AFAB Volume 5 Issue 2

Volume 5 Issue 22015

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Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 53

Sooyoun Ahn University of Florida, USA

Walid Q. Alali University of Georgia, USA

Kenneth M. Bischoff NCAUR, USDA-ARS, USA

Debabrata Biswas University of Maryland, USA

Claudia S. Dunkley University of Georgia, USA

Michael Flythe USDA, Agricultural Research Service

Lawrence Goodridge McGill University, Canada

Leluo Guan University of Alberta, Canada

Joshua Gurtler ERRC, USDA-ARS, USA

Yong D. Hang Cornell University, USA

Armitra Jackson-Davis Alabama A&M University, USA

Divya Jaroni Oklahoma State University, USA

Weihong Jiang Shanghai Institute for Biol. Sciences, P.R. China

Michael Johnson University of Arkansas, USA

Timothy Kelly East Carolina University, USA

William R. Kenealy Mascoma Corporation, USA

Hae-Yeong Kim Kyung Hee University, South Korea

Woo-Kyun Kim University of Georgia, USA

M.B. Kirkham Kansas State University, USA

Todd Kostman University of Wisconsin, Oshkosh, USA

Y. M. Kwon University of Arkansas, USA

Maria Luz Sanz Murias Instituto de Quimica Organic General, Spain

Byeng R. Min Tuskegee University in Tuskegee, AL

Melanie R. Mormile Missouri University of Science and Tech., USA

Rama Nannapaneni Mississippi State University, USA

Jack A. Neal, Jr. University of Houston, USA

Benedict Okeke Auburn University at Montgomery, USA

John Patterson Purdue University, USA

Toni Poole FFSRU, USDA-ARS, USA

Marcos Rostagno LBRU, USDA-ARS, USA

Roni Shapira Hebrew University of Jerusalem, Israel

Kalidas Shetty North Dakota State University, USA

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Ok-Kyung KooKorea Food Research Institute, South Korea

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A Surveillance of Cantaloupe Genotypes for the Prevalence of Listeria and Salmonella

G. Dev Kumar, K. Crosby, D. Leskovar, H. Bang, G.K. Jayaprakasha, B. Patil, and S. Ravishankar

73

The Efficacy of a Commercial Antimicrobial for Inhibiting Salmonella in Pet Food C. A. O’Bryan, C. L. Hemminger, P. M. Rubinelli, O. Kyung Koo, R. S. Story, P. G. Crandall, and S. C. Ricke

65

ARTICLES

The Mutating Gastrointestinal Flora, Multidrug Resistant Enterococcus faeciumA. Limayem

56

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Instructions for Authors87

Introduction to Authors

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TABLE OF CONTENTS


www.afabjournal.comCopyright © 2015

Agriculture, Food and Analytical Bacteriology

ABSTRACT

Currently, 80% of the antibiotics used in the United States (U.S.) are dedicated to agricultural systems,

primarily to promote animal livestock growth and control microbial contaminant load at slaughter. More-

over, 87% of drug resistant microorganisms including Enterococcus faecium were detected in ground tur-

key products due to antibiotic agent usage namely, virginiamycin. Given that quinupristin/dalfopristin (QD),

like virginiamycin (VIR), another mixture of streptogramin has been used in hospitals as a last alternative to

treat immunocompromised population infected by vancomycin-resistant E. faecium (VRE). Consequently,

understanding the epidemiology and the antibiotic resistance in some E. faecium strains from food ani-

mals’ to humans is a matter of great concern that urges effective strategies to intervention. This review

encompasses the most prominent knowledge on the ecology and dissemination of the multidrug resistant

E. faecium (MEF). Beneficial attributes of some E. faecium strains are also reviewed. Future directions in-

cluding mitigation strategies through systemic and molecular approaches are suggested.

Keywords: Multidrug resistant E. faecium, Fecal indicators, Farm animals, Food chain, Hospitals,

Antimicrobials, Virginiamycin, Vancomycin, Immunocompromised

INTRODUCTION

Increase in multi-drug resistance jeopardizes hu-

man and animal health in the U.S. and worldwide

(Centers for Disease Control (CDC), 2013). Vanco-

mycin resistant strains of Enterococcus including pri-

Correspondence: Alya Limayem, [email protected]: +1 -813-974-7404

marily E. faecium causes hospital-acquired infections

of approximately 20,000 and the death of 1,300 per

year in the U.S (CDC, 2013). Some strains of Entero-

coccus faecium, previously known as Streptococcus

faecium started to emerge as a nosocomial VRE in

1984 (Nannini and Murray, 2006). Clonal Complex

strains (CCs), predominantly (CC17) cause urinary

tract and bloodstream infections among patients

in hospitals (CDC, 2013). Resistant (CC17) could

The mutating gastrointestinal flora, multidrug resistant Enterococcus faecium

A. Limayem1

1 Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620, USA

Agric. Food Anal. Bacteriol. 5: 56-64, 2015


further lead to endocarditis and death in immuno-

compromised populations (Lebreton et al., 2013).

Enterococci can be carried on the hands of health

care workers, transferred from one patient to anoth-

er and can persist for up to 60 minutes (Gilmore et

al., 2002). Transmission from a health care worker’s

hands to the patient could take place upon contact

with the patient’s intravenous or urinary catheters.

This can result in colonization of the patient’s GI

tract with the acquired strain, which then becomes

part of the patient’s endogenous flora. The acquired

strain, carrying antibiotic resistance genes, is able to

live in the GI tract. Infections then arise from these

newly acquired strains, most commonly of the uri-

nary tract producing cystisis, prostatitis, and epididy-

mitis (Gilmore et al., 2002). This study demonstrated

that Enterococci are also found in intra-abdominal,

pelvic, and soft tissue infections and can cause noso-

comial bacteremia. However, endocarditis is consid-

ered the most serious enterococcal infection, as it

causes inflammation of the heart valves. In many cas-

es of endocarditis, antibiotic treatment fails and sur-

gery to remove the infected valve is necessary. Due

to the substantial multidrug resistance of the leading

nosocomial E. faecium, treatment of these infections

at an early stage is difficult. An estimated of 10,000

of vancomycin resistant E. faecium infections and

650 deaths occur in the U.S. each year (CDC, 2013).

Over the past 20 years, the incidence of multidrug

resistant E. faecium has significantly increased; 77%

of enterococcal bloodstream infections involve this

microorganism (CDC, 2013). The immune-deficient

populations including patients affected by hema-

tologic malignancies are considered at high-risk of

MEF exposure. Patients subjected to Intensive Care

Unit (ICU), organ transplants and prolonged stays

in hospitals are also of prime concern (Zhou et al.,

2013). MEF infections have also been associated

with surgical wounds from indwelling catheter use

(Ryan, 2004). In the U.S., the level of VRE has dramat-

ically climbed in the last 27 years to achieve an un-

precedented increase of 80% (CDC, 2013). As early

as 1980, the VRE epidemic began in Europe and was

partially correlated to the extensive use of avoparcin

in farm animals (Arias and Murray, 2012). Despite the

ban on the usage of avoparcin in livestock, there has

been a noticeable increase of VRE infections in some

European hospitals (Lebreton et al., 2013; Top et al.,

2008). In the U.S., in spite of focused awareness from

federal administrations for controlling drug use in

livestock, there is no action for similar ban in agricul-

ture use. Virginiamycin has been used in agricultural

system for growth promotion and bacterial control of

farm animals (Barton et al., 2003; Claycamp and Hoo-

berman, 2004). As such, without a similar strepto-

gramin ban, MEF strains would disseminate in food

animal products (Barton et al., 2003). As such, anti-

biotic resistance would disseminate in farming ani-

mals and subsequently pass through the food chain

to humans (Busani et al., 2004; Hayes et al., 2004;

Tejedor-Junco et al., 2005). Recently Limayem et al.

(2015) reported that out of 30 ground turkey samples

collected from multiple grocery stores, there were

27.7% multidrug resistant E. faecium strains. All the

isolated MEF strains were resistant to QD and its

homologous virginiamycin. Several factors impact

this threat of further dissemination of resistant E.

faecium. This strain has an unrelenting ability to mu-

tate and generate intrinsic and extrinsic mechanisms

of resistance to a broad continuum of antibiotics.

(Tremblay et al., 2011). Moreover, MEF has a tremen-

dous capability to gain drug resistance via conjuga-

tion mechanisms or gene transfer from other micro-

organisms (Werner et al., 2000). Aside from its gene

permutation properties, the phenotypic versatility of

MEF enables the gene to occupy different locations

in the cell including plasmids, integrons and operons

(Tremblay et al., 2011).

Such alarming evidence for antibiotic resistance

spread, propelled the need to ensure greater knowl-

edge on the ecology, epidemiology and antibiotic

resistance of some E. faecium strains from food ani-

mals to clinical settings. Beneficial attributes of some

E. faecium strains are also discussed. Further stud-

ies on the risk from resistant E. faecium strains are

suggested to ensure greater preventive actions and

public health safety.


ECOLOGY AND SPECIFIC CHARACTER-ISTICS

E. faecium has a spherical shape and is a Gram-

positive facultative anaerobic microbe that grows

over a large temperature range, from 10 to over

45°C, where the optimal temperature for growth

is 42.7°C. Some strains of E. faecium survive harsh

environmental conditions that include acidic en-

vironments and exposure to detergents (Jackson

et al., 2005). Enterococci in general exhibit similar

physiology to Streptococci with the discrimination

established primarily by the Lancefield group D

antigen and secondarily by growth in high salt con-

centrations (Fisher and Phillips, 2009). E. faecium’s

remarkable phenotypic elasticity and intrinsic ability

to generate resistance or virulence genes through

chromosomal exchanges, plasmids transfers, and

mutations, give rise to the possibility of pathoge-

nicity (Davies et al., 2010). Moreover, E. faecium is

able to survive the heating process used during the

making of foods such as sausage and cheese. With

its high resistance to salt (6.5%), the ubiquitous E.

faecium strain can survive in marine environments for

a long period of time (Hardwood et al., 2000). It has

been reported by Fisher and Phillips (2009) that sub-

stantial multidrug resistant E. faecium strains have

been observed in numerous aquatic environment

and pristine waters (Rice et al., 1995; Valenzuela et

al., 2010). Consequently, several strains of E. faecium

have been found in seafood including shellfish along

with fishes and brined nordic shrimps (Mejlholm et

al., 2008; Thapa et al., 2006; Valenzuela et al., 2010).

Although most of the E. faecium strains inhabit the

warm-blooded animal gut, there are some strains

that have been isolated from oral cavities and vagi-

nal tracts of humans and animals (Rice et al., 1995).

E. faecium was also isolated from a wide range of

surfaces including water, soil, and mechanical equip-

ment such as medical and agricultural devices (klein,

2003). There are some strains of E. faecium that can

survive on inanimate objects for up to four months

(Kramer et al., 2006; Lebreton et al., 2013). In Europe,

E. faecium strains have been commonly isolated

from pets, farm animals, and food products (Frei-

tas et al., 2011; Garcia-Migura et al., 2005; Garcia-

Migura et al., 2007). Furthermore, Enterococci have

been isolated from the runoff of farms that used pig

manure as well as urban sewage (Khun et al., 2003).

E. faecium can be found in concentrations of 104 to

105 per gram of human fecal material, making it a key

indicator for fecal contamination (Franz et al., 1999).

While E. faecium can be used to indicate contamina-

tion of water supplies from farmland, its isolation is

less prevalent from livestock than from human feces

(Franz et al., 1999).

BENEFICIAL ATTRIBUTES

Some strains of E. faecium are also lactic acid

bacteria and are known for their probiotic attributes.

They have been extensively added in food for their

fermentative ability and health benefits. It has been

shown that rabbits in animal husbandries that were

given water containing E. faecium as a probiotic had

higher average weight gains as well as a healthier

natural intestinal flora (Laukova et. al., 2012). While

E. faecium helps prevent antibiotic-associated diar-

rhea, enhance the immune system, and lower the

cholesterol level (Franz et al., 2011), other strains are

used for their food safety attributes in limiting zoo-

notic pathogens from food animals through bacte-

riocin production (Franz et al., 2011). As evidenced

by De Kwaadsteniet et al. (2005), E. faecium P21 iso-

lated from sausage produces both enterocins A and

B. Enterocins proved to be active against a substan-

tial range of Gram-positive bacteria including pri-

marily Listeria species and Staphylococcus aureus.

E. faecium, RZS C5 strain, has been isolated from

natural cheese and also demonstrates antilisterial

properties without exhibiting virulence factors (Le-

roy et al., 2003). Nonvirulent strains of E. faecium

have been suggested as a possible probiotic against

microbes possessing antimicrobial resistances (Franz

et al., 2011; Lund and Edlund, 2001). A substantial

review on the antibacterial properties of bacteriocins

has been implemented by Fisher and Phillips (2009).

However, the tremendous ability of some strains to

acquire virulence genes from other strains and con-


vert into pathogenic strains would hinder the benefi-

cial attributes of E. faecium. This is increasingly more

problematic due to the considerable ability of E. fae-

cium to mutate and acquire virulent genes in mul-

tiple types of environment (Arias and Murray, 2012).

EPIDEMIOLOGY

The previously named Streptococcus faecium

started to evolve as a nosocomial vancomycin resis-

tant E. faecium in 1984. In the U.S., the level of VRE

has substantially increased from approximately 1 % to

80% throughout the past three decades (CDC, 2013).

Currently, the opportunistic strains of E. faecium are

being considered among the second leading causes

of hospital-associated infections (Arias and Murray,

2012). The transmission of E. faecium is via fecal-oral

route with transfer primarily through contaminated

food or water and catheter-related E. faecium in-

oculation and colonization in hospitals (Austin et al.,

1999; Sydnor and Perl, 2011). The E. faecium strain

can also form a niche in the human gut and constitute

a fetal reservoir for the susceptible immunocompro-

mised human population that have a hematological

malignancy (Zhou et al., 2013). E. faecium can cause

serious health outcomes related to wound infections,

bacteremia, and urinary tract infections that can fur-

ther lead to septicemia and endocarditis in some

cases (Teixeira et al., 2007). Symptoms, depending on

the infective dose and ranging from mild to severe

causes, most commonly involve fever and chills along

with flank pain and shortness of breath for the patient

who is affected by endocarditis (Chan et al., 2012).

As previously mentioned, in Europe, the VRE epi-

demic began in the late 1980s and was partially as-

sociated with the extensive use of avoparcin in farm-

ing animals (Arias and Murray, 2012). Wegener et al

(1999) have reported that glycopeptide avoparcin

contained high levels of vanA encoding resistance

to glycopeptide agent found on the Tn1546 trans-

poson (Wegener, 1999; Biavasco et al., 2007). The

spread of VRE infections within human populations

led to avoparcin prohibition in European countries

(Lebreton et al., 2013).

Currently the MEF strains are proliferating at a

considerable rate thus impacting the downstream

food chain as well as surrounding pristine aquatics

systems (Rice et al., 1995; Valenzuela et al., 2010).

E faecium strains that are multi-drug resistant are

evolving as the leading cause of nosocomial patho-

gens constituting a serious level of threat that has

caused almost 10,000 infections and 650 deaths

each year in the U.S. (CDC, 2013).

Typically, there are two subpopulations of E. fae-

cium (Freitas et al., 2011): (1) the commensal/com-

munity-associated (CA) strains and (2) the hospital-

associated (HA) strains, Clonal Complex 17 (CC17).

The CC17 strains gave rise to the worldwide noso-

comial VRE as the most prevalent source of entero-

coccus bloodstream and urinary tract infections. It

has putative pathogenicity island-carrying putative

glycoside hydrolase (hyl) and enterococcal surface

protein (esp) genes that enables it to adhere to the

host tissue, aggregate, form persistent biofilms, and

cause infections (Freitas et al., 2010). Additionally,

Arias and Murray (2012) extensively reviewed the

main enterococcal E. faecium’s pathogenesis and

the components causing virulence phenotypes in

vivo. It is also worth mentioning that the virulence

expression depends on both strains and the host cell

tissue. Currently, there are an increasing number of

the MEF strains beyond VRE that are growing at a

rapid pace and cause severe complications primarily

for the immune-deficient human population. It could

also lead to sudden death if the appropriate choice

of antibiotic is not determined at an early stage.

ANTIBIOTIC RESISTANCE

The high intrinsic capability and phenotypic

elasticity of the MEF strain enable it to acquire ex-

ogenous genes from the environment, mutate

continuously, and transfer resistant genes to other

pathogens within harsh environmental conditions

(Lund and Edlund, 2001). Several MEF strains are

intrinsically resistant to a wide range of antibiotics

including the aminoglycosides. As thoroughly re-

viewed by Arias and Murray (2012), the main antibi-


otic resistance mechanisms of enterococcal strains

encompass the alteration of the target binding site

and the reduction of binding affinity (Franz et al.,

2011). Furthermore, as being homologous to QD,

virginiamycin is the last alternative used to treat VRE

in hospitals. Virginiamycin is used in large scale to

promote animal growth in farm animals and to sup-

press microbial contaminant load at slaughter (Do-

nabedian et al., 2006; Kasimoglu-Dogru et al., 2010).

The mechanisms of resistance that are used by MEF

strains against streptogramin agents involve mainly

the alteration of ribosomal sites, active efflux and the

inactivation of enzymes or drug modification (Soltani

et al., 2001). The most common enzyme inactivation

mechanism against streptogramin type A includes

O-acetyltransferase. Acetyltransferase of virginiamy-

cin agent is encoded by the gene vat. Among the

most prevalent vat genes (Simjee et al., 2006) encod-

ing streptogramin A in E. faecium are vat (D) and vat

(E) (Werner and Witte, 1999). Changes in vat (E) gene

in streptogramin resistant E. faecium due to single

base replacement, has been reported by Soltani et

al. (2001). The active efflux is noted to extrude strep-

togramins via ABC porters (Sletvold et al., 2008) en-

coded by vga (A) or vgb (B) alleles. Several studies

have reported that the erm (B) genes in enterococci

have already been found widely disseminated in the

environment (De Leener et al., 2005; Hayes et al.,

2005; Werner et al., 2000).

CONCLUSIONS-FUTURE DIRECTIONS

Extensive research studies have elucidated the

antibiotic resistance profile of VRE in human popu-

lations and hospitals (Acharya et al., 2007; Haris-

berger et al., 2011; Getachew et al., 2013; Ghidán

et al., 2008a ; Ghidán et al., 2008b ; González et al.,

2009; Jung et al., 2007; Novais et al., 2006; Poeta et

al., 2006). Additional studies have also evidenced

the resistance profile of Enterococcus genera in

food animals (Persoons et al., 2010; Pesavento et al.,

2014; Simjee et al., 2007) primarily in poultry (Fra-

calanzza et al., 2007; Ghidán et al., 2008a ; Ghidán et

al., 2008b; Schwaiger et al., 2010; Usui et al., 2014).

While numerous molecular investigations are con-

ducted on Enterococcus strains, (Aslam et al., 2012;

Cha et al., 2012; Garnier et al., 2004; Getachew et

al., 2013) including their genetic relatedness (Frei-

tas et al., 2010; González et al., 2009; Hwang et al.,

2010; Oh et al., 2007; Tremblay et al., 2011), there is

an exigency for a deeper genomic analysis to trace

connections between drug resistance profile shared

between humans and food animals in the U.S. Fur-

thermore, the rising MEF strain of its type beyond

VRE from food animals to hospitals (Getachew et al.,

2013; Pesavento et al., 2014) broad continuum is of

paramount threat level. Hence, there is an urgent

need to trace the genetic profile of MEF strains from

the source to hospitals within a comprehensive mod-

eling approach.

Further investigations including a complete char-

acterization of the MEF strain revealing rapid detec-

tion and quantification within a comprehensive risk

model will enable the development of effective miti-

gation strategies for the emerging drug resistance

in food. It thus, offers a clear insight to managers to

track the contamination pathways and set preventive

actions to ensure food and public health safety.

ACKNOWLEDGEMENTS

The author thanks the Moffitt Cancer Center

(MCC) in Florida State for their kind donation of the

multidrug resistant clinical isolates of E. faecium. The

author also extends her thanks to the National Sani-

tary Foundation Laboratory for their support in pro-

viding the poultry samples and the clinical isolates

from Michigan State.

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ABSTRACT

A commercially available antimicrobial consisting of a proprietary mixture of 5-25% (wt/vol) nonanoic

acid, 1-25% (wt/vol) butyric acid, 1-50% (wt/vol) trans-2-hexenal and water was tested for efficacy against

Gram-negative and Gram-positive bacteria, some isolates of Salmonella spp in vitro and activity against

Salmonella in pet food. The in vitro efficacy of the antimicrobial was found to be generally effective against

both Gram-positive and Gram-negative bacteria. Minimal inhibitory concentrations (MICs) were deter-

mined for isolates of Salmonella serotypes. Isolates of Heidelberg, Montevideo and Enteritidis had MICs

of 1.5 μl/ml while the other five tested isolates had MICs of 2.0 μl/ml. The effectiveness of the antimicrobial

in ground pet food artificially contaminated with a high level of Salmonella was assessed at 0, 1.0, 1.5, or

2.0 ml/kg of feed. Contaminated feed was sampled on days 0, 1, 4, 7 and 14 after treatment. All levels of

antimicrobial resulted in nearly a 1.0 log CFU/g reduction of Salmonella numbers at time of treatment, and

Salmonella levels were 2.0 log CFU/g lower at day 14 as compared to the untreated control. This antimicro-

bial would be useful in extending the shelf life of dried pet foods as well as limiting survival and growth of

Salmonella.

Keywords: Salmonella; pet food; organic acids; butyric acid; nonanoic acid; trans-2-hexenal; anti-

microbial; food safety; foodborne illness; companion animals

INTRODUCTION

Companion animals have become an increasing

aspect of the family unit in most societies. In the US

alone there are an estimated 37% of households with

at least one dog and 30% of households with a cat

Correspondence: Steven C. Ricke, [email protected]: +1 -479-575-4678

(AVMA, 2014). Most of these households feed their

pets dry food for at least part of the diet (Buchanan

et al., 2011). Many of these dry pet foods contain in-

gredients of animal origin, and thus are at risk for

contamination with Salmonella spp. Dry pet foods

are made using extrusion manufacturing in which the

combined ingredients are heated and formed into

the final product of various shapes and sizes. The

extrusion process takes place at very high tempera-

The Efficacy of a Commercial Antimicrobial for Inhibiting Salmonella in Pet Food

C. A. O’Bryan1, C. L. Hemminger1, P. M. Rubinelli1, O. Kyung Koo2, R. S. Story1, P. G. Crandall1, and S. C. Ricke1

1 Center for Food Safety and Department of Food Science, University of Arkansas, Fayetteville, AR 727042 Current address: Food Safety Research Group, Korea Food Research Institute, Seongnam-si,

Gyeonggi-do, Republic of Korea.



tures which acts as a kill step for pathogens. How-

ever, high temperatures also destroy some of the nu-

trients present in the food, so flavor enhancers and

fat, both of animal origin, are then sprayed on after

extrusion. However there is no additional kill step for

pathogens after this process (Thompson, 2008).

White et al. (2003) sampled randomly collected

dog treats derived from pig ears and other animal

parts in the United States and cultured them for the

presence of Salmonella. Forty-one percent of the

samples were found to be positive for Salmonella

and 24 different serotypes were isolated from the

positive samples. They isolated S. Infantis with PFGE

patterns indistinguishable from the strains respon-

sible for the 1999 Canadian outbreak from several

products. Li et al. (2012) reported on the prevalence

of Salmonella spp. in animal feeds. They isolated

Salmonella from 6.1% of pet foods and treats, and

from 7.1% of supplement-type pet products. More

recently, Nemser et al. (2014) found only 1 of 670

dry pet foods or treats were positive for Salmonella

spp. Nevertheless, Salmonella infections have been

found both in pets and in humans, and were deter-

mined to be linked to contaminated pet foods and

treats (Clark et al. 2001; CDC 2005; Behravesh et al.

2010; Imanishi et al. 2014).

In 1999 in Canada, an outbreak of Salmonella se-

rotype Infantis infections in humans was found to be

associated with pet treats for dogs produced from

processed pig ears. Phage typing and pulsed-field

gel electrophoresis (PFGE) determined that Salmo-

nella enterica serotype Infantis isolated from pig ear

pet treats as well as isolates from humans exposed

to the pig ears were the same (Clark et al., 2001).

Schotte et al. (2007) reported on a large outbreak of

canine salmonellosis in German military watch dogs.

The outbreak was recognized by a monitoring pro-

gram and was found to be due to 2 serotypes of Sal-

monella, Montevideo and Give. Dogs in 4 kennels

were exposed and 63.8% of the dogs had positive

fecal samples, although only 9 dogs exhibited clini-

cal disease. Two commercial dehydrated dog foods

were implicated by risk analysis as the suspected

infectious sources and S. Montevideo and S. Give

with similar plasmid profiles and PFGE-restriction

patterns were isolated from the suspected foods

and fecal samples. In 2012 in the United States a

routine sample collected of dry dog food was found

to be positive for S. Infantis (Imanishi et al., 2014).

The Centers for Disease Control and Prevention was

able to link the genetic fingerprint of this isolate with

humans with infections caused by S. Infantis. The

subsequent outbreak investigation identified 53 ill

humans infected with the outbreak strain in 21 states

and 2 provinces in Canada. Traceback investigations

identified one production plant as the source of the

contaminated food, and the outbreak strain was iso-

lated from unopened bags of dry dog food and fecal

specimens from dogs that had eaten the food and

lived with ill people.

These outbreaks confirm that large outbreaks of

salmonellosis occur after feeding contaminated dry

pet foods and pet treats. This also puts pet owners

and vulnerable members of their households at risk

as they often live in close contact with their animals.

These highly publicized salmonellosis outbreaks and

recalls of dry pet foods due to contamination with S.

enterica have caused a major review of microbiologi-

cal control programs, and have reinforced the idea

that food safety should extend beyond traditional

factory quality management processes. As in food

for human consumption, ensuring the microbiologi-

cal integrity of pet foods must cover the entire pro-

duction pipeline (‘farm-to-fork approach’). The study

reported in this paper was developed to determine

whether a commercial antimicrobial would restrict

the survival and growth of Salmonella in dry dog

food. The antimicrobial contains butyric acid, nona-

noic acid and trans-2-hexenal.

MATERIALS AND METHODS

Determination of antimicrobial spec-trum

Lyophilized cultures of test organisms (Salmonella

Typhimurium, Escherichia coli, Staphylococcus aure-

us, Clostridium perfringens, Lactobacillus plantarum,

Streptococcus agalactiae and Campylobacter jejuni)


were obtained from the American Type Culture Col-

lection (ATCC; Manassas, VA). Cultures were resus-

citated according to ATCC recommended methods

and transferred to Standard Methods Agar (SMA; BD

Diagnostics, Franklin Lakes, NJ). Agar plates were in-

cubated for 24 hours at 35°C.

After incubation, bacterial colonies were trans-

ferred to individual test tubes containing 10 mL of

Trypticase Soy Broth (TSB; BD Diagnostics). Test

tubes were incubated at 35°C for 18 to 24 hours. The

level of bacteria in the broth culture was determined

by serial dilution and plating on SMA. Cultures were

diluted to a final concentration of 105 cfu ml-1 with

Butterfield’s phosphate buffer.

One mL of CO-60 surfactant and 1 mL of the an-

timicrobial (Preserv-8®; Anitox Corp., Lawrenceville,

GA) were mixed together (the surfactant was used to

allow the antimicrobial to be soluble in water for test

purposes). A 0.2 ml aliquot of the mixture was added

to 9.8 mL of sterile, deionized water to prepare a 1%

stock solution (10 ml kg-1). The stock solution was di-

luted with sterile deionized water to the equivalent of

5, 1, 0.5, 0.1 and 0.05 ml kg-1.

A 100 μL aliquot of the 105 cfu ml-1 inoculum was

added to each of the dilution tubes containing the

different concentrations of antimicrobial. Tubes were

vortexed for 30 seconds every hour for four hours.

A 1 mL aliquot was removed from each tube at 24

hours and serially diluted in Butterfield’s phosphate

buffer. Dilutions were plated on selective agars as

recommended for each type of bacteria. Plates were

incubated at 35°C for 48 hours prior to enumeration.

Clostridium, Lactobacillus and Campylobacter plates

were incubated under anaerobic conditions.

Determination of minimal inhibitory concentration

Isolates of 8 serovars of Salmonella were tested

(Heidelberg, Montevideo, Enteritidis, Typhimurium,

Worthington, Kentucky, Senftenberg and Infantis); all

isolates were obtained from the culture collection of

the Center for Food Safety of the University of Arkan-

sas. Overnight cultures were prepared by inoculating

10 ml of sterile LB broth (EMD Millipore, Billerica,

MA) with a single isolate of a serotype of Salmonella

and incubating at 37°C for 18 to 24 h. Minimal inhibi-

tory concentration (MIC) levels were determined in

96-well clear microtiter plates (NUNC, Rochester,

N.Y., U.S.A.) with lids. A stock solution of the antimi-

crobial was prepared at 1%. Prepared sterile LB broth

was aseptically pipetted (100 μl) into all wells of the

microtiter plate. A 100 μl aliquot of the antimicrobi-

al was pipetted into the first row of wells and serial

2-fold dilutions were performed to the end point of

0.25% of the antimicrobial, and 100 μl of excess solu-

tion was discarded from the last row to keep well vol-

umes equal. One row was used as a positive control

and contained 5 μl ml-1 of butyric acid; another row of

wells was used as a negative control and contained

bacteria and LB only. A 100 μL aliquot (containing

approximately 105 cfu) of a single Salmonella culture

was pipetted into each well. Microtiter plates were

incubated statically at 37°C for 18 hours and optical

density (OD) was read at 600 nm. The MIC was de-

fined as the first well that had an OD no greater than

the wells containing butyric acid. The experiment

was repeated in triplicate.

Efficacy of antimicrobial in animal feed

A culture of each serovar of Salmonella was pre-

pared by individually inoculating into 10 ml of sterile

TSB with a single serovar and incubating in a shaking

incubator at 37°C for 24 hours. One ml aliquots from

each of the 8 cultures were mixed to form a cocktail.

Cell density of the inoculum was adjusted to approxi-

mately 108 cfu ml-1.

Meat and bone meal (MBM) was used as the carri-

er matrix to inoculate the feed. The autoclaved MBM

was weighed out into 20 g aliquots and each aliquot

was mixed with 90 ml of 0.01% peptone water and

autoclaved. Four of the five samples were inoculated

with the cocktail and shaken well. All samples were

subsequently centrifuged (Beckman JR-21, Beckman

Coulter, Indianapolis, IN) for 15 minutes at 27,000 x g,

the excess peptone was poured off and the MBM was

placed into deep petri dishes, covered with a sterile

filter paper, and allowed to dry at ambient tempera-

ture for 48 hours in a biosafety cabinet.


The inoculated MBM was scraped out from the

deep plates and placed in a stomacher bag and

stomached to a powder. An aliquot of 10 g of the

inoculum was placed with 990 g of ground dog food

(from a commercial source) in a lab scale mixer (Fig-

ure 1) and mixed for 2 minutes. The antimicrobial was

added to the inoculated feed using a nebulizer fit-

ted into the mixer and at a positive air pressure of

8 PSI. The antimicrobial was injected through a sep-

tum with a 19-gauge needle. The mixer was set to a

speed of 60 rpm and allowed to mix for 2 minutes.

Levels of antimicrobial were equivalent to 0, 5.0, 7.5

and 10 m kg-1 of feed. Each group was sampled on

days 0 (immediately after treatment), 1, 4, 7 and 14

after treatment.

For each sample of inoculated ground dog food

mixed with antimicrobial, 1 g was placed in 9 mL of

sterile 0.1 % peptone water (initial 1:10 dilution) and

further diluted to the appropriate end point by serial

dilution. An aliquot of 0.1 ml of each dilution was dis-

pensed onto duplicate xylose lysine desoxycholate

(XLD; BD Diagnostics) agar plates and spread with

a sterile spreader. Uninoculated MBM (UMBM) was

used as a negative Salmonella control, which was

cultured as described for samples. Plates were incu-

bated at 37°C for 24 hours and then enumerated for

the amount of Salmonella remaining. The entire ex-

periment was replicated three times.

RESULTS

Antimicrobial spectrum

The antimicrobial was observed to have efficacy

against both Gram-positive and Gram-negative bac-

teria (Table 1). The degree of efficacy was similar to

that obtained with formaldehyde and formic acid

under similar test conditions (Carrique-Mas et al.

2006). A 0.05% dilution (0.5 ml kg-1) of the antimicro-

bial gave 100% reduction of S. Typhimurium, E. coli,

S. aureus, S. agalactiae and C. jejuni after 24 hours

of exposure. C. perfringens and L. plantarum were

observed to be more resistant than the other organ-

isms, with C. perfringens reduced 100% at 0.1% (1

ml kg-1) and L. plantarum reduced 100% at 0.5% (5

ml kg-1).

Figure 1. Lab scale mixer used to mix antimicrobial with feed


Minimal inhibitory concentration

Minimal inhibitory concentrations of Salmonella

serotypes varied between 1.5 μl ml-1 to 2.0 μl ml-1,

which is equivalent to 1.5 ml kg-1 and 2.0 ml kg-1 of

feed respectively.

Efficacy in pet food

The antimicrobial inhibited Salmonella survival in

the feed at all levels of application (Fig 2). Numbers

of Salmonella in the untreated control increased

from 8.1 log cfu g-1 at time 0 to 8.2 log cfu g-1 at 4

days. Numbers of Salmonella in the untreated con-

trol decreased from day 4 to day 14 with the final

number being 7.3 log cfu g-1. At time 0 all levels of

treatment had a lower count by almost 1 log cfu g-1

and within 24 hours all levels of antimicrobial were a

full 1 log cfu g-1 lower than the untreated control. At

the end of 14 days all levels were close to 2.0 log cfu

g-1 lower than the untreated feed. The UMBM was

negative for Salmonella growth.

DISCUSSION

Organic acids are often used as preservatives of

human foods (Brul and Coote, 1999) and have also

been used in poultry feed to control mold and bac-

teria (Paster et al., 1987). Treatment of poultry feed

with organic acids has been shown to have the po-

tential to reduce infection levels of Salmonella (Khan

and Katamay, 1969; Matlho et al., 1997). Any chemical

used to control Salmonella in feeds must also either

be metabolized by the animal or excreted without

absorption (Carrique-Mas et al., 2007). Hume et al.

(1993) found that organic acids used to treat poultry

feed were rapidly metabolized by the birds.

Researchers have suggested that small chain fatty

acids exhibit antimicrobial activity in the undissoci-

ated form because they are lipid permeable in this

form and can cross the microbial cell wall and dis-

sociate in the more alkaline interior of the microor-

ganism making the cytoplasm unstable for survival.

(Paster, 1979; Van Immerseel et al., 2006). Butyric

acid when used alone was been found to inhibit Sal-

Table 1. Results of efficacy testing of antimicrobial on various bacteria regularly found in pet food and animal feed. Initial inoculum was 5.0 log cfu/mL of bacterial culture. Exposure time was 24 hours

Treatment

Level

Percent reduction compared to the control

S. Ty-phimurium

E. coliS.

aureus

Cl. perfrin-gens

L.

planta-rum

S. agalac-tiae

C.

jejuni

0.005% 36 7 43 0 0.0 92.3 6.1

0.01% 59 29 39 4 0.0 98.3 59.2

0.05% 100 100 100 85 75 100 100

0.1% 100 100 100 100 99 100 100

0.5% 100 100 100 100 100 100 100


monella (Khan and Katamay, 1969). Khan and Khata-

may (1969) found that butyric acid completely inhib-

ited the growth of Salmonella in media, and when it

was used to treat meat and bone meal artificially in-

oculated with Salmonella no viable organisms were

recovered even after a week. Nonanoic acid (also

known as pelargonic acid) is a naturally occurring

fatty acid with a faint odor compared to butyric acid

and is almost insoluble in water (EPA, 2004). Nona-

noic acid is found in a variety of fruits as well as in

dairy products, and is on the FDA generally recog-

nized as safe (GRAS) list as a synthetic food flavoring

agent, as an adjuvant, production aid and sanitizer

to be used on food contact surfaces. Very few have

studied the effects of nonanoic acid as an antimicro-

bial, but Khan and Khatamay (1969) found essentially

no activity against Salmonella artificially inoculated

into meat and bone meal.

Another volatile compound contained in the stud-

ied antimicrobial is trans-2-hexenal, which is present

in many edible plants such as apples, pears, grapes,

strawberries, kiwi, and tomatoes and has been an ef-

fective antimicrobial against Helicobacter pylori and

S. Cholerasuis (Kubo et al., 1999; 2001). Kim and Shin

(2004) found that trans-2-hexenal (247 mg/L) was

effective against Bacillus cereus, S. Typhimurium,

Vibrio parahemolyticus, Listeria monocytogenes,

Staphylococcus aureus and Escherichia coli 0157:H7.

Nakamura and Hatanaka (2002) demonstrated that

trans-2-hexenal was effective in controlling S. Ty-

phimurium at a level of 3 - 30 ug ml-1. The suggested

mode of action of trans-2 hexenal is the destruction

of electron transport systems and the perturbation

of membrane permeability (Gardini et al., 2001).

Previous research has shown that the reduction of

Salmonella in feed by treatment with organic acids

may require up to a week of contact to achieve re-

sults (Iba and Berchieri, 1995). Our results suggest a

Figure 2. Efficacy of antimicrobial against a cocktail of Salmonella inoculated into pet food. Antimicrobial was added at 0, 5.0 ml/kg, 7.5 ml/kg or 10 ml/kg of pet food. Error bars represent standard deviation from the mean.

4.5

5

5.5

6

6.5

7

7.5

8

8.5

9

9.5

0 1 4 7 14

Log

CFU

Sal

mo

nella

Day

0

5.0 mL/kg

7.5 mL/kg

10 mL/kg


reduction of a high level of contamination with Sal-

monella within 24 hours of application by this com-

bination of organic acids with trans-2-hexenal. Addi-

tionally, the reduction was much greater after 4 days

of contact as compared to the control, where Salmo-

nella growth actually increased. Wales et al. (2013)

studied various feed treatment formulations contain-

ing organic acids and found reductions in Salmonel-

la of around 1 log unit after 7 days. They also found

that those formulations that ultimately had greater

reductions also reduced Salmonella numbers much

sooner, often within 24 hours of incorporation.

The tested antimicrobial was effective in feed at

all levels tested regardless of MIC determined in

vitro. All components are generally recognized as

safe (GRAS) in the US, and thus are approved for

use in animal feeds. This antimicrobial is a promising

new treatment to reduce Salmonella carriage in pet

foods.

ACKNOWLEDGEMENTS

This research was funded by a grant from Anitox

Corp., Lawrenceville, GA.

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ABSTRACT

Netting is a common characteristic in predominant cantaloupe (Cucumis melo L) varieties. Over the

past several years, Listeria and Salmonella outbreaks associated with cantaloupes have become a subject

of concern to consumers. It is hypothesized that unlike non-netted melons, the netted structure of the

cantaloupe rind could be a host for pathogens. Therefore, we investigated whether pathogen contamina-

tion in the field setting is closely associated with the netted rind. Twenty one netted cantaloupe genotypes

consisting of experimental F1 hybrids, inbred lines from the Texas A&M melon breeding program and com-

mercial cultivars were tested for the presence of Listeria spp. and Salmonella serotypes. Pathogen isolation

was performed using selective/differential media after pre-enrichment and selective enrichment. Use of

selective media resulted in the occurrence of 36.36% false positives for Listeria spp. and 16.25% false posi-

tives for Salmonella serovars. Isolates were confirmed using biochemical tests (Listeria API and API 20E) for

both pathogens and real time PCR for Listeria. Testing resulted in one of the triplicates in the cantaloupe

breeding line, ‘1405’ being positive for Listeria innocua. None of the genotypes were positive for Salmo-

nella serovars indicating that there was a low prevalence of the pathogens in the melon genotypes tested

in our study. The occurrence of false positives on selective/differential media highlights the importance

of developing sound selective protocols for the detection and isolation of pathogens from cantaloupes.

Understanding the natural prevalence of foodborne pathogens under growing conditions will help in de-

veloping field-based risk assessments for cantaloupes.

Keywords: cantaloupe lines, foodborne pathogens, field level assessment, rind netting, contamination, Salmonella. Listeria, false positives, PCR, chromogenic media

Correspondence: Sadhana Ravishankar, [email protected], Tel: +1 -520-626-1499

BRIEF COMMUNICATIONA Surveillance of Cantaloupe Genotypes

for the Prevalence of Listeria and Salmonella

G. Dev Kumar1, K. Crosby2, D. Leskovar2.3, H. Bang2, G.K. Jayaprakasha2, B. Patil2, and S. Ravishankar1

1School of Animal and Comparative Biomedical Sciences, University of Arizona, 1117, E. Lowell Street, Tucson, AZ 85721, USA

2 Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843, USA

3Texas A&M AgriLife Research and Extension Center, Texas A&M System, Uvalde, TX 78801, USA



INTRODUCTION

Fresh fruits and vegetables are profitable com-

modities for growers and distributors as consump-

tion trends are on the rise (Hanning et al., 2009).

Cantaloupes and honeydew melons are popular

among the United States (US) consumers because of

their sweetness and nutritional content (Ukuku and

Sapers, 2007). The high antioxidant value of canta-

loupes and the convenience of prepackaged ready

to eat (RTE) fruits have further contributed to the

popularity of this type of melon in the US (Lester and

Hodges, 2008). However, sales of cantaloupes are

often severely affected during and after an outbreak

implication, making it important to mitigate contam-

ination by pathogenic bacteria (Ribera et al., 2012).

Cantaloupes contaminated by foodborne patho-

gens such as Listeria monocytogenes and Salmonel-

la enterica have resulted in widespread diseases and

associated economic losses. In 2011, a multistate

outbreak caused by L. monocytogenes contaminat-

ed cantaloupes resulted in 146 cases, 30 fatalities and

one miscarriage (Laksanalamai et al., 2012). In 2012,

another cantaloupe outbreak caused by Salmonella

serotypes Typhimurium and Newport involving 24

states resulted in 261 cases, three deaths and 94 hos-

pitalizations (CDC, 2012). These outbreaks highlight

the susceptibility of cantaloupes to contamination

by foodborne pathogenic bacteria. Various factors

such as netting of the rind, presence of pathogens in

soil, water and manure, hydrophobicity and attach-

ment appendages of the pathogens, as well as bio-

film formation could result in colonization of enteric

pathogens on cantaloupe rinds (Ukuku and Sapers,

2007; Hanning et al., 2009). Rainfall, water runoff, un-

derground water, and surface water currents can all

aid in the dissemination of foodborne pathogens in

soils and sediments (Bech et al., 2010).

Sweet melons such as the netted cantaloupe

(Cucumis melo L. reticulatus) can get contaminated

during pre-harvest operations in the field or during

post-harvest processing (Ukuku and Sapers, 2007).

Aggregates of foodborne pathogens on cantaloupe

rinds could result in contamination of the fruit in the

field and consequently cross-contamination of other

fruits in packaging houses (Morris and Monier, 2003).

The factors responsible for the 2011 L. monocyto-

genes contaminated cantaloupe outbreak were at-

tributed to packing house design, ineffective equip-

ment sanitation and lack of pre-cooling of melons

before cold storage (Laksanalamai et al., 2012). Mel-

ons with a contaminated rind are a food safety risk,

as pathogenic bacteria can potentially be transferred

from the surface to the flesh by cutting tools (Lin and

Wei, 1997), especially in RTE pre-cut products.

A study by Gagliardi et al. (2003) indicated that

following post-harvest processing, there were higher

bacterial counts on cantaloupe rind compared to

those still in the field (Gagliardi et al., 2003). The ineffi-

ciency of washing is most likely due to the porous sur-

face characteristics of cantaloupe and the increased

roughness resulting from the microstructures present

in the netting which could favor bacterial attachment

(Webster and Craig, 1976; Chen et al., 2012). Averting

on-field contamination of melons by pathogenic bac-

teria could be preemptive, as it is known that melon

rinds retain bacteria even after washing and chemical

sanitizer treatments (Sapers et al., 2001).

Using cantaloupe genotypes that have lower re-

tention of foodborne pathogenic bacteria on their

rinds could help reduce the risk of fruit contamination

in the field. Determining genotypes of cantaloupes

that are less susceptible to bacterial attachment and

contamination could contribute to enhanced micro-

bial safety of cantaloupes. Hence, the objective of

this study was to survey the prevalence of Listeria

and Salmonella spp. amongst various genotypes of

cantaloupes harvested directly from the field.

MATERIALS AND METHODS

Cantaloupe genotypes

The test cantaloupes consisted of 14 experimen-

tal hybrids, three inbred lines and four commercial

cultivars. These cantaloupe genotypes had been

selected for high yield, disease resistance, and firm,

high quality fruit (Crosby et al., 2006). Seeds of all

genotypes were sown directly in a silty-clay soil at


the Texas A&M AgriLife Research Center, Uvalde, TX

(long. 29º1’N, lat. 99º5’W, elevation 283 m) on March

30, 2012. Plants were grown with standard com-

mercial practices of sub-surface drip irrigation and

fertigation, on black plastic mulch at a spacing of 2

m between beds and 0.30 m between plants on the

bed. All fruits were allowed to reach half slip matu-

rity before harvesting. Slip is considered as the ab-

scission zone between the fruit and peduncle. Half

slip maturity is a standard commercial harvest pro-

cedure used by growers in Texas and other southern

regions of the US. Net characteristics ranged from

complete coverage, high off the epidermis (ropy) to

sparse coverage of short netting. Some genotypes

had a higher incidence of splitting in the net tracts

than others. The majority had some resistance to

Fusarium induced rind lesions, but some genotypes

did exhibit this damage when fruit contacted the

clay loam soil. Fruits were harvested between July

1 and July 7, 2012 and shipped to the Ravishankar

laboratory in the Department of Veterinary Science

and Microbiology (Currently School of Animal and

Comparative Biomedical Sciences) at the University

of Arizona within 2 days. No post-harvest methods

were performed on the cantaloupes before analysis.

Storage and inspection

Upon arrival to the laboratory, cantaloupes were

initially inspected for any visible damage or spoilage.

The longitudinal circumference from the stem scar

of each fruit was measured using a measuring tape.

Cantaloupe fruits were given alternate numerical

codes to prevent bias and maintain anonymity. Can-

taloupes were stored at 4°C for 24 h and were evalu-

ated for the presence of Listeria and Salmonella.

Surveillance of cantaloupes for Listeria spp.

Plugs of cantaloupe (20 mm length) were ob-

tained from the stem scar, the bottom of the fruit

and the sides of each fruit using a sterile cork borer

(20 mm diameter) in order to collect both rind and

flesh tissues. A total of 25 g of tissue was taken

from each cantaloupe for sampling. Isolation of Lis-

teria spp. was performed based on the procedure

adapted from the “FDA-Bacteriological Analytical

Manual (BAM) for the isolation of L. monocytogenes

from foods” (Hitchins and Jinneman, 2011). Briefly,

tissue samples were mixed with 225 ml of basal Buff-

ered Listeria Enrichment Broth (BLEB) (EMD Chemi-

cals Inc, Gibbstown, NJ) in a stomacher (Stomacher

Lab-Blender 400, Tekmar Co., Cincinnati, OH) for 2

min and incubated for 4 h at 30°C. Following this,

cycloheximide (Sigma-Aldrich, St. Louis, MO) was

added and the suspension was incubated at 30°C

for 48 h. After incubation, loopful of the suspensions

were streaked on to petri dishes containing modified

Oxford formulation (MOX; Becton, Dickinson and

Co, Sparks, MD) agar and Listeria CHROMagarTM

(CHROMagar, Paris, France) which were incubated at

37°C for 48 h. Typical black colonies formed on MOX

and blue colonies on CHROMagar irrespective of

halo formation were Gram stained and streaked for

isolation to account for Listeria spp. Those colonies

that were Gram positive were further confirmed us-

ing Listeria API strips (bioMerieux, Hazelwood, MO),

accessory tests (catalase, oxidase and hemolysis) ac-

cording to the manufacturer’s instructions, and real

time PCR (iQ Check Listeria spp. Kit, Bio-Rad labora-

tories, Hercules, CA)

Real time PCR confirmation of Listeria spp.

One isolated colony from a MOX plate was added

to 100 μl of lysis buffer (Bio-Rad Laboratories) and

incubated for 15 min at 95°C. To 45 μL of the PCR

amplification master mix, 5 μL of the lysed DNA

sample was added along with 5 μL fluorogenic oli-

gonucleotide molecular beacon probe solution (iQ

Check Listeria spp. Kit, Bio-Rad Laboratories). The

thermocycler (MiniOpticon™ real-time PCR detec-

tion system, Bio-Rad Laboratories) was programmed

as follows: 50°C for 2 min, 95°C for 5 min followed

by 95°C for 20 s, 55°C for 30 s, 72°C for 30 s for 50

cycles, and 72°C for 5 min. An increase in fluores-

cence from the amplification of the target sequence

resulting in a Ct value ≥10 was considered positive.


Surveillance of cantaloupes for Salmo-nella serovars

A total of 25 g of tissue was taken from each can-

taloupe using techniques similar to those described

earlier for Listeria. Isolation of Salmonella spp. was

performed based on the procedure adapted from the

“FDA-BAM for the isolation of Salmonella spp. from

foods” (Andrews and Hammack, 2007). Briefly, tissue

samples were mixed with 225 ml sterile universal pre-

enrichment broth (UPB; Becton, Dickinson and Co.)

for 2 min using a stomacher (Stomacher Lab-Blender

400, Tekmar Co.). The suspension was incubated for

24 h at 37°C following which 100 μl was transferred

to 10 ml Rappaport-Vassiliadis (RV; EMD Chemicals

Inc.) medium and another 1 ml to 10 ml tetrathionate

(TT; EMD Chemicals Inc.) broth. The RV suspension

was vortexed and incubated at 42°C for 24 h in a wa-

ter bath. The TT broth suspension was vortexed and

incubated at 37°C for 24 h. Following incubation,

the suspensions from the broths were streaked on

to xylose lysine desoxycholate (XLD) agar (Becton,

Dickinson and Co.) and CHROMagarTM Salmonella

(CHROMagar) and incubated for 48 h. Typical colo-

nies were Gram stained and Gram negative isolates

were confirmed as Salmonella using API 20E strips

(bioMerieux), and by conducting biochemical tests

(catalase, oxidase) according to the manufacturer’s

recommendations.

Statistical Analysis

Geometric means and standard deviations were

calculated for the incidences of false positives on

selective plating media. A t-test was performed to

determine significant differences (p<0.05) between

false positive rates on different selective media. Sta-

tistical analysis was performed using Microsoft Excel

2007 (Microsoft Corp., Seattle, WA). Means and stan-

dard deviations were calculated for the longitudinal

circumference values of melons.

RESULTS

Sizes of cantaloupes based on their di-ameter

A total of 21 cantaloupe genotypes (3 fruits each

for most genotypes) were surveyed for the pres-

ence of Listeria and Salmonella spp. The cantaloupe

genotypes with the maximum average longitudinal

circumference were lines 18 and 20 with 60.53±4.06

and 62.23±4.58 cm, respectively (Table 1). Canta-

loupe breeding line 6 had the smallest melons with

an average longitudinal circumference of 46.57±1.50

cm. Cantaloupe genotypes 17 and Oro Duro were

also some of the smaller lines tested with an average

longitudinal circumference of 50.37±2.61 cm and

49.97±1.44 cm, respectively.

Surveillance of cantaloupes for Listeria spp.

None of the 21 cantaloupe genotypes were posi-

tive for the presence of L. monocytogenes. One

cantaloupe sample from the breeding line 1405 was

positive for the presence of L. innocua after enrich-

ment and plating on selective media (Table 1). This

sample was further confirmed through Listeria API

tests and real Time PCR. Real Time PCR analysis re-

sulted in one isolate from cantaloupe line 1405 hav-

ing an increase in the fluorescence curve indicating

amplification and a Ct>10, indicating a positive result

for Listeria spp. Sixteen of the 21 melon lines tested

demonstrated black and blue colored colonies on

MOX and Listeria CHROMagar, respectively. The

blue colonies were chosen to determine the pres-

ence of other Listeria spp. All these colonies were

Gram positive. However, the results of API Listeria

test and real-time PCR indicated that 15 of these iso-

lates were negative for L. monocytogenes.

Surveillance of cantaloupes for Salmo-nella

None of the 21 cantaloupe genotypes were posi-

tive for the presence of Salmonella serovars. Out of


Table 1. Rind net characteristics and surveillance tests for the presence of Listeria spp. and Sal-monella on various cantaloupe genotypes using API Listeria for Listeria and API 20E for Salmo-nella.

C a n t a -loupe

g e n o -type

Rind net characteristics Pathogen surveil-lance testz

PhotographLongitudinal c i r c u m f e r -ence (cm)

Net

Coverage (%)

Splitting

/corkinessAPI Liste-ria strip

API 20E strip

Experimental Hybrid

1 50.37±2.62 100 Low Neg Neg

2 52.90±0.69 100 Low Neg Neg

3 54.60±1.30 100 Low Neg Neg

6 46.57±1.50 90 Low Neg Neg


C a n t a -loupe

g e n o -type



Net

Coverage (%)

Splitting


API 20E strip

7* 58.40 100 Low Neg Neg

9 53.95±6.29 100 Low Neg Neg

10 59.70±5.86 100 Low Neg Neg

11 50.20±2.69 100 Low Neg Neg


C a n t a -loupe

g e n o -type



Net

Coverage (%)

Splitting


API 20E strip

12* 53.30 100 Low Neg Neg

14 54.17±1.50 100 Low Neg Neg

15* 67.30 100 Low Neg Neg

17 50.37±2.61 100 Low Neg Neg


C a n t a -loupe

g e n o -type



Net

Coverage (%)

Splitting


API 20E strip

18 60.53±4.06 100 Med Neg Neg

20 62.23±4.58 100 Med Neg Neg

Inbred

146 51.67±3.87 100 Low Neg Neg

F39 49.53±4.58 100 Low Neg Neg

1405 57.60±1.93 100 Med Pos+Neg Neg


C a n t a -loupe

g e n o -type



Net

Coverage (%)

Splitting


API 20E strip

Commercial variety

Mission 53.33±2.55 100 Low Neg Neg

Oro Duro 49.97±1.44 100 Low Neg Neg

Sol Real 52.07±2.19 100 Low Neg Neg

Journey 54.60±5.86 90 Med Neg Neg

z Positive colonies isolated from selective media were tested for the presence of pathogen using an API Listeria strip for Listeria and an API 20E strip for Salmonella. Results were Negative (Neg) or Postive (Pos) for Listeria or Salmonella. One of the triplicates in 1405 showed positive for Listeria while two other replicates came out nega-tive.

* Only one sample was available in experimental hybrid 7, 12 and 15, because it is difficult to synchronize the maturity of all genotypes and open pollinated fruits in a field trial or some fruits may have aborted during fruit development.


the 21 cantaloupe genotypes tested, 8 genotypes- 3,

9, 10, 18, 146, 1405, Mission and Oro Duro showed

typical black colonies on XLD agar indicative of H2S

production and typical mauve colonies on Salmo-

nella CHROMagar. All the isolates from the 8 lines

were Gram negative rods. However, when API 20E

tests were conducted, all 8 were negative for the

presence of Salmonella serovars (Table 1). A total of

10 and 8 melons (from the 8 genotypes tested) re-

sulted in false positives on XLD agar and Salmonella

CHROMagar, respectively. The same melons from

genotypes- 9, 18, 146, 1405, and Oro Duro resulted

in false positives on both selective media, while dif-

ferent melons from genotypes 3, 10 and Mission re-

sulted in false positives on XLD agar and Salmonella

CHROMagar.

DISCUSSION

A total of 21 cantaloupe genotypes were surveyed

for the presence of Salmonella and Listeria spp. Of

all the lines surveyed, line 1405 was positive for the

presence of L. innocua (Table 1). This is an inbred

line with a typical, complete net of medium height

and low incidence of splitting. The heavier net com-

pletely covers the rind (most Western shipper canta-

loupes) and could result in improved attachment of

microbiota.

L. innocua has been used as a surrogate for L.

monocytogenes (Buchholz et al., 2011), because of

similar growth and survival characteristics (McKinney

et al., 2009). L. monocytogenes might be capable

of surviving in similar or harsher environments than

L. innocua (Buchholz et al., 2011). The presence of

L. innocua on cantaloupe could indicate conditions

suitable for the possible survival and contamination

by L. monocytogenes.

L. monocytogenes is commonly found in the envi-

ronment and on plant material (Laksanalamai et al.,

2012) and can survive under adverse environmental

conditions (Tompkin, 2002). Johnston et al., (2005)

surveyed cantaloupes and other produce from the

southern regions of the US for the presence of patho-

gens (L. monocytogenes and Salmonella serovars)

and indicator organisms. Of the 398 produce items

sampled, none were positive for L. monocytogenes.

Of all the produce tested for Salmonella, three of the

90 cantaloupes were positive for Salmonella Monte-

video (Johnston et al., 2005). While lower numbers

of contaminated produce can occur in the field,

cross-contamination in the packing house may result

in higher volumes of product getting contaminated

and thereby causing outbreaks.

In our study, the cantaloupe genotypes tested

were not positive for Salmonella. The use of manure,

or presence of wild life, birds, or compost piles in the

field vicinity could serve as reservoirs of contamina-

tion. While the research farm at the Texas A&M AgriL-

ife Research Center did not contain these pathogen

reservoirs, farms could potentially be subjected to

pathogen introduction through environmental con-

tamination and animal or bird intrusion. Previous

studies have indicated that foodborne pathogens

can survive in soil and water for extended periods of

time and can be transferred to fruit tissue (Baloda et

al., 2001; Gupta et al., 2007; Barak and Liang, 2008).

Factors that affect the survival of the pathogen in soil

include soil type, nutrient availability, manure and

temperature (Andrews-Polymenis et al., 2010).

Selective isolation of pathogens from cantaloupes

resulted in false positive samples for both Listeria

spp. and Salmonella on selective media. In a study

to evaluate chromogenic agar media for the recov-

ery and detection of L. monocytogenes in foods, it

was observed that natural microbiota in foods are

capable of overgrowing pathogenic target microor-

ganisms (Michael, 2004). In our study, the sensitivity

of non-chromogenic plating media was not signifi-

cantly different (P>0.05) from chromogenic plating

media for distinguishing false positives, in case of

both pathogens.

Cantaloupes are rich in sugars and the rinds of

cantaloupes are capable of harboring high amounts

of microbiota because of the naturally present net-

ting (Ukuku and Sapers, 2007). The breakage or

rupture of the rinds could potentially result in cross-

contamination of the microorganisms from one fruit

to another, due to spilling of the juice, which is rich in

antioxidants and sugars (Lester and Hodges, 2008).


While our study indicated that the various genotypes

did not result in significant differences in pathogen

attachment, mitigation strategies should be ex-

plored to reduce the risk of on-field contamination

of cantaloupes.

CONCLUSIONS

A survey of 21 cantaloupe genotypes resulted in

a single line of netted cantaloupe being positive

for Listeria spp., which was confirmed as L. innocua.

The presence of L. innocua on cantaloupe indicates

the existence of conditions wherein pathogenic L.

monocytogenes could survive. More research is

needed to understand the role of cantaloupe net-

ting on microbial attachment and persistence.

ACKNOWLEDGEMENTS

The authors would like to thank Libin Zhu and Jen-

nifer Todd of the Ravishankar lab for their technical

support.

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15

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Hops (Humulus lupulus) ß-Acid as an Inhibitor of Caprine Rumen Hyper-Ammonia-Produc-ing Bacteria In VitroM. D. Flythe, G. E. Aiken1, G. L. Gellin, J. L. Klotz, B. M. Goff, K. M. Andries

29

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at first use in the abstract and again in the body of

the manuscript. If abbreviations are extensive au-

thors may need to provide a list of abbreviations

at the beginning of the manuscript. In vivo, in vitro

and bacterial names must be italicized (obligatory).

Authors must avoid single sentence paragraphs and

merge such paragraphs appropriately. Authors must

not begin sentences with “Figure or Table shows…”

as these are inanimate objects and cannot “show”

anything. When number are reported in text or in ta-

bles, always put a zero in front of decimal numbers:

“0.10” instead of “.10”.

MANUSCRIPT SECTIONS

Abstract

The abstract provides an abridged version of the

manuscript. Please submit your abstract on a sepa-

rate page after the title page. The abstract should

provide a justification of your work, objectives, meth-

ods, results, discussion and implications of study or

review findings . Your abstract must consist of com-

plete sentences without references to other work or

footnotes and must not exceed 250 words. On the

same page as your abstract, please provide at least ten (10) keywords to be used for linking and index-

ing. Ideally, these keywords should include signifi-

cant words from the title.

Introduction

The introduction should clearly present the foun-

dation of the manuscript topic and what makes the

research or the review unique. The introduction

should validate why this topic is important based on

previously published literature, and the relevance of

the current research. Overall goals and project ob-

jectives must be clearly stated in the final sentence

of the last paragraphs of the introduction.

Materials and Methods

Information on equipment and chemicals used

must include the full company name, city, and state

(country if outside the United States or Province if

in Canada) [i.e., (Model 123, ACME Inc., Afab, AR)].


Variability, Replication, and Statistical Analysis

To properly assess biological systems indepen-

dent replication of experiments and quantification

of variation among replicates is required by AFAB.

Reviewers and/or editors may request additional

statistical analysis depending on the nature of the

data and it will be the responsibility of the authors

to respond appropriately. Statistical methods com-

monly used in the bacteriology do not need to be

described in detail, but an adequate description

and/or appropriate references should be provided.

The statistical model and experimental unit must

be designated when appropriate. The experimen-

tal unit is the smallest unit to which an individual

treatment is imposed. For bacterial growth stud-

ies, the average of replicate tubes per single study

per treatment is the experimental unit; therefore,

individual studies must be replicated. Repeated

time analyses of the same sample usually do not

constitute independent experimental units. Mea-

surements on the same experimental unit over time

are also not independent and must not be consid-

ered as independent experimental units. For analy-

sis of time effects, assess as a rate of change over

time. Standard deviation refers to the variability

in the biological response being measured and is

presented as standard deviation or standard error

according to the definitions described in statistical

references or textbooks.

Results

Results represent the presentation of data in

words and all data should be described in same

fashion. No discussion of literature is included in

the results section.

Discussion

The discussion section involves comparing the

current data outcomes with previously published

work in this area without repeating the text in the

results section. Critical and in-depth dialogue is

encouraged.

Results and Discussion

Results and discussion can be under combined or

separate headings.

Conclusions

State conclusions (not a summary) briefly in one

paragraph.

Acknowledgments

Acknowledgments of individuals should include

institution, city, and state; city and country if not U.S.;

and City or Province if in Canada. Copies being re-

viewed shall have authors’ institutions omitted to re-

tain anonymity.

References

a) Citing References In Text

Authors of cited papers in the text are to be pre-

sented as follows: Adams and Harry (1992) or Smith

and Jones (1990, 1992). If more than two authors of

one article, the first author’s name is followed by the

abbreviation et al. in italics. If the sentence structure

requires that the authors’ names be included in pa-

rentheses, the proper format is (Adams and Harry,

1982; Harry, 1988a,b; Harry et al., 1993). Citations to a

group of references should be listed first alphabeti-

cally then chronologically. Work that has not been

submitted or accepted for publication shall be listed

in the text as: “G.C. Jay (institution, city, and state,

personal communication).” The author’s own un-

published work should be listed in the text as “(J.

Adams, unpublished data).” Personal communica-

tions and unsubmitted unpublished data must not

be included in the References section. Two or more

publications by the same authors in the same year

must be made distinct with lowercase letters after

the year (2010a,b). Likewise when multiple author ci-

tations designated by et al. in the text have the same

first author, then even if the other authors are differ-

ent these references in the text and the references

section must be identified by a letter. For example


“(James et al., 2010a,b)” in text, refers to “James,

Smith, and Elliot. 2010a” and “James, West, and Ad-

ams. 2010b” in the reference section.

b) Citing References In Reference Section

In the References section, references are listed in

alphabetical order by authors’ last names, and then

chronologically. List only those references cited in the

text. Manuscripts submitted for publication, accepted

for publication or in press can be given in the refer-

ence section followed by the designation: “(submit-

ted)”, “(accepted)’, or “(In Press), respectively. If the

DOI number of unpublished references is available,

you must give the number. The year of publication fol-

lows the authors’ names. All authors’ names must be

included in the citation in the Reference section. Jour-

nals must be abbreviated. First and last page num-

bers must be provided. Sample references are given

below. Consult recent issues of AFAB for examples

not included in the following section.

Journal manuscript:

Examples:

Chase, G., and L. Erlandsen. 1976. Evidence for a

complex life cycle and endospore formation in the

attached, filamentous, segmented bacterium from

murine ileum. J. Bacteriol. 127:572-583.

Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van

Doesburg, and A. J. M. Stams. 2009. A typical

one-carbon metabolism of an acetogenic and

hydrogenogenic Moorella thermioacetica strain.

Arch. Microbiol. 191:123-131.

Book:

Examples:

Hungate, R. E. 1966. The rumen and its microbes

Academic Press, Inc., New York, NY. 533 p.

Book Chapter:

Examples:

O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010.

Assessing consumer concerns and perceptions

of food safety risks and practices: Methodologies

and outcomes. In: S. C. Ricke and F. T. Jones. Eds.

Perspectives on Food Safety Issues of Food Animal

Derived Foods. Univ. Arkansas Press, Fayetteville,

AR. p 273-288.

Dissertation and thesis:

Maciorowski, K. G. 2000. Rapid detection of Salmo-

nella spp. and indicators of fecal contamination

in animal feed. Ph.D. Diss. Texas A&M University,

College Station, TX.

Donalson, L. M. 2005. The in vivo and in vitro effect

of a fructooligosacharide prebiotic combined with

alfalfa molt diets on egg production and Salmo-

nella in laying hens. M.S. thesis. Texas A&M Uni-

versity, College Station, TX.

Van Loo, E. 2009. Consumer perception of ready-to-

eat deli foods and organic meat. M.S. thesis. Uni-

versity of Arkansas, Fayetteville, AR. 202 p.

Web sites, patents:

Examples:

Davis, C. 2010. Salmonella. Medicinenet.com.

http://www.medicinenet.com/salmonella /article.

htm. Accessed July, 2010.

Afab, F. 2010, Development of a novel process. U.S.

Patent #_____

Author(s). Year. Article title. Journal title [abbreviated].

Volume number:inclusive pages.

Author(s) [or editor(s)]. Year. Title. Edition or volume (if

relevant). Publisher name, Place of publication. Number

of pages.

Author(s) of the chapter. Year. Title of the chapter. In:

author(s) or editor(s). Title of the book. Edition or vol-

ume, if relevant. Publisher name, Place of publication.

Inclusive pages of chapter.

Author. Date of degree. Title. Type of publication, such

as Ph.D. Diss or M.S. thesis. Institution, Place of institu-

tion. Total number of pages.


Abstracts and Symposia Proceedings:

Fischer, J. R. 2007. Building a prosperous future in

which agriculture uses and produces energy effi-

ciently and effectively. NABC report 19, Agricultural

Biofuels: Tech., Sustainability, and Profitability. p.27

Musgrove, M. T., and M. E. Berrang. 2008. Presence

of aerobic microorganisms, Enterobacteriaceae and

Salmonella in the shell egg processing environment.

IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10)

Vianna, M. E., H. P. Horz, and G. Conrads. 2006. Op-

tions and risks by using diagnostic gene chips. Pro-

gram and abstracts book , The 8th Biennieal Con-

gress of the Anaerobe Society of the Americas. p.

86 (Abstr.)

Data Presentation in Tables and Figures

Figures and tables to be published in AFAB must

be constructed in such a fashion that they are able

to “stand alone” in the published manuscript. This

means that the reader should be able to look at

the figure or table independently of the rest of the

manuscript and be able to comprehend the experi-

mental approach sufficiently to interpret the data.

Consequently, all statistical analyses should be very

carefully presented along with variation estimates

and what constitutes an independent replication

and the number of replicates used to calculate the

averages presented in the table or figure.

Each table and figure must be on a separate

page in the submitted paper. In addition, you will

need to submit all data for charts, tables and

figures in native format when possible (e.g., Mi-

crosoft Excel, Powerpoint). Photographs should

be submitted as high-resolution (600 dpi) .jpg or

tif. files. All figures should be clearly presented with

well defined axis and units of measurement. Sym-

bols, lines, and bars must be made distinct as “stand

alone” black and white presentations. Stippling,

dashed lines etc. are encouraged for multiple com-

parison but shades of gray are discouraged. Color

images, micrographs, pictures are recommended

and there is no additional fee for their submission.

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AFAB Volume 5 Issue 2

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Transcript of AFAB Volume 5 Issue 2