Thesis

THE NOTTINGHAM TRENT UNIVERSITY

Determination of death rate of Cronobacter and related organisms.

by

CHARLOTTE CARNEIRO (N0289704)

Project Report submitted in partial fulfilment of the M.Sc in Biotechnology

Supervisor : Professor Stephen Forsythe

Year in which the study is completed2010

DECLARATION OF OWNERSHIP This submission is the result of my work. All help and advice, other than that received from tutors, has been acknowledged and primary and secondary sources of information have been properly attributed. Should this statement prove to be untrue, I recognise the right and duty of the Board of Examiners to recommend what action should be taken in line with the University’s regulations on assessment contained in the Handbook.

Signed: .......................................................... Date: .........................................

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CONTENTS

Page No.

Abstract ………………………………………………………………………… 4

Abbreviations used …………………………………………………………….. 5

List of figures……………………………………………………………………..6

List of table ………………………………………………………………………9

Chapter 1. Introduction

1.1 History of the organism................................................................................10

1.2 Characteristics of the Organism…………………………………………...11

1.3 Virulence of Cronobacter.............................................................................12

1.4 Pathogenesis of Cronobacter........................................................................13

1.5 Anti microbial resistance...............................................................................15

1.6 Isolation of Cronobacter................................................................................15

1.7 The growth kinetics of Cronobacter ............................................................17

1.8 PIF and Cronobacter......................................................................................171.9 Reconstitution of PIF’s...................................................................................18

1.10 Temperature Profiling……………………………………………………....18

1.11 Strategies for prevention of an infection........................................................19

1.12 Aims and objectives of the research................................................................20

Chapter 2. Materials and Methods

2.1 Samples collection for the isolation of Cronobacter…………………………21

2.2 Isolation of Cronobacter……………………………………………………...23

Gram Stain

ID 32 E assay

API ZYM assay

16S rRNA gene sequencing

Pulsed field gel electrophoresis

2.3 Death Kinetics based on temperature………………………………………....30

2.4 Bacterial Survival in PIFs………………………………………………………31

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Chapter 3. Results

3.1 Isolation of Cronobacter………………………………………………….32 3.2 Death kinetics of the strains with respect to temperature………………..44 3.3 Bacterial Survival in PIFs…………………………………………………53

Chapter 4. Discussion and Conclusion

4.1 Identification of Cronobacter and other related organisms………………...69

4.2 Survival potential and the D value………………………………………….72 4.3 Temperature profiling at room temperature (250C)………………………...72

4.4 Bacteriocidal affect of reconstitution………………………………………..73

Future Prospects....................................................................................................75

Acknowledgements ……………………………………………………………....76

References…………………………………………………………………………77

ABSTRACT

3

Cronobacter is a gram negative opportunistic food-borne pathogenic organism that is found in

powdered infant formulas (PIF). In recent times the organism has also been isolated from a

wide range of food samples as well as environmental samples. Cronobacter has been known to

cause meningitis, sepsis and necrotizing enterocolitis in neonates and in the last couple of years

has also been known to cause infections in adults and immunodeficient individuals.

The organism is ubiquitous and on the basis of this 15 samples which included ready to eat mix

powders, fresh herbs and dry herbs were examined for the presence of Cronobacter and related

organisms. The strains obtained were subjected biochemical analysis, pulsed-field gel

electrophoresis and to 16S rRNA gene sequencing to identify the organism and the death

kinetics of the strains were studied by conventional microbiology techniques. The Survival

potential of Cronobacter, with respect to temperature was also determined in reconstituted

PIFs.

Out of 15 samples screened for Cronobacter, 2 strains of Cronobacter sakazakii were isolated.

Both the strains were isolated from ready to eat mix powders. The death kinetics of the

organism was determined at both 700C and 550C. However, faster death of the organism was

observed at 700C. The D value at 550C varied between 0.63-2.60 minutes. While, the D value at

700C was between 0.05- 0.83 minutes.

The reconstitution of PIFs with sterile boiled water at an initial temperature of 70 0C reduced

the bacterial load while, a small amount of bacterial growth was observed when the initial

temperature was recorded at 550C.

The study displays the diversity of the organism and its phenotypic closeness to the other

strains in the Enterobacteriaceae family. Therefore the sequencing of the 16s rRNA gene is

vital for identifying the organism. It can also be concluded that reconstitution of PIFs at high

temperature reduces the survival of bacteria.

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ABBREVIATIONS USED

PIF- Powdered infant formula

WHO- World Health Organization

FAO- Food and Agriculture Organization

Omp- Outer membrane protein

DFI- Druggan Forsythe Iversen

VRBGA- Violet Red Bile Glucose Agar

EE broth- Enterobacteriaceae enrichment broth

TSA- Trypticase soy agar

PCA- Plate Count Agar

BPW- Buffered Peptone Water

ESIA- Enterobacter Sakazakii isolation chromogenic agar

MLST- Multi locus sequence typing

BMEC- Brain microvascular endothelial cells

PFGE- Pulsed field gel electrophoresis

TEB- TE Buffer

TSB- Tryptic soy broth

PCR- Polymerase chain reaction

TMTC- To many to count

ICMSF- International Commission for Microbiological Specifications for Foods

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LIST OF FIGURES

Fig.1.1 False colour image of Cronobacter sakazaki

Fig.1.2 Electron micrograph of the rat intestinal epithelium with an adherent

Cronobacter

Fig.1.3 Isolation of Cronobacter

- Fig.1.3 (a) Isolation of Cronobacter on DFI agar plate.

- Fig.1.3 (b) Isolation of Cronobacter on ESIA agar plate.

Fig.2.1 Herbs from Suk Chuen Dai Po Ton Soup Stock

Fig.2.2 API ZYM assay kit

Fig.2.3 Setting of plugs

Fig.2.4 Lysis of cells in agarose plugs

Fig.2.5 Agarose plugs placed on the comb

Fig.2.6 Infant formulas for colony inoculation

Fig.3.1 Colonies observed on the VRBGA plate

Fig.3.2 Colonies observed on the PCA plate

Fig.3.3 Black coloured colonies on the DFI plate.

Fig.3.4 Gram stain

Fig.3.5 ID 32 E Results

- Fig.3.5 (a) ID 32 E assay on strain C1

- Fig.3.5(b) ID 32 E assay on strain C2

- Fig.3.5(c) ID 32 E assay on strain C3

- Fig.3.5(d) ID 32 E assay on strain C4

- Fig.3.5(e) ID 32 E assay on strain C5

Fig.3.6 The phylogenetic tree of strains C1, C2, C3, C4 and C5.

Fig.3.7 Pulse field gel electrophoresis

- Fig.3.7 (a) Pulse field gel electrophoresis of strains C1, C2, C3 and C4.

- Fig.3.7 (b) Pulse field gel electrophoresis of strains C1, C2, C3, C4 and C5.

Fig.3.8 Thermal inactivation of Klebsiella oxytoca at 70ºC.

- Fig.3.8(A) Thermal inactivation of Klebsiella oxytoca at 70ºC in whey milk.

- Fig.3.8(B) Thermal inactivation of Klebsiella oxytoca at 70ºC in casein milk.

Fig.3.9 Thermal inactivation of Cronobacter sakazakii (C2) at 70ºC.

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- Fig.3.9(A) Thermal inactivation of Cronobacter sakazakii at 70ºC in whey milk.

- Fig.3.9(B) Thermal inactivation of Cronobacter sakazakii at 70ºC in casein milk.




Fig.3.11 Thermal inactivation of Enterobacter helveticus at 70ºC.

- Fig.3.11(A) Thermal inactivation of Enterobacter helveticus at 70ºC in whey milk.

- Fig.3.11(B) Thermal inactivation of Enterobacter helveticus at 70ºC in casein milk.


- Fig.3.12(A) Thermal inactivation of Klebsiella oxytoca at 55ºC in whey milk.

- Fig.3.12(B) Thermal inactivation of Klebsiella oxytoca at 55ºC in casein milk.








- Fig.3.15(A) Thermal inactivation of Enterobacter helveticus at 55ºC in whey milk.

- Fig.3.15(B) Thermal inactivation of Enterobacter helveticus at 55ºC in casein milk.

Fig.3.16 Survival capacity of Cronobacter in Pregestimil when the initial temperature

was 550C.

- Fig.3.16 (A) Survival potential of strain Cronobacter sakazakii 1 in Pregestimil.

- Fig.3.16 (B) Survival potential of strain Cronobacter sakazakii 658 in Pregestimil.

- Fig.3.16 (C) Survival potential of strain Cronobacter turicensis in Pregestimil.

Fig.3.17 Survival capacity of Cronobacter in Pepti Junior when the initial temperature

was 550C.

- Fig.3.17 (A) Survival potential of strain Cronobacter sakazakii 1 in Pepti Junior.

- Fig.3.17 (B) Survival potential of strain Cronobacter sakazakii 658 in Pepti Junior.

- Fig.3.17 (C) Survival potential of strain Cronobacter turicensis in Pepti Junior.

Fig.3.18 Survival capacity of Cronobacter in LCP Neocate when the initial temperature

was 550C.

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- Fig.3.18 (A) Survival potential of strain Cronobacter sakazakii 1 in LCP Neocate.

- Fig.3.18 (B) Survival potential of strain Cronobacter sakazakii 658 in LCP Neocate.

- Fig.3.18 (C) Survival potential of strain Cronobacter turicensis in LCP Neocate.

Fig.3.19 Survival capacity of Cronobacter in SHS Caprilon when the initial

temperature was 550C.

- Fig.3.19 (A) Survival potential of strain Cronobacter sakazakii 1 in SHS Caprilon.

- Fig.3.19 (B) Survival potential of strain Cronobacter sakazakii 658 in SHS Caprilon.

- Fig.3.19 (C) Survival potential of strain Cronobacter turicensis in SHS Caprilon.

Fig.3.20 Survival capacity of Cronobacter in Nutramigen when the initial temperature

was 550C.

- Fig.3.20 (A) Survival potential of strain Cronobacter sakazakii 1 in Nutramigen.

- Fig.3.20 (B) Survival potential of strain Cronobacter sakazakii 658 in Nutramigen.

- Fig.3.20 (C) Survival potential of strain Cronobacter turicensis in Nutramigen.

Fig.3.21 Survival capacity of Cronobacter turicensis in Pepti Junior when the initial


Fig.3.22 Survival capacity of Cronobacter turicensis in LCP Neocate when the initial


Fig.4.1 Growth of Klebsiella oxytoca on ESIA.

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LIST OF TABLES

TABLE1. Samples collected for Cronobacter determination.

TABLE2. Media employed for the isolation of Cronobacter

TABLE3. The plate count obtained on VRBGA and PCA plates.

TABLE4. ID 32 E results

TABLE5. 16S rRNA results

TABLE6. API ZYM assay results

TABLE7. D value of strains C1, C2, C3 and C4.

TABLE8. The temperature profile of reconstituted PIFs LCP neocate, C+G premium

and SHS Caprilon

TABLE9. The temperature profile of reconstituted PIFs LCP neocate, C+G premium

and SHS Caprilon when the kettle was boiled at different volumes.

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1. INTRODUCTION

1.1 History of the organism

Cronobacter spp. is an opportunistic organism and has been known to cause several

infections in neonates especially premature infants and infants with a low birth weight.

The infections include meningitis, necrotizing enterocolitis and septicaemia which can

be fatal. It is also known to cause infections in adults and immunodeficient individuals

(Jaradat et al., 2009). Most of the patients die within a week on being infected, while

the survivors show neurological complexities. Cronobacter is a ubiquitous organism

and is a contaminant of various dried PIFs. The organism along with Salmonella has

been categorized by the WHO and FAO as a category ‘A’ organism as they have been

found in PIFs and have displayed clear evidence of causality through the ingestion of

reconstituted PIF (WHO/FAO Meeting report MRA Series 6).

Cronobacter spp was formerly known as Enterobacter sakazakii by Farmer et al until

it was taxonomically reclassified into a genus in June 2007 (FSA Project., 2009). Prior

to this it was also known at ‘yellow pigmented Enterobacter cloacae’ until 1970

(Mullane et al., 2006). Apart from PIF’s, Cronobacter spp. has been isolated and

cultured from a variety of environments. Due to its ubiquitous nature it is found in

vegetables, herbs, manufacturing plants, kitchen equipments, meats, insects, crude oil

etc. Interestingly, it can also be isolated from humans. It is found in cerebrospinal

fluid, blood, skin wounds, breast abscess, respiratory secretions and digestive tract

samples (Jaradat et al., 2009).

The first case of infant infection due to Cronobacter was observed in 1958 in an

outbreak in England. This outbreak led to the death of two infants (Drudy et al., 2005).

Since then 120 cases of infections and 27 deaths have been reported in association with

the organism.

The WHO and FAO paid extra attention to infections caused by Cronobacter after it

was found to affect ~1 per 100,000 infants which increased to 9.4 per 100,000. A

major outbreak due to the organism was observed in the neonatal intensive care unit in

France in 1994 and the strains have been studied and published. (Forsythe et al., 2007).

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1.2 Characteristics of the Organism

Fig.1.1 False colour image of Cronobacter sakazakii

Fig .1.1 False colour image of Cronobacter sakazakii adhered to an enteral feeding tube (Forsythe., 2010).

Cronobacter spp. formerly known as Enterobacter sakazakii is an opportunistic pathogen. It is

a Gram negative facultative anaerobe. It is capable of moving i.e. it is motile and cannot form

spores (Jaradat et al., 2009). The organism belongs to the Enterobacteriaceae family. The

genus Cronobacter comprises of five species - C.sakazakii, C.malonaticus, C.turicensis,

C.muytjensii and C.dublinensis (Forsythe., 2010). Of these, C. sakazakii, C.malonaticus, C.

turicensis are known to have caused infections in neonates (Iversen et al., 2008). Cronobacter

is found to be closely related phenotypically to the other organisms in Enterobacteriaceae

family, especially E. cloacae and Citrobacter species and therefore identity confirmation using

PCR primers, chromogenic or biochemical techniques like ID 32 E may provide false positive

results. Hence it is essential to carry out sequencing of 16S rRNA gene for identity

confirmation of the organism as the 16S rRNA sequencing is known to be the most reliable and

accurate in the identification of micro organisms (Iversen et al., 2006).

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The classification of Cronobacter strains was initially based on biochemical traits. DNA

analysis came about later. However the 16S rRNA sequencing, in the case of C. sakazakii and

C. malonaticus was not reasonable enough as the species were closely related to each other.

They were thought to be one species at first. Multi locus sequence typing (MLST) was carried

out on the strains to study the DNA variation with respect to different strains. MLST is

dependent on 7 housekeeping genes. MLST provided better results in terms of classification.

This organism is catalase positive and oxidase negative. They are also found to be exhibit α-D-

glucosidase activity (Iversen and Forsythe, 2006).

Cronobacter is the most thermotolerant organism in the Enterobacteriaceae family and the

thermotolerance is linked to the presence of the KT gene (Oravcova et al., 2008). It is seen to

grow over a several different temperatures. The minimum temperature growth was observed at

~50 C while, the maximum growth temperature was noted at 44-470 C. The temperature may

however vary in different species (Forsythe et al., 2004). It displays best growth at 370 C.

Generation time of the organism at 210 C i.e. room temperature is 40-94 minutes. Cronobacter

can also form biofilms which are unaffected by disinfectants (Hunter et al., 2008).

Cronobacter also display resistance to osmotic stress and dryness. Some species of

Cronobacter are encapsulated and therefore it is resistant to extreme conditions. The organism

has been recovered from desiccated PIF after 2.5 years of storage.

Recently the strain Cronobacter sakazakii strain BAA-894 in the Cronobacter genus was first

sequenced. The genome size of Cronobacter sakazakii strain BAA-894 was found to be

4.36837 Mb long with 57% GC content (Kucerova et al., 2010). It displayed the presence of

two plasmids which were 31 kb and 131 kb respectively. The propghage and variable regions

were shown to possess virulence factors.

1.3 Virulence of Cronobacter

Virulence factors are produced by the organism and it bestows the organism with the ability to

cause an infection. (Brock biology of micro organisms 12th edition, 2009). The virulence

factors with respect to Cronobacter still remain unknown and have not yet been identified. The

most important factor contributing to the virulence of the organism is suspected to be the

production of endotoxin. The genes involved in the production of endotoxin have not yet been

identified (Drudy et al., 2005).

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Cronobacter possesses endotoxins which play a vital role in adhesion to cells and establishing

pathogenicity. The organism also displays an outer protective capsule composed of

heteropolysaccharide. The heteropolysaccharide is made up of glucuronic acid, D-glucose, D-

galactose, D-fucose and D-mannose (Forsythe, 2010). The capsule contributes the adhesion of

the organism to plant cells. The gene ompA is known to cause virulence in C.turicensis and

C.sakazakii. OmpA is a fibronectin binding protein which is expressed on the organism

surface. The fibronectin binding protein is essential for the invasion of the brain microvascular

endothelial cells and causing neonatal meningitis. (Nair et al., 2009).

1.4 Pathogenesis of Cronobacter

The virulence factors are directly involved with the ability of the organism to establish an

infection. Pathogenesis takes place in the following stages- exposure, adherence, invasion,

colonization and growth. Out of these stages adherence and colonization are most important.

Cronobacter displays clustered adhesion (Hunter et al., 2008). Cronobacter can adhere itself

instantaneously to surfaces like epithelial cells, endothelial cells as well as surfaces like plastic

and silicon enabling the formation of a biofilm. Most of the Cronobacter strains attach

themselves to the Caco-2 epithelial cells and it is followed by invasion (Forsythe et al., 2008).

Endotoxins play a vital role in adherence. The outer membrane protein A (ompA) which is

expressed by Cronobacter, plays an important role in the invasion of the brain endothelial cells

and a moderate role in the invasion of the human intestinal epithelial cells.

In rats, Cronobacter attaches itself onto the intestinal epithelial cells in the midst of an

infection. This attachment causes the apoptosis of the epithelium in a dose dependent manner

(Hunter et al., 2008). Further studies on the organism revealed the capability of Cronobacter to

invade brain capillary endothelial cells. The organism holds on to the human macrophages and

this leads to an increase in the IL-10/ IL-12 secretion. Due to this a Type-2 immune response is

activated but it is unable to inhibit the infection (Forsythe et al., 2008)

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Fig.1.2 Electron micrograph of the rat intestinal epithelium with an adherent Cronobacter

Fig.1.2 Electron micrograph of the rat intestinal epithelium with an adherent Cronobacter cell.

Adherence is an important step to establish pathogenesis (Hunter et al., 2008).

1.4.1 Motility

Cronobacter like other Gram negative micro organisms are motile. Motility is of great aid to

the organism in establishing pathogenesis as it adhere the organism to the host cells. It also

helps the organism to seek nutrients required for growth (Josenhans and Suerbaum, 2002).

Cronobacter utilizes its peritrichous flagella for motility. The flagella are composed of the

protein flagellin.

The flagellum enables the organism to move from one place to another. The flagella play a key

role in contributing to the ability of the organism to cause a disease. In C.turicensis the

expression of the fliP gene encodes the flagella proteins. The motor switch protein in

C.turicensis is fliM.

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1.4.2 Biofilm formation

Biofilm is defined as a population of bacterial cells which adhere to and grow on a contact

surface. The bacterial biofilm is confined within a definite matrix (Costerton et al., 1995).The

heteropolysaccharide capsule produced by Cronobacter, provides the organism with the

potential to form a biofilm on contact surface. The contact surfaces may include feeding

equipments like feeding tubes or the human cells. The biofilm provides protection and

enhances the survival of the organism by protecting it from osmotic, thermal and other types of

stress.

1.5 Anti microbial resistance

The biofilm formation protects the bacterial population and bestows them with resistance to

antibiotics lincomycin, clindamycin, streptogramins, rifampicin and fosfomycin. However,

Cronobacter is vulnerable to certain antibiotics. These antibiotics may include tetracyclines,

aminoglycosides, chloramphenicol etc. The resistance and susceptibility to antibiotics may

vary depending upon the strain of the organism. Most of the infections caused by Cronobacter

are treated with ampicillin-gentamicin or ampicillin-chloramphenicol. Another crucial factor

due to which the organism displays resistance to antibiotics like penicillins and cephalosporins

is the synthesis of β-lactamase. This enzyme inactivates antibiotics (Drudy et al., 2005).

1.6 Isolation of Cronobacter

The organism exhibits a fairly reasonable growth on both selective and non selective media.

The best growth is observed at 37 0 C. However, it does not display adequate growth in

enrichment broths. The isolation and detection of Cronobacter involves the exploitation of

various chromogenic and fluorogenic agar media which are dependent on the enzyme α-

glucosidase activity. This enzyme activity is well expressed in Cronobacter and a few

organisms belonging to the Enterobacteriaceae family (Iversen et al., 2008). The incorporation

of 5-bromo-4-chloro-3indolyl α D-glucopyranoside in the medium is useful for the detection of

Cronobacter. Cronobacter possesses α- glucosidase enzyme which breakdown sugar and

hydrolyzes the chromogen which leads to the production of coloured colonies (Iversen et al.,

2008)

15

Fig.1.3 Isolation of Cronobacter

Fig.1.3 (a) A DFI agar plate displaying isolated green coloured colonies of Cronobacter. After

streaking a loopful from EE broth inoculated with the sample on the agar, the plate was incubated at

37oCfor 24-hrs.

Fig.1.3 (b) Turquoise coloured colonies of Cronobacter growing on an ESIA agar plate. After streaking

a loopful from EE broth inoculated with the sample on the agar, the plate was incubated at 37 oCfor 24-

hrs.

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1.7 The growth kinetics of Cronobacter

The orderly increase in the chemical component of a cell is defined as growth. The growth

curve of any organism displays four stages lag phase, the exponential or the log phase, the

stationary phase and the decline phase. Cronobacter grows efficiently between 60 C to 450C in

all types of media. A few strains may be sensitive to the temperature at 45 0C while a few

display stable growth even at 470C (Iversen et al., 2004). It has a doubling time of 13hours.

The growth kinetic of Cronobacter is studied using impedance and conventional microbiology

techniques. Cronobacter has been known to generate gas on metabolizing sugars in the

medium. The generation of gas is also dependent on the temperature, as majority of

Cronobacter strains generate gas at 370 C. The rest of the strains produce gas at 440C. The

organism also displays thermotolerance and can be destroyed by pasteurization treatment.

Cronobacter is capable of growth even during refrigeration.

1.7.1 Decimal reduction time

Decimal reduction time which is also known as the D value is the time required to reduce the

bacterial load by 90% when the temperature is consistent. The presence of a capsule results in a

higher D value (Iversen et al., 2004). High fat, protein and carbohydrate in the medium of

growth also contribute to an increase in the D value. Extremely high D values were calculated,

when PIFs with a high percentage of fat in them were used (Nazarowec-White and Farber,

1997). Early studies show the variation in the D value between different Cronobacter strains.

The D value calculated at 550C was found to be between 2-49 minutes and a higher D value

was observed in whey milk (FSA report, 2009).

1.8 PIF and Cronobacter

PIFs are of three types – whey based, casein based and soy based formulas. As stated earlier,

Cronobacter is placed in the category ‘A’ organisms on the basis of strength of evidence in

PIF’s. They are categorised along with Salmonella, since they are epidemiologically and

microbiologically known to cause infections like meningitis, necrotizing enterocolitis,

septicaemia etc. The contamination of PIF’s can be either intrinsic or extrinsic. The intrinsic

contamination may be due to the induction of the organism during some stage of the

manufacturing process i.e. the wet mix process, the dry mix process or the combined process.

17

On the other hand, extrinsic contamination may result during reconstitution and use. E.g.

Contaminated utensils, blenders, spoons. A variety of other closely related organisms have also

been isolated from PIFs. These organisms include Enterobacter cloacae, Klebsiella

pneumoniae, K. oxytoca, E. hormaechei, E. coli and Citrobacter freundii. (Muytjens et al,

1988).

1.9 Reconstitution of PIF’s

The reconstitution of PIF’s plays a critical role in enabling organism growth. The ingestion of

reconstituted PIF containing contaminants may lead to infection. The organism growth is

directly related to the temperature of the water added and the holding time. The addition of

water to the PIF may facilitate the growth of micro organisms at a favourable temperature. At

high temperatures like 50-700C no appreciable growth is observed. Inactivation of Cronobacter

spps. is observed at 30-40 0C. However, reactivation of the organism takes place at a long

holding time. While at 10-20 0C, minimal organism growth is observed (FSA PROJECT,

2009). According to the international guidelines, the temperature of water used in the

reconstitution of PIF’s should be over 700C while the FSA advises the boiling of water in the

kettle and using the water after cooling it for half an hour. The hospital prepares a minimum of

30 ml of reconstituted PIF.

1.10 Temperature Profiling

Innumerable aspects have to be taken into consideration during the preparation of

powdered infant formulas. Lack of knowledge and biological variations are the major

factors which may lead to an infection. The cooling response of the reconstituted PIF’s is

largely dependent on the temperature of the external environment and the volume of water

boiled in the kettle.

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1.11 Strategies for prevention of an infection

Since Cronobacter is ubiquitous it is not possible to completely restrain its presence. However,

a few strategies can be used in order to minimize an infection. With respect to infants the best

and most effective strategy is to promote breast feeding. Essential precautions should be taken

during the production and packaging of PIF’s. Well sterilized feeding equipment and vessels

should be used by hospitals and infant care takers. The holding time of reconstituted PIF in the

feeding tubes should be minimized. The prevention of infections in adults and immune

compromised individuals is difficult as the source of the infection is uncertain. The use of

reconstituted dairy products should be avoided. With respect to other food products,

pasteurisation should be carried in the last step of production before the products are available

in local markets. Pasteurisation leads to the eradication of potential pathogens (Iversen C., and

Forsythe S, 2004).

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1.12 Aims and objectives of the research

The aims and objectives of the research were also divided into two parts.

The main aim of the first part of the study was:

The primary isolation and determination of Cronobacter and related organisms

obtained from 15 different food samples.

The study followed by the death rate determination at 700C as well as 550 C.

The D value was calculated for the organisms at 55 0C and 700 C.

The study focuses on obtaining more data on the isolation of infection causing

organisms from food products other than PIFs.

The main aim of the second part of the project was:

To determine the survival of the organism at different reconstitution temperatures to

clear the concerns of a local hospital.

The study began with temperature profiling of reconstituted PIF and was followed by

spiking the reconstituted PIFs with infection causing strains of Cronobacter and

observing the organism survival potential. The experiment was carried out on 5

important PIFs commonly used in hospitals.

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2. MATERIALS AND METHODS

2.1 Samples collection for the isolation of Cronobacter

A wide variety of samples were used in order to obtain versatile results. 15 different samples

ranging from soup sachets, instant casserole mix, cake mix, fresh herbs and dried herbs from

supermarkets and the local market were obtained and utilized to determine the presence of

Cronobacter and related organisms.

TABLE 1. Samples collected for Cronobacter determination.

SAMPLE INGREDIENTS NUTRITIONAL INFORMATION

Batchelors Slim a SoupMediterranean Tomato

Water, tomato, leek, onion, potato starch, glucose syrup, sugar, vegetable oil, salt, flavour enhancers, yeast stabilisers, parsley, garlic, colours, emulsifiers, antioxidants, wheat flour, milk powder.

Per 100g as servedEnergy 110kJ / 26kcalProtein 0.5gCarbohydrates 4.9gFat 0.5gFibre 0.4gSodium 0.26g

Colman’s of NorwichSausage Casserole

Wheat flour, dried tomato 20%, corn flour, sugar, herbs and spices, salt, dried onion, yeast, flavouring, vegetable oil, dried lemon juice, traces of egg and milk.

Per 100g as soldEnergy 1531kJ / 361kcalProtein 8.9gCarbohydrates 77.7gFat 1.6gFibre 5.5gSodium 2.2g

ASDA Chocolate Sponge mix Wheat flour, sugar, vegetable oil, cocoa powder (6%),raising agents.

Per 100g as soldEnergy 1333 kJProtein 7.0gCarbohydrates 45.5gFat 11.9gFibre 2.8gSodium 0.26g

BART Basil 15 grams Basil

Sainsbury fresh coriander Coriander leaves

21

SAMPLE INGREDIENTS NUTRITIONAL INFORMATION

Suk Chuen Dai Po Ton Soup Stock

HerbsCassaveChinese foxglove rhizomePilose asiabell rootAttractylodes rhizomeSzechwan lovage rhizomeWhite peony rootAngelica rootLiquorice rootCassia barkUnnamed herb

Per 120g servingEnergy 837.5 kJProtein 5gFat 0gCarbohydrate 55gSodium 750mg

Fig.2.1 Herbs from Suk Chuen Dai Po Ton Soup Stock

Fig.2.1 Herbs from Suk Chuen Dai Po Ton Soup Stock packet which were used to isolate Cronobacter and related organisms.


22

All 15 samples were examined for Cronobacter and related organisms. Out of 15 samples, 12

were herbs. The Iversen and Forsythe method of detection was adopted (Iversen and Forsythe,

2006). Maintenance of sterile conditions was an important factor. The sample source and

description were recorded. Approximately 10g of each sample were measured and added to

90ml of BPW under sterile conditions. In the case of hard samples like dried herbs, the

samples were homogenized with the help of a stomacher. Homogenization of the sample aids

the microbial flora to disperse throughout the solution. Serial dilutions were then carried out.

The homogenized solution was labelled as the 10-1 dilution. 1ml of the 10-1 dilution was pipette

into 9ml of saline; 10-2 dilution. This was continued till the 10-5 dilution. 0.1ml of each dilution

were transferred onto Plate Count Agar and Violet Red Bile Glucose Agar plates respectively

and spread with the help of a sterile spreader. The plates and sample in BPW were incubated

overnight at 370 C. The plates were checked for colony growth the next day and the Aerobic

plate count and Enterobacieriaceae colony count were determined.

10ml of the overnight incubated sample in BPW was transferred into 90ml EE broth and was

incubated overnight at 370 C. After incubation the inoculated EE broth was streaked onto DFI

and ESIA plates respectively and the plates were incubated overnight. The colony

characteristics were recorded and depending on the results they were streaked onto TSA plates

and incubated overnight at 370 C. The colonies harvested from the TSA plates were then used

to carry out other tests.

TABLE 2. Media employed for the isolation of Cronobacter

23

MEDIA COMPISITION (g/l)

Violet Red Bile Glucose Agar (VRBGA)Peptone 7.0; Yeast extract 3.0; Sodium chloride 5.0; Bile salts 1.5; Glucose 10.0; Neutral red 0.03;Crystal violet 0.002; Agar 13.0.(pH 7.2 +/- 0.2)

Plate Count Agar (PCA)

Yeast extract 2.5; Pancreatic digest of casein 5.0; Glucose 1.0; Agar 15.0. (pH 7.0 ± 0.2)

Druggan Forsythe Iversen Agar (DFI)Tryptone 15s Soya peptone 5.0; Sodium chloride 5.0; Ferric

ammonium citrate 1.0; Sodium desoxycholate 1.0; Sodium thiosulphate 1.0; Chromogen 0.1; Agar 15.0 ;(pH 7.3 ± 0.2)

Enterobacter Sakazakii isolation chromogenic agar (ESIA)

Pancreatic peptone of casein 7.0;Yeast extract 3.0; Sodium chloride 5.0; Sodium desoxycholate 0.6; 5-Bromo-4-chloro-3-indolyl α-D-glucopyranoside 0.15; Crystal violet 2; Agar 12.0. (pH 7.0 ± 0.2)

TABLE 2. Media used in the isolation of Cronobacter from food samples.

2.2.1 Gram Stain

Gram stain was carried in order to observe the colony characteristics and to determine if the

organisms were Gram positive or negative. Respective colonies from the TSA plate were

harvested and bacterial smears were prepared on glass slides. The slides were then flooded with

crystal violet for 2 minutes. After 2 minutes the crystal violet was poured off and the slides

were flooded with Gram's iodine for another 2 minutes. The Gram’s iodine was the poured off

and washed. The slides were then decolourized with alcohol and then washed and stained with

safranin for 2 minutes. The slides were then washed, dried and observed for Gram negative

organisms.

2.2.2 ID 32 E assay

24

The ID 32 E assay designed by Biomerieux is employed for the identification of organisms

belonging to the Enterobacteriacece family and non fastidious organisms. The assay comprises

of a strip containing 32 cupules. Each cupule is composed of a dehydrated test substrate. A few

colonies were harvested from the overnight incubated TSA plates which were streaked with the

organism. The colonies were picked with the aid of a sterile loop and suspended in sterile

saline in order to obtain a turbidity of 0.5. The organisms were homogenized in the saline with

the help of a test tube shaker. The suspension was then immediately utilized. 55µl was pipetted

into each of the cupule and the strips was covered with lids and incubated overnight. After

incubation the expression of the reactions were read visually and the entered into a database.

The database enables the identification of the organism as it possesses a list of organisms that

are capable of identification by this assay.

2.2.3 API ZYM assay

The API ZYM assay, is another semi - quantitative assay designed by Biomerieux. It enables

the detection and rapid study of enzymatic activity in micro organisms. The assay comprises of

a strip containing 20 cupules or micro wells. The base of each cupule is composed of synthetic

fibres. Each cupule contains an enzymatic substrate and its buffer. The assay is capable of

carrying out 19 enzymatic reactions with extremely small amount of sample as well as complex

samples. The enzymes assayed for are alkaline phosphatase, esterase, esterase lipase, lipase,

leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α- chymotrypsin, acid

phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β- galactosidase, β-

glucuronidase, α-glucosidase, β-glucosidase, N-acteyl- β-glucosaminidase, α-mannosidase and

α-fucosidase. Around 5 colonies were picked from TSA plates which were streaked with the

organism and incubated overnight. The colonies were suspended in sterile saline and

homogenized. 55 µl was pipette in each cupule and the strips were covered and incubated for

around 4 -4 ½ hours at 370C. The suspension causes the rehydration of enzymatic substrates.

After incubation, 1 drop of ZYM A reagent and 1 drop of ZYM B reagent were added to each

cupule and the strips were left for colour development for 5-7 minutes. This was followed by

reading the strips which depended on the intensity of the reaction and data interpretation.

Fig.2.2 API ZYM assay kit

25

Fig.2.2 API ZYM assay kit employed for the determination of enzymatic activity. (Source:

http://www.fishersci.com/wps/portal/PRODUCTDETAIL?

LBCID=11516104&productId=1327569&catalogId=29103&pos=13&catCode=HC_SC&from

Cat=yes&keepSessionSearchOutPut=true&brCategoryId=56622&hlpi=y&fromSearch).

2.2.4 16S rRNA gene sequencing

As stated in the introduction Cronobacter is closely related to the other species in the

Enterobacteriaceae family and therefore sequencing of the 16S rRNA gene is essential. A few

colonies were harvested from TSA agar plates on which the organism were streaked and

incubated at 370C overnight. A loopful of the colonies were harvested from agar plate and

suspended in sterile saline and homogenized using a test tube shaker. A small amount was

pipette onto FTA Elute Micro Cards. These cards comprise of a specialized matrix which

causes microbial inactivation. The matrix leads to the lysis of cellular material and leaves

behind the organism DNA which is analyzed. An anti- desiccant was applied onto the cards.

The FTA Elute Micro Cards were then shipped to the Accugenix laboratory for the 16S rRNA

sequencing and they produce results on the organism within duration of 7 hours. The

Accugenix laboratory extracts the DNA from the cards and amplifies the DNA using PCR with

the aid of universal primers. DNA extraction was followed by cycle sequencing where the

DNA was sequenced and the nucleotides were labeled fluorescently. The fluorescently labeled

nucleotides were then examined with the help of an automated sequencer and the DNA

sequence was obtained which was compared to the Accugenix laboratory database. The data

was then analyzed and checked for quality.

2.2.5 Pulsed field gel electrophoresis

26

http://www.fishersci.com/wps/portal/PRODUCTDETAIL?LBCID=11516104&productId=1327569&catalogId=29103&pos=13&catCode=HC_SC&fromCat=yes&keepSessionSearchOutPut=true&brCategoryId=56622&hlpi=y&fromSearch



PFGE enables the separation of large molecules of DNA by changing the field direction within

regular intervals of time. The isolates were streaked on TSA and incubated overnight at 370C.

PFGE was carried out systematically. The initial step involved the preparation of a cell

suspension buffer in which colonies harvested from a TSA plates were suspended in order to

attain cell concentration of 1.35 ± 0.05. This was followed by pipetting 300µl out a solution

comprising of T.E buffer + 1% agarose + 1% SDS (TEB agarose) in eppendorf tubes and

placing them in a thermo block for 15minutes at 550C. The next step involved the casting of

plugs. 300 µl of suspension was mixed with 300µl TEB agarose and 15 µl of proteinase K. 100

µl of the above mix was dispensed into each plug and the plugs were left to set.

Fig.2.3 Setting of plugs

Fig.2.3 The setting of plugs after casting with suspension, TEB agarose and proteinase K.

For cells lysis in the agarose plugs, a mix of cell lysis buffer and proteinaseK was prepared and

5ml was added to polypropylene tubes. This was followed by adding the plugs to it. The tubes

were then placed in a water bath shaker for 1 ½ - 2 hours. The water bath was set at 550C.

Fig.2.4 Lysis of cells in agarose plugs

27

Fig.2.4 Lysis of cells in agarose plug by cell lysis buffer and proteinase K.

After cell lysis, the lysis buffer was discarded and plugs were washed twice with ultra pure

water for 15 minutes by incubating the tubes in the water bath shaker. It was then washed

thrice with T.E buffer for 15minutes and the tubes were placed in a water bath shaker at 500C.

After the last wash 5ml of TE was added to the plugs in the tube. The tubes were stored at 4 0C

overnight.

1/3 the plugs were then cut and transferred in an eppindorff tube containing 135 µl of grade

water and 15 µl of buffer. The tubes were left to stand at room temperature for 30 minutes.

This was followed by discarding the mixture and adding 150 µl of restriction enzyme mixture

containing restriction enzyme XbaI and incubating at 370C for 1 ½ to 2 hrs. The plugs were

then placed in the required order on the comb and 100ml of 0.5 TEB buffer and the enzyme

were poured.

Fig.2.5 Agarose plugs placed on the comb

28

Fig.2.5 Agarose plugs placed of the comb before the pouring of enzyme and TEB buffer.

100ml of 0.5X TEB 1% agarose which was incubated at 600C was then poured and left to

solidify. The gel is then placed into a running tank containing 2litres of TBE buffer and was

run for 20hours at 6V. After 20hours the gel was stained with ethidium bromide. Ethidium

bromide enhanced the visibility of the bands and the gel was observed under UV light.

2.3 Death Kinetics based on temperature

29

After the above mentioned assays the identity and purity of the strains were confirmed and the

strains were further used in the death rate studies. Isolates of interest were streaked on TSA

overnight and incubated at 370C. A few colonies were then harvested and inoculated into 10ml

of casein and 10 ml of whey infant milk i.e. Cow and gate 1 st infant milk and SMA extra

hungry infant milk respectively. The milk provides nutrients for growth and therefore

Cronobacter is normally found in infant formulas.

Fig.2.6 Infant formulas for colony inoculation

Fig.2.6 Cow and Gate infant formula and SMA extra hungry infant formula were under to inoculate

organisms of interest.

The tubes containing infant milk with inoculated organisms were then incubated overnight.

This was followed by pipetting 9ml of fresh whey and casein milk into sterile tubes. These

tubes were placed in a water bath for 30 minutes at the desired temperature which was either

700C or 550C depending on the experiment. 1ml of the overnight inoculated milk was then

added to the preheated milk. Following this serial dilutions were carried out in a 96 well which

comprised of 135 µl of sterile saline. The dilutions were carried out at an interval of every five

minutes for 30minutes and the dilutions were pipette onto PCA plates using Miles and Misra

method. The plates were incubated at 370C overnight and the colony count was determined.

Following the colony count, the D value was determined. Log-linear relationship was

employed for D value calculation and the formula used to calculate the D value was X = (Y-b)/

a.

30

2.4 Bacterial Survival in PIFs

2.4.1 Reconstitution temperature analysis

In order to determine the survival potential of bacteria in reconstituted PIFs it is necessary to

carry out temperature profiling without spiking it with bacteria. It is essential to record the

room temperature. Pregestimil, Nutramigen, LCP Neocate, Pepti junior and SHS Caprilon are

PIFs that are used by hospitals, were measured under sterile conditions and transferred to a

sterile bottle. A kettle containing water was boiled and different volumes of boiled water and

sterile cold water were added to the PIFs in order to obtain temperatures above 700C and a total

volume of 30ml. The bottles containing reconstituted PIF was left at room temperature and the

temperature was recorded at 30 second intervals for 30minutes. The temperature of

reconstituted PIFs was recorded for 30 minutes as most of the instruction in the PIF can

recommend boiling water and cooling it for 30 minutes. Fluctuations in the room temperature

were also recorded throughout the duration. Temperature profiling was also carried out on

reconstituted PIFs when different volumes of water were boiled in the kettle.

2.4.2 Bacteriocidal affect of reconstitution

To verify the survival of microorganisms in reconstituted PIF the above mentioned experiment

was repeated but 0.1ml of overnight culture of the desired organism in TSB was added before

adding the boiled water. Three strains were studies Cronobacter sakazakii1, Cronobacter

sakazakii 658 and Cronobacter turicensis. The study was carried out at two initial

reconstitution temperatures 700C and 550C. The temperature was measured every 2 minutes for

16 minutes at 700C and 10 minutes at 550C respectively. This was followed by serial dilutions

were carried out. The dilutions were pipette onto PCA plates by Miles and Misra method and

the colony count was recorded after incubation. The plates were incubated overnight at 370C. A

graph of the log cfu/ml was plotted against time.

3. RESULTS


31

The isolation of Cronobacter from a large variety of samples was carried out in order to

pinpoint their versatile nature in terms of source and ability to survive desiccation. It also

approaches the different chromogenic media used, the biochemical assays and molecular tests

exploited for the identification of the organism.

3.1.1 VRBGA and PCA plate count results.

TABLE3. The plate count obtained on VRBGA and PCA plates.

SAMPLE AND AGAR USED

DILUTION

10-1

DILUTION

10-2

DILUTION

10-3

DILUTION

10-4

DILUTION

10-5

1) Colman’s of NorwichSausage Casserole

VRBGA 9 53 TMTC 0 0PCA TMTC 16 TMTC 109 31

2) Saintsbury fresh coriander

VRBGA TMTC TMTC TMTC TMTC 46PCA TMTC TMTC TMTC TMTC 212

3) ASDA Chocolate Sponge mix

VRBGA 0 0 TMTC 0 0PCA TMTC TMTC 0 0 0

4) BART Basil 15 grams

VRBGA TMTC TMTC 22 TMTC 98PCA 78 7 28 TMTC 104

5) Batchelors Slim a SoupMediterranean Tomato

VRBGA 3 0 0 0 0PCA 62 18 5 0 0

6) Cassia barkVRBGA 0 0 0 0 0PCA 3 0 2 0 0

7) CassaveVRBGA 0 0 0 0 0

32

PCA 3 0 0 0 08) Attractylodes

rhizome

VRBGA 0 0 0 0 0PCA 6 0 0 0 0

9) Unnamed herbVRBGA 0 0 0 0 0PCA 0 0 0 0 0

10) Liquorice rootVRBGA 0 0 0 0 0PCA 15 6 1 0 0

11) Pilose asiabell root

VRBGA 0 0 0 0 0PCA 0 0 0 0 0

12) Chinese foxglove rhizome

VRBGA 0 0 0 0 0PCA TMTC 31 6 1 0

13) Szechwan lovage rhizome

VRBGA 0 0 0 0 0PCA 12 1 0 0 0

14) White peony root

VRBGA 0 0 0 0 0PCA 6 2 0 0 0

15) Angelica root

VRBGA 0 0 0 0 0PCA 5 0 0 0 0

TABLE3. The plate count on VRBGA and PCA plates onto which 0.1 ml of the serially diluted samples were pipette and incubated overnight at 370 C.

The table displays that the growth of organisms in the ready to eat mix was abundant in comparison with the Chinese herbs form Suk Chuen Dai Po Ton Soup Stock.

33

The colonies on the VRBGA plates were light pink in colour and when tested for oxidase

potential, they proved to be oxidase negative. A few samples like Colman’s of Norwich

Sausage Casserole, Sainsbury fresh coriander, ASDA Chocolate Sponge mix and BART

Basil, exhibited dense colony growth on VRBGA plates and a change in the media colour was

also observed in a few samples. The media colour changed from pink to yellow on the plates

which were streaked with the Sausage Casserole and Cake mix. The VRBGA plates for

Sausage Casserole also displayed the presence of a few purple colonies. However, the colonies

on the PCA plates were off white in colour and shapeless.

Fig.3.1 Colonies observed on the VRBGA plate

Fig.3.1 Photograph of a VRBGA plate on which the 10-3 dilution of the ASDA chocolate sponge cake mix was pipetted. The plate also displays light pink coloured colonies and change in the agar colour. The plate was incubated at 370 C for 24hrs.

Fig.3.2 Colonies observed on the PCA plate

Fig.3.2 Photograph of a PCA plate on which the 10-2 dilution of the ASDA chocolate sponge cake mix was pipetted. The plate also displays the off white colour coloured shapeless colonies. The plate was incubated at 370 C for 24hrs.

34

3.1.2 DFI and ESIA plate results

Out of fifteen samples tested for the presence of Cronobacter, only four samples had shown

necessary growth on DFI and ESIA Plates. The samples that displayed growth were Colman’s

of Norwich Sausage Casserole, ASDA Chocolate Sponge mix, Chinese herb liquorice,

Sainsbury fresh coriander and BART Basil. The DFI and ESIA plates for all the samples

except Sainsbury fresh coriander, exhibited green coloured colonies on the DFI plate and

turquoise coloured colonies on ESIA plates. However, coriander sample had shown the growth

of black coloured pinpoint colonies on the DFI plate.

Fig.3.3 Black coloured colonies on the DFI plate.

Fig.3.3 Black colony growth seen on the DFI plate onto which a loopful of EE broth inoculated with Sainsbury fresh coriander was streaked. The plate was incubated overnight at 370C.

35

3.1.3 Gram Stain

The gram stain results of most strains displayed reddish pink colour. The organisms were rod

shaped indicating the strains were gram negative. Thus, this indicates that organisms were

unable to retain crystal violet and took up safranin which was the counter stain.

Fig.3.4 Gram stain

Fig.3.4 Photograph of a gram stain of the organism obtained from the Chinese herb Liquorice, the organism was found to be reddish pink in colour and rod shaped.

The strains were assigned the following NTU id.

NTU id SOURCE AGAR OBTAINED FROMC1 BART Basil ESIAC2 ASDA Chocolate Sponge mix ESIAC3 Colman’s of Norwich Sausage

CasseroleESIA

C4 Chinese herb Liquorice DFIC5 BART Basil DFI

36

3.1.4 ID 32 E Assay

On being subjected to the ID 32 E assay the strains C1, C2, C3, C4 and C5 produced the following result.

Fig.3.5 (a) ID 32 E assay on strain C1

Fig.3.5 (a) The ID 32 E assay for strain C1 determines that the significant taxon for the strain is Enterobacter sakazakii and has a 99% ID matched. The next significant taxa for the strain C1 is Enterobacter cloacae.

Fig.3.5(b) ID 32 E assay on strain C2

Fig.3.5 (b) The ID 32 E assay for strain C2 determines that the significant taxon for the strain is Enterobacter sakazakii and has a 99.9% ID matched. The next significant taxa for the strain C2 isAeromonas hydrophila/caviae/sorbia.

37

Fig.3.5 (c) ID 32 E assays on strain C3

Fig.3.5(c) The ID 32 E assay for strain C3 determines that the significant taxon for the strain is Enterobacter sakazakii and has a 99.9% ID matched.

Fig.3.5 (d) ID 32 E assays on strain C4

Fig.3.5 (d) The ID 32 E assay for strain C4 determines that the significant taxon for the strain could either be Escherichia vulneris which has a 55.6% ID matched or Buttiauxella agrestis which has a 44.2% ID matched. The next close taxon may be Enterobacter sakazakii which displays 0.1% ID match.

38

Fig.3.5 (e) ID 32 E assays on strain C5

Fig.3.5 (e) The ID 32 E assay for strain C5 determines that the significant taxon for the strain is Enterobacter cloacae and has a 99.5% ID matched. The next significant taxon for the strain C5 isEnterobacter sakazaki which displays 0.2% ID match.

TABLE4. ID 32 E results

NTU id SOURCE AGAR OBTAINED

FROM

ID 32 E PROFILE

ID 32 E RESULT

C1 BART Basil ESIA 34275767051 Enterobacter sakazaki

C2 ASDA Chocolate Sponge mix

ESIA 34256363050 Enterobacter sakazaki

C3 Colman’s of Norwich Sausage Casserole

ESIA 34256367040 Enterobacter sakazaki

C4 Chinese herb Liquorice

DFI 04674563051 Escherichia vulneris

C5 BART Basil DFI 34074747210 Enterobacter cloacae

TABLE4. The table displays the ID 32 E result, profile number and information about the source if the strain.

39

3.1.5 16S rRNA

The following results were obtained on sequencing the 16S rRNA gene.

TABLE5. 16S rRNA results

NTU id SOURCE AGAR OBTAINED

FROM

ACCUGENIX CODE 16S rRNA sequencing results

C1 BART Basil ESIA C1014689 Klebsiella oxytoca

C2 ASDA Chocolate Sponge mix

ESIA C1014690 Cronobacter sakazakii

C3 Colman’s of Norwich Sausage Casserole

ESIA C1014691 Cronobacter sakazakii

C4 Chinese herb Liquorice

DFI C1014692 Enterobacter helveticus

C5 BART Basil DFI C1023741 Enterobacter hormaechei

TABLE5. Information on the organism source and strain identity after 16S rRNA sequencing can be obtained from the table.

Fig.3.6 The phylogenetic tree of strains C1,C2,C3,C4 and C5.

Fig.3.6 Displays the phylogenetic relationship between the strains. From the phylogenetic tree it can be observed that the strains Cronobacter sakazakii and Enterobacter hormaechei are closely related. While the strain Klebsiella oxytoca is not related to the other three strains.

40

3.1.6 API ZYM Assay

TABLE6. API ZYM assay results

ENZYMES NTU IDC2 C3 C1 C4 C5

Control 0 0 0 0 0Alkaline phosphatase 0 0 3 0 0Esterase 0 2 0 0 0Esterase lipase 2 4 0 1 0Lipase 0 0 0 0 0Leucine arylamidase 0 2 0 0 1Valine arylamidase 0 0 0 0 0Cystine arylamidase 0 0 0 0 2Trypsin 0 0 0 0 0α- chymotrypsin 0 0 0 0 0Acid phosphatase 0 2 5 0 2Naphthol-AS-BI-phosphohydrolase

0 0 0 0 2

α-galactosidase 0 1 0 0 0β- galactosidase 0 3 1 0 0β-glucuronidase 0 0 0 0 0α-glucosidase 0 0 0 0 0β-glucosidase 0 0 0 0 3N-acteyl- β-glucosaminidase 1 0 3 0 0α-mannosidase 0 0 0 0 0α-fucosidase 0 0 0 0 3

TABLE6. The table displays the API ZYM assay result. The list of enzymes is seen at the left of the table and the values assigned depending in the intensity of the colour produced based on the strain is seen on the right of the table. The value 0 determines a negative reaction, while 5 indicates a positive reaction of high intensity.

In comparison with the other strains the Cronobacter sakazakii strains i.e. strain C3 and C2

exhibits Esterase lipase activity. Strain C2 is a produces a reaction of a very low intensity with

enzymes N-acteyl- β-glucosaminidase. While, strain C3 produces a low intensity reaction with

enzymes Leucine arylamidase and α-galactosidase. C3 produces a moderate reaction with β-

galactosidase.

41

3.1.7 Pulse field gel electrophoresis

Pulse field gel electrophoresis aids in discriminating between strains C1, C2, C3, C4 and C5.

Fig.3.7 (a) Pulse field gel electrophoresis of strains C1, C2, C3 and C4.

DNA C1 C2 C3 C4 DNA MARKER MARKER ( Salmonella 2.16s-63.8s)

Fig.3.7 (a) Pulse field gel electrophoresis discriminated between strains C1, C2, C3 and C4. The DNA fragments produced on being digested by enzyme XbaI aids in determining that the strains C2 and C3 are closely related confirming the results obtained by sequencing the 16S rRna gene.

42

Fig.3.7 (b) Pulse field gel electrophoresis of strains C1, C2, C3, C4 and C5.

DNA C1 C2 C3 C4 C5 DNAMARKER MARKER

Fig.3.7 (b) Pulse field gel electrophoresis discriminated between strains C1, C2, C3, C4 and C5. The DNA fragments produced were digested by enzyme XbaI. However, the gel did not take up ethidium bromide and the bands are not clearly visible. However it can be concluded that strain C5 is not related to strain C2 and C3.

43

3.2 Death kinetics of the strains with respect to temperature.

3.2.1 Death kinetics of the strains C1, C2, C3 and C4 AT 70 0 C.

Fig 3.8 Thermal inactivation of Klebsiella oxytoca at 70ºC. (A)

0 1 2 3 4 5 60

0.5

1

1.5

2

2.5

3

3.5

4

4.5

f(x) = − 0.222802671328572 x + 3.9453486351275R² = 0.854998304074881

Death Kinetics of Klebsiella oxytoca at 70 degrees in whey milk.(C1)

WHEY

Linear (WHEY)

Time (mins)

log

cfu

/ml

Fig.3.8(A) Thermal inactivation of Klebsiella oxytoca at 70ºC in whey milk. The linear log was employed for D value calculation.

(B)

Fig.3.8(B) Thermal inactivation of Klebsiella oxytoca at 70ºC in casein milk. The linear log was employed for D value calculation.

44

0 1 2 3 4 5 6

0

1

2

3

4

5

6

7

f(x) = − 1.16901960800285 x + 6.088652537937R² = 0.97959040082266

Death Kinetics of Klebsiella oxytoca at 70 degrees in casein milk(C1)

CASEIN

Linear (CASEIN)

Time (mins)

log

cfu

/ m

l


(A)

0 1 2 3 4 5 6

0

1

2

3

4

5

6

f(x) = − 1.13064250275507 x + 5.71101042814612R² = 0.998747232303942

Death Kinetics of Cronobacter sakazakii at 70 degrees in whey milk(C2)

WHEY

Linear (WHEY)

Time (mins)

log

cfu

/ml

Fig.3.9(A) Thermal inactivation of Cronobacter sakazakii at 70ºC in whey milk. The strain C2 was isolated from ASDA Chocolate Sponge mix. The linear log was employed for D value calculation.

(B)

0 1 2 3 4 5 60

1

2

3

4

5

6

f(x) = − 0.995544721057776 x + 4.1481030044074R² = 0.75

Death Kinetics of Cronobacter sakazakii at 70 degrees in casein milk(C2)

CASEIN

Linear (CASEIN)

Time (mins)

log c

fu/m

l

Fig.3.9(B) Thermal inactivation of Cronobacter sakazakii at 70ºC in casein milk. The strain C2 was isolated from ASDA Chocolate Sponge mix. The linear log was employed for D value calculation.

45


(A)

0 1 2 3 4 5 60

1

2

3

4

5

6

f(x) = − 0.995544721057776 x + 5.04775967251941R² = 0.99763008522158

Death Kinetics of Cronobacter sakazakii at 70 degrees in whey milk(C3)

WHEY

Linear (WHEY)

Time (mins)

log

cfu

/ml

Fig.3.10(A) Thermal inactivation of Cronobacter sakazakii at 70ºC in whey milk. The strain C3 was isolated from Colman’s of Norwich Sausage Casserole. The linear log was employed for D value calculation.

(B)

0 1 2 3 4 5 60

1

2

3

4

5

6

7

f(x) = − 1.15563025007673 x + 6.03286354657815R² = 0.977212726211745

Death Kinetics of Cronobacter sakazakii at 70 degrees in casein milk(C3)

Time (mins)

log

cfu

/ml

Fig.3.10(B) Thermal inactivation of Cronobacter sakazakii at 70ºC in casein milk. The strain C3 was isolated from Colman’s of Norwich Sausage Casserole. The linear log was employed for D value calculation.

Fig.3.11 Thermal inactivation of Enterobacter helveticus at 70ºC .

46

(A)

0 1 2 3 4 5 60

1

2

3

4

5

6

f(x) = − 1.00827853703165 x + 5.2598576573171R² = 0.977962316559106

Death Kinetics of Enterobacter helveticus at 70 degrees in whey milk(C4)

WHEY

Linear (WHEY)

Time (mins)

log

cfu

/ml

Fig.3.11(A) Thermal inactivation of Enterobacter helveticus at 70ºC in whey milk. The linear log was employed for D value calculation.

(B)

0 1 2 3 4 5 60

1

2

3

4

5

6

7

f(x) = − 1.18588378514286 x + 4.94118243809524R² = 0.75

Death Kinetics of Enterobacter helveticus at 70 degrees in casein milk(C4).

CASEIN

Linear (CASEIN)

Time (mins)

log c

fu/m

l

Fig.3.11(B) Thermal inactivation of Enterobacter helveticus at 70ºC in casein milk. The linear log was employed for D value calculation.

3.2.2 Death kinetics of the strains C1, C2, C3 and C4 AT 55 0 C.

47


(A)

0 5 10 15 20 250

1

2

3

4

5

6

7

8

9

f(x) = − 0.34725289932934 x + 7.15200307536099R² = 0.90036597370517

Death Kinetics of Klebsiella oxytoca at 55 degrees in whey milk

WHEY

Linear (WHEY)

Time (mins)

log

cfu

/ml

Fig.3.12(A) Thermal inactivation of Klebsiella oxytoca at 55ºC in whey milk. The linear log was employed for D value calculation.

(B)

0 5 10 15 20 250

1

2

3

4

5

6

7

8

9

f(x) = − 0.278207479247556 x + 7.63033065325872R² = 0.907957410216379

Death Kinetics of Klebsiella oxytoca at 55 degrees in casein milk (C1).

CASEIN

Linear (CASEIN)

Time(min)

log

cfu

/ml

Fig.3.12(B) Thermal inactivation of Klebsiella oxytoca at 55ºC in casein milk. The linear log was employed for D value calculation.


48

(A)

0 5 10 15 20 250

1

2

3

4

5

6

7

8

9

f(x) = − 0.233368383805013 x + 7.87311690334222R² = 0.958995016550931

Death Kinetics of Cronobacter sakazakii at 55 degrees in whey milk

WHEY

Linear (WHEY)

Time (mins)

log

cfu

/ml

Fig.3.13(A) Thermal inactivation of Cronobacter sakazakii at 55ºC in whey milk. The strain C2 was isolated from ASDA Chocolate Sponge mix. The linear log was employed for D value calculation.

(B)

0 5 10 15 20 250

1

2

3

4

5

6

7

8

9

f(x) = − 0.253617573629649 x + 8.30243678213883R² = 0.964988804142341

Death Kinetics of Cronobacter sakazakii at 55 degrees in casein milk (C2).

CASEINLinear (CASEIN)

Time(mins)

log

cfu

/ml

Fig.3.13(B) Thermal inactivation of Cronobacter sakazakii at 55ºC in casein milk. The strain C2 was isolated from ASDA Chocolate Sponge mix. The linear log was employed for D value calculation.


49

(A)

0 5 10 15 20 250

1

2

3

4

5

6

7

8

9

f(x) = − 0.175362006789145 x + 7.54540156065372R² = 0.934551139758547

Death Kinetics of Cronobacter sakazakii at 55 degrees in whey milk (C3)

WHEY

Linear (WHEY)

Time (mins)

logcfu/ml

Fig.3.14(A) Thermal inactivation of Cronobacter sakazakii at 55ºC in whey milk. The strain C3 was isolated from Colman’s of Norwich Sausage Casserole. The linear log was employed for D value calculation.

(B)

0 5 10 15 20 250

1

2

3

4

5

6

7

8

9

f(x) = − 0.210217478880945 x + 8.17529867808698R² = 0.957893451040918

Death Kinetics of Cronobacter Sakazakii at 55 drgrees in casein milk (C3).


Time(mins)

log cfu/ ml

Fig.3.14(B) Thermal inactivation of Cronobacter sakazakii at 55ºC in casein milk. The strain C3 was isolated from Colman’s of Norwich Sausage Casserole. The linear log was employed for D value calculation.


50

(A)

0 5 10 15 20 250

1

2

3

4

5

6

7

8

f(x) = − 0.155210155732084 x + 7.00957537871541R² = 0.917668005338427

Death Kinetics of Enterobacter helveticus at 55 degrees in whey milk (C4).

WHEYLinear (WHEY)

Time (mins)

logcfu/ml

Fig.3.15(A) Thermal inactivation of Enterobacter helveticus at 55ºC in whey milk. The linear log was employed for D value calculation.

0 5 10 15 20 250

1

2

3

4

5

6

7

8

9

f(x) = − 0.178281010445752 x + 7.57726738685757R² = 0.922590142627018

Death Kinetics of Enterobacter helveticus at 55 degrees in casein milk (C4).


Time (mins)

logcfu/ml

Fig.3.15(B) Thermal inactivation of Enterobacter helveticus at 55ºC in casein milk. The linear log was employed for D value calculation.

TABLE7. D value of strains C1, C2, C3 and C4.

NTU Organism Infant formula D value D value

51

ID 550C 700CC1 Klebsiella oxytoca WHEY 2.164265 0.56364

CASEIN 1.329856 0.20787

C2 Cronobacter sakazakii

WHEY 0.12017 0.051327

CASEIN 0.88142 0.833166

C3 Cronobacter sakazakii

WHEY 2.466286 0.070352

CASEIN 0.6381 0.21991

C4 Enterobacter helveticus

WHEY 0.451613 0.21627

CASEIN 2.606742 0.833755

TABLE7. Show the calculated D. It also provides information on the type of infant formula used.

3.3 Bacterial Survival in PIFs

3.3.1 Reconstitution temperature analysis

52

Table8. The temperature profile of reconstituted PIFs LCP neocate, C+G premium and SHS Caprilon

53

54

Time (min)

Time (sec) Temp (°C)

LCP

Neocate

C+G Premium

1stSHS

Caprilon 30 70 66 471 60 68 65 47 90 66 64 462 120 64 63 45 150 63 61 453 180 62 60 44 210 61 59 434 240 59 58 43 270 59 56 425 300 59 56 42 330 58 54 416 360 57 53 41 390 57 52 407 420 56 52 40 450 56 51 398 480 55 50 39 510 54 50 389 540 54 48 38 570 53 48 37

10 600 53 47 37 630 52 46 37

11 660 51 45 36 690 50 45 36

12 720 50 44 36 750 49 44 35

13 780 48 43 35 810 48 43 35

14 840 48 42 35 870 47 41 34

15 900 46 41 34 930 46 41 33

16 960 45 40 33 990 45 40 33

17 1020 44 40 33 1050 44 39 32

18 1080 44 39 32 1110 43 38 32

19 1140 43 38 32 1170 43 37 31

20 1200 42 37 31 1230 42 37 31

21 1260 42 37 31 1290 41 36 31

22 1320 41 36 30 1350 41 36 30

23 1380 41 36 30 1410 40 36 30

24 1440 40 35 30 1470 40 35 30

25 1500 40 35 30 1530 40 35 30

26 1560 39 34 30 1590 39 34 30

27 1620 39 34 29 1650 38 33 29

28 1680 38 33 29

Table8. The temperature profile of reconstituted PIFs LCP neocate, C+G premium and SHS Caprilon at room temperature 250C. The ratio of boiled water to cold sterile water was 29:1. The temperature after 30 minutes was found to be between 280C to 370C depending upon the PIF.

Table9. The temperature profile of reconstituted PIFs LCP neocate, C+G premium and SHS Caprilon when the kettle was boiled at different volumes.

Time (min)

Time (sec)

VOLUME 500ml

VOLUME 1000ml VOLUME 1500ml

TEMP (0C) TEMP (0C) TEMP (0C)

LCP

Neocate

C+G Premium

1stSHS

CaprilonLCP

Neocate

C+G Premium

1st

SHS Caprilo

nLCP

Neocate

C+G Premium

1stSHS

Caprilon 30 70 70 71 71 73 71 75 72 721 60 64 67 67 65 68.5 62 67 67 67 90 60 65 65 63 65.5 61 65 65 652 120 58 64 62 61 64 60 62 64 63 150 56 62 60 61 62.5 59 61 62.5 623 180 54 61 59 60 61 58 60 61 61 210 52 59 58 58 60 58 58 60 604 240 51 58 57 57 59 57 57.5 59 59 270 50 57 56 56 57.5 55 57 58 575 300 50 56 55 55 56 55 56 57 57 330 49 55 54 54 55 53 55 56 566 360 49 55 53 53 54 52 54 55 55.5 390 48 54 52 52 53 51 54.5 54 557 420 48 52 52 52 52 51 52 53 54 450 47 52 50 51 51 50 51.5 52 538 480 47 52 50 50 50 50 51 51.5 52.5

55

510 47 51 50 49 49 49 50.5 51 529 540 46 50 49 49 49 49 50 50 51 570 46 50 48 48 48 48 50 50 50

10 600 45 49 47 48 47 48 49 49 50 630 45 49 47 47 46.5 47 48.5 48.5 49

11 660 44 48 46 47 46.5 47 48 48 49 690 44 47 45 46 46 46 47 47 48

12 720 43 47 45 46 45.5 46 47 47 47.5 750 43 46 45 45 45 45 46 46 47

13 780 42 46 44 45 44.5 45 45.5 45.5 46 810 42 45 43 44.5 44 44.5 45 45 46

14 840 42 45 43 44 44 44 45 44.5 45.5 870 41 45 42 44 43.5 43.5 44 44 45

15 900 41 44 42 43 43 43 44 44 44.5 930 41 43 41 43 42.5 43 43 44 44.5

16 960 40 43 41 42.5 42 42.5 43 43 44 990 40 43 41 42 41.5 42 42.5 42.5 43

17 1020 40 42 41 41.5 41 41.5 42 42 42.5 1050 39 42 40 41 41 41 42 42 42

18 1080 39 41 40 41 40.5 40.5 42 41 42 1110 38 41 39 40.5 40 40 41 41 41.5

19 1140 37 41 39 40 40 40 41 40.5 41 1170 37 40 39 40 39.5 40 41 40 41

20 1200 37 40 38 40 39 39.5 40.5 40 40

123036 40 38 39.5 39 39.5 40 39.5 40

21 1260 36 39 38 39 38 39 40 39.5 40 1290 36 39 37 38 38 38 40 39 40

22 1320 36 39 37 38 38 38 40 39 39.5 1350 35 38 37 38 37.5 38 40 38.5 39

23 1380 35 38 36 37 37.5 38 39.5 38 39 1410 35 38 36 37 37 38 39 38 39

24 1440 35 38 36 37 37 37.5 39 38 38 1470 35 37 36 37 37 37 39 37.5 38

25 1500 35 37 36 37 36.5 37 38 37 38 1530 35 37 36 37 36.5 37 38 37 38

26 1560 34 37 35 36.5 36 36 38 37 37 1590 34 36 35 36.5 36 36 38 37 37

27 1620 34 36 35 36 36.5 36 37.5 36.5 37 1650 34 36 35 36 35.5 36 37 36 37

28 1680 34 36 35 36 35 35.5 37 36 36.5 1710 34 36 35 35.5 35 35.5 37 36 36.5

29 1740 33 35 34 35.5 35 35 36.5 35.5 36 1770 33 35 34 35 35 35 36.5 35 36

30 1800 33 35 34 35 34.5 35 36 35 36

56

Table9. Shows the different temperatures obtained on the reconstitution of PIFs LCP neocate, C+G premium and SHS Caprilon. Different volumes i.e. 500 ml, 1000ml and 1500ml of distilled were boiled in the kettle and employed for reconstitution. The room temperature recorded was 250C and the ratio of boiled water to sterile water was 29:1. The temperature after 30 minutes was found to be between 300C to 400C depending upon the volume in the kettle.

3.3.2 Bacteriocidal affect of reconstitution

Bacteriocidal affect of reconstitution when the initial temperature was 55 0 C.

1) Pregestimil

Fig.3.16 Survival capacity of Cronobacter in Pregestimil when the initial temperature was 55 0 C.

A

0 2 4 6 8 100

1

2

3

4

5

6

0

10

20

30

40

50

60

C. sakazakii 1 in Pregestimil

C sakazakii 1 Temperature

Time in minutes

log cfu/mL

57

B

0 2 4 6 8 10

0

1

2

3

4

5

6

7

8

9

0

10

20

30

40

50

60

C. sakazakii 658 in Pregestimil

C sakazakii 658Temperature

Time in minutes

log cfu/mL

C

0 2 4 6 8 100

1

2

3

4

5

6

0

10

20

30

40

50

60

C. Turicensis in Pregestimil

C. turicensis Temperature

Time in minutes

log cfu/mL

Fig.3.16 (A) Survival potential of strain Cronobacter sakazakii 1 in Pregestimil. (B) Survival potential of strain Cronobacter sakazakii 658 in Pregestimil. (C) Survival potential of strain Cronobacter turicensis in Pregestimil. The initial temperature of reconstitution was 550C. The kettle was boiled and cooled for 10mins before using the boiled water for reconstitution of PIF.

58

2) Pepti Junior

Fig.3.17 Survival capacity of Cronobacter in Pepti Junior when the initial temperature was 55 0 C.

A

0 2 4 6 8 100

1

2

3

4

5

6

7

0

10

20

30

40

50

60

C. sakazakii 1 in Pepti Junior.


Time in minutes

log cfu/mL

B

0 2 4 6 8 100

1

2

3

4

5

6

7

8

0

10

20

30

40

50

60C. sakazakii 658 in Pepti Junior

C. sakazakii 658Temperature

Time in minutes

log cfu/mL

60

C

0 2 4 6 8 100

1

2

3

4

5

6

7

0

10

20

30

40

50

60C. Turicensis in Pepti Junior

C. turicensisTemperature

Time in minutes

log cfu/mL

Fig.3.17 (A) Survival potential of strain Cronobacter sakazakii 1 in Pepti Junior. (B) Survival potential of strain Cronobacter sakazakii 658 in Pepti Junior. (C) Survival potential of strain Cronobacter turicensis in Pepti Junior. The initial temperature of reconstitution was 550C. The kettle was boiled and cooled for 10mins before using the boiled water for reconstitution of PIF.

3) LCP Neocate

61

Fig.3.18 Survival capacity of Cronobacter in LCP Neocate when the initial temperature was 55 0 C.

A

0 2 4 6 8 104

4.1

4.2

4.3

4.4

4.5

4.6

4.7

4.8

4.9

5

0

10

20

30

40

50

60

C. Sakazakii 1 in LCP Neocate


Time in minutes

log cfu/mL

B

0 2 4 6 8 100

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0

10

20

30

40

50

60C. Sakazakii 658 in LCP Neocate


Time in minutes

log cfu/mL

C

62

0 2 4 6 8 103.8

4

4.2

4.4

4.6

4.8

5

0

10

20

30

40

50

60C. Turicensis in LCP Neocate

C.turicensis Temperature

Time in minutes

log cfu/mL

Fig.3.18 (A) Survival potential of strain Cronobacter sakazakii 1 in LCP Neocate. (B) Survival potential of strain Cronobacter sakazakii 658 in LCP Neocate. (C) Survival potential of strain Cronobacter turicensis in LCP Neocate. The initial temperature of reconstitution was 550C. The kettle was boiled and cooled for 10mins before using the boiled water for reconstitution of PIF.

4) SHS Caprilon

63

Fig.3.19 Survival capacity of Cronobacter in SHS Caprilon when the initial temperature was 55 0 C.

A

0 2 4 6 8 100

1

2

3

4

5

6

7

0

10

20

30

40

50

60

C. Sakazakii 1 in SHS Caprilon


Time in minutes

log cfu/mL

B

0 2 4 6 8 100

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0

10

20

30

40

50

60C. Sakazakii 658 in SHS Caprilon


Time in minutes

log cfu/mL

C

64

0 2 4 6 8 100

1

2

3

4

5

6

7

0

10

20

30

40

50

60C. Turicensis in SHS Caprilon


Time in minutes

log cfu/mL

Fig.3.19 (A) Survival potential of strain Cronobacter sakazakii 1 in SHS Caprilon. (B) Survival potential of strain Cronobacter sakazakii 658 in SHS Caprilon. (C) Survival potential of strain Cronobacter turicensis in SHS Caprilon. The initial temperature of reconstitution was 550C. The kettle was boiled and cooled for 10mins before using the boiled water for reconstitution of PIF.

5) Nutramigen

65

Fig.3.20 Survival capacity of Cronobacter in Nutramigen when the initial temperature was 55 0 C.

A

0 2 4 6 8 100

1

2

3

4

5

6

0

10

20

30

40

50

60

Cronobacter sakazakii 1 in Nutramigen


Time in minutes

log cfu/mL

B

0 2 4 6 8 100

1

2

3

4

5

6

0

10

20

30

40

50

60

Cronobacter sakazakii 658 in Nutramigen

C. Sakazakii 658Temperature

Time in minutes

log cfu/mL

C

66

0 2 4 6 8 100

1

2

3

4

5

6

0

10

20

30

40

50

60

C. Turicensis in Nutramigen


Time in minutes

log cfu/mL

Fig.3.20 (A) Survival potential of strain Cronobacter sakazakii 1 in Nutramigen. (B) Survival potential of strain Cronobacter sakazakii 658 in Nutramigen. (C) Survival potential of strain Cronobacter turicensis in Nutramigen. The initial temperature of reconstitution was 550C. The kettle was boiled and cooled for 10mins before using the boiled water for reconstitution of PIF.

Bacteriocidal affect of reconstitution when the initial temperature was 70 0 C.

67

The strains Cronobacter sakazakii 1, Cronobacter sakazakii 658 and Cronobacter turicensis

did not display growth in Pregestimil when the initial reconstitution temperature was 70 0C

and therefore the graph could not be plot. No growth was observed for the strains

Cronobacter sakazakii 1and Cronobacter sakazakii 658 in Pepti junior. However, growth

was observed for Cronobacter turicensis in Pepti junior.

Fig.3.21 Survival capacity of Cronobacter turicensis in Pepti Junior when the initial temperature was 70 0 C.

0 2 4 6 8 10 12 14 16 180

1

2

3

4

5

6

0

10

20

30

40

50

60

70

80Cronobacter turicensis in Pepti Junior at 70 °C

Cronobacter turicensis Temperature

Time in minutes

Log cfu/mL

Fig.3.21 Shows the survival potential of strain Cronobacter turicensis in Pepti Junior. The initial temperature of reconstitution was 700C which was obtained by adding boiled water and sterile cold water in the ratio of 29:1 for 30 ml.

68

Cronobacter turicensis also displayed growth in LCP Neocate while, the other two strains did not.

Fig.3.22 Survival capacity of Cronobacter turicensis in LCP Neocate when the initial temperature was 70 0 C.

0 2 4 6 8 10 12 14 16 180

1

2

3

4

5

6

0

10

20

30

40

50

60

70

80Cronobacter turicensis in LCP Neocate at 70 °C

Cronobacter turicensisTemperature

Time in minutes

Log cfu/mL

Fig.3.22 Determines the survival potential of strain Cronobacter turicensis in LCP Neocate. The initial temperature of reconstitution was 700C which was obtained by adding boiled water and sterile cold water in the ratio of 29:1 for 30 ml.

The strains did not show any growth in the PIFs, SHS Caprilon and Nutramigen and therefore the graph could not be plot.

4. DISCUSSION AND CONCLUSION

69

Cronobacter has been long associated with infections like meningitis, sepsis and enterocolitis

in neonates across the world. Neonates are found to be susceptible to gram negative

organisms. The Organism has been termed as 'Severe hazard for restricted populations, life

threatening or substantial chronic sequelae or long duration' by the ICMSF (Iversen et al.,

2006). Although the epidemiology of the organism was initially unknown recently studies

have been carried out on it. Various phenotypic features of the organism were studied. The

sequencing of Cronobacter sakazakii strain BAA-894 has shed light on the genotypic

features of Cronobacter.

The study was carried out for duration of four months. It began with the isolation and

identification of organisms, especially Cronobacter from a variety of food samples.

4.1 Identification of Cronobacter and other related organisms.

Out of 15 samples which were screened for the presence of Cronobacter, 2 samples displayed

positive results. Both the samples were ready to eat mix powders. It is important to mention

that none of the fresh and dried herbs contained Cronobacter spp since, plant material is

known to be the major source of the organism. Difficulty is encountered in identity

confirmation of the organism, a wide range of cromogenic and biochemical techniques were

employed. Molecular techniques were also used. The chromogenic and biochemical

techniques were of great aid in studying the phenotypic features while the molecular

techniques produced information on the genotypic features.

A total of 4 isolates were obtained out of the samples tested. The isolates displayed green

coloured and turquoise coloured colonies on the DFI and ESIA plates (Fig.1.3(a) and

Fig.1.3(b). They were presumptive Cronobacter colonies. However, when sub-cultured on

TSA plates, most the colonies picked from DFI media did not display growth. The reason for

no growth remains unknown. On carrying out the ID 32 E test the organisms isolated from 3

samples were identified as Enterobacter sakazaki while the isolates from the other two

samples were identified as Escherichia vulneris and Enterobacter cloacae respectively.

Therefore it could be recorded that the DFI and ESIA media did not produce false negatives.

However, 3 false positives were observed.

The ID 32 E tests were found to be accurate for the identification of Cronobacter from 2

samples but were unable to identify the other isolates correctly. The accuracy of the ID 32 E

70

test may not always produce accurate results but it can be used as an alternate test for the

identification of clinical organisms (O’Hara C and Miller, 1999). The 16s results helped in

verifying the accuracy of ID 32 E results.

The 16S rRNA sequencing analysis identified 2 isolates out of 5 as Cronobacter sakazakii.

The rest of the isolates were identified as Klebsiella oxytoca, Enterobacter helveticus and

Enterobacter hormaechei. The 16S rRNA sequencing analysis was found to be rapid, reliable

and reduces data complexity, enhancing the accuracy in strain identification. 16S rRNA

sequencing is vital for organism identification and it out beats the other techniques for in

identifying and confirming the presence of Cronobacter in samples.

The Pulsed-field gel electrophoresis (PFGE) aided genotyping and the bands produced

confirmed that both the isolates belonged to the same species as they displayed a high degree

of similarity.

The API ZYM test is a biochemical assay which determined the enzyme activity of

Cronobacter sakazakii with respect to the other isolates. Both the Cronobacter sakazakii

isolates displayed a high and moderate intensity reaction for the enzyme esterase lipase.

Cronobacter is known to produce enzyme α-D glucosidase. However, both the Cronobacter

sakazakii strains did not show reaction for the enzymes α-glucosidase and β-glucosidase.

Both the Cronobacter sakazakii strains were isolated from ready to eat mix powders as the

organism is capable of survival in dry conditions and is thermotolerant and osmotolerant. The

samples were ASDA Chocolate Sponge mix and Colman’s of Norwich Sausage Casserole.

The samples may be contaminated with Cronobacter during the manufacture process. Both

the samples contain plant material which is known to be a major source of Cronobacter

(Forsythe S, 2005). The samples required the powders to be mixed with other ingredients and

are cooked by baking at a temperature of 2000C or above. This eliminates the risk of an

infection. However there is a possibility of cross contamination with PIF while cooking. This

may lead to an infection.

The isolation of Klebsiella oxytoca from the ESIA plate of BART basil was also found to be

interesting. The ESIA plate of BART basil had two types of colonies- purple colonies and

turquoise colonies.

Fig.4.1 Growth of Klebsiella oxytoca on ESIA

71

Fig4.1 Klebsiella oxytoca displayed purple and turquoise coloured colonies on ESIA agar which was streaked with a loopful of EE broth inoculated with BART basil. The plate was incubated for 24hrs at 370C.

Klebsiella oxytoca is a gram negative organism which also belongs to the Enterobacteriaceae

family. The capsule of the organism has high polysaccharide content and has been examined

for its antigen specificity (Baldi et al, 2009). Klebsiella oxytoca is capable of causing

hemorrhagic colitis and sepsis. The hemorrhagic colitis is antibiotic associated i.e. it is

caused after treatment with an antibiotics, specifically antibiotics like quinolones and

cephalosporins (Hogenauer C et al, 2006). The presence of the organism in BART basil may

pose as a threat to individuals on antibiotic treatment as basil is a common herb employed in

food seasoning. Klebsiella oxytoca was not found to be related to any of the 3 other strains

obtained from the samples. However, Cronobacter sakazakii and Enterobacter hormaechei

were found to be closely related to each other and were also found to be related with

Enterobacter helveticus.

4.2 Survival potential and the D value.

The survival potential of the strains was analyzed at temperatures 550C and 700C for duration

of 20 minutes. At 700C most of the Strains did not display growth after duration of 5 minutes.

72

The D value at 700C could not be calculated due to unequal time intervals but when the graph

was trimmed to 5 minutes at an interval of 2.5 minutes the D value could be calculated. At

700C the D value was calculated to be between 0.05- 0.83 minutes. The D values were found

to be higher in casein milk than whey milk while, the strains analyzed in the FSA report

showed higher D values in whey milk. At 550C very few cell from all the strains could be

recovered. On calculating the D value of the strains in both whey and casein milk, the D

values of the strains found were similar in both. The highest thermotolerance was displayed

by Enterobacter helveticus in casein milk as the D value was 2.606742 minutes. The D

values Cronobacter sakazakii isolated from ASDA Chocolate Sponge mix were found to be

0.12017 minutes and 0.88142minutes in whey milk and casein milk. The D value in casein

milk was higher than that of whey milk. This was different compared to the FSA report since;

most the strains displayed a higher D value in whey milk than casein milk. The

thermotolerance of the other Cronobacter sakazakii strain was higher in whey milk than

casein milk. The D value was 2.466286 minutes.

The D values for both the Cronobacter sakazakii at 550C strains were between 0.12 - 2.46

minutes. The thermotolerance of the organism was less compared to the Cronobacter strains

in the FSA report as the thermotolerance of the organisms was found to be between 2-49

minutes. The thermotolerance of the organism may be low due to the absence of the KT gene.

4.3 Temperature profiling at room temperature (25 0 C).

The cooling profiles for different volumes PIFs were analyzed for 30 minutes (TABLE9.)

and the temperatures after 30 minutes were between 300C to 400C. The temperatures

obtained were influenced by the volume of water boiled in the kettle. Lesser the volume of

water boiled in the kettle, the faster was the cooling of the reconstituted PIF. The temperature

difference with respect to the volumes was found to be between 1-20C.

4.4 Bacteriocidal affect of reconstitution

Bacteriocidal affect of reconstitution was studied using 3 strains C. sakazakii 1, C. sakazakii

658 and C. turicensis when the initial temperatures were 550C and 700C. The strain

73

C.turicensis may pose as a threat and cause infections as it is capable of growth at both 70 0C

and 550C. C.sakazakii 658 displayed a greater survival potential than the other strains at 550C.

The temperature was found to be around 450C after 10 minutes when the initial temperature

of reconstitution was 550C and was around 500C after 18 minutes when the initial temperature

was 700C. Therefore the, reconstitution of PIFs should be carried out at a temperature above

700C as stated by the FSA report.

CONCLUSION

Cronobacter sakazakii strains were isolated and identified from ready to eat powders. The

identification was carried out by phenotypic and genotypic techniques. The sequencing of

74

the 16S rRNA gene was found to be the most reliable technique for identification of the

organism. Both the Cronobacter sakazakii strains possessed a lower thermotolerance

compared to other strains in previous studies on the organism. Studies should further be

carried out in order to detect the function of the KT gene in the isolated strains and its relation

with thermotolerance. Also the food safety can be maintained by regularly screening the

manufacturing and production environment for Cronobacter spps.

FUTURE PROSPECTS

The studies carried out on the isolation of Cronobacter spp. and other organisms from food samples and the Death and survival determination has huge scope in future research.

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Many more ready to eat food samples can be further probed for the presence of Cronobacter spp and precautions can be taken to limit contamination with the organisms and avoid infections. The samples can also be tested of other organism belonging to the Enterobacteriaceae family which possess the potential to cause infections.

Studies could be carried out to detect the presence of Klebsiella oxytoca in other samples of culinary herbs used on daily basis.

Both the strains of Cronobacter sakazakii obtained from food samples which display low thermotolerance, can be analyzed for the presence of the KT gene as it is known to affect the thermotolerance of the organism.

The D value of the strains which could not be calculated due to irregular time intervals could be determined by checking the milk for the presence of the organism every 30 seconds at 700C.

ACKNOWLEDGEMENTS

I would like to express my gratitude to my supervisor Professor Stephen Forsythe for selecting me to work on this project and for being extremely supportive. He has been a

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constant source of encouragement and granted me the liberty to express my views and suggestions. He has helped me think out of the box and working under the guidance of Professor Forsythe has been an extremely fulfilling experience.

I would like to thank Amal Hoddoon, Halima Alsadeg and Maria Gillett for helping me throughout the course of my project and for patiently teaching me all the techniques and

skills required for my project. I would also like to thank Téva Aleins and Emmanuelle Le Neuder for all the constant help.I would like to extend my gratitude to the Microbiology lab staff for creating an extremely enthusiastic working atmosphere which helps in individual betterment and for being ever so helpful.

Last but not the least, I would like to thank the Almighty and my lovely friends and family,

especially my parents for making my dream of studying in the UK come true.

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Meeting report:Enterobacter sakazakii and other microorganisms in powdered infant formula: meeting report (2004, 2006 and 2008). MRA Series 6.

Web sites used:1) Accugenixvs.MicroSeq

http://www.accugenix.com/PDF/White%20Papers/White%20Paper%20- %20Accugenix%20vs%20MicroSeq.pdf . Last used on 20th August 2010

2) Oxoid.http://www.oxoid.com/uk/blue/index.asp . Last used on 21st August 2010

3) Accugenixhttp://www.accugenix.com/serv_Genotypic.php?p=1 . Last used on 21st August 2010

4) Food microbe websitehttp://www.foodmicrobe.com .Last used on 27th August 2010.

5) NCBI nucleotide homehttp://www.ncbi.nlm.nih.gov/nucleotide . Last used on 25th August 2010.

6) ClustalW2http://www.ebi.ac.uk/Tools/clustalw2/index.html . Last used on 25th August 2010.

Books used:

Madigan, M., Martinko, J., Clark, J., et al. Brock book of microorganisms 12th edition. Pearson education. Inc

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http://www.ebi.ac.uk/Tools/clustalw2/index.html

http://www.ncbi.nlm.nih.gov/nucleotide

http://www.foodmicrobe.com/

http://www.accugenix.com/serv_Genotypic.php?p=1

http://www.oxoid.com/uk/blue/index.asp

http://www.accugenix.com/PDF/White%20Papers/White%20Paper%20-%20%20%20%20%20Accugenix%20vs%20MicroSeq.pdf

http://www.accugenix.com/PDF/White%20Papers/White%20Paper%20-%20%20%20%20%20Accugenix%20vs%20MicroSeq.pdf

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