forms
via email to [email protected]. All registration forms must be submitted electronically. To
complete the registration, print page 1 of this form, PI sign, date, and mail to: Compliance
Coordinator-IBC, 210 Admin. Building, Fayetteville, AR 72701, or FAX it to 479-575-3846.
As Principal Investigator:
I attest that the information in the registration is accurate and complete and I will submit changes to the Institutional Biosafety Committee (IBC) in a timely manner.
I am familiar with and agree to abide by the current, applicable guidelines and regulations governing my research, including, but not limited to: the NIH Guidelines for Research Involving Recombinant DNA Molecules and the Biosafety in Microbiological and Biomedical Laboratories manual.
I agree to accept responsibility for training all laboratory and animal care personnel involved in this research on potential biohazards, relevant biosafety practices, techniques, and emergency procedures.
If applicable, I have carefully reviewed the NIH Guidelines and accept the responsibilities described therein for principal investigators (Section IV-B-7). I will submit a written report to the IBC and to the Office of Recombinant DNA Activities at NIH (if applicable) concerning: any research related accident, exposure incident, or release of rDNA materials to the environment; problems implementing biological and physical containment procedures; or violations of NIH Guidelines. I agree that no work will be initiated prior to project approval by the IBC.
I will submit my annual progress report to the IBC in a timely fashion.
154
Principal Investigator Typed/Printed Name: Steven C. Ricke
Signature (PI): ____________________________________ Date: ____________________
CONTACT INFORMATION:
Principal Investigator:
Name: Steven C. Ricke
Department: FDSC
Title: Professor
Campus Address: FDSC E 27
Telephone: 575-4678
*After Hours Phone:
Fax: 575-6936
E-Mail: [email protected]
Co-Principal Investigator:
Name: Philip G. Crandall
Department: FDSC
Title: Professor
Campus Address: FDSC N221
Telephone: 575-7686
*After Hours Phone:
Fax: 575-6936
E-Mail: [email protected]
*Required if research is at Biosafety Level 2 or higher
PROJECT INFORMATION:
Have you registered ANY project previously with the IBC? Yes
Is this a new project or a renewal?
Project Title: Production of a New Vaccine for Poultry to Prevent Salmonella
Project Start Date: 11/1/2012
Project End Date: 10/31/2013
Granting Agency: USDA/SBIR
Indicate the containment conditions you propose to use (check all that apply):
Biosafety Level 1 Ref: 1
2
Biosafety Level 1A Ref: 1
2
Biosafety Level 1P Ref: 1
2
Biosafety Level 2 Ref: 1
2
Biosafety Level 2A Ref: 1
2
Biosafety Level 2P Ref: 1
2
Biosafety Level 3 Ref: 2
Biosafety Level 3A Ref: 2
Biosafety Level 3P Ref: 2
References:
155
1: Biosafety in Microbiological and Biomedical Laboratories (BMBL) 4th Edition
2: NIH Guidelines for Research Involving Recombinant DNA Molecules
3: University of Arkansas Biological Safety Manual
If you are working at Biosafety Level 2 or higher, has your laboratory received an onsite
inspection by the Biosafety Officer or a member of the IBC?
If yes, enter date if known: 1/9/2012
If no, schedule an inspection with the Biological Safety Officer.
Please provide the following information on the research project (DO NOT attach or insert
entire grant proposals unless it is a Research Support & Sponsored Programs proposal).
Project Abstract:
Avirulent live Salmonella vaccines are considered to be more effective for preventing the spread
of Salmonella in poultry flocks. A live Salmonella vaccine should be completely genetically
stable and avirulent for both animals and humans. We have developed a double deletion mutant
that is deficient in its ability to synthesize lysine (lysA-) and is defective in its virulence
properties (hilA-).The goal of this project is to determine the survival and control of this vaccine
strain and differences in attachment or invasiveness in human Caco-2 cell model.
Specific Aims:
a. Determine motility of the vaccine strain.
b. Determine survival of vaccine strain under acid conditions.
c. Determine the ability of the vaccine strain to withstand thermal treatment.
d. Evaluate the attachment and invasiveness of the vaccine strain in human Caco-2 cell
model.
Relevant Materials and Methods (this information should be specific to the research
project being registered and should highlight any procedures that involve biohazardous or
recombinant materials):
Motility (Shah et al, 2011; Vikram et al., 2011; Wang et al. 2007): Grow Salmonella overnight
(~16 h). Stab-inoculate 1 µl of culture in the middle of semi-solid media (0.3% LB agar).
Incubate at 37 °C for 7-8 hr and measure the diameter of the halo of growth.
Survival under acid conditions (Malheiros et al., 2008): Prepare overnight Salmonella culture in
LB broth. Wash the cells with PBS three times and add the cells to PBS acidified to pH 3.5, 4.0,
4.5, and 5.0 with HCl. Incubate the cells in 30°C water bath and take 1 ml at each interval time
(20~30 min, up to 3 h) to plate on LB agar with appropriate dilutions for enumeration.
Survival under thermal condition (Malheiros et al., 2008; Alvarez-Ordonez et al., 2008): Prepare
overnight bacteria culture. Wash the cells with PBS three times and add the bacterial cells to
156
PBS. Incubate the cells in 52, 56, 60°C water bath and take 1 ml at each interval time (10 – 20
min up to 2h and 5 min up to 30 min for 60°C) to plate on LB agar with appropriate dilution for
enumeration.
Adhesion and Invasion:
Caco-2 cells preparation: Media: Dulbecco’s modified Eagle medium (DMEM) (Invitrogen)
supplemented with 10 % (v/v) fetal bovine serum (FBS; Invitrogen) in 95 % air/5 % CO2 at
37°C. Cell condition: Caco-2 human colon adenocarcinoma cells with passage of 20-35. When
confluence is achieved (~10 days), harvest the cells by trypsinization (add trypsin for 10-15min,
disrupt the cells) and resuspend in D10F. Plate the resuspeneded cells onto 12-well plates
(Cellstar) at 1x10E5 ~10E6 cells per well. Change the medium every other day until confluency
(10~12 or up to 14 days until differentiated). Cell differentiation can be monitored by estimation
of the production of intestinal alkaline phosphatase using SensoLyte pNPP alkaline phosphatase
assay kit (AnaSpec). (Intestinal alkaline phosphatase is a brush border enzyme expressed
exclusively in villus-associated enterocytes, and expression indicates the development of
digestive and absorptive function (Goldberg et al., 2008; Hara et al., 1993).)
Adhesion assay: Prepare overnight bacteria culture (~16 h, grown in LB broth). Wash the cells
with PBS three times and dilute the bacterial cells to MOI ration of 10: 1 (10E6 Salmonella:
10E5 Caco-2 cells) Add the bacteria to Caco-2 cells and incubate for 2 h. Wash the cells with
cell-PBS (137 mM NaCl, 5.4 mM KCl, 3.5 mM Na2HPO4, 4.4 mM NaH2PO4, 11 mM glucose,
pH 7.2) four times. Treat the adherent cells with 0.1% Triton-X 100 (Sigma, St. Louis, MO) in
cell-PBS for 10 min at 37°C and collect the disrupted cells. Plate the bacteria on LB agar with
appropriate dilution for enumeration.
Invasion assay: Prepare overnight bacteria culture (~16 h, grown in LB broth). Wash the cells
with PBS three times and dilute the bacterial cells to MOI ration of 10: 1 (Salmonella: Caco-2
cells). Add the bacteria to Caco-2 cells and incubate for 2 h. Wash the cells with cell-PBS for
four times. Add 100 µg gentamicin ml/1 in D10F in each well and incubate for 2 h to kill
extracellular bacteria. Wash the cells with cell-PBS four times, add 0.1% Triton-X 100 in cell-
PBS to the Caco-2 cells for 10 min at 37°C and collect the disrupted cells. Plate the bacteria on
LB agar with appropriate dilution for enumeration (Shah et al, 2011).
The information requested above can be entered directly or cut & pasted into the space
provided, or can be provided as an attached word document. If you provide an
attachment, please indicate “See Attached” and list the file name(s) in the space below:
Click here to enter text.
PERSONNEL QUALIFICATIONS & FACILITY INFORMATION:
List all personnel (including PI and Co-PI) to be involved in this project:
Name (First and Last) - Position
(Title, academic degrees,
certifications, and field of
expertise)
Qualifications/Training/Relevant Experience (Describe
previous work or training with biohazardous and/or
recombinant DNA; include Biosafety Levels )
Example: Bob Biohazard - 14 yrs working with E. coli at BL1, Salmonella enterica at
157
Associate Professor, PhD-
Microbiology
BL2, 8 yrs working with transgenic mice.
Steven C. Ricke, Professor, PhD
Gut Microbiology
20+ years with BSL 1 and 2
Philip Crandall, Professor, PhD,
Food Science
15+ years with BSL 1 and 2
Peter Rubinelli, Sr. Scientist, Sea
Star Int., PhD Cell Biology
10 years working with cell cultures and BSL2 organisms
Ok Kyung Koo, Post Doc, PhD
Food Science
8 years working with BSL2 organisms and cell culture
Corliss O’Bryan, Post Doc, PhD
Med Micro
30+ years with BSL1 and 2
Si Hong Park, Grad student, MS,
Micro
3 years with BSL 1 and 2
Juliany Rivera, Grad student, BS,
Micro
1 year with BSL 1 and 2
Click here to enter text. Click here to enter text.
Additional Personnel Information (if needed):
Click here to enter text.
List all the laboratories/facilities where research is to be conducted:
Building: Room #: Category: *Signage Correct?
Biomass 132 Laboratory Yes
Biomass 136 Tissue Culture Yes
Biomass 101 Autoclave/BioStorage Yes
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* Biohazard signs are required for entrances to Biosafety Level 2 (including Animal
Biosafety Level 2) areas. EH&S will supply these signs. If an updated biohazard sign is
required, please indicate the location and what agents/organisms/hazards should be listed
on the sign:
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Additional Facility Information (if needed):
Click here to enter text.
158
SAFETY PROCEDURES:
Please indicate which of the following personal protective equipment (PPE) will be used to
minimize the exposure of laboratory personnel during all procedures that require handling or
manipulation of registered biological materials.
Gloves:
Latex Vinyl Nitrile Leather Other
Specify: Click here to enter text.
Face & Eye Protection:
Face Shield Safety Goggles Safety Glasses
Other Specify: Click here to enter text.
Clothing Protection:
Re-usable Lab Coat Re-usable Coverall Disposable Clothing Protection
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Dirty or contaminated protective clothing cleaning procedures: (Check all that apply)
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glassware and liquid containing infectious materials. Autoclaving or using fresh 10%
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exceptions:
Work surfaces will be decontaminated with a freshly prepared 10% bleach solution before and
after working. Exception is biosafety cabinets which will be disinfected before and after use with
Lysol® No Rinse Sanitizer in order to avoid the corrosiveness of the bleach on the metal of the
biosafety cabinets. Instruments and equipment will be decontaminated by wiping down with
10% bleach. Paper towels used for these purposes will be discarded in biohazard bags.
Glassware, waste, and disposable tubes will be autoclaved under standard conditions (15 psi, 121
C, 20 min). Disposable items (pipette tips, pipets, etc) will be discarded into 10% bleach. After
30 minutes it will be permissible to place these items in a biohazard bag for autoclaving before
disposal.
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Describe waste disposal methods to be employed for all biological and recombinant
materials. Include methods for the following types of waste: (ref: UofA BiosafetyManual )
Sharps:
Placed into 10% bleach solution for decontamination followed by discarding into sharps waste
container
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Placed into biohazard bags and autoclaved before disposal. Liquids will be disposed of in drains
after autoclaving. Disposable glass will be placed in glass disposal after autoclaving.
Pathological Waste:
Liquid biological waste will always be discarded into freshly made 10% bleach and then
autoclaved for decontamination treatment before it is discarded. Other biological waste will be
placed carefully into biohazard waste bags, autoclaved at 15 psi, 121C for 20 min.
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Biomass Res. Ctr. Room 101: Autoclaves are checked monthly using SteriGage test strips (3M)
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spores.
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Describe the procedures/equipment that will be used to prevent personnel exposure during
aerosol-producing procedures:
All pipetting of infectious material will take place in the biological safety cabinet. Mechanical
pipetting devices will be used. Lab coats buttoned over street clothes, gloves and goggles will be
worn. All materials needed will be placed in the biological safety cabinet before work begins.
Sash of the cabinet will be lowered and all movements will be slow to avoid disruption of the air
currents. Centrifuged cultures will be contained in a closed Eppendorf tube or contained in
screw-capped polypropylene or polystyrene tubes with gasket seals to prevent aerosol exposure.
Cultures to be vortexed will be contained in screw-capped polypropylene or polystyrene tubes,
and vortexing will be done within the biological safety cabinets. Sonicating will be done within
the biosafety cabinet or within an enclosure on the bench top.
EMERGENCY PROCEDURES:
In the event of personnel exposure (e.g. mucous membrane exposure or parenteral
inoculation), describe what steps will be taken including treatment, notification of proper
supervisory and administrative officials, and medical follow up evaluation or treatment:
In the event of accidental exposure of personnel the person exposed should notify the laboratory
supervisor immediately. Treatable exposures will be treated by use of the first aid kit containing
antimicrobial agents. Mucous membrane exposure or puncture with contaminated material will
result in the person being taken to the Health Center for prophylactic antibiotic therapy. In the
event of environmental contamination, describe what steps will be taken including a spill
response plan incorporating necessary personal protective equipment (PPE) and decontamination
procedures.
161
In the event of environmental contamination, describe what steps will be taken including a
spill response plan incorporating necessary personal protective equipment (PPE) and
decontamination procedures.
For a spill inside the biological safety cabinet, alert nearby people and inform laboratory
supervisor. Safety goggles, lab coat buttoned over street clothes and latex gloves should be worn
during clean up. If there are any sharps they will be picked up with tongs, and the spill covered
with paper towels. Carefully pour disinfectant (freshly made 10% bleach) around the edges of
the spill, then into the spill without splashing. Let sit for 20 minutes. Use more paper towels to
wipe up the spill working inward from the edge. Clean the area with fresh paper towels soaked in
disinfectant. Place all contaminated towels in a biohazard bag for autoclaving. Remove personal
protective clothing and wash hands thoroughly. For a spill in the centrifuge turn off motor, allow
the machine to be at rest for 30 minutes before opening. If breakage is discovered after the
machine has stopped, re close the lid immediately and allow the unit to be at rest for 30 minutes.
Unplug centrifuge before initiating clean up. Wear strong, thick rubber gloves and other personal
protective equipment (PPE) before proceeding with clean up. Flood centrifuge bowl with
disinfectant. Place paper towels soaked in a disinfectant over the entire spill area. Allow 20
minute contact time. Use forceps to remove broken tubes and fragments. Place them in a sharps
container for autoclaving and disposal as infectious waste. Remove buckets, trunnions and rotor
and place in disinfectant for 24 hours or autoclave. Unbroken, capped tubes may be placed in
disinfectant and recovered after 20 minute contact time or autoclaved. Use mechanical means to
remove remaining disinfectant soaked materials from centrifuge bowl and discard as infectious
waste. Place paper towels soaked in a disinfectant in the centrifuge bowl and allow it to soak
overnight, wipe down again with disinfectant, wash with water and dry. Discard disinfectant
soaked materials as infectious waste. Remove protective clothing used during cleanup and place
in a biohazard bag for autoclaving. Wash hands whenever gloves are removed.
For a spill outside the biological safety cabinet or centrifuge have all laboratory personnel
evacuate. Close the doors and use clean up procedures as above.
TRANSPORTATION/SHIPMENT OF BIOLOGICAL MATERIALS:
Transportation of Biological Materials: The Department of Transportation regulates some
biological materials as hazardous materials; see 49 CFR Parts 171 - 173. Transporting any of
these regulated materials requires special training for all personnel who will be involved in the
shipping process (packaging, labeling, loading, transporting or preparing/signing shipping
documents).
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Cultures of Human or Animal Pathogens Environmenatl samples known or suspected to contain a human or anumal pathogen
162
Human or animal material (including excreta, secreta, blood and its components, tissue, tissue fluids, or cell lines) containing or suspected of containing a human or animal pathogen.
Transportation/Shipment Training: Have any project personnel who will be involved in
packaging, labeling, completing, or signing shipping documents received formal training to ship
infectious substances or diagnostic specimens within the past 3 years?
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163
CHAPTER 4
Antimicrobial Activity of Essential Oils against Salmonella Heidelberg Strains from
Multiple Sources
Juliany Rivera Calo1,2
and Steven C. Ricke1,2
1Department of Food Science, University of Arkansas, Fayetteville, AR
2Center for Food Safety and Department of Food Science, University of Arkansas,
Fayetteville, AR
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1. Abstract
Salmonella spp. is one of the more prominent foodborne pathogens that represent a major
health risk to humans. Salmonella serovar Heidelberg strains are increasingly becoming an
important public health concern, since they have been identified as one of the primary
Salmonella serovars responsible for human outbreaks. Over the years, Salmonella Heidelberg
isolates have exhibited higher rates of resistance to multiple antimicrobial agents compared to
other Salmonella serovars. Essential oils (EOs) have been widely used as alternatives to
chemical-based antimicrobials. In the current research, five EOs were screened to determine their
antimicrobial activity against 15 Salmonella Heidelberg strains from different sources. Oils
tested were R(+)-limonene, orange terpenes, cold compressed orange oil, trans-cinnamaldehyde
and carvacrol. EOs were stabilized in nutrient broth by adding 0.15% (w/v) agar. Tube dilution
assays and minimal inhibitory concentrations (MIC) were determined by observing any color
change in the samples. Carvacrol and trans-cinnamaldehyde completely inhibited the growth of
Salmonella Heidelberg strains, while R(+)-limonene and orange terpenes did not show any
inhibitory activity against these strains. Cold compressed orange oil only inhibited growth of two
of the strains exhibiting a MIC of 1%. No relationship was found between the activity of the EOs
tested and the sources of the strain. The use of all natural antimicrobials, such as EOs, offers the
potential for improving the safety of foods contaminated with S. Heidelberg, but there are strain
differences, which must be taken into account.
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2. Introduction
Foodborne illnesses continue to be one of the primary public health concerns in the
United States. Annually, it is estimated that over 1 million Americans contract Salmonella
(Scallan et al., 2011). Yearly costs for Salmonella control efforts are estimated to be up to $14.6
billion (Scharff, 2010; Heithoff, et al., 2012). Salmonella is not only a public health concern due
to the significant number of cases per year, but also because many strains have developed
resistance to antimicrobial agents (Kim et al., 2005; Foley and Lynne, 2008; Bajpai et al., 2012)
due to the unrestricted use of antimicrobials in feeds and increased therapeutic use (Su et al.,
2004; Kim et al., 2005).
Salmonella enterica serovar Heidelberg (S. Heidelberg) ranks fourth among the top five
serovars associated with human infections and is responsible for causing an estimated 84,000
illnesses in the United States annually (CDC, 2008; FDA, 2009; Foley et al., 2011; Han et al.,
2011). This serovar is one of the most commonly isolated in the United States and Canada from
clinical cases of salmonellosis, retail meats and livestock (Zhao et al., 2008; Hur et al., 2012).
While most Salmonella infections are self-limiting and become resolved within a few days, S.
Heidelberg tends to cause a significantly higher percentage of invasive infections (Vugia et al.,
2004; Han et al., 2011). As a result, antimicrobial therapy is often necessary, making
antimicrobial resistance a significant concern. Because of the tendency of this serovar to cause
severe extra-intestinal infections (Wilmshurst and Sutcliffe, 1995) such as myocarditis and
septicemia (Vugia et al., 2004), the occurrence of S. Heidelberg MDR strains is of extreme
importance. Salmonella Heidelberg strains exhibiting antimicrobial resistance have been isolated
from humans, retail meats and food animals (Logue et al., 2003; Nayak et al., 2004; Kaldhone et
al., 2008; Zhao et al., 2008; Lynne et al., 2009; Oloya et al., 2009; Han et al., 2011). Studies have
166
shown poultry-associated Salmonella Heidelberg strains to harbor IncFIB, IncA/C, IncH2, and
IncI1 plasmids, which may contain genes that confer resistance to several antibiotics such as
tetracycline, kanamycin, streptomycin, and sulfonamides (Han et al., 2012). Because S.
Heidelberg is responsible for causing more invasive infections when compared to other serovars,
it is important to monitor its prevalence and resistance. Novel and unique intervention strategies
are a priority to reduce or eliminate its presence.
Aromatic plants and their extracts have been examined for their effectiveness in food
safety and preservation applications (Fisher and Phillips, 2008). Essential oils for example,
exhibit antimicrobial properties that may make them suitable alternatives to antibiotics (Chaves
et al., 2008). These potential attributes and an increasing demand for natural medicinal treatment
options have brought the attention to the use of EOs as potential alternative antimicrobials
(Fisher and Phillips, 2008; Solórzano-Santos and Miranda-Novales, 2012). Essential oils derived
as by-products of the citrus industry have been screened for antimicrobial properties against
common foodborne pathogens and several have been shown to possess antimicrobial properties
(Dabbah et al., 1970). This study reports the results of testing various EOs against several
Salmonella Heidelberg strains from different sources using tube dilution assays.
3. Materials and Methods
3.1. DNA extraction
One colony of each of the 15 S. Heidelberg strains were inoculated in 5 mL Luria Bertani
(LB) broth and incubated at 37°C for 16 h, shaking at 190 rpm using a C76 Water Bath Shaker
(New Brunswick Scientific, Edison, NJ, USA). One mL of bacterial cells was transferred to a
microcentrifuge tube, samples were centrifuged at 14,000 x g for 10 min and supernatant was
167
discarded. Genomic DNA was extracted using the Qiagen DNeasy Blood Tissue kit (Qiagen,
Valencia, CA, USA) according to the manufacturer’s instruction. The isolated genomic DNA
concentration and purity were measured using a NanoDrop ND-1000 (Thermo Scientific,
Wilmington, DE, USA) and DNA samples were subsequently stored at -20°C until used.
3.2. Confirmatory PCR for S. Heidelberg strains
The conventional PCR assay was conducted using MJ Mini Personal Thermal Cycler
(Bio-Rad, Hercules, CA, USA). Salmonella Heidelberg specific primers were used to confirm
that all 15 strains belong to the serovar Heidelberg (Table 4.1). A 25 μL total reaction volume
composed of 1 μL of template DNA, 1 μL of each primer (IDT, Coralville, IA, USA), 12.5 μL of
SYBR Green (Cambrex Bioscience, Walkersville, MD, USA), and 9.5 μL of DNase-RNase free
water. The PCR conditions consisted of pre-denaturation at 94°C for 5 min, then 35 cycles of
denaturation at 94°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 30 s, with a final
extension cycle at 72°C for 5 min. The PCR products were confirmed on 1.5% of agarose gel
and visualized on a transilluminator (Bio-Rad, Hercules).
3.3. Essential oils and cultures
All of the EOs tested, R(+)-limonene, orange terpenes, cold compressed orange oil, trans-
cinnamaldehyde and carvacrol, were obtained from Sigma Aldrich (St. Louis, MO, USA).
Fifteen S. Heidelberg strains from different sources were examined (Table 4.2). One colony of
each strain was inoculated into Luria-Bertani broth and incubated for 18 h at 42°C, shaking at
190 rpm. This experiment was repeated growing S. Heidelberg strains at 37°C.
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3.4. Modified tube dilution assay
To maintain the EOs in a homogeneous mixture, 0.15% agar was added to nutrient broth
(NB), and boiled for 1 min before autoclaving. When the media reached room temperature
triphenyl tetrazolium chloride (TTC) was added at a rate of 1mL/100mL to act as a growth
indicator. As previously described by O’Bryan et al., (2008), serial dilutions were made by
placing 10 mL of the NB with 0.15% agar (NBA) in the first tube and 5 mL in the remaining
tubes for a total of four tubes. One hundred μL of oil was added to the 1st (10 mL) tube for an
initial concentration of 10 μL/mL. Serial dilutions were done by transferring 5 mL of the
emulsion to the next tube, the procedure was repeated for a total of four dilutions. Five mL were
removed from the last tube and discarded so that all tubes had the same volume (5 mL). All tubes
were inoculated with 50 μL of an overnight culture of each S. Heidelberg strain. All tubes were
incubated for 24 h at 37°C. A change in color from light yellow to pink/red indicated growth.
The MIC was determined to be the lowest concentration of EOs that showed no growth, no color
in the medium.
3.5. Statistical analysis
MIC tests were repeated as three independent experiments. Mean ± standard deviation
was conducted using JMP Pro Software Version 11.0 (SAS Institute Inc., Cary, NC).
4. Results and Discussion
We used S. Heidelberg specific primers to confirm that all 15 strains used in this study
(Table 4.1) belong to S. Heidelberg (Figure 4.1). Minimum inhibitory concentrations (Table 4.3)
were determined by observing the tubes for any color change to pink or red. When S. Heidelberg
169
strains were grown at 42°C, results showed that trans-cinnamaldehyde (Figure 4.2) and carvacrol
(Figure 4.3) exhibited MICs of 131 μg/mL and 122 μg/mL respectively, by completely inhibiting
the growth of each S. Heidelberg strain, while orange terpenes (Figure 4.4) and R-(+)-limonene
(Figure 4.5) did not exhibit any inhibitory activity against any of these strains. Cold compressed
orange oil only inhibited growth on two of the strains (945 and 114), with MIC’s of 843 μg/mL
(Figure 4.6). No relationship was observed between the source of the strain and the EOs tested.
Similar results were obtained when strains were grown at 37°C, although under this condition
cold compressed orange oil showed no antimicrobial activity for all strains, while at 42°C it
inhibited strains 945 and 114 with an MIC of 843 μg/mL. Temperature did not seem to have an
impact in terms of the effectiveness of each EOs as an antimicrobial.
Studies have shown the existence of differences between serovars as well as strains
within the same serovar of Salmonella enterica. In their research, González et al., (2012)
compared hilA gene expression in response to acid stress different Salmonella serovars and
strains. Results showed that there are serovar and strain differences in virulence gene expression
and acid tolerance; regulation of hilA showed to be serovar and strain dependent as well as
dependent on acid type (González et al., 2012). In an extensive genome analysis comparison
between UK-1 and other S. Typhimurium strains, Luo et al., (2012) reported that virulence
factors pertaining to one strain might increase or decrease virulence when present in a different
strain. Shah et al., (2011) reported that isolates from S. Enteritidis that have been recovered from
poultry or poultry environment are not equally pathogenic, nor do they have similar
invasiveness.
Previous research has shown that different EOs confer antimicrobial activity pre and post
harvest on Salmonella. The antimicrobial effect of EOs has been reported to be concentration
170
dependent (Sivropoulou et al. 1996). Ravishankar et al., (2010) reported that at a 0.2%
concentration, antimicrobials, carvacrol and cinnamaldehyde completely inactivated the
antibiotic-resistant and -susceptible isolates of S. enterica. Zhou et al., (2007) investigated the
antimicrobial activity of cinnamaldehyde, thymol and carvacrol individually and in combination
against S. Typhimurium, and reported the lowest concentrations of cinnamaldehyde, thymol and
carvacrol inhibiting the growth of S. Typhimurium significantly were 200, 400 and 400 mg/L,
respectively. When combined, cinnamaldehyde/thymol, cinnamaldehyde/carvacrol and
thymol/carvacrol showed the concentration of cinnamaldehyde, thymol and carvacrol could be
decreased from 200, 400 and 400 mg/L to 100, 100 and 100 mg/L, respectively (Zhou et al.,
2007). In this project we used cinnamaldehyde in its trans isomer, which is the form that is
present as a major component of bark extract of cinnamon (Kollanoor Johny et al., 2008).
Using the MIC method to study the effectiveness of orange essential oils against different
Salmonella serovars, O’Bryan et al., (2008) reported that it appeared not to be a significant
difference in response between strains of the same serovar or between different Salmonella
serovars tested. From seven citrus EOs tested in their research, three of them: high purity orange
terpenes, d-limonene and terpenes from orange essence, showed antimicrobial activity against
Salmonella. Despite the fact that limonene is one of the most well known and characterized of
the EOs from citrus products (Dabbah et al., 1970; Caccioni et al., 1998), which can exert potent,
broad-spectrum antimicrobial activity (Di Pasqua et al., 2006), this essential oil, in the form of
R-(+)-limonene did not exhibit any antimicrobial activity in this study (Figure 4.5).
Essential oils have also been studied to have antimicrobial activity against Salmonella
when used post harvest. In their research, Alali et al., (2013) determined the effect of non-
pharmaceuticals (a blend of organic acids, a blend of EOs, lactic acids, and a combination of
171
levulinic acid and sodium dodecyl sulfate) on weight gain, feed conversion ratio, mortality of
broilers and their ability to reduce colonization and fecal shedding of Salmonella Heidelberg.
Their results showed that the broilers that received the EOs had significantly increased weight
gain and mortality was lower compared to other treatments. Salmonella Heidelberg
contamination in crops was significantly lower in challenged and unchallenged broilers that
received EOs and lactic acids in drinking water, when compared to other treatments (Alali et al.,
2013). Results of our study shows that some EOs could be used as an antimicrobial agent against
the foodborne pathogen Salmonella. Further studies will be needed to confirm the antimicrobial
effect of these EOs in vivo.
5. Acknowledgments
We thank Dr. Steven L. Foley, Division of Microbiology, NCTR, U.S. Food and Drug
Administration, Jefferson, AR, and Dr. W. Florian Fricke, University of Maryland, School of
Medicine, Baltimore, MD for providing Salmonella Heidelberg strains used in this study. Dr.
Young M. Kwon, Poultry Science Department, University of Arkansas, Fayetteville, AR for
providing the EOs for the study. We also thank the Department of Food Science, University of
Arkansas, Fayetteville, AR for providing graduate stipend support.
172
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Ravishankar, S. L. Zhu, J. Reyna-Granados, B. Law, L. Joens, and M. Friedman. 2010.
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on celery and oysters. J. Food Prot. 73:234-240.
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Accessed at: http://publichealth.lacounty.gov.
Shah, D. H., X. Zhou, T. Addwebi, M. A. Davis, L. Orfe, D. R. Call, J. Guard, and T. E.
Besser. 2011. Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates
is associated with pathogenicity, motility and proteins secreted by the type III secretion system.
Microbiology. 157:1428-1445.
Sivropoulou, A., E. Papanikolaou, C. Nikolaou, S. Kokkini, T. Lanaras, and M.
Arsenakis. 1996. Antimicrobial and cytotoxic activities of Origanum essential oils. J. Agric.
Food Chem. 44:1202-1205.
Solórzano-Santos, F., and M. G. Miranda-Novales. 2012. Essential oils from aromatic
herbs as antimicrobial agents. Curr. Opin. Biotechnol. 23:136-141.
Su, L.-H., C.-H., Chiu, C. Chu, and J. T. Ou. 2004. Antimicrobial resistance in
nontyphoid Salmonella serotypes: a global challenge. Clin. Infect. Dis. 39:546-551.
Vugia, D. J., M. Samuel, M. M. Farley, R. Marcus, B. Shiferaw, S. Shallow, K. Smith,
and F. J. Angulo. 2004. Invasive Salmonella infections in the United States, FoodNet, 1996-
1999: incidence, serotype distribution, and outcome. Clin. Infect. Dis. 38:S149-156.
Watson, P. R., S. M. Paulin, A. P. Bland, P. W. Jones, and T. S. Wallis. 1995.
Characterization of intestinal invasion by Salmonella typhimurium and Salmonella dublin and
effect of a mutation in the invH gene. Infect. Immun. 63:2743-2754.
Wilmshurst, P., and H. Sutcliffe. 1995. Splenic abscess due to Salmonella Heidelberg.
Clin. Infect. Dis. 21:1065.
Zhao, S., D. G. White, S. L. Friedman, A. Glenn, K. Blickenstaff, S. L. Ayers, J. W.
Abbott, E. Hall-Robinson, and P. F. McDermott. 2008. Antimicrobial resistance in Salmonella
enterica serovar Heidelberg isolates from retail meats, including poultry, from 2002 to 2006.
Appl. Environ. Microbiol. 74:6656-6662.
Zhou, F., B. Ji, H. Zhang, H. Jiang, Z. Yang, J. Li, and W. Yan. 2007. The antibacterial
effect of cinnamaldehyde, thymol, carvacrol and their combinations against the foodborne
pathogen Salmonella Typhimurium. J. Food Saf. 27:124-133.
176
Table 4.1. Salmonella Heidelberg specific primer pair used in this study.
Primer Target Sequence PCR product
size (bp)
Reference
SH_SHP-2f Salmonella Heidelberg 5’-GCATA GTTCC AAAGC
ACGTT-3’ 180 In this study
SH_SHP-1r 5’-GCTCA ACATA AGGGA
AGCAA-3’
177
Table 4.2. Salmonella Heidelberg strains used in this study
Strain Source Reference
ARI-14 poultry products In this study
SL 476 ground turkey Fricke et al., 2011
SL 486 human Fricke et al., 2011
692 chicken egg house Lynne et al., 2009
945 human Han et al., 2011
114 cattle Lynne et al., 2009
163 turkey Kaldhone et al., 2008
136 swine Lynne et al., 2009
1148 human Han et al., 2012
824 turkey Kaldhone et al., 2008
130 chicken Lynne et al., 2009
937 human Han et al., 2011
118 cattle Lynne et al., 2009
144 swine Lynne et al., 2009
146 swine Lynne et al., 2009
178
Figure 4.1. Gel electrophoresis of PCR products to confirm Salmonella Heidelberg strains. Lane
1: DNA ladder, lanes 2 to16: Salmonella Heidelberg strains, and NC: negative control.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 NC
180bp
179
Table 4.3. Minimum inhibitory concentrations (in μg/mL) of five different EOs against
Salmonella Heidelberg strains.
Strain Essential oils tested
carvacrol
R-(+)-
limonene
orange
terpenes orange oil
trans-
cinnamaldehyde
SL 486 122 ± 0 No effect No effect No effect 131 ± 0
SL 476 122 ± 0 No effect No effect No effect 131 ± 0
ARI-14 122 ± 0 No effect No effect No effect 131 ± 0
692 122 ± 0 No effect No effect No effect 131 ± 0
945 122 ± 0 No effect No effect 843 ± 0 131 ± 0
114 122 ± 0 No effect No effect 843 ± 0 131 ± 0
163 122 ± 0 No effect No effect No effect 131 ± 0
136 122 ± 0 No effect No effect No effect 131 ± 0
1148 122 ± 0 No effect No effect No effect 131 ± 0
824 122 ± 0 No effect No effect No effect 131 ± 0
130 122 ± 0 No effect No effect No effect 131 ± 0
937 122 ± 0 No effect No effect No effect 131 ± 0
118 122 ± 0 No effect No effect No effect 131 ± 0
144 122 ± 0 No effect No effect No effect 131 ± 0
146 122 ± 0 No effect No effect No effect 131 ± 0
180
(Photo taken by author)
Figure 4.2. Modified tube dilution assay for trans-cinnamaldehyde. This EO exhibited MICs of
131 μg/mL for all 15 strains, inhibiting their growth. Tubes 1 through 4 represent treatment with
different EOs concentrations. Tube 5 contains NAB with the correspondent Salmonella
Heidelberg strain (positive control), and tube 5 contains NAB with trans-cinnamaldehyde
(negative control).
1 2 3 4 5 6
181
(Photo taken by author)
Figure 4.3. Modified tube dilution assay for carvacrol. For all 15 strains carvacrol exhibited
MICs of 122 μg/mL. Tubes 1 through 4 represent treatment with different EOs concentrations
Tube 5 contains NAB with the correspondent Salmonella Heidelberg strain (positive control),
and tube 5 contains NAB with carvacrol (negative control).
1 2 3 4 5 6
182
(Photo taken by author)
Figure 4.4. Modified tube dilution assay for orange terpenes. This EO did not exhibit any
antimicrobial activity on these strains. Tubes 1 through 4 represent treatment with different EOs
concentrations. Tube 5 contains NAB with the correspondent Salmonella Heidelberg strain
(positive control), and tube 5 contains NAB with orange terpenes (negative control).
1 2 3 4 5 6
183
(Photo taken by author)
Figure 4.5. Modified tube dilution assay for R-(+)-limonene. No antimicrobial activity was
observed for any of the strains. Tubes 1 through 4 represent treatment with different EOs
concentrations. Tube 5 contains NAB with the correspondent Salmonella Heidelberg strain
(positive control), and tube 5 contains NAB with R-(+)limonene (negative control).
1 2 3 4 5 6
184
(Photo taken by author)
Figure 4.6. Modified tube dilution assay for cold compressed orange oil. Orange oil did not
exhibit any antimicrobial activity for 13 of the Salmonella Heidelberg strains, but strains 945
(human) and 114 (cattle) showed an MIC of 843 μg/mL. Tubes 1 through 4 represent treatment
with different EOs concentrations. Tube 5 contains NAB with the correspondent Salmonella
Heidelberg strain and no EOs (positive control), and tube 5 contains NAB with orange oil, no S.
Heidelberg (negative control).
1 2 3 4 5 6
1 2 3 4 5 6
185
7. Authorship Statement for Chapter 4
Juliany Rivera Calo is the first author of the paper and completed at least 51% of the studies
among coauthors, which the title is “Antimicrobial Activity of Essential Oils Against
Salmonella Heidelberg Strain from Multiple Sources” in chapter 4.
Major Advisor: Dr. Steven C. Ricke
186
8. Appendix
8.1. Institutional Biosafety Committee (IBC) Number
187
8.2. Institutional Biosafety Committee (IBC) Number
IBC#: 13008
Please check the boxes for each of the forms that are applicable to the research project you
are registering. The General Information Form - FORM 1 (this form) MUST be completed
on all submitted project registrations, regardless of the type of research.
Recombinant DNA (EVEN IF IT IS EXEMPT from the NIH Guidelines.) (FORM 2) Pathogens (human/animal/plant) (FORM 3) Biotoxins (FORM 4) Human materials/nonhuman primate materials (FORM 5) Animals or animal tissues and any of the above categories; transgenic animals or tissues; wild vertebrates or tissues (FORM 6)
Plants, plant tissues, or seed and any of the above categories; transgenic plants, plant tissues, or seeds (FORM 7)
CDC regulated select agents (FORM 8)
Notice to Pat Walker Health Center (FORM 9)
To initiate the review process, you must attach and send all completed registration forms
via email to [email protected]. All registration forms must be submitted electronically. To
complete the registration, print page 1 of this form, PI sign, date, and mail to: Compliance
Coordinator-IBC, 210 Admin. Building, Fayetteville, AR 72701, or FAX it to 479-575-3846.
As Principal Investigator:
I attest that the information in the registration is accurate and complete and I will submit changes to the Institutional Biosafety Committee (IBC) in a timely manner.
I am familiar with and agree to abide by the current, applicable guidelines and regulations governing my research, including, but not limited to: the NIH Guidelines for Research Involving Recombinant DNA Molecules and the Biosafety in Microbiological and Biomedical Laboratories manual.
I agree to accept responsibility for training all laboratory and animal care personnel involved in this research on potential biohazards, relevant biosafety practices, techniques, and emergency procedures.
If applicable, I have carefully reviewed the NIH Guidelines and accept the responsibilities described therein for principal investigators (Section IV-B-7). I will submit a written report to the IBC and to the Office of Recombinant DNA Activities at NIH (if applicable) concerning: any research related accident, exposure incident, or release of rDNA materials to the environment; problems implementing biological and physical containment procedures; or violations of NIH Guidelines. I agree that no work will be initiated prior to project approval by the IBC.
I will submit my annual progress report to the IBC in a timely fashion.
188
Principal Investigator Typed/Printed Name: Steven C. Ricke
Signature (PI): ____________________________________ Date: ____________________
CONTACT INFORMATION:
Principal Investigator:
Name: Steven C. Ricke
Department: FDSC
Title: Professor
Campus Address: FDSC E 27
Telephone: 575-4678
*After Hours Phone:
Fax: 575-6936
E-Mail: [email protected]
Co-Principal Investigator:
Name: Philip G. Crandall
Department: FDSC
Title: Professor
Campus Address: FDSC N221
Telephone: 575-7686
*After Hours Phone:
Fax: 575-6936
E-Mail: [email protected]
*Required if research is at Biosafety Level 2 or higher
PROJECT INFORMATION:
Have you registered ANY project previously with the IBC? Yes
Is this a new project or a renewal?
Project Title: Production of a New Vaccine for Poultry to Prevent Salmonella
Project Start Date: 11/1/2012
Project End Date: 10/31/2013
Granting Agency: USDA/SBIR
Indicate the containment conditions you propose to use (check all that apply):
Biosafety Level 1 Ref: 1
2
Biosafety Level 1A Ref: 1
2
Biosafety Level 1P Ref: 1
2
Biosafety Level 2 Ref: 1
2
Biosafety Level 2A Ref: 1
2
Biosafety Level 2P Ref: 1
2
Biosafety Level 3 Ref: 2
Biosafety Level 3A Ref: 2
Biosafety Level 3P Ref: 2
References:
189
1: Biosafety in Microbiological and Biomedical Laboratories (BMBL) 4th Edition
2: NIH Guidelines for Research Involving Recombinant DNA Molecules
3: University of Arkansas Biological Safety Manual
If you are working at Biosafety Level 2 or higher, has your laboratory received an onsite
inspection by the Biosafety Officer or a member of the IBC?
If yes, enter date if known: 1/9/2012
If no, schedule an inspection with the Biological Safety Officer.
Please provide the following information on the research project (DO NOT attach or insert
entire grant proposals unless it is a Research Support & Sponsored Programs proposal).
Project Abstract:
Avirulent live Salmonella vaccines are considered to be more effective for preventing the spread
of Salmonella in poultry flocks. A live Salmonella vaccine should be completely genetically
stable and avirulent for both animals and humans. We have developed a double deletion mutant
that is deficient in its ability to synthesize lysine (lysA-) and is defective in its virulence
properties (hilA-).The goal of this project is to determine the survival and control of this vaccine
strain and differences in attachment or invasiveness in human Caco-2 cell model.
Specific Aims:
a. Determine motility of the vaccine strain.
b. Determine survival of vaccine strain under acid conditions.
c. Determine the ability of the vaccine strain to withstand thermal treatment.
d. Evaluate the attachment and invasiveness of the vaccine strain in human Caco-2 cell
model.
Relevant Materials and Methods (this information should be specific to the research
project being registered and should highlight any procedures that involve biohazardous or
recombinant materials):
Motility (Shah et al, 2011; Vikram et al., 2011; Wang et al. 2007): Grow Salmonella overnight
(~16 h). Stab-inoculate 1 µl of culture in the middle of semi-solid media (0.3% LB agar).
Incubate at 37 °C for 7-8 hr and measure the diameter of the halo of growth.
Survival under acid conditions (Malheiros et al., 2008): Prepare overnight Salmonella culture in
LB broth. Wash the cells with PBS three times and add the cells to PBS acidified to pH 3.5, 4.0,
4.5, and 5.0 with HCl. Incubate the cells in 30°C water bath and take 1 ml at each interval time
(20~30 min, up to 3 h) to plate on LB agar with appropriate dilutions for enumeration.
Survival under thermal condition (Malheiros et al., 2008; Alvarez-Ordonez et al., 2008): Prepare
overnight bacteria culture. Wash the cells with PBS three times and add the bacterial cells to
190
PBS. Incubate the cells in 52, 56, 60°C water bath and take 1 ml at each interval time (10 – 20
min up to 2h and 5 min up to 30 min for 60°C) to plate on LB agar with appropriate dilution for
enumeration.
Adhesion and Invasion:
Caco-2 cells preparation: Media: Dulbecco’s modified Eagle medium (DMEM) (Invitrogen)
supplemented with 10 % (v/v) fetal bovine serum (FBS; Invitrogen) in 95 % air/5 % CO2 at
37°C. Cell condition: Caco-2 human colon adenocarcinoma cells with passage of 20-35. When
confluence is achieved (~10 days), harvest the cells by trypsinization (add trypsin for 10-15min,
disrupt the cells) and resuspend in D10F. Plate the resuspeneded cells onto 12-well plates
(Cellstar) at 1x10E5 ~10E6 cells per well. Change the medium every other day until confluency
(10~12 or up to 14 days until differentiated). Cell differentiation can be monitored by estimation
of the production of intestinal alkaline phosphatase using SensoLyte pNPP alkaline phosphatase
assay kit (AnaSpec). (Intestinal alkaline phosphatase is a brush border enzyme expressed
exclusively in villus-associated enterocytes, and expression indicates the development of
digestive and absorptive function (Goldberg et al., 2008; Hara et al., 1993).)
Adhesion assay: Prepare overnight bacteria culture (~16 h, grown in LB broth). Wash the cells
with PBS three times and dilute the bacterial cells to MOI ration of 10: 1 (10E6 Salmonella:
10E5 Caco-2 cells) Add the bacteria to Caco-2 cells and incubate for 2 h. Wash the cells with
cell-PBS (137 mM NaCl, 5.4 mM KCl, 3.5 mM Na2HPO4, 4.4 mM NaH2PO4, 11 mM glucose,
pH 7.2) four times. Treat the adherent cells with 0.1% Triton-X 100 (Sigma, St. Louis, MO) in
cell-PBS for 10 min at 37°C and collect the disrupted cells. Plate the bacteria on LB agar with
appropriate dilution for enumeration.
Invasion assay: Prepare overnight bacteria culture (~16 h, grown in LB broth). Wash the cells
with PBS three times and dilute the bacterial cells to MOI ration of 10: 1 (Salmonella: Caco-2
cells). Add the bacteria to Caco-2 cells and incubate for 2 h. Wash the cells with cell-PBS for
four times. Add 100 µg gentamicin ml/1 in D10F in each well and incubate for 2 h to kill
extracellular bacteria. Wash the cells with cell-PBS four times, add 0.1% Triton-X 100 in cell-
PBS to the Caco-2 cells for 10 min at 37°C and collect the disrupted cells. Plate the bacteria on
LB agar with appropriate dilution for enumeration (Shah et al, 2011).
The information requested above can be entered directly or cut & pasted into the space
provided, or can be provided as an attached word document. If you provide an
attachment, please indicate “See Attached” and list the file name(s) in the space below:
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PERSONNEL QUALIFICATIONS & FACILITY INFORMATION:
List all personnel (including PI and Co-PI) to be involved in this project:
Name (First and Last) - Position
(Title, academic degrees,
certifications, and field of
expertise)
Qualifications/Training/Relevant Experience (Describe
previous work or training with biohazardous and/or
recombinant DNA; include Biosafety Levels )
Example: Bob Biohazard - 14 yrs working with E. coli at BL1, Salmonella enterica at
191
Associate Professor, PhD-
Microbiology
BL2, 8 yrs working with transgenic mice.
Steven C. Ricke, Professor, PhD
Gut Microbiology
20+ years with BSL 1 and 2
Philip Crandall, Professor, PhD,
Food Science
15+ years with BSL 1 and 2
Peter Rubinelli, Sr. Scientist, Sea
Star Int., PhD Cell Biology
10 years working with cell cultures and BSL2 organisms
Ok Kyung Koo, Post Doc, PhD
Food Science
8 years working with BSL2 organisms and cell culture
Corliss O’Bryan, Post Doc, PhD
Med Micro
30+ years with BSL1 and 2
Si Hong Park, Grad student, MS,
Micro
3 years with BSL 1 and 2
Juliany Rivera, Grad student, BS,
Micro
1 year with BSL 1 and 2
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Additional Personnel Information (if needed):
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List all the laboratories/facilities where research is to be conducted:
Building: Room #: Category: *Signage Correct?
Biomass 132 Laboratory Yes
Biomass 136 Tissue Culture Yes
Biomass 101 Autoclave/BioStorage Yes
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* Biohazard signs are required for entrances to Biosafety Level 2 (including Animal
Biosafety Level 2) areas. EH&S will supply these signs. If an updated biohazard sign is
required, please indicate the location and what agents/organisms/hazards should be listed
on the sign:
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Additional Facility Information (if needed):
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192
SAFETY PROCEDURES:
Please indicate which of the following personal protective equipment (PPE) will be used to
minimize the exposure of laboratory personnel during all procedures that require handling or
manipulation of registered biological materials.
Gloves:
Latex Vinyl Nitrile Leather Other
Specify: Click here to enter text.
Face & Eye Protection:
Face Shield Safety Goggles Safety Glasses
Other Specify: Click here to enter text.
Clothing Protection:
Re-usable Lab Coat Re-usable Coverall Disposable Clothing Protection
Other Specify: Click here to enter text.
Dirty or contaminated protective clothing cleaning procedures: (Check all that apply)
Autoclaved prior to laundering or disposal Laundered on site using bleach Laundered by qualified commercial service
Other Specify: Click here to enter text.
Outline procedures for routine decontamination of work surfaces, instruments, equipment,
glassware and liquid containing infectious materials. Autoclaving or using fresh 10%
bleach as a chemical disinfectant are preferred treatments; please specify and justify any
exceptions:
Work surfaces will be decontaminated with a freshly prepared 10% bleach solution before and
after working. Exception is biosafety cabinets which will be disinfected before and after use with
Lysol® No Rinse Sanitizer in order to avoid the corrosiveness of the bleach on the metal of the
biosafety cabinets. Instruments and equipment will be decontaminated by wiping down with
10% bleach. Paper towels used for these purposes will be discarded in biohazard bags.
Glassware, waste, and disposable tubes will be autoclaved under standard conditions (15 psi, 121
C, 20 min). Disposable items (pipette tips, pipets, etc) will be discarded into 10% bleach. After
30 minutes it will be permissible to place these items in a biohazard bag for autoclaving before
disposal.
193
Describe waste disposal methods to be employed for all biological and recombinant
materials. Include methods for the following types of waste: (ref: UofA BiosafetyManual )
Sharps:
Placed into 10% bleach solution for decontamination followed by discarding into sharps waste
container
Cultures, Stocks and Disposable Labware:
Placed into biohazard bags and autoclaved before disposal. Liquids will be disposed of in drains
after autoclaving. Disposable glass will be placed in glass disposal after autoclaving.
Pathological Waste:
Liquid biological waste will always be discarded into freshly made 10% bleach and then
autoclaved for decontamination treatment before it is discarded. Other biological waste will be
placed carefully into biohazard waste bags, autoclaved at 15 psi, 121C for 20 min.
Other:
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Autoclave(s), to be used in this project, location(s) and validation procedures:
Biomass Res. Ctr. Room 101: Autoclaves are checked monthly using SteriGage test strips (3M)
and SporAmpule vials to ensure autoclaves completely sterilize all bacterial life forms including
spores.
Will biological safety cabinet(s) be used?
Choose an item.
If yes, please provide the following information:
Make/Model Serial Number Certification
Expiration
Location (bldg/room)
Thermo Forma 1186
100663
11/30/2012
BIOR 132
Forma Scientific
1000
13324-539
11/30/2012
BIOR 132
Forma Scientific
1284
104294-5978
11/30/2012
BIOR 136
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text.
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text.
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date.
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text.
194
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text.
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text.
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date.
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text.
Additional Biological Safety Cabinet Information (if needed):
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Indicate if any of the following aerosol-producing procedures will occur: (check all that
apply)
Centrifuging Grinding Blending Vigorous Shaking or Mixing Sonic Disruption Pipetting Dissection Innoculating Animals Intranasally Stomacher
Other Describe: Click here to enter text.
Describe the procedures/equipment that will be used to prevent personnel exposure during
aerosol-producing procedures:
All pipetting of infectious material will take place in the biological safety cabinet. Mechanical
pipetting devices will be used. Lab coats buttoned over street clothes, gloves and goggles will be
worn. All materials needed will be placed in the biological safety cabinet before work begins.
Sash of the cabinet will be lowered and all movements will be slow to avoid disruption of the air
currents. Centrifuged cultures will be contained in a closed Eppendorf tube or contained in
screw-capped polypropylene or polystyrene tubes with gasket seals to prevent aerosol exposure.
Cultures to be vortexed will be contained in screw-capped polypropylene or polystyrene tubes,
and vortexing will be done within the biological safety cabinets. Sonicating will be done within
the biosafety cabinet or within an enclosure on the bench top.
EMERGENCY PROCEDURES:
In the event of personnel exposure (e.g. mucous membrane exposure or parenteral
inoculation), describe what steps will be taken including treatment, notification of proper
supervisory and administrative officials, and medical follow up evaluation or treatment:
In the event of accidental exposure of personnel the person exposed should notify the laboratory
supervisor immediately. Treatable exposures will be treated by use of the first aid kit containing
antimicrobial agents. Mucous membrane exposure or puncture with contaminated material will
result in the person being taken to the Health Center for prophylactic antibiotic therapy. In the
event of environmental contamination, describe what steps will be taken including a spill
response plan incorporating necessary personal protective equipment (PPE) and decontamination
procedures.
195
In the event of environmental contamination, describe what steps will be taken including a
spill response plan incorporating necessary personal protective equipment (PPE) and
decontamination procedures.
For a spill inside the biological safety cabinet, alert nearby people and inform laboratory
supervisor. Safety goggles, lab coat buttoned over street clothes and latex gloves should be worn
during clean up. If there are any sharps they will be picked up with tongs, and the spill covered
with paper towels. Carefully pour disinfectant (freshly made 10% bleach) around the edges of
the spill, then into the spill without splashing. Let sit for 20 minutes. Use more paper towels to
wipe up the spill working inward from the edge. Clean the area with fresh paper towels soaked in
disinfectant. Place all contaminated towels in a biohazard bag for autoclaving. Remove personal
protective clothing and wash hands thoroughly. For a spill in the centrifuge turn off motor, allow
the machine to be at rest for 30 minutes before opening. If breakage is discovered after the
machine has stopped, re close the lid immediately and allow the unit to be at rest for 30 minutes.
Unplug centrifuge before initiating clean up. Wear strong, thick rubber gloves and other personal
protective equipment (PPE) before proceeding with clean up. Flood centrifuge bowl with
disinfectant. Place paper towels soaked in a disinfectant over the entire spill area. Allow 20
minute contact time. Use forceps to remove broken tubes and fragments. Place them in a sharps
container for autoclaving and disposal as infectious waste. Remove buckets, trunnions and rotor
and place in disinfectant for 24 hours or autoclave. Unbroken, capped tubes may be placed in
disinfectant and recovered after 20 minute contact time or autoclaved. Use mechanical means to
remove remaining disinfectant soaked materials from centrifuge bowl and discard as infectious
waste. Place paper towels soaked in a disinfectant in the centrifuge bowl and allow it to soak
overnight, wipe down again with disinfectant, wash with water and dry. Discard disinfectant
soaked materials as infectious waste. Remove protective clothing used during cleanup and place
in a biohazard bag for autoclaving. Wash hands whenever gloves are removed.
For a spill outside the biological safety cabinet or centrifuge have all laboratory personnel
evacuate. Close the doors and use clean up procedures as above.
TRANSPORTATION/SHIPMENT OF BIOLOGICAL MATERIALS:
Transportation of Biological Materials: The Department of Transportation regulates some
biological materials as hazardous materials; see 49 CFR Parts 171 - 173. Transporting any of
these regulated materials requires special training for all personnel who will be involved in the
shipping process (packaging, labeling, loading, transporting or preparing/signing shipping
documents).
Will you be involved in transporting or shipping human or animal pathogens off campus?
No
If yes, complete the remaining:
Cultures of Human or Animal Pathogens Environmenatl samples known or suspected to contain a human or anumal pathogen
196
Human or animal material (including excreta, secreta, blood and its components, tissue, tissue fluids, or cell lines) containing or suspected of containing a human or animal pathogen.
Transportation/Shipment Training: Have any project personnel who will be involved in
packaging, labeling, completing, or signing shipping documents received formal training to ship
infectious substances or diagnostic specimens within the past 3 years?
Choose an item.
If yes, please provide the following information:
Name Date Trained Certified Shipping Trainer
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197
CHAPTER 5
Salmonella spp. Adhesion and Invasion Comparisons to Caco-2 and HD11 Cell Lines
Juliany Rivera Calo1,2
and Steven C. Ricke1,2
1Department of Food Science, University of Arkansas, Fayetteville, AR
2Center for Food Safety and Department of Food Science, University of Arkansas,
Fayetteville, AR
198
1. Abstract
Salmonella enterica are important facultative intracellular pathogens that cause
gastroenteritis in humans. Four different strains from three of the most predominant Salmonella
serovars in poultry, Typhimurium, Enteritidis, and Heidelberg were studied. Agar disc diffusion
test and tube dilution assays were used to determine gentamicin susceptibility of S. Typhimurium
ATCC 14028 and S. Heidelberg ARI-14. Both strains were susceptible to the presence of
gentamicin disc on the plate. Minimum inhibitory concentration (MIC) of gentamicin was 1% for
the strains tested. The adhesion and invasion abilities of these strains were determined using two
different cell lines: HD11, a chicken macrophage cell line and Caco-2, a human intestinal
epithelial cell line. Results showed that attachment percentages for each Salmonella strain were
higher than the ability of the strain to invade the cells. For Caco-2 cells, Salmonella
Typhimurium and Salmonella Heidelberg showed similar attachment percentages. For HD11
cells, attachment percentages were lower than for Caco-2 but Salmonella exhibited higher
percentages for invasion, presumably due to phagocytosis by HD11. Salmonella Enteritidis
exhibited lower percentages for adhesion and invasion in HD11. The type of cell line and the
different serovars studied appeared to be a factor for the differences in adhesion and invasion.
Understanding the ability and mechanisms of this pathogen to attach and invade different cell
lines could be helpful for the reduction of Salmonella infections.
199
2. Introduction
Infections by Salmonella enterica subspecies enterica are one of the leading causes of
food borne gastroenteritis in humans (WHO, 2007). Annually, it is estimated that over 1 million
Americans contract Salmonella (Scallan et al., 2011). Yearly costs for Salmonella control efforts
are estimated to be up to $14.6 billion (Scharff, 2010; Heithoff, et al., 2012). Many of its
serovars have been associated with the consumption of poultry products, one of the main
reservoirs of Salmonella (CDC, 2009). Salmonella Enteritidis, S. Typhimurium and S.
Heidelberg are the three most frequent serovars recovered from humans and isolated from
poultry (Foley et al., 2011; Finstad et al., 2012; Howard et al., 2012).
Some of the genes necessary for invasion of intestinal epithelial cells and induction of
intestinal secretory and inflammatory responses are encoded in Salmonella Pathogenicity Island
1 (SPI-1) (Watson et al., 1995; Hur et al., 2012). Salmonella Pathogenicity Island 2 (SPI-2) is
key for the establishment of systemic infection beyond the intestinal epithelium; it also encodes
essential genes for intracellular replication (Cirillo et al., 1998; Ohl and Miller, 2001; Hur et al.,
2012). Adhesion of Salmonella to the intestinal epithelial surface is an important first step in
pathogenesis and is central to its colonization of the intestine. Once Salmonella is attached to the
intestinal epithelium, normally it expresses a multiprotein complex, known as T3SS, that
facilitates endothelial uptake and invasion (Foley et al., 2008, 2013; Winnen et al., 2008). This
T3SS is associated with SPI-1, which contains virulence genes involved in Salmonella adhesion,
invasion and toxicity (Foley et al., 2013). The human intestinal Caco-2 cell line has been
extensively used over the past twenty years as a model of the intestinal barrier. The parental cell
line, originally obtained from a human colon adenocarcinoma, goes through a spontaneous
differentiation process that leads to the formation of a monolayer of cells, expressing several
200
morphological and functional characteristics of the mature enterocyte (Sambuy et al., 2005).
HD11 cells are a macrophage-like immortalized cell line derived from chicken bone marrow and
transformed with the avian myelocytomatosis type MC29 virus (Beug et al., 1979). In this
experiment, adhesion and invasion of different Salmonella serovars to Caco-2 and HD11 cell
lines were studied. Several pathogens and host factors may be an important key to determine the
mechanisms for the differences in Salmonella responses within different cell types (Bueno et al.,
2012; Foley et al., 2013). Understanding the ability and mechanisms of this pathogen to attach
and invade different cell lines could be helpful for the reduction of Salmonella infections.
3. Materials and Methods
3.1. Bacterial strains for antibiotic susceptibility testing
One colony of S. Typhimurium ATCC 14028, S. Heidelberg ARI-14 and E. coli 25922
(negative control) was inoculated into 5 mL of Luria Bertani (LB) Broth (Alfa Aesar, Ward Hill,
MA, USA) and incubated for 16 h at 37°C, 190 rpm shaking.
3.2. Antibiotic susceptibility testing
Susceptibility of Salmonella Typhimurium ATCC 14028 and S. Heidelberg ARI-14 to
gentamicin was determined by two methods, the agar disc diffusion test and the modified tube
dilution assay. For the agar disc diffusion test, a sterile cotton swab was dipped into the
overnight bacterial culture and gently squeezed against the tube to remove any excess fluid. The
cotton swab subsequently streaked on Mueller Hinton Agar (BD Biosciences, Franklin Lakes,
NJ, USA) plate at different angles to provide even growth. One 6-mm gentamicin paper disc
(Becton Dickson, Sparks, MD, U.S.A.) was aseptically placed on the center of each agar plate,
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followed by incubation for 16 to 24 h at 37°C. Zone inhibition diameters were measured for
each plate and averages were calculated. Plates were streaked per triplicate and each experiment
was repeated three times.
The minimum inhibitory concentration for each bacterial strain was determined using a
modified tube dilution assay method. Serial dilutions were made by placing 10 mL of nutrient
broth (EMD Millipore, Billerica, MA, USA) in the first tube and 5 mL in the remaining tubes for
a total of five tubes. One hundred μL of gentamicin (Life Technologies, Grand Island, NY, USA)
at a stock concentration of 500 μg/mL was added to the 1st (10 mL) tube and serial dilutions were
done by transferring 5 mL of the mixture to the next tube, the procedure was repeated for a total
of five dilutions. Five μL were removed and discarded from the last tube so that all tubes had the
same volume (5 mL). All tubes were inoculated with 50 μL of the bacterial overnight culture.
Fifty microliters of triphenyl tetrazolium chloride (TTC) was added to each tube to act as a
growth indicator. All tubes were incubated for 24 h at 37°C. A change in color from light yellow
to pink/red indicated growth. The MIC was determined to be the lowest concentration of
gentamicin that showed no growth, no color in the medium. After the 24 h incubation, 100 μL of
each dilution for each bacterial strain was inoculated into Mueller-Hinton agar (BD Biosciences)
plates to determine CFU/mL and confirm MIC results. Plates were done per triplicate and each
experiment was repeated three times.
3.3. Cell cultures
Human epithelial (Caco-2) cells and chicken macrophage (HD11) cells were maintained
in HyClone™ Classical Liquid Media: Dulbeccos Modified Eagles Medium (MEM), High
Glucose (Thermo Scientific, Logan, UT, USA) with 10% fetal bovine serum (FBS) and non
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essential amino acids (NEAA) and grown routinely in a 75 cm2 flask at 37°C in a 5% CO2
incubator (New Brunswick, Eppendorf, Enfield, CT, USA). Once the cells in flask were
approximately 80% confluent, they were treated with 0.25% Trypsin-EDTA (Life Technologies)
to release the attached cells and new stock cultures were inoculated with 104
cells per mL. For
the adhesion and invasion assays, 104 Caco-2 and HD11 cells per mL were inoculated in 24-well
tissue culture plates (Greiner Bio-One, Monroe, NC, USA) and incubated at 37°C in a 5% CO2
incubator until a semi-confluent monolayer was obtained.
3.4. Bacterial cultures for adhesion and invasion assays
Salmonella strains used for this experiment are listed in Table 5.1. One colony of each
strain, S. Typhimurium ATCC 14028, S. Typhimurium UK-1, S. Heidelberg ARI-14 and S.
Enteritidis ATCC 13076, was inoculated on 8 mL LB broth and incubated for 16 to 18 h at 37°C.
3.5. Adhesion assays
Cells were enumerated in three representative wells by trypsinizing each well with 0.3
mL trypsin-EDTA, and incubating at 37°C in a 5% CO2 incubator for 5 min and adding 0.7 mL
D10F. Overnight bacterial cultures were washed three times with PBS (137 mM NaCl, 2.7 mM
KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). Bacterial cells were diluted to an MOI ratio of
10:1 (106
Salmonella: 105 HD11 or Caco-2 cells). Washed bacteria were diluted 10
-6 with PBS
and 100 μL were plated on LB agar plates for determining CFU/mL. The diluted bacteria were
added to the cell lines, Caco-2 or HD11, and plate was incubated at 37°C in a 5% CO2 incubator
(Thermo/Forma Scientific, Marietta, OH, USA) for 2 h. Cells were subsequently washed three
times with cell-PBS (137 mM NaCl, 5.4 mM KCl, 3.5 mM Na2HPO4, 4.4 mM NaH2PO4, 11 mM
203
glucose, pH 7.2), and treated with 1 mL of 0.1% Triton X-100 in cell-PBS. The plate was
incubated for 10 min, at 37°C in a 5% CO2 incubator. The disrupted cells were collected, serially
diluted and plated on LB agar plates, in duplicate, to determine adhesion percentage. Plates were
incubated at 37°C for 16 h. All LB agar plates to determine CFU/mL were inoculated per
duplicate. Each strain was tested in triplicate in three independent experiments.
3.6. Invasion assays
Cells were enumerated in three representative wells by trypsinizing each well with 0.3
mL trypsin-EDTA, and incubating at 37°C in a 5% CO2 incubator for 5 min and adding 0.7 mL
D10F. Overnight bacterial cultures were washed three times with PBS. Bacterial cells were
diluted to obtain an MOI ratio of 10:1 (Salmonella: animal cells). Washed bacteria were diluted
10-6
with PBS and 100 μL were plated on LB agar plates for determining CFU/mL. The diluted
bacteria was added to the cell lines, Caco-2 or HD11, and plate was incubated at 37°C in a 5%
CO2 incubator for 2 h. Cells were subsequently washed three times with cell-PBS and treated
with 1 mL DMEM with 100 μg/mL gentamicin per well to kill cell-adherent extracellular
bacteria. Plate was incubated for 2 h, at 37°C in a 5% CO2 incubator. Cells were then washed
three times with cell-PBS and treated with 1 mL of 0.1% Triton X-100 in cell-PBS. The plate
was incubated for 10 min, at 37°C in a 5% CO2 incubator. Serial dilutions of suspensions were
made in PBS and inoculated onto LB agar plates, in duplicate, to determine the number of
organisms that survived treatment with gentamicin and hence had invaded the Caco-2 or HD11
cells. Plates were incubated at 37°C for 16 h. Each strain was tested in triplicate in three
independent experiments.
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3.7. Statistical analysis
All statistical analyses were conducted using JMP Pro Software Version 11.0 (SAS
Institute Inc., Cary, NC). Mean ± standard deviation was calculated for each antibiotic
susceptibility test. One-way ANOVA and Tukey-Kramer HSD test were performed on the
adhesion and invasion percentages of each bacterial strain to Caco-2 and HD11 cells. Statistical
significance was established at P < 0.05.
4. Results and Discussion
The disc diffusion assay results are presented in Table 5.2. Salmonella Heidelberg ARI-
14 and S. Typhimurium ATCC 14028 produced an average inhibition zone of 20.7 ± 1.1 and
20.7 ± 0.1 respectively. Escherichia coli strain 25922 used as a control, showed an inhibition
zone of 19.0 ± 0. All three strains appeared to be susceptible to the presence of gentamicin
(Figure 5.1). Results from the tube dilution method are listed in Table 5.3. For all three strains,
the MIC of gentamicin was 500 μg/mL; no change in color was observed (Figures 5.2 to 5.4). To
further confirm results from this method, CFU/mL was calculated for each strain. Contrary to
what was observed on the tubes, where there was no change in color that should have indicated
the presence of bacteria in the media, growth was observed on the agar plates for all three strains
after the third dilution (0.25%) (Figure 5.5). Based on the CFU/mL the MIC of gentamicin at
which no growth was observed for any of the strains was 250 μg/mL. Minimum inhibitory
concentrations results provide confirmation for using gentamicin during invasion assays to kill
cell-adherent extracellular bacteria. Andrews (2001) published expected MIC ranges for
determining the susceptibility of several bacteria to a wide selection of antibiotics, along with a
list of appropriate controls that must be included when determining MIC. The suggested MIC
205
range for Enterobacteriaceae susceptibility against gentamicin was 0.03 to 128 mg/L using E.
coli 25922 as a control (Andrews, 2001). Shah et al (2011) studied cell invasion of several
poultry-associated Salmonella Enteritidis isolates and they reported a MIC of gentamicin of
<0.125 μg for all the isolates used in the study.
The ability of several Salmonella strains from different serovars to attach and invade two
cell lines: Caco-2, a human intestinal epithelial cell line; and HD11, a chicken macrophage cell
line and was studied. As expected, attachment percentages for each Salmonella strain were
higher than the ability of the strain to invade the cells. Results showed that S. Heidelberg and S.
Typhimurium exhibited adhesion percentages of 28.8 ± 6.37 and 18.1 ± 6.25 respectively, to
Caco-2 cells (Table 5.4). In contrast, the ability of these strains to invade Caco-2 cells was lower,
1.37 ± 0.25 for S. Heidelberg and 1.52 ± 0.02 for S. Typhimurium (Table 5.4). The underlying
mechanisms used by intracellular pathogens such as Salmonella to penetrate the host epithelium
are not well understood. As a result, researchers have used cultured mammalian cells as in vitro
models to study interaction and internalization of Salmonella (Gianella et al., 1973; Finlay et al.,
1988; Durant et al., 1999). Invasion in cultured epithelial cells is commonly used to measure the
pathogenicity of Salmonella (van Asten et al., 2000, 2004; Shah et al., 2011). Previous studies
have reported that Salmonella invasion into cultured mammalian cells can be influenced by
several environmental stimuli such as osmolarity (Galán and Curtis, 1990; Tartera and Metcalf,
1993) carbohydrate availability (Schiemann, 1995), and oxygen availability (Ernst et al., 1990;
Lee and Falkow, 1990; Francis et al., 1992). In their research, Durant et al., (1999) reported that
Salmonella can encounter high concentrations of short-chain fatty acids (SCFA) in the lower
regions of the gastrointestinal tract, this in addition to changes in pH, oxygen tension, and
osmolarity. Shah et al., (2011) reported that in cultured Caco-2 cells, isolates with high
206
invasiveness were able to invade and/or survive in significantly higher numbers within chicken
macrophage cells than other isolates with low invasiveness.
For HD11 cells, attachment percentages were higher than for Caco-2 (Table 5.5), and
Salmonella exhibited higher percentages for invasion, ranging from 2.9 to 17.6%. Two other
strains, S. Typhimurium UK-1 and S. Enteritidis ATCC 13076, were used for experiments with
HD11 cells. From all strains, Salmonella Typhimurium strain UK-1 invaded HD11 cells with
high percentages, 17.6 ± 3.29, which is not surprising given the high virulence of this strain.
Salmonella Typhimurium UK-1, (UK stands for universal killer) is a chicken-passaged isolate of
a highly virulent strain that was originally isolated in 1991 from an infected horse (Curtis et al.,
1991). Invasion results from Salmonella Typhimurium ATCC 14028 (9.3 ± 0.57) and UK-1
(17.6 ± 3.29) to HD11 cells demonstrate that there are strain differences among the same
serovar; different strains can have different invasiveness and virulence. Luo et al., (2012)
conducted an extensive genome analysis comparison between UK-1 and other S. Typhimurium
strains and reported that virulence factors pertaining to one strain may increase or decrease
virulence when are found present in a different strain (Luo et al., 2012). Once the polymorphic
genomic regions of the strains are identified and analyzed, even highly similar strains of S.
Typhimurium could be differentiated (Luo et al., 2012).
From all three serovars studied, Salmonella Enteritidis showed the lowest adhesion and
invasion percentage, 2.9 ± 1.48. In their research, He et al., (2012) compared Salmonella cell
invasion and intracellular survival of five different poultry-associated serovars (S. Heidelberg, S.
Enteritidis, S. Typhimurium, S. Senftenberg, and S. Kentucky). Their results showed that
compared to the other four serovars, S. Enteritidis was more resistant to intracellular killing,
leading them to believe that the intracellular survival ability of this serovar may be related with
207
systemic invasion in chickens (He et al., 2012). In their research, Shah et al., (2011) tested the
ability of S. Enteritidis isolates with low and high invasiveness to invade chicken macrophage
HD11 cells. Results showed that invasion percentages of 4.88 ± 0.13 and 6.07 ± 0.08 for isolates
with low and high percentages respectively (Shah et al., 2011). These results are comparable to
our findings of low invasiveness of S. Enteritidis. Matulova et al., (2012) also reported lower
invasion after pre-treating the HD11 cells with avidin prior infection with S. Enteritidis. Shah et
al., (2011) suggested that not all isolates of S. Enteritidis recovered from poultry might be
equally pathogenic or have similar potential to invade cells; and that S. Enteritidis pathogenicity
is related to both the secretion of Type III secretion system effector proteins and motility. Saeed
et al., (2006) reported that compared to isolates of S. Enteritidis recovered from chicken ceca,
isolates recovered from eggs or from human clinical cases showed a greater adherence and
invasiveness of chicken ovarian granulosa cells.
In conclusion, the type of cell line and the different serovars studied appeared to be a
factor for the differences in adhesion and invasion. Further studies should be conducted in order
to understand the properties that make some serovars more able to attach and invade the cells
than others. Understanding the ability and mechanisms of this pathogen to attach and invade
different cell lines could be helpful for the reduction of Salmonella infections.
5. Acknowledgments
We thank Dr. Peter Rubinelli, Center for Food Safety, University of Arkansas,
Fayetteville, AR, for his training and insights in conducting adhesion and invasion assays. We
also thank the Department of Food Science, University of Arkansas, Fayetteville, AR for
providing graduate stipend support to Juliany Rivera Calo.
208
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Table 5.1. Salmonella strains used in this study.
Adhesion and Invasion to Caco-2 cells Adhesion and Invasion to HD11 cells
Salmonella Typhimurium ATCC 14028 Salmonella Typhimurium ATCC 14028
Salmonella Heidelberg ARI-14 Salmonella Typhimurium UK-1
Salmonella Heidelberg ARI-14
Salmonella Enteritidis ATCC 13076
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Table 5.2. Zones of inhibition (in millimeters) of gentamicin against S. Heidelberg and S.
Typhimurium. (mean ± standard deviation).
Strain Zone Diameter
(mm) Susceptibility
S. Heidelberg ARI-14 20.7 ± 1.1 Susceptible
S. Typhimurium ATCC 14028 20.7 ± 0.9 Susceptible
E. coli 25922 (negative control) 19.0 ± 0 Susceptible
214
(Photo taken by author)
Figure 5.1. Antibacterial activity of gentamicin against strains of S. Typhimurium, S. Heidelberg
and E. coli. All three strains studied showed to be sensitive against gentamicin.
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Table 5.3. MIC of gentamicin against strains of S. Heidelberg, S. Typhimurium, and E. coli
examined.
Bacterial strain MIC (μg/mL)
S. Heidelberg ARI-14
500 ± 0
S. Typhimurium ATCC 14028
500 ± 0
E. coli 25922 500 ± 0
216
(Photo taken by author)
Figure 5.2. Gentamicin minimum inhibitory concentration against Salmonella Typhimurium
ATCC 14028. No change in color was observed for any of the dilutions treated with gentamicin.
217
(Photo taken by author)
Figure 5.3. Gentamicin minimum inhibitory concentration against Salmonella Heidelberg ARI-
14. No change in color was observed for any of the dilutions treated with gentamicin.
Negative control:
no gentamicin
218
(Photo taken by author)
Figure 5.4. Gentamicin minimum inhibitory concentration against Escherichia coli 25922. No
change in color was observed for any of the dilutions treated with gentamicin. Tube labeled as
NB: nutrient broth (positive control, no bacteria).
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a)
b)
c)
(Photo taken by author)
Figure 5.5. MIC of gentamicin based on CFU/mL for each strain. The minimum inhibitory
concentration for all three bacterial cultures showed to be 0.5% of the stock gentamicin
concentration (500 μg/mL). a) Salmonella Typhimurium ATCC 14028, b) Salmonella
Heidelberg ARI-14, and c) Escherichia coli 25922.
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Table 5.4. Salmonella Typhimurium and S. Heidelberg adhesion and invasion to human epithelial, Caco-2, cells.
Bacterial strain Log
Dilution
# of colonies # bacteria
added
# bacteria adhering % Adhesion
S. Typhimurium
ATCC 14028
4 17.67 ± 2.31 9.83x106 ± 0 1.78x10
6 ± 0.25 18.1 ± 6.25
a
S. Heidelberg
ARI-14
4 26.0 ± 2.83 9.13x106 ± 0 2.63x10
6 ± 0.32 28.8 ± 6.37
a
# bacteria invading
% Invasion
S. Typhimurium
ATCC 14028
2
124.75 ± 10.69
8.26x106 ± 0
1.25x105 ± 0.01
1.52 ± 0.02a
S. Heidelberg
ARI-14
2
63.17 ± 17.54
5.24x106 ± 0
5.96 x104
± 1.42
1.37 ± 0.25a
*Percentages not connected by the same letter are significantly different between each assay (P < 0.05).
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Table 5.5. Adhesion and invasion of four Salmonella strains from different serovars to chicken macrophage, HD11, cells.
Bacterial strain Log Dilution # of colonies # bacteria
added
# bacteria
adhering % Adhesion*
S. Heidelberg 4 59.6 ± 15.96 1.3x107 ± 0.47 5.5x10
6 ± 1.98 43.4 ± 7.46
a
S. Typhimurium
ATCC 14028 4 61.8 ± 17.58 1.6x10
7 ± 0.47 6.2x10
6 ± 1.15
38.7 ± 4.57a
S. Typhimurium
UK-1 4 69.2 ± 19.85 1.8x10
7 ± 0.71 7.0x10
6 ± 1.93 38.9 ± 9.24
a
S. Enteritidis 4 39.2 ± 9.24 2.1x107 ± 0 3.9x10
6 ± 0.50 18.6 ± 2.42
b
# bacteria
invading
% Invasion*
S. Heidelberg 4 33.2 ± 9.04 6.91x10
7 ± 0 3.3x10
6 ± 0.50 4.8 ± 0.73
c
S. Typhimurium
ATCC 14028
4 37.3 ± 4.19 4.0x107 ± 0.28 3.7x10
6 ± 0.08 9.3 ± 0.57
b
S. Typhimurium
UK-1 4 25.3 ± 4.99 1.4x10
7 ± 0.15 2.5x10
6 ± 0.40
17.6 ± 3.29a
S. Enteritidis 4 4.05 ± 3.06 1.7x107 ± 0.04 4.9x10
5 ± 2.30 2.9 ± 1.48
c
*Percentages not connected by the same letter are significantly different between each assay (P < 0.05).
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7. Authorship Statement for Chapter 5
Juliany Rivera Calo is the first author of the paper and completed at least 51% of the studies
among coauthors, which the title is “Salmonella spp. Adhesion and Invasion Comparisons to
Caco-2 and HD11 Cell Lines” in chapter 5.
Major Advisor: Dr. Steven C. Ricke
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8. Appendix
8.1. Institutional Biosafety Committee (IBC) Number
224
8.2. Institutional Biosafety Committee (IBC) Number
IBC#: 13008
Please check the boxes for each of the forms that are applicable to the research project you
are registering. The General Information Form - FORM 1 (this form) MUST be completed
on all submitted project registrations, regardless of the type of research.
Recombinant DNA (EVEN IF IT IS EXEMPT from the NIH Guidelines.) (FORM 2) Pathogens (human/animal/plant) (FORM 3) Biotoxins (FORM 4) Human materials/nonhuman primate materials (FORM 5) Animals or animal tissues and any of the above categories; transgenic animals or tissues; wild vertebrates or tissues (FORM 6)
Plants, plant tissues, or seed and any of the above categories; transgenic plants, plant tissues, or seeds (FORM 7)
CDC regulated select agents (FORM 8)
Notice to Pat Walker Health Center (FORM 9)
To initiate the review process, you must attach and send all completed registration forms
via email to [email protected]. All registration forms must be submitted electronically. To
complete the registration, print page 1 of this form, PI sign, date, and mail to: Compliance
Coordinator-IBC, 210 Admin. Building, Fayetteville, AR 72701, or FAX it to 479-575-3846.
As Principal Investigator:
I attest that the information in the registration is accurate and complete and I will submit changes to the Institutional Biosafety Committee (IBC) in a timely manner.
I am familiar with and agree to abide by the current, applicable guidelines and regulations governing my research, including, but not limited to: the NIH Guidelines for Research Involving Recombinant DNA Molecules and the Biosafety in Microbiological and Biomedical Laboratories manual.
I agree to accept responsibility for training all laboratory and animal care personnel involved in this research on potential biohazards, relevant biosafety practices, techniques, and emergency procedures.
If applicable, I have carefully reviewed the NIH Guidelines and accept the responsibilities described therein for principal investigators (Section IV-B-7). I will submit a written report to the IBC and to the Office of Recombinant DNA Activities at NIH (if applicable) concerning: any research related accident, exposure incident, or release of rDNA materials to the environment; problems implementing biological and physical containment procedures; or violations of NIH Guidelines. I agree that no work will be initiated prior to project approval by the IBC.
I will submit my annual progress report to the IBC in a timely fashion.
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Principal Investigator Typed/Printed Name: Steven C. Ricke
Signature (PI): ____________________________________ Date: ____________________
CONTACT INFORMATION:
Principal Investigator:
Name: Steven C. Ricke
Department: FDSC
Title: Professor
Campus Address: FDSC E 27
Telephone: 575-4678
*After Hours Phone:
Fax: 575-6936
E-Mail: [email protected]
Co-Principal Investigator:
Name: Philip G. Crandall
Department: FDSC
Title: Professor
Campus Address: FDSC N221
Telephone: 575-7686
*After Hours Phone:
Fax: 575-6936
E-Mail: [email protected]
*Required if research is at Biosafety Level 2 or higher
PROJECT INFORMATION:
Have you registered ANY project previously with the IBC? Yes
Is this a new project or a renewal?
Project Title: Production of a New Vaccine for Poultry to Prevent Salmonella
Project Start Date: 11/1/2012
Project End Date: 10/31/2013
Granting Agency: USDA/SBIR
Indicate the containment conditions you propose to use (check all that apply):
Biosafety Level 1 Ref: 1
2
Biosafety Level 1A Ref: 1
2
Biosafety Level 1P Ref: 1
2
Biosafety Level 2 Ref: 1
2
Biosafety Level 2A Ref: 1
2
Biosafety Level 2P Ref: 1
2
Biosafety Level 3 Ref: 2
Biosafety Level 3A Ref: 2
Biosafety Level 3P Ref: 2
226
References:
1: Biosafety in Microbiological and Biomedical Laboratories (BMBL) 4th Edition
2: NIH Guidelines for Research Involving Recombinant DNA Molecules
3: University of Arkansas Biological Safety Manual
If you are working at Biosafety Level 2 or higher, has your laboratory received an onsite
inspection by the Biosafety Officer or a member of the IBC?
If yes, enter date if known: 1/9/2012
If no, schedule an inspection with the Biological Safety Officer.
Please provide the following information on the research project (DO NOT attach or insert
entire grant proposals unless it is a Research Support & Sponsored Programs proposal).
Project Abstract:
Avirulent live Salmonella vaccines are considered to be more effective for preventing the spread
of Salmonella in poultry flocks. A live Salmonella vaccine should be completely genetically
stable and avirulent for both animals and humans. We have developed a double deletion mutant
that is deficient in its ability to synthesize lysine (lysA-) and is defective in its virulence
properties (hilA-).The goal of this project is to determine the survival and control of this vaccine
strain and differences in attachment or invasiveness in human Caco-2 cell model.
Specific Aims:
a. Determine motility of the vaccine strain.
b. Determine survival of vaccine strain under acid conditions.
c. Determine the ability of the vaccine strain to withstand thermal treatment.
d. Evaluate the attachment and invasiveness of the vaccine strain in human Caco-2 cell
model.
Relevant Materials and Methods (this information should be specific to the research
project being registered and should highlight any procedures that involve biohazardous or
recombinant materials):
Motility (Shah et al, 2011; Vikram et al., 2011; Wang et al. 2007): Grow Salmonella overnight
(~16 h). Stab-inoculate 1 µl of culture in the middle of semi-solid media (0.3% LB agar).
Incubate at 37 °C for 7-8 hr and measure the diameter of the halo of growth.
Survival under acid conditions (Malheiros et al., 2008): Prepare overnight Salmonella culture in
LB broth. Wash the cells with PBS three times and add the cells to PBS acidified to pH 3.5, 4.0,
4.5, and 5.0 with HCl. Incubate the cells in 30°C water bath and take 1 ml at each interval time
(20~30 min, up to 3 h) to plate on LB agar with appropriate dilutions for enumeration.
227
Survival under thermal condition (Malheiros et al., 2008; Alvarez-Ordonez et al., 2008): Prepare
overnight bacteria culture. Wash the cells with PBS three times and add the bacterial cells to
PBS. Incubate the cells in 52, 56, 60°C water bath and take 1 ml at each interval time (10 – 20
min up to 2h and 5 min up to 30 min for 60°C) to plate on LB agar with appropriate dilution for
enumeration.
Adhesion and Invasion:
Caco-2 cells preparation: Media: Dulbecco’s modified Eagle medium (DMEM) (Invitrogen)
supplemented with 10 % (v/v) fetal bovine serum (FBS; Invitrogen) in 95 % air/5 % CO2 at
37°C. Cell condition: Caco-2 human colon adenocarcinoma cells with passage of 20-35. When
confluence is achieved (~10 days), harvest the cells by trypsinization (add trypsin for 10-15min,
disrupt the cells) and resuspend in D10F. Plate the resuspeneded cells onto 12-well plates
(Cellstar) at 1x10E5 ~10E6 cells per well. Change the medium every other day until confluency
(10~12 or up to 14 days until differentiated). Cell differentiation can be monitored by estimation
of the production of intestinal alkaline phosphatase using SensoLyte pNPP alkaline phosphatase
assay kit (AnaSpec). (Intestinal alkaline phosphatase is a brush border enzyme expressed
exclusively in villus-associated enterocytes, and expression indicates the development of
digestive and absorptive function (Goldberg et al., 2008; Hara et al., 1993).)
Adhesion assay: Prepare overnight bacteria culture (~16 h, grown in LB broth). Wash the cells
with PBS three times and dilute the bacterial cells to MOI ration of 10: 1 (10E6 Salmonella:
10E5 Caco-2 cells) Add the bacteria to Caco-2 cells and incubate for 2 h. Wash the cells with
cell-PBS (137 mM NaCl, 5.4 mM KCl, 3.5 mM Na2HPO4, 4.4 mM NaH2PO4, 11 mM glucose,
pH 7.2) four times. Treat the adherent cells with 0.1% Triton-X 100 (Sigma, St. Louis, MO) in
cell-PBS for 10 min at 37°C and collect the disrupted cells. Plate the bacteria on LB agar with
appropriate dilution for enumeration.
Invasion assay: Prepare overnight bacteria culture (~16 h, grown in LB broth). Wash the cells
with PBS three times and dilute the bacterial cells to MOI ration of 10: 1 (Salmonella: Caco-2
cells). Add the bacteria to Caco-2 cells and incubate for 2 h. Wash the cells with cell-PBS for
four times. Add 100 µg gentamicin ml/1 in D10F in each well and incubate for 2 h to kill
extracellular bacteria. Wash the cells with cell-PBS four times, add 0.1% Triton-X 100 in cell-
PBS to the Caco-2 cells for 10 min at 37°C and collect the disrupted cells. Plate the bacteria on
LB agar with appropriate dilution for enumeration (Shah et al, 2011).
The information requested above can be entered directly or cut & pasted into the space
provided, or can be provided as an attached word document. If you provide an
attachment, please indicate “See Attached” and list the file name(s) in the space below:
Click here to enter text.
PERSONNEL QUALIFICATIONS & FACILITY INFORMATION:
List all personnel (including PI and Co-PI) to be involved in this project:
Name (First and Last) - Position
(Title, academic degrees,
certifications, and field of
Qualifications/Training/Relevant Experience (Describe
previous work or training with biohazardous and/or
recombinant DNA; include Biosafety Levels )
228
expertise)
Example: Bob Biohazard -
Associate Professor, PhD-
Microbiology
14 yrs working with E. coli at BL1, Salmonella enterica at
BL2, 8 yrs working with transgenic mice.
Steven C. Ricke, Professor, PhD
Gut Microbiology
20+ years with BSL 1 and 2
Philip Crandall, Professor, PhD,
Food Science
15+ years with BSL 1 and 2
Peter Rubinelli, Sr. Scientist, Sea
Star Int., PhD Cell Biology
10 years working with cell cultures and BSL2 organisms
Ok Kyung Koo, Post Doc, PhD
Food Science
8 years working with BSL2 organisms and cell culture
Corliss O’Bryan, Post Doc, PhD
Med Micro
30+ years with BSL1 and 2
Si Hong Park, Grad student, MS,
Micro
3 years with BSL 1 and 2
Juliany Rivera, Grad student, BS,
Micro
1 year with BSL 1 and 2
Click here to enter text. Click here to enter text.
Additional Personnel Information (if needed):
Click here to enter text.
List all the laboratories/facilities where research is to be conducted:
Building: Room #: Category: *Signage Correct?
Biomass 132 Laboratory Yes
Biomass 136 Tissue Culture Yes
Biomass 101 Autoclave/BioStorage Yes
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* Biohazard signs are required for entrances to Biosafety Level 2 (including Animal
Biosafety Level 2) areas. EH&S will supply these signs. If an updated biohazard sign is
required, please indicate the location and what agents/organisms/hazards should be listed
on the sign:
Click here to enter text.
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Additional Facility Information (if needed):
Click here to enter text.
SAFETY PROCEDURES:
Please indicate which of the following personal protective equipment (PPE) will be used to
minimize the exposure of laboratory personnel during all procedures that require handling or
manipulation of registered biological materials.
Gloves:
Latex Vinyl Nitrile Leather Other
Specify: Click here to enter text.
Face & Eye Protection:
Face Shield Safety Goggles Safety Glasses
Other Specify: Click here to enter text.
Clothing Protection:
Re-usable Lab Coat Re-usable Coverall Disposable Clothing Protection
Other Specify: Click here to enter text.
Dirty or contaminated protective clothing cleaning procedures: (Check all that apply)
Autoclaved prior to laundering or disposal Laundered on site using bleach Laundered by qualified commercial service
Other Specify: Click here to enter text.
Outline procedures for routine decontamination of work surfaces, instruments, equipment,
glassware and liquid containing infectious materials. Autoclaving or using fresh 10%
bleach as a chemical disinfectant are preferred treatments; please specify and justify any
exceptions:
Work surfaces will be decontaminated with a freshly prepared 10% bleach solution before and
after working. Exception is biosafety cabinets which will be disinfected before and after use with
Lysol® No Rinse Sanitizer in order to avoid the corrosiveness of the bleach on the metal of the
biosafety cabinets. Instruments and equipment will be decontaminated by wiping down with
10% bleach. Paper towels used for these purposes will be discarded in biohazard bags.
Glassware, waste, and disposable tubes will be autoclaved under standard conditions (15 psi, 121
C, 20 min). Disposable items (pipette tips, pipets, etc) will be discarded into 10% bleach. After
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30 minutes it will be permissible to place these items in a biohazard bag for autoclaving before
disposal.
Describe waste disposal methods to be employed for all biological and recombinant
materials. Include methods for the following types of waste: (ref: UofA BiosafetyManual )
Sharps:
Placed into 10% bleach solution for decontamination followed by discarding into sharps waste
container
Cultures, Stocks and Disposable Labware:
Placed into biohazard bags and autoclaved before disposal. Liquids will be disposed of in drains
after autoclaving. Disposable glass will be placed in glass disposal after autoclaving.
Pathological Waste:
Liquid biological waste will always be discarded into freshly made 10% bleach and then
autoclaved for decontamination treatment before it is discarded. Other biological waste will be
placed carefully into biohazard waste bags, autoclaved at 15 psi, 121C for 20 min.
Other:
Click here to enter text.
Autoclave(s), to be used in this project, location(s) and validation procedures:
Biomass Res. Ctr. Room 101: Autoclaves are checked monthly using SteriGage test strips (3M)
and SporAmpule vials to ensure autoclaves completely sterilize all bacterial life forms including
spores.
Will biological safety cabinet(s) be used?
Choose an item.
If yes, please provide the following information:
Make/Model Serial Number Certification
Expiration
Location (bldg/room)
Thermo Forma 1186
100663
11/30/2012
BIOR 132
Forma Scientific
1000
13324-539
11/30/2012
BIOR 132
Forma Scientific
1284
104294-5978
11/30/2012
BIOR 136
231
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text.
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text.
Click here to enter a
date.
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text.
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text.
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text.
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date.
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text.
Additional Biological Safety Cabinet Information (if needed):
Click here to enter text.
Indicate if any of the following aerosol-producing procedures will occur: (check all that
apply)
Centrifuging Grinding Blending Vigorous Shaking or Mixing Sonic Disruption Pipetting Dissection Innoculating Animals Intranasally Stomacher
Other Describe: Click here to enter text.
Describe the procedures/equipment that will be used to prevent personnel exposure during
aerosol-producing procedures:
All pipetting of infectious material will take place in the biological safety cabinet. Mechanical
pipetting devices will be used. Lab coats buttoned over street clothes, gloves and goggles will be
worn. All materials needed will be placed in the biological safety cabinet before work begins.
Sash of the cabinet will be lowered and all movements will be slow to avoid disruption of the air
currents. Centrifuged cultures will be contained in a closed Eppendorf tube or contained in
screw-capped polypropylene or polystyrene tubes with gasket seals to prevent aerosol exposure.
Cultures to be vortexed will be contained in screw-capped polypropylene or polystyrene tubes,
and vortexing will be done within the biological safety cabinets. Sonicating will be done within
the biosafety cabinet or within an enclosure on the bench top.
EMERGENCY PROCEDURES:
In the event of personnel exposure (e.g. mucous membrane exposure or parenteral
inoculation), describe what steps will be taken including treatment, notification of proper
supervisory and administrative officials, and medical follow up evaluation or treatment:
In the event of accidental exposure of personnel the person exposed should notify the laboratory
supervisor immediately. Treatable exposures will be treated by use of the first aid kit containing
antimicrobial agents. Mucous membrane exposure or puncture with contaminated material will
result in the person being taken to the Health Center for prophylactic antibiotic therapy. In the
event of environmental contamination, describe what steps will be taken including a spill
232
response plan incorporating necessary personal protective equipment (PPE) and decontamination
procedures.
In the event of environmental contamination, describe what steps will be taken including a
spill response plan incorporating necessary personal protective equipment (PPE) and
decontamination procedures.
For a spill inside the biological safety cabinet, alert nearby people and inform laboratory
supervisor. Safety goggles, lab coat buttoned over street clothes and latex gloves should be worn
during clean up. If there are any sharps they will be picked up with tongs, and the spill covered
with paper towels. Carefully pour disinfectant (freshly made 10% bleach) around the edges of
the spill, then into the spill without splashing. Let sit for 20 minutes. Use more paper towels to
wipe up the spill working inward from the edge. Clean the area with fresh paper towels soaked in
disinfectant. Place all contaminated towels in a biohazard bag for autoclaving. Remove personal
protective clothing and wash hands thoroughly. For a spill in the centrifuge turn off motor, allow
the machine to be at rest for 30 minutes before opening. If breakage is discovered after the
machine has stopped, re close the lid immediately and allow the unit to be at rest for 30 minutes.
Unplug centrifuge before initiating clean up. Wear strong, thick rubber gloves and other personal
protective equipment (PPE) before proceeding with clean up. Flood centrifuge bowl with
disinfectant. Place paper towels soaked in a disinfectant over the entire spill area. Allow 20
minute contact time. Use forceps to remove broken tubes and fragments. Place them in a sharps
container for autoclaving and disposal as infectious waste. Remove buckets, trunnions and rotor
and place in disinfectant for 24 hours or autoclave. Unbroken, capped tubes may be placed in
disinfectant and recovered after 20 minute contact time or autoclaved. Use mechanical means to
remove remaining disinfectant soaked materials from centrifuge bowl and discard as infectious
waste. Place paper towels soaked in a disinfectant in the centrifuge bowl and allow it to soak
overnight, wipe down again with disinfectant, wash with water and dry. Discard disinfectant
soaked materials as infectious waste. Remove protective clothing used during cleanup and place
in a biohazard bag for autoclaving. Wash hands whenever gloves are removed.
For a spill outside the biological safety cabinet or centrifuge have all laboratory personnel
evacuate. Close the doors and use clean up procedures as above.
TRANSPORTATION/SHIPMENT OF BIOLOGICAL MATERIALS:
Transportation of Biological Materials: The Department of Transportation regulates some
biological materials as hazardous materials; see 49 CFR Parts 171 - 173. Transporting any of
these regulated materials requires special training for all personnel who will be involved in the
shipping process (packaging, labeling, loading, transporting or preparing/signing shipping
documents).
Will you be involved in transporting or shipping human or animal pathogens off campus?
No
If yes, complete the remaining:
233
Cultures of Human or Animal Pathogens Environmenatl samples known or suspected to contain a human or anumal pathogen Human or animal material (including excreta, secreta, blood and its components, tissue, tissue fluids, or cell lines) containing or suspected of containing a human or animal pathogen.
Transportation/Shipment Training: Have any project personnel who will be involved in
packaging, labeling, completing, or signing shipping documents received formal training to ship
infectious substances or diagnostic specimens within the past 3 years?
Choose an item.
If yes, please provide the following information:
Name Date Trained Certified Shipping Trainer
Click here to enter text.
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234
CONCLUSIONS
Salmonella spp. continues to be one of the major foodborne pathogens of great concern
for public health, for the food industry, and in general all around the world. Understanding the
basic growth, mechanisms, pathogenesis, and antimicrobial resistance of this pathogen is of great
importance in order to be able to develop effective, accurate and rapid ways for detection and
isolation, and being able to find ways to reduce the presence of Salmonella.
In this research we studied the basic growth of multiple Salmonella serovars, which were
found to be both serovar and strain dependent. We observed the effect of spent media, more
specifically from S. Heidelberg, to the growth of S. Typhimurium and found an intriguing effect
for which more studies will be needed in order to determine the cause of it. Several factors could
play a role into this, metabolism, nutrients, virulence, and genetic traits, among many others that
have yet to be determined. The idea of competitive interaction has been investigated by many
researchers. In this research we studied the ability of two Salmonella serovars to compete
directly with each other, for which we found that the growth of S. Typhimurium is inhibited by S.
Heidelberg, the same characteristic that we observed on our spent media studies. Selective and
non-selective enrichment media methods were tested to determine the more effective and rapid
way to detect and isolate Salmonella, for which non-selective enrichment methods seemed to be
more effective.
The constant problem of increasing drug resistance has created a more urgent demand to
develop new and improved antimicrobials against resistant microorganisms (Spellberg et al.,
2004; Berghman et al., 2005). We tested the antimicrobial activity of different EOs against S.
Heidelberg strains from multiple sources. Lastly, the ability of Salmonella serovars to attach and
invade human epithelial and chicken macrophage cell lines was studied. Understanding the
235
mechanisms involved in bacterial response, could facilitate the refining and optimization of
antimicrobial formulations or discover new ways to overcome potential tolerance mechanisms,
leading to an improvement in biosecurity measures and to enhance the protection of farm-animal
and as a result, public health (Condell et al., 2012a).