ugwu, celestina chibuzo - university of nigeria … · mrs. ugwu, celestina chibuzo a postgraduate...

1

ISOLATION AND

CHARACTERIZATION OF Bacillus

cereus STRAINS FROM VARIOUS

FOODS IN NSUKKA.

BY

UGWU, CELESTINA CHIBUZO

PG/M.Sc./03/34129

DEPARTMENT OF MICROBIOLOGY

UNIVERSITY OF NIGERIA, NSUKKA

MARCH, 2009.

2

TITLE PAGE

ISOLATION AND CHARACTERIZATION OF Bacillus cereus

STRAINS FROM VARIOUS FOODS IN NSUKKA.

BY

UGWU, CELESTINA CHIBUZO

PG/M.Sc./03/34129

SUBMITTED TO THE SCHOOL OF POST GRADUATE

STUDIES,

UNIVERSITY OF NIGERIA, NSUKKA

IN PARTIAL FULFILLMENT OF THE REQUIREMENT

FOR THE AWARD OF MASTER’S DEGREE

(M.Sc.) IN MEDICAL MICROBIOLOGY

SUPERVISOR: DR. (MRS.) I.M. EZEONU

MARCH, 2009.

3

CERTIFICATION

Mrs. Ugwu, Celestina Chibuzo a postgraduate student in the

Department of Microbiology, majoring in medical Microbiology, has

satisfactorily completed the requirements for course work and research for

the degree of Master in Science (M.Sc.) in Microbiology. The work is

embodied in her dissertation original and has not been submitted in part

or full for either diploma or degree of this University or any other

University.

Prof. J.C. Ogbonna I.M. Ezeonu Ph.D.

Head, Supervisor

Department of Microbiology Department of MCB

University of Nigeria, Nsukka University of Nigeria, Nsukka.

4

DEDICATION

I dedicate this work to God Almighty and Mother Mary

5

ACKNOWLEDGEMENT

It is my wish to express my profound gratitude to God Almighty for

affording me good health, wisdom and understanding to accomplish this

work.

I am highly indebted to my project supervisor Dr. (Mrs.) I.M.

Ezeonu for her patience, encouragement, time spent with me and

corrections from the initial stage to the conclusions of this work despite

her crowded involvement in other things.

My gratitude also goes to Dr. Emeka Nweze for his contributions

and encouragement throughout this work. I am grateful to him for helping

me get the reference strains which I used in this work. I am also thankful

to Dr. Rooney, Alejandro P. a Research Geneticist, Microbial Genomics and

Bioprocessing Research Unit, USA for his kindness in taking his pains to

send me the reference strains.

My gratitude also goes to the following people for their help and

encouragement throughout this work; Prof. (Mrs.) J.I. Okafor, Prof. C.U.

Iroegbu, Prof. J.O. Ugwuanyi, Rev. Sr. Dr. M.C.U. Dibua, Dr. C.U.

Anyanwu, Mr. J.A.C. Ugonabo, Dr. E.A. Eze, Mr. Chibuzo Eze, Mrs. B.N.

Nwike, Mrs. J.O. Etonyeaku, Mr. E.I Oyibo, Mr. P.U. Ugwu, Mrs. M.O.

Ugboaja, Mr. J.O. Mbawuike, Mrs. T.I. Onyia, Mrs. E.E. Ilukwe, and Mrs.

M. Ayogu.

I am grateful to my lovely husband Mr. Ugwu, Francis E. for his

moral, financial support and encouragement throughout the period of my

studies. I am also thankful to my lovely mother, siblings and my children

6

Ogechi, Nnanna, Ifeanyi, Chinedum and Emeka for encouragement and

patience.

I am also grateful to Miss Onugwu Nwakaego of Our Redeemer

Computers and Mr. Chibuzo of Ez Computers who did their best to see this

work through.

7

TABLE OF CONTENTS

Title Page ------------------------------------------------------------------------------

-------i

Certification ---------------------------------------------------------------------------

-------ii

Dedication ----------------------------------------------------------------------------

------iii

Acknowledgement --------------------------------------------------------------------

------iv

Table of Contents --------------------------------------------------------------------

------vi

List of Tables --------------------------------------------------------------------------

------x

List of Figures -------------------------------------------------------------------------

------xi

Abstract---------------------------------------------------------------------------------

----xiii

CHAPTER ONE: INTRODUCTION ----------------------------------------------

------1

CHAPTER TWO: LITERATURE REVIEW

2.1 Taxonomy of Bacillus cereus -----------------------------------------------

------6

8

2.2 History of Bacillus cereus ---------------------------------------------------

-------7

2.2.1 Characteristics of Bacillus cereus ------------------------------------------

-------8

2.2.2 Serotyping of Bacillus cereus -----------------------------------------------

------9

2.2.3 The Genome of Bacillus cereus --------------------------------------------

-------9

2.3 The Ecology of Bacillus cereus----------------------------------------------

-----10

2.3.1 Bacillus cereus in the Environment ----------------------------------------

-----10

2.3.2 Bacillus cereus in Food ------------------------------------------------------

-----10

2.3.3 Reservoir of Bacillus cereus ------------------------------------------------

-----13

2.4 Foods Implicated in Growth and Poisoning ------------------------------

-----14

2.5 Symptoms of Bacillus cereus Food Poisoning ----------------------------

-----15

2.5.1 The Diarrhoeal Syndrome ---------------------------------------------------

----15

2.5.2 The Emetic Syndrome -------------------------------------------------------

-----16

9

2.6 Epidemiology of Bacillus cereus --------------------------------------------

----17

2.6.1 The Incidence of Bacillus cereus Food Poisoning -----------------------

-----17

2.6.2 Transmission of Bacillus cereus --------------------------------------------

-----18

2.6.3 Nature of Bacillus cereus Cells in Foods at Consumption --------------

-----20

2.6.4 Fate of Bacillus cereus Cells in the Digestive Tract ---------------------

-----20

2.6.5 Dose Response Relations ---------------------------------------------------

-----21

2.7 Enterotoxines and Virulence Factors of Bacillus cereus ----------------

-----22

2.7.1 Haemolysin BL HBL ----------------------------------------------------------

-----22

2.7.2 Non Haemolytic Enterotoxin (NHE) ----------------------------------------

----23

2.7.3. Enterotoxin T -----------------------------------------------------------------

-----24

2.7.4 Enterotoxin FM ---------------------------------------------------------------

-----25

2.7.5 Haemolysin 1 -----------------------------------------------------------------

-----26

10

2.7.6 Haemolysin 2 -----------------------------------------------------------------

-----26

2.7.7 Phospholipases C -------------------------------------------------------------

-----26

2.7.7.1 Phosphatidyl Inositol Hydrase ---------------------------------------------

----27

2.7.7.2 Phosphatidyl Choline Hydrolase ------------------------------------------

-----27

2.7.7.3 Sphingomyelinase -----------------------------------------------------------

-----27

2.7.8 Cytotoxin-K --------------------------------------------------------------------

----28

2.7.9 Vacuolation Factor -----------------------------------------------------------

-----28

2.7.10 Spore ---------------------------------------------------------------------------

----29

2.8 Physical and Chemical Factors Affecting Bacillus cereus Growth -----

-----30

2.8.1 Temperature ------------------------------------------------------------------

-----30

2.8.2 pH ------------------------------------------------------------------------------

-----31

2.8.3 Water Activity -----------------------------------------------------------------

-----32

11

2.8.4 Sodium Chloride -------------------------------------------------------------

-----32

2.8.5 Effect of Heating -------------------------------------------------------------

-----33

2.8.6 Effect of Food Additives -----------------------------------------------------

-----34

CHAPTER THREE: MATERIALS AND METHODS

3.1 Media and Media Preparation ----------------------------------------------

-----35

3.2 Samples collection -----------------------------------------------------------

-----36

3.3 Isolation of Organisms ------------------------------------------------------

-----36

3.4 Identification Tests ----------------------------------------------------------

-----37

3.4.1 Motility -------------------------------------------------------------------------

-----37

3.4.2 Catalase -----------------------------------------------------------------------

-----37

3.4.3 Hemolysis ---------------------------------------------------------------------

-----38

3.4.4 Gelatin Liquefaction ----------------------------------------------------------

----38

12

3.4.5 Starch Hydrolysis -------------------------------------------------------------

-----39

3.4.6 Spore Staining ----------------------------------------------------------------

-----39

3.5 Standardization of inoculum ------------------------------------------------

-----40

3.6 Determination of the effect of Temperature on the

Vegetative Growth and Spore Germination of Bacillus cereus Strains -------------------------------------------------------------------------

-----41

3.7 Determination of the effect of pH on the Growth of Bacillus cereus Strains ------------------------------------------------------------41

3.8 Evaluation of Heat Resistance of Vegetative Cells ----------------------

-----41

3.9 Determination of the effect of Carbon Source on the Growth of Bacillus cereus -------------------------------------------------------------

----42

3.10 Plasmid DNA Profiling -------------------------------------------------------

-----43

3.11 Data Analysis -----------------------------------------------------------------

-----45

CHAPTER FOUR: RESULTS

4.1 Isolation and Characterization of Bacillus cereus -----------------------

-----46

4.1.1 Morphological and Metabolic Characterization of all the

Isolates -----------------------------------------------------------------------------46

13

4.1.2 Morphological and Metabolic Characterization of Reference Strains -------------------------------------------------------------------------

-----46

4.1.3 Selection of Classical Bacillus cereus Isolates ---------------------------

-----51

4.1.4 Plasmid Profile of Bacillus cereus Strains Isolated from Foods --------

---51

4.2 Effect of Temperature on the Vegetative Growth of Bacillus cereus Strains ----------------------------------------------------------------

----58

4.3 Effect of Temperature on the Spore Germination of Bacillus

cereus Strains ---------------------------------------------------------------------58

4.4 Effect of pH on the Growth Bacillus cereus Strains ---------------------

-----63

4.5 Heat Resistance of Bacillus cereus Strains--------------------------------

-----69

4.6 Effect of Carbon Source on the Growth of Bacillus cereus Strains -------------------------------------------------------------------------

-----78

CHAPTER FIVE: DISCUSSION

Conclusion -----------------------------------------------------------------------------

-----88

Recommendation ---------------------------------------------------------------------

-----89

References ----------------------------------------------------------------------------

-----90

Appendixes ----------------------------------------------------------------------------

---102

14

LIST OF TABLES

Table 1: Incidence of Bacillus cereus in Different Food Samples -------

-----47

Table 2: Morphological and Metabolic Characterization of all the

Isolates ----------------------------------------------------------------

-----48

Table 3: Morphological and Metabolic Characterization of Reference

Strains -----------------------------------------------------------------

-----49

Table 4: Morphological and Metabolic Characterization of Bacillus

cereus Strains --------------------------------------------------------

-----52

Table 5: The Decimal Reduction Times (D-value) for the Test Strains

–77

15

LIST OF FIGURES

Figure 1: Plasmid Profile of Bacillus cereus Strains Isolated from Foods------------------------------------------------------------

-----57

Figure 2: Vegetative growth of Bacillus cereus Strains at 370C----------

-----59

Figure 3: Vegetative growth of Bacillus cereus Strains at 280C-----------

-----60

Figure 4: Spore germination of Bacillus cereus Strains at 370C-----------

-----61

Figure 5: Spore germination of Bacillus cereus Strains at 280C-----------

-----62

Figure 6: Effect of pH on the Growth of NRRL-B14724 strain of Bacillus cereus ---------------------------------------------------------64

Figure 7: Effect of pH on the Growth of BC7 strain of Bacillus cereus-----------------------------------------------------------------------65

Figure 8: Effect of pH on the Growth of BC9 strain of Bacillus

cereus ----------------------------------------------------------------------66

Figure 9: Effect of pH on the Growth of NBC strain of Bacillus cereus ----------------------------------------------------------------------67

Figure 10: Effect of pH on the Growth of BC10 strain of Bacillus

cereus-----------------------------------------------------------------------68

Figure 11: Heat resistance of isolates at 40 degrees ------------------------

-----70

16

Figure 12: Heat resistance of isolates at 50 degrees-------------------------

-----71


-----72


-----73


-----74


-----75

Figure 17: Heat resistance of isolates at 100 degrees ----------------------

-----76

Figure 18: Growth of Bacillus cereus Strains on 1% Glucose

as sole carbon source ----------------------------------------------------79

Figure 19: Growth of Bacillus cereus Strains on 5% Glucose

as sole carbon source------------------------------------------------

-----80

Figure 20: Growth of Bacillus cereus Strains on 10%

Glucose as sole carbon source ------------------------------------------81

Figure 21: Growth of Bacillus cereus Strains on rice as sole carbon source --------------------------------------------------------

-----82

17

ABSTRACT

This study was carried out to determine the prevalence of Bacillus cereus in

various foods and characterize the Bacillus cereus strains isolated. All tested food

samples yielded Bacillus cereus as determined by growth on B. cereus selective

medium. A total of 350 B. cereus strains were isolated, comprising 123 (35.14%)

from rice, 111 (31.71%) from meat and 116 (33.14%) from fish. Morphological

and metabolic characterization of the isolates showed that 259 (74.00%) of the

B. cereus strains were haemolytic, 58 (16.57%) hydrolyzed starch, 252 (72.00%)

liquefied gelatin and 253 (72.29%) fermented mannitol. All isolates were motile,

catalase positive and contained endospores. Of the 350 strains, 67 (19.14%) had

the same morphological and biochemical characteristics as the two reference

strains used in the study and were therefore confirmed to be typical B. cereus

strains. Evaluation of the effect of temperature on growth showed that both

vegetative growth and spore germination occurred at 28oC and 37oC but not at

4oC. Growth of the cells occurred from pH 5 to pH 9 at 37oC with optimum at pH

7. There was no growth at pH 4. The B. cereus strains were significantly (P <

0.05) different in their responses to heat. Spores of all strains were resistant to

heat up to 60oC. At 80oC, the decimal reduction times (D-values) for the test

strains were: BC7, 208 min; BC9, 383 min; NBC, 23 min; NRRL-B14724, 250 min

and NRRL-B14725, 192 min. At 90oC, the D-values were: BC7, 64 min; BC9, 110

min; NRRL-B14724, 134 min and NRRL-B14725, 15 min and at 100oC, the D-

values were: BC7, 17 min; BC9, 16 min; and NRRL-B14724, 21 min. Differences

were also observed in the use of carbon source by the strains. Only the negative

control, NBC, grew on glucose while there was no significant (P > 0.05) growth

of the reference strain and classical test isolates. All strains grew well on rice in

contrast to the reference strain. The results of plasmid profiling showed that the

classical, negative control and reference strains contained multiple plasmids,

characteristic of B. cereus, except Bacillus thuringiensis, which contained only

one plasmid. The similarity in plasmid profiles of the strains suggested that they

are all B. cereus strains, including the atypical NBC strain.

18

CHAPTER ONE

INTRODUCTION

Bacillus cereus is an aerobic spore former that is commonly found

in soil, on vegetation and in many raw and processed foods. Consumption

of foods that contain large numbers of Bacillus cereus (> 106/g) may

result in foods poisoning, especially when foods are prepared and held for

several hours without adequate refrigeration before serving. Cooked meat,

vegetables, boiled or fried rice, vanilla sauce, custards, soups and raw

vegetable sprouts have been incriminated in past outbreaks (Bennett and

Harmon, 1988; Adams and Moss, 1995).

Two types of illnesses are attributed to the consumption of foods

contaminated with Bacillus cereus. The first is the diarrhoeal form, which

is characterized by abdominal pain, nausea and watery diarrhoea. It has

an incubation period of 4 – 16 hours and symptoms last for 12 – 24 hours.

The second type of illness is the emetic form, which is characterized by

acute attack of nausea and vomiting that occurs within 1 – 5 hours after a

meal. Desserts, meat dishes, and dairy products are the foods most

frequently associated with the diarrhoeal form, whereas rice and pasta are

the most common vehicles of the emetic form (Kramer and Gilbert, 1989).

Bacillus cereus is ubiquitous and can be found in a wide range of

foodstuffs, soil, raw materials, raw fruits and vegetables, raw herbs, dry

foods and processed foods. Almost all kinds of foods have been implicated

in B. cereus food borne poisoning and these foods may be contaminated

from any of many sources. Soil can contain between 103 spores of B.

19

cereus per gram (Guinebretiere and Nguyen-the, 2003; Te Giffel et al.,

1995; Christiansson et al., 1999). Spores of B. cereus persist on the

surface of processing equipment because these spores have strong

adhesion properties and might form biofilms (Anderson et al., 1995). Raw

milk is contaminated by B. cereus strains that persist in milk silo tanks

(Svensson et al., 2004). In complex foods, some ingredients have been

identified as important sources of contamination with B. cereus spores.

These include texturing agents, (Guinebretiere and Nguyen-the, 2003),

liquid eggs, herbs and spices (ICMSF, 2003). Spores of B. cereus were also

found in paper mill industries and in packaging materials (Pirttijarvi et al.,

2000) which could represent an additional route of contamination for

foods. Furthermore, because the organism is a spore former, complete

elimination of this organism from foods may present a difficulty. This may

explain why outbreaks of illness due to Bacillus cereus continue to be

recorded.

B. cereus has a long history of association with food poisoning. An

early report associating food poisoning with Bacillus species was made in

1906 when Lubenau described an outbreak in a sanatorium where 300

inmates and staff developed symptoms of profuse diarrhoea, stomach

cramps and vomiting. A spore forming Bacillus was isolated from meatballs

from the incriminated meal. Although Lubeanau (1906) named the

organism Bacillus peptonificans, the properties he described resemble

those of Bacillus cereus. Subsequently, aerobic spore formers were

implicated in a number of outbreaks in Europe between 1943 and 1945.

20

They were suspected of causing 117 of 367 cases investigated by the

Stockholm Board of Health. Bacillus cereus was not conclusively

established as a cause of food poisoning until 1950, after the taxonomy of

the genus had been clarified.

Hauge (1950) described four outbreaks in Norway involving 600

people. The food vehicle was a vanilla sauce which had been prepared a

day in advance and stored at room temperature before serving. Samples

of the sauce later tested contained from 1.1 x 106 to 2.5 x 107 cells of

Bacillus cereus. This classic report and many of the early ones from

Europe described an illness in which diarrhoea was the predominant

symptom.

Since 1975, a number of other Bacillus species have been

associated with food borne illness. In these episodes, tests have failed to

find known pathogens but food remnants and / or clinical specimens have

yielded high numbers of Bacillus spp. Far more common than outbreaks

featuring Bacillus cereus, they usually feature Bacillus species belonging to

the same morphological group, predominantly Bacillus subtills but also

Bacillus licheniformis and Bacillus pumilus. B. cereus food poisoning may

sometimes be misdiagnosed due to symptomatic similarities to

Staphylococcus aureus intoxication (Bacillus cereus vomiting type) or

Clostridium perfringens food poisonging (B. cereus diarrhoeal type). In

1977, 35 diarrhoeal food poisoning outbreaks were reported in the US,

following consumption of meat loaf, cooked rice, mashed potatoes, green

bean salad, chicken pot pie, vanilla sauce, and vegetable sprouts turkey

21

loaf. The symptoms exhibited include watery diarrhea, cramps, abdominal

pain, nausea but not vomiting, and no fever. The onset was from 12 – 18

hr and duration was greater than 24 hrs. These symptoms are similar to

those observed in Clostridium perfringens food poisoning. By 1979, 110

outbreaks of emetic food poisoning occurred in Chinese restaurants or

carry-outs and followed the consumption of boiled rice (boiled rice kept at

room temperature). The symptoms included acute nausea and vomiting,

no diarrhoea, no fever and the onset was from 1 – 5 h while duration was

for 6 – 24 hr. These are similar to symptoms of Staphylococcus aureus

food poisoning. In 1980, 9 outbreaks were reported to the Centres for

Disease Control and included such foods as beef, turkey and Mexican

foods. In 1981, 8 outbreaks were reported which primarily involved rice

and shellfish. On September 22, 1985, the Marine Bureau of Health was

notified of a gastrointestinal illness among patrons of a Japanese

restaurant. The customers exhibited symptoms of illness while still on the

restaurant premises, while the question of the specific vehicle remains

incompletely resolved; the clinical and laboratory findings substantially

support Bacillus cereus as the cause of the outbreak. On July 21, 1993,

the Lord fair-fax (Virginia) Health District received reports of acute

gastrointestinal illness that occurred among children and staff at two

jointly owned child day care centres following a catered lunch. Of the 80

persons, 67 ate the catered lunch. Chicken fried rice prepared at a local

restaurant was the only food significantly associated with illness. Illness

22

occurred in 14 (29%) of 48 persons who ate the chicken fried rice,

compared with none of 16 who did not.

In Nigeria, there is limited information on outbreaks of food

poisoning due to Bacillus cereus.

The objectives of this study were:

• To determine the incidence of Bacillus cereus strains in different food

samples.

• To characterize the Bacillus cereus strains isolated.

• To determine the effect of temperature on the vegetative growth and

spore germination.

• To determine the effect of pH on the growth

• To determine the heat resistance of both vegetative cells and spores of

Bacillus cereus.

• To determine the effect of carbon sources [glucose versus complex

carbohydrate (rice)] on the growth.

23

CHAPTER TWO

LITERATURE REVIEW

2.1 Taxonomy of Bacillus cereus

Bacillus cereus belongs to the family Bacillaceae. The family’s

distinguishing feature is the production of endospores, which are highly

refractive resting structures formed within the bacterial cells. The

members of the genus Bacillus are characterized as Gram-positive, rod-

shaped, aerobic or facultative, endospore-forming bacteria.

Bacillus cereus and the five species Bacillus thuringiensis, Bacillus

anthracis, Bacillus weihenstephanensis, Bacillus mycoides and Bacillus

pseudomycoides form a very homogenous group with 99% similarity in

their 16S r RNA sequences (Ash et al., 1991; Ash and Collins, 1992).

These six species can, however, be distinguished by phenotypic and

genotypic features (Claus and Berkeley, 1986; Lechner et al., 1998;

Nakamura, 1998).

Bacillus cereus is currently considered as a food borne disease

agent, B. thuringiensis produces a parasporal crystal toxin against insect

and is used as a bio-pesticide, B. anthracis is the agent of anthrax and B.

weihenstephanensis is able to grow at 4–5oC. However, the distinction of

the species B. thuringiensis, B. cereus and B. anthracis is not supported by

recent results in taxonomy (Guinebretiere and Sanchis, 2003; Helgason et

al., 2004). In particular, the ability to produce the parasporal crystal, the

distinctive feature between Bacillus thuringiensis and Bacillus cereus can

24

be carried by a plasmid. Thus, loss of the plasmid would turn a Bacillus

thuringiensis into a Bacillus cereus and vice versa.

2.2 History and Description of Bacillus cereus

Outbreaks of food poisoning due to Bacillus spp. have been

described since the beginning of the century (Lund, 1990). The first

confirmed outbreak of Bacillus cereus food poisoning occurred in Norway

in 1950. The food vehicle was vanilla sauce, which had been prepared a

day in advance and stored at room temperature before serving.

Consumption of the contaminated vanilla sauce resulted in a diarrhoeal

illness. The sauce was later found to contain 2.5 x 107 to 1.1 x 108 cells of

Bacillus cereus. Four related outbreaks were described involving more

than 600 people. To provide further evidence that Bacillus cereus was the

causative agent, Hauge, (1950) inoculated sterile sauce with Bacillus

cereus, incubated it for 24 hours and then consumed the sauce. The onset

of symptoms occurred 12 hours later. Subsequently, Bacillus cereus was

recognized as an important cause of food poisoning world wide. From

1980 to 1995, Bacillus cereus accounts for 7.4% of all bacterial food-borne

outbreaks of known aetiology in Australia. However, this number is

doubtlessly a minor representation of the true incidence of food poisoning

caused by this organism due to underreporting and the mildness of the

majority of cases.

25

2.2.1 Characteristics of Bacillus cereus

Bacillus cereus is a Gram-positive, aerobic, spore forming bacillus

with large vegetative cells, typically 1.0 µm by 3.0 – 5.0 µm in chains. This

organism grows over a temperature range of 8 to 55oC, with optimum

growth around 28 – 40oC, and does not have any marked tolerance for

low pH (minimum 5.0 – 6.0, depending on the acidulant) or water activity

(minimum 0.95).

Spores are central, ellipsoidal in shape and do not cause swelling in

the sporangium. As a spore former; Bacillus cereus is widely distributed in

the environment and can be isolated from soil, water and vegetation. This

ubiquity means that it is also a common component of the transient gut

flora in humans. The spores show a variable heat resistance, recorded D

values at 95oC in phosphate buffer range between around 1 min up to 36

min.

Bacillus cereus produces typical crenated colonies which retain the

turquoise-purple of the pH Indicator (bromocresol purple) on Bacillus

cereus selective medium due to their inability to ferment mannitol. They

are surrounded by a zone of egg-yolk precipitation caused by lecithinase

activity. This organism also produces lavender colonies with beta or apha

hemolysis on blood agar.

26

2.2.2 Serotyping of Bacillus cereus

Bacillus cereus may be differentiated into 18 serotypes based upon

flagellar (H) antigens. Eight of these serotypes are responsible for food

borne illness caused by this organism. Serotypes 1, 3, 4, 8 and 12 have

been associated with emetic illness while serotypes 1, 6, 8, 9, 10 and 12

have been associated with diarrhoeal (Serotypes, 1, 8 and 12 have been

associated with both syndromes (Graham and Paul, 2006). Shinagawa et

al., (1992) also discovered serotype 1 to be the most commonly isolated

serotype from emetic outbreaks of Bacillus cereus (Serotype 1: 23%;

serotype 8: 2%; untypable, 2%).

Parry and Gilbert (1980) conducted a study to determine the heat

resistance of Bacillus cereus spores at 95oC. The researchers found that

isolates of serotype 1 were the most resistant. They suggested that the

preparation of rice might select for serotype 1 and therefore explain why

this serotype is most commonly implicated in food-borne outbreaks.

2.2.3 The Genome of Bacillus cereus

Genetic maps of Bacillus cereus strains have indicated that the

chromosomal size may vary from 2.4 to more than 5.5 Mb. It is believed

that the Bacillus cereus genome may exist either as one large

chromosome with smaller extrachromosomal elements, or as a small

chromosome with large extra chromosomal elements. The genome seems

to have a constant region of 2.4 Mb and a less stable region which is more

easily mobilized into other genetic elements. The less stable part of the

27

genome is localized to one region of the chromosome and is believed to

be subject to frequent relocations between the chromosome and episomal

elements (Carlson and Kolso, 1993). They found plasmids in five Bacillus

cereus strains with an apparent size of 730 kb (ATCC 6464), 600 kb (F

2038/78), 450 kb (Strain 41), 400 kb (ATCC 33018) and 290 kb

(F4810/72). These large plasmids were separated in a pulse-time-

dependent manner, indicating they were linear (large circular plasmids will

not enter the gel).

2.3 The Ecology of Bacillus cereus

2.3.1 Bacillus cereus in the Environment

Bacillus cereus may be found in soil, dust and water that have run

off the soil. Bacillus cereus occurs in soils containing low levels of organic

matter. The presence of this organism in plant foods is due to soil

contamination rather than a specific association between the micro-

organism and plants.

2.3.2 Bacillus cereus in Food

Bacillus cereus is widespread in the environment and enters the

food chain through contaminated food and water. The organism is present

in most raw foods of plant origin with numbers especially high in some

samples of spices and cereals. The resistance to desiccation of the spores

allows the organism to survive on most dried food products. Rice is the

most commonly implicated food in cases of Bacillus cereus gastroenteritis.

28

Most samples of rice have low levels of Bacillus cereus present. Fried or

cooked rice has been implicated in approximately 95% of cases of Bacillus

cereus food poisoning with emetic symptoms indicating that there is a

relationship between substrate and emetic toxin production. Many food

poisoning cases have occurred where bulk rice is prepared in advance.

Resistant spores (particularly serotype 1) may survive cooking to

germinate, grow and produce emetic toxin during storage. This scenario

commonly occurs in Chinese restaurants and take-away establishments.

Leftover portions of boiled rice from bulk cooking are allowed to “dry off”

at room temperature and when required are either reheated or, more

usually flash fried before service. Parry and Gilbert, (1980) found Bacillus

cereus in 25 of 252 samples of boiled rice and 49 of 204 samples of fried

rice. Levels of the bacterium ranged from 102 – 105 cfu per gram in boiled

and fried rice.

Bacillus cereus is of particular concern in the dairy industry for

several reasons. Firstly the spores are very hydrophobic and attach to the

surfaces of the pipelines of the dairy processing plant, where they might

germinate, multiply and resporulate. Secondly, pasteurization heating is

insufficient to kill the spores, while competition from vegetative bacteria is

eliminated. Thirdly several strains of Bacillus cereus are psychrotrophic

and are capable of growth in milk at temperatures as low as 4 – 6oC

(Anderson et al., 1995). Granum (1997) suggested that milk drinkers may

be partially protected against Bacillus cereus food poisoning through

immunity acquired by continuous consumption of this organism. Despite

29

the frequency at which dairy products are found to be contaminated with

Bacillus cereus, no outbreak have occurred from consumption of milk and

milk products, except for a few cases involving cream and certain

desserts. Presumably the high numbers of the organism required to elicit

symptoms causes visible spoilage of milk and other dairy products and this

deters consumption of the product (Andersson et al., 1995).

Bacillus cereus has been isolated from a variety of other foods

including beans, cocoa, fish, dried potatoes, lentils, oil and meat. An

outbreak of diarrhoeal food poisoning occurred after a University field day

in South Carolina, U.S.A. People at the field day who ate barbecued pork

were five times more likely to develop symptoms than those who did not

eat the pork. The pork was un-refrigerated for 18 hours after cooking.

Subsequent tests showed that the leftover pork contained > 105 cfu/g of

enterotoxigenic Bacillus cereus.

Raw fruits and vegetables, raw herbs, dry foods, and processed

foods before storage usually contain < 100 spores/g. However, in some

herbs and spices, presence of > 100 spores/g was reported (ICMSF,

2003). Bacillus cereus is frequently present as spores either because

vegetative cells have been killed by preparation of ingredients (for

instance mild heat treatment, drying) or because conditions do not permit

spore germination and growth (dry product such as spices and herbs for

instant). The presence of spores in such raw materials is usually not a

concern. However Bacillus cereus spores would survive cooking and high

numbers of spores in spices and herbs can be a problem whenever they

30

are used in processed foods allowing growth of the organism. Storage of

the processed product or use of raw materials in complex foods, with

conditions suitable for Bacillus cereus growth, permit spore germination

and growth of Bacillus cereus to numbers that might represent a hazard

for consumers.

Additional contamination during processing may occur because

spores of Bacillus cereus have strong adhesion properties, might form

biofilms and may persist on the surface of processing equipment

(Andersson et al., 1995). For instance raw milk can be contaminated by

Bacillus cereus strains that persist in milk silo tanks (Svensson et al.,

2004). Contamination of pasteurized and powdered milk by Bacillus cereus

strains persisting in pasteurized and drying equipment was also suspected

(Eneroth et al., 2001; Svensson et al., 1999; Te Giffel et al., 1996). In

complex foods some ingredients have been identified as important source

of contamination with Bacillus cereus spores such as texturing agents,

(Guinebretiere and Nguyen-the, 2003) liquid eggs, herbs and spices

(ICMSF, 2003).

2.3.3 Reservoir of Bacillus cereus

Soil is the primary source of contamination of foods with spores of

Bacillus cereus. For instance, the same genotypes were found in the soil of

dairy farms and in the milk (Christiansson et al., 1999), and in the soil on

which vegetables were grown and in cooked chilled foods containing the

vegetables (Guinebretiere and Nguyen-the, 2003). Soil can contain

31

between 103 and 105 spores of Bacillus cereus per gram (Guinebretiere

and Nguyen-the, 2003; Te Giffel et al., 1995; Christiansson et al., 1999).

Development of some strains of Bacillus cereus and of some strains of the

very close species Bacillus thuringiensis was observed in the rhizosphere

of plants and in the gut of earthworms (Hendriksen and Hansen, 2002;

Halverson et al., 1993). Climate presumably influences the Bacillus cereus

population in soil, psychrotrophic strains being more frequent in soil from

cold regions (Vonstetten et al., 1999).

2.4 Foods Implicated in Poisoning

Because Bacillus cereus is ubiquitous and might be present in

several foods consumed by patients, it is particularly difficult to trace the

food involved in food borne poisoning.

Food categories implicated are frequently heat-treated foods (recipe

dishes, stews, purees). However, other food categories (salads, sprouts,

orange juice, mayonnaise dressing) have been implicated. Food borne

poisoning frequently occurs in restaurant or in catering services. Failure in

refrigeration and/or too long delay before consumption are presumably

factors that lead to poisoning (EFSA, 2005).

Emetic Bacillus cereus food borne intoxications are frequently linked

to rice and pasta dishes. In particular, cooking rice and keeping it

unrefrigerated several hours before frying or re-heating may lead to

several emetic intoxication outbreaks. The emetic toxin, cereulide, may be

32

produced during storage of the cooked rice but not destroyed by the

frying or re-heating step (Kramer and Gilbert, 1989).

A wide range of foods have been implicated in poisoning from other

Bacillus spp, including various recipe dishes, meat products, pastries, dairy

products, infant food formulae, sandwich, pizzas, canned tomato juice. In

all cases, suspected foods usually contained between 106 – 109 cfu/g

(Kramer and Gilbert, 1989; Salkinoja-Salonen et al., 1999).

2.5 Symptoms of Bacillus cereus food poisoning

2.5.1 The Diarrhoeal Syndrome

The diarrhoeal illness caused by Bacillus cereus is characterized by

abdominal pain, profuse watery diarrhoea and rectal tenesmus. The illness

is usually quite mild. However, cases that require hospitalization have

occurred. The incubation period for this syndrome is 8 – 16 hours after the

consumption of the food and symptoms last for 12 – 24 hours. Nausea

and vomiting are not commonly associated with the diarrhoeal illness.

Levels of Bacillus cereus found in the implicated foods responsible for this

syndrome range from 5 x 105 to 9 x 108 cfu/g (Adams and Moss, 1995).

The diarrhoeal syndrome of Bacillus cereus food poisoning can be

mediated by at least three distinct enterotoxins. The pH and proteolytic

enzyme of the gut digests these toxins if they are preformed in foods.

Therefore, the diarrhoeal illness is proposed to be due to bacteria growing

in the intestine. Spores, which have survived indigestion, are believed to

sporulate in the intestine and grow to produce viable cells, which then

33

produce toxin. The action of these toxins at the molecular level is not well

established. However, they are known to reverse the absorption of fluids,

sodium, and calcium and to cause mal-absorption of glucose and amino

acids (Mantynen and Lindstrom, 1998).

2.5.2 The Emetic Syndrome

The emetic illness is more acute than the diarrhoeal illness and

symptoms occur only 1–5 hours after the ingestion of the contaminated

food. The illness is characterized by nausea and vomiting, which lasts for 6

– 24 hours. Levels of Bacillus cereus in foods associated with the emetic

illness range from 1.0 x 103 to 5.0 x 1010 cfu/g (Adams and Moss, 1995).

The emetic toxin is regarded as the most dangerous of the toxins

produced by Bacillus cereus. This is highlighted in a case where a 17-year

old boy and his father developed acute gastroenteritis after eating

spaghetti and pesto that had been prepared four days earlier. The boy

died within two days due to fulminant liver failure and rhabdomyolysis.

The father developed hyperbilirubinemia and rhabdomyolsis but

recovered. High concentrations of the emetic toxin were found in both the

residue from the pan used to reheat the food and the boy’s liver and bile

(Mahler et al., 1997). In most cases of emetic toxin food poisoning, illness

occurs following ingestion of pre cooked food held for too long at

unsatisfactory storage temperatures.

34

2.6 Epidemiology of Bacillus cereus

2.6.1 The Incidence of Bacillus cereus Food Poisoning

The reported incidence of Bacillus cereus food poisoning varies

widely between different countries. It is apparent that a major factor in

this variation is the differing reporting procedures between countries. In

most cases the true prevalence of food poisoning is grossly underreported.

Moreover, the reporting rate of illness caused by Bacillus cereus may be

underestimated due to the relatively short duration of both disease

syndromes (< 24 hours). In addition, the frequency at which single people

are affected is usually not monitored. Consequently the full extent of

Bacillus cereus food poisoning is unknown.

In the USA, from 1988 to 1992, a total of 2,423 food-borne disease

outbreaks affecting 77,375 people were reported. In approximately 40%

of these cases the aetiology was confirmed. Bacillus cereus was

responsible for 21 outbreaks, affecting 433 people. Chinese food was the

most commonly implicated vehicle of transmission (Notermans and Batt,

1998).

In the Netherlands, from 1992 to 1994, a total of 1,543 outbreaks

and 1,087 single cases of food-borne disease were reported, involving a

total of 7,567 people. The causative agent was identified in only 8.3% of

the cases. Bacillus cereus was the most frequently isolated pathogen and

was implicated in 40 incidences. The most commonly implicated food

vehicle was Chinese foods (Notermans and Batt, 1998). The Netherlands

and Norway experienced the highest reported incidence of outbreaks from

35

this organism. However, Bacillus cereus has been the focus of much

research in these countries and this may explain the high isolation rate

from food poisoning outbreaks (Granum, 1997).

The type of illness most commonly encountered in countries also

vary. In Japan, the emetic illness is reported about 10 times more

frequently than the diarrhoeal form of the disease. In Europe and North

America the diarrhoeal illness is reported more frequently. This variation is

presumably due to the differences in diet and nutrition that exist between

these countries (Granum, 1997).

2.6.2 Transmission of Bacillus cereus

Asymptomatic carriage of Bacillus cereus has been reported in 14 to

43% of people. In addition, during the acute phase of the illness, faeces

may contain up to 109 organisms/gram and bacteria can also be isolated

from the vomitus. This suggests that human contacts such as food

handlers may be an important source of Bacillus cereus in foods. The

belief, however, is that the organism most commonly enters the food

chain through contaminated soil or water (Graham and Paul, 2006).

Raw foods of plant origin are the major source of Bacillus cereus.

The majority of outbreaks associated with this organism occur when food

has been held for too long at unsatisfactory storage temperatures. For

instance, Johnson et al., (1983) found that the numbers of Bacillus cereus

can double in 25 – 60 minutes in boiled rice held at 30oC. This rate is

reported to be higher if protein source such as chicken, beef or eggs are

36

present. Milk is frequently found contaminated with Bacillus cereus

particularly if cows are fed with silage or are housed in barns.

Contamination is often linked to the cow’s udder or equipment at the farm

and the dairy factory. Bacillus cereus and Bacillus licheniformis were the

most commonly isolated species of Bacillus found in milk at all stages of

processing. Bacillus cereus was associated with cattle feed throughout the

year. However, the bacteria were more common in raw milk during the

summer months. Although Bacillus licheniformis was more frequently

found in higher numbers, Bacillus cereus grew to dominate the bacterial

population when grown at ambient temperatures. In all but three months

Bacillus cereus was isolated from raw or heat-treated milks after

incubation (30oC for 24 h) regardless of it being undetected in the non-

incubated samples (Graham and Paul, 2006).

Foods implicated in Bacillus cereus food poisoning illnesses usually

contain at least 105 cfu/g. However, 10% of outbreaks have been

associated with food containing less than 105 cfu/g. Kramer and Gilbert

(1989) surveyed the data from a large number of Bacillus cereus disease

outbreaks. Levels of Bacillus cereus involved in the diarrhoeal syndromes

studied varied from 1.2 x 103 to 1.0 x 108 organisms/gram with a median

value of approximately 1 x 107 organisms/gram. The data from the emetic

outbreaks showed that the numbers of Bacillus cereus in implicated foods

ranged from 1.0 x 103 to 5.0 x 1010 organisms/gram with a median value

of 1 x 107 organisms. Granum (1997) suggested that food with more than

104 Bacillus cereus cells/spores may not be safe for consumption.

37

2.6.3 Nature of Bacillus cereus Cells Present in Foods at

Consumption

Diarrhoeal poisoning is most probably due to Bacillus cereus cells

ingested with the food. Most foods will be contaminated with spores of

Bacillus cereus and whenever conditions are not favourable to spore

germination and growth, Bacillus cereus would be ingested as spores. In

contrast, whenever conditions in the food permit germination and growth,

Bacillus cereus would be ingested as vegetative cells. In particular,

refrigeration at 4-7oC, would select the psychrotrophic strains of Bacillus

cereus (Notermans et al., 1997). The number of viable Bacillus cereus cells

and the nature of the organisms (vegetative or spores, respectively;

psychrotrophy or mesophily) that may be ingested is determined by the

combination of initial contamination and the way the food is handled prior

to ingestion. In the case of emetic poisonings, production of emetic toxin

in the food would depend on the presence of strains carrying the genes

responsible for emetic toxin production and of food conditions.

2.6.4 Fate of Bacillus cereus cells in the Digestive Tract

After consumption, food reaches the stomach where the ingested

Bacillus cereus cells and toxins are exposed to low pH and the action of

pepsin. In case where the food was handled properly only spores would

be present and irrespective of the pH of the stomach, spores would reach

the small intestine. If, on the other hand, the food was handled

improperly, spores, vegetative cells and toxins would be present. The

38

emetic toxin, depending on the concentration, would bind to the 5-HT3

receptors in the stomach and cause the emetic intoxication. Diarrhoeal

toxins are destroyed by enzymatic activity in the stomach. Whether only

spores or spores and vegetative cells pass the stomach to reach the small

intestine would depend on the pH of the stomach. A sufficiently low pH

will kill the vegetative cells, but higher pH values give vegetative cells the

opportunity to reach the small intestine (Clavel et al., 2004).

Thus, pH of the stomach during digestion of the contaminated food

(depending on the consumer and on the composition of the meal) and

nature of Bacillus cereus in the food (vegetative cells or spores) would

certainly influence the number of Bacillus cereus cells required to cause

diarrhoea.

2.6.5 Dose Response Relations

Bacillus cereus spores occur widely in foods and survive extended

storage (Setlow et al., 1997). The levels of Bacillus cereus reported in food

poisoning range from 102 – 108 cfu/g (Beattie and Williams, 2000). It is

generally believed that any food exceeding 104 to 105 cells or spores per

gram may not be safe for consumption (Beattie and Williams, 2000;

Granum and Lund, 1997; Notermans and Batt, 1998). This number is

often exceeded in a wide range of foods that are actually consumed.

Nonetheless, illness is relatively rare considering the high levels (> 103

cfu) of Bacillus cereus that are consumed (Notermans et al., 1997;

Notermans and Batt, 1998). This probably reflects the wide variation of

39

pathogenic potential and overall diversity among Bacillus cereus strains

(Helgason et al., 2000). Bacillus cereus toxico-infections and intoxications

with a short incubation period contains 105 – 108 Bacillus cereus per gram.

The infective dose may vary from 105 – 108 viable cells or spores in

part because of the large differences in the amount of enterotoxin

produced by different strains. Hence food containing > 104 cells/spores

per g may sometimes pose a risk (Granum and Lund, 1997).

2.7 Enterotoxins and Virulence Factors of Bacillus cereus

Enterotoxins and virulence factors of Bacillus cereus remain

uncertain, partly because it produces a large number of proteins that

potentially possess toxigenic activity and partly because these proteins are

difficult to isolate. Illness associated with this organism may be mediated

by the synergistic effect of a number of products (Graham and Lund,

2006).

2.7.1 Haemolysin BL (HBL)

Haemolysin BL (HBL) is a haemolysin consisting of three

proteinaceous subunits: B, L1 and L2, a binding factor and two “lytic”

factors, respectively. These three subunits have been purified and

characterized. The toxin shows dermonecrotic activity as well as activity

towards vascular permeability, and causes fluid accumulation in ligated

rabbit ileal loops (Granum and Lund, 1997). Beecher and Macmillan (1990)

identified a three component toxin that was only active when all three

40

components were present. The researchers found that the individual

components of HBL were not haemolytic. However, when the components

were recombined, haemolytic activity was restored. All three components

are necessary for maximal enterotoxic activity (Beecher et al., 1995;

Thompson et al., 1984; Beecher and Macmillan, 1991).

The B component of HBL has a molecular mass of 35 KDa and is

encoded by the gene hbla. The component’s role as a binding protein was

verified by an immunofluorescent staining procedure that detected the

attachment of purified B component to sheep erythrocytes (Beecher and

Macmillan, 1991). The L1 component of HBL has a molecular mass of 36

KDa and L2 has a molecular mass of 45KDa and these mediate lysis of

cells.

2.7.2 Non-haemolytic Enterotoxin (NHE)

Non-haemolytic Enterotoxin (NHE) has been characterized (Lund

and Granum, 1996) and, like HBL, consists of three proteinaceous subunits

as well; NHE A, NHE B and NHE C, two lytic factors and a binding factor

respectively. Although binary combinations of the subunits show some

biological effect, maximal activity is achieved when all three components

are present (Lund and Granum, 1997). Here too, a single gene codes for

each subunits, and the genes of all three subunits are grouped in one

operon. There is substantial similarity between the proteins of the

haemolytic and the non-haemolytic enterotoxin (Granum et al., 1999).

41

Transcription of the operon for the haemolytic enterotoxin HBL, and

the operon for the non-haemolytic enterotoxin NHE seems to be positively

regulated by plcR, a gene that also regulates Phospholipase C expression

(Agaisse et al., 1999; Granum et al., 1999). This plcR gene has also been

described in connection with enterotoxic properties of Bacillus

thuringiensis, which also belongs to the Bacillus cereus group (Agaisse et

al., 1999). It has been proven that plcR- strains are not able to produce

the enterotoxin HBL, while their plcR+ equivalent did show HBL-

production (Salamitou et al., 2000)

2.7.3 Enterotoxin T

Enterotoxin T is a single component protein enterotoxin with

activity towards vascular permeability. Also it exhibits cytotoxicity to vero

cells, causes fluid accumulation in the ligated rabbit ileal loop test, and is

lethal to mice after intravenous injection (Agata et al., 1995a). Such

properties were found also by Shinagawa after the production and

subsequent purification of a single non-haemolytic protein (Shinagawa et

al., 1992a). The Enterotoxin T has a molecular weight of 41 KDa.

Studies utilizing PCR to determine the prevalence of enterotoxin T

in strains of Bacillus cereus vary in their results. Agata et al., (1995a)

found the gene bce-T in 100% (10/10) of the isolates tested. The bacteria

included four strains obtained from diarrhoeal syndrome food born

illnesses, three strains from emetic syndrome food-borne illness and three

strains from soil and from raw and cooked rice. Granum et al., (1995)

42

detected the bceT gene in only 40% (37/71) of the isolates tested. Hsieh

et al., (1999) reported that the gene was present in 50% (14/28) of

Bacillus cereus food isolates and 57% (17/30) outbreak-associated strains.

Mantynen and Lindstrom (1998) only found the gene in the model strain

(B – 4ac) of 58 strains of Bacillus cereus. These discrepancies may be due

to the use of different primers or to varying populations in different

regions.

2.7.4 Enterotoxin FM

Enterotoxin F M is a three component enterotoxin complex

composed of three protein with molecular masses of approximately 39, 45

and 105 kDa. This three component enterotoxin complex was first

discovered by Granum et al., (1996). Similar to HBL, all components are

required for maximal cytotoxic activity. The complex is highly cytotoxic to

vero cells but is non-haemolytic. The 45 kDa component is the same

protein as the main antigen detected in the Bacillus Diarrhoeal Visual

Immunoassay (Tecra). The sequence of the 39 kDa protein overlaps with

five of the six amino acids in the N-terminal of the L1 protein of HBL

(Beecher and Wong, 1994). Furthermore, the three toxic component were

all recognized by a polyclonal antiserum for detection of enterotoxin from

Bacillus cereus (Lund and Granum, 1996).

43

2.7.5 Haemolysin 1

Haemolysin 1 (or cereolysin) is a thiol activated protein that cross-

reacts with streptolysin-O and has a molecular weight of about 5 kDa. This

protein is responsible for the main haemolysis observed in Bacillus cereus

and is lethal when injected into mice. Haemolysin 1 is heat labile but is not

susceptible to proteolysis. It is inhibited by cholesterol and serum

(Granum, 1994).

2.7.6 Haemolysin 2

Haemolysin 2 is heat labile and susceptible to proteolytic enzymes.

The molecular weight of the protein is approximately 30 kDa. It is not

susceptible to cholesterol. The in vivo toxicity of this protein has not yet

been established (Granum, 1994).

2.7.7 Phospholipases C

Bacillus cereus produces three different phospholipases C. They

have all been cloned and are well characterized. Phospholipases C specific

for various phospholipids have been isolated from several bacteria, such

as Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus cereus

(Yamada et al., 1988).

44

2.7.7.1 Phosphatidyl inositol Hydrase

Phosphatidylinositol Hydrase (PIH) is a 34 kDa enzyme which

hydrolyses phosphatidylinositol (P1) and P1 – glycan – containing

membrane anchors, which are important structural components of one

class of membrane proteins. Phosphatidylinositol Hydrase is non-

haemolytic and does not require any ions for biological activity. This

enzyme is not generally linked to the two other phospholipases C

(Granum, 1994).

2.7.7.2 Phosphatidylcholine Hydrolase

Hydrolysis of lecithinin, (egg yolk reaction) is a major criterion for

the identification of Bacillus cereus spp. Most strains of the Bacillus cereus

group, such as Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides,

possess lecithinase activity. The reaction is catalyzed by the phospholipase

C, phosphatidylcholine hydrolase (PCH) which hydrolyses lecithin (Schraft

and Griffiths, 1995). PCH also hydrolyses phosphatidylethanolamine and

phosphatidylserine. PCH requires the presence of either zinc or calcium for

activity (Granum, 1994).

2.7.7.3 Sphingomyelinase

Sphingomyelinase hydrolyses sphingomyelin. The enzyme requires

magnesium for activity and is inhibited by zinc and calcium. Hsieh et al.,

(1999) designed primers based on the reported sequence of four different

strains of Bacillus cereus.

45

Positive PCR results were obtained for all Bacillus cereus group

strains tested. This suggests that sphingomyelinase alone cannot be

responsible for the symptoms elicited by this organism as not all strains

are capable of causing disease.

2.7.8 Cytotoxin-K

Cytotoxin-K is a single component protein enterotoxin showing

necrotic and haemolytic activity and is highly toxic to epithelial cells as

shown with human CaCo-2 cells (Hardy et al., 2001). Cytotoxin-K is the

most recently described enterotoxin from Bacillus cereus. It was detected

after a food poisoning outbreak in an elderly home in France. In total, 44

people were ill, 6 of these patients had bloody diarrhoea, and three of the

six died (Lund et al., 2000).

2.7.9 Vacuolation Factor

The vacuolation factor is a peptide produced by Bacillus cereus

which was named Emetic toxin of Bacillus cereus and was shown to cause

vacuolation of mitochondria when exposed to HEP-2 (human carcinoma of

the larynx) cells. The HEP-2 cell vacuolation factor was extracted and

purified by Agata et al., (1994). The emetic toxin is an extremely stable

compound. It can survive trypsin and pepsin treatments, pH 2 – 11 and

heating at 121oC for 90 min (Granum and Lund, 1997). The toxin

responsible for vomiting type was a 5 kDa dodecadepeptide and was

named cereulide. Agata et al., (1994) also determined the structure of the

46

toxin: (D – O – Leu – D – Ala – L – O – Val – I – Val). The emetic toxin

was shown to have a very similar structure to the potassium ionophore

valinomycin. Like the emetic toxin valinomycin also inflicts mitochondrial

damage when added to HEP-2 cells and induces emesis in Suncus murinus

(Agata et al., 1995b). Mikkola et al., (1999) found that the action of the

emetic toxin was complementary to that of valinomycin over a wide range

of tests. They concluded that the toxic effects of the emetic toxin was due

to its being a potassium ionophore. The toxin may be the translation

product of a Bacillus cereus gene or the result of enzymatic conversion of

a certain substrate in the growth medium. The production of the emetic

toxin has been suggested to be related to sporulation (Shinagawa et al.,

1992b).

2.7.10 Spore

The spores produced by Bacillus cereus are highly resistant to heat,

radiation and chemicals due to their metabolic dormancy and tough

physical nature. The vegetative cells of Bacillus cereus are not particularly

resistant to these environmental stresses.

Generally, adherence of enteropathogens to the intestinal

epithelium is an essential step required for colonization (Contrepois,

1993). It has been suggested that appendages on the spore surface

contribute to the adhesion of Bacillus cereus spores to epithelial cells

(Andersson et al., 1998). Also hydrophobicity is believed to play a role in

the adherence not only to solid surfaces (Husmark and Ronner, 1990), but

47

also to epithelial cells (Andersson et al., 1998). However, the

hydrophobicity of vegetative cells is much less than that of spores

(Wiencek et al., 1990). The Bacillus cereus spores are known to be more

hydrophobic than spores from other Bacillus species (Husmark, 1993;

Ronner et al., 1990). It is believed that the hydrophobicity is one of the

main reasons for the adhesion problems caused by Bacillus cereus spores

in the daily industry (Andersson et al., 1995). Hence, spore adhesion could

be an important virulence factor for some Bacillus cereus strains

(Andersson et al., 1998).

2.8 Physical and Chemical Factors Affecting Bacillus cereus

Growth

2.8.1 Temperature

Mesophilic strains of Bacillus cereus can grow between 10oC and

42oC, some strains being able to grow at 50 – 55oC (Kramer and Gilbert

1989, Nguyen-the and Carlin, 2003), with an optimal growth temperature

between 30 and 37oC. Psychrotrophic strains would grow below 10oC, at

temperatures as low as 4oC (Kramer and Gilbert 1989, Nguyen-the and

Carlin, 2003; Anderson et al., 1995, Van Netten et al., 1990; Te Giffel et

al., 1996, 1995, 1997; Dufrenne et al., 1994; Francis et al., 1998;

Andersen-Borge et al., 2001).

Refrigeration reduces growth of Bacillus cereus by increasing the

generation time. For instance, doubling times of a cocktail of 5 strains of

Bacillus cereus, including psychrotrophic and mesophilic strains in

48

laboratory media were 1.6 h at 19.5oC, 2.9 h at 14.2oC, 4 h at 9.6oC and

6.7 h at 6.5oC (Choma et al., 2000). Refrigeration also considerably

increases lag time. Valero et al., (2000) reported lag time of 148.77 h and

1.96 h at 5 and 30oC respectively.

Growth of some strains of Bacillus cereus was observed at 55oC,

but growth kinetics at this temperature, have not been elucidated. At 50oC

and below, important variations were observed among strains. Generation

times for Bacillus cereus in laboratory media were 0.3 to 3.6 h at 50oC (for

strains able to grow) 0.3 to 0.7 h at 40oC, 0.4 to 1.3 h at 35oC (Rajkowski

and Mikolajcik, 1987; Johnson et al., 1983). These generation times are

similar or slightly lower than those reported for Clostridium perfringens.

0.5 h at 51oC and 0.2 h at 40oC in cooked meat (Willardsen et al., 1978),

0.3 to 0.4 h at 350C in laboratory media (Beuchat et al., 1980).

2.8.2 pH

Bacillus cereus is not a particularly acid tolerant bacterium. The pH

limit for growth in carrot substrate acidified with citric acid was between

4.5 and 4.75 (Valero et al., 2000). In milk acidified with HCl, slight growth

was observed at pH 4.1 at 37oC (Clavel et al., 2004). At 25oC, growth rate

of strains of Bacillus cereus were approximately constant between pH 7

and pH 5.5 but dropped dramatically below this level (Lundsay et al.,

2000).

49

2.8.3 Water Activity

Bacillus spp. are more sensitive to low water activity than yeast and

moulds. In the particular case of Bacillus cereus the water activity must be

higher than 0.92 for growth (Kramer and Gilbert, 1989). For other Bacillus

spp, at 20oC and pH 6.4 in Tryptose caseine – Soja broth, mixture of 9

strains isolated from bakery products had lag times of 10 days and 15 –

18 days respectively for aw of 0.92 and 0.91 (Quintavalla and Parolari,

1993).

2.8.4 Sodium Chloride

Sodium chloride has effect on the growth of Bacillus cereus

depending on the concentration. Raevuori and Genigeorgis (1975) and

Claus and Berkeley (1986) reported that 11 – 89% of strains of Bacillus

cereus grow in 7% of NaCl. Mossel et al., (1967) suggested that 5% NaCl

included in isolation agars was a useful selective agent for Bacillus cereus

but that 10% was too inhibitory. Peters et al., (1991) identified the

temperature range over which Bacillus cereus could grow at different NaCl

concentrations. These ranged from growth at all temperatures examined

(14 – 41oC) at NaCl concentration of 0.5% (w/v) with pH 4.7 the lowest

permitting growth to growth at 21 – 39oC at 5% (w/v) NaCl, with minimum

pH of 5.5. At 7% (w/v) NaCl no growth was recorded at any temperature.

50

2.8.5 Effect of Heating.

Heat is the most common method used to kill bacterial spores in

foods. However, spores of Bacillus cereus have a broad range of heat

resistance. This has been a problem for producers in developing consistent

cooking or pasteurization processes. For instance, among a set of strains

from various origins, time to reduce 10 fold, the number of cultivable

spores at 90oC, pH 7 (D-value) varied from a few minutes to > 100 min

(Dufrenne et al., 1994). Strains from food borne outbreaks had D values

at 100oC ranging from 6 to 27 minutes (Rajkowski and Mikolajcik, 1987).

Strains isolated from spoiled canned vegetables had D values at 130oC

around 0.3 min (Bradshaw et al., 1975). Spores isolated from vegetables

showed a D105oC value or 0.63 min in reference substrate (pH 7)

(Fernandez et al., 1999).

Heat resistance of Bacillus cereus spores can be modified by the pH

(EFSA, 2005). Survival of Bacillus cereus spores at 95oC decreased by

three fold when the pH of the heating substrate was decreased from 6.2

to 4.7 (Fernandez et al., 2002). Mazas et al., (1998) found that

acidification from pH 7 to 4 produced a five fold decrease in D103oC values.

In low acid foods, the process used to eliminate Clostridium botulinum,

121oC for 3 min, would also eliminate spores of Bacillus cereus. Foods

subjected to less severe heat treatments will occasionally carry spores of

Bacillus cereus.

51

2.8.6 Effect of Food Additives.

Food additives such as nisin induce the inactivation of Bacillus

cereus vegetative cells while others such as Carvacrol have a very little

effect (Ray 1992; Pol and Smith 1999; Periago and Moezelaar 2001).

52

CHAPTER THREE

MATERIALS AND METHODS

3.1 Media and media preparation

A total of six different media were used throughout the study. They

are: Bacillus cereus selective agar, Bacillus cereus selective broth, rice

medium, nutrient agar, nutrient broth and SIM medium. The Bacillus

cereus selective agar is composed of: agar, 2.0 g; D-mannitol, 1.0 g;

(NH4)2PO4, 0.1 g; KCl, 0.02 g; MgSO4. 7H2O, 0.02 g; Yeast extract, 0.02 g;

Bromocresol purple, 0.004 g and 10 mI of 20% egg yolk emulsion

(Ranald, 1996). All these were dissolved in 100 ml of distilled water.

To prepare the egg yolk emulsion, 12 eggs were soaked in 1:100

dilution of saturated mercuric chloride solution for 1 min. Eleven eggs

were cracked and yolks were separated from whites. The egg yolks were

then mixed with the 12th egg (white and yolk) and mixed. Twenty

milliliters of egg yolk mixture was added to 80 ml of 0.9% NaCl solution.

This was then mixed thoroughly. The emulsion was warmed to 45 – 50oC.

The medium components except egg yolk emulsion were added to

100 ml of distilled water. The mixture was autoclaved at 121oC for 15 min

and then cooled to 45 – 50oC. Ten milliliters of sterile egg yolk emulsion

(20%) was added aseptically and mixed thoroughly. The medium was

then poured into sterile Petri dishes and incubated for 24 h at 37oC to

ensure sterility.

53

The Bacillus cereus selective broth is composed of the above

components except the agar. Other media were prepared according to the

manufacturers and users specification.

3.2 Sample Collection

Different food samples such as fried fish, cooked rice, cooked fish,

raw meat and raw fish were collected for this study from restaurants in

University of Nigeria, Nsukka and vendors in Nsukka market. A total of 325

food samples including 115 rice, 103 meat and 107 fish were collected.

These samples were wrapped in sterile white polythene bags as soon as

they were bought and brought to the laboratory. They were used

immediately.

3.3 Isolation of Organism

After the samples were collected and brought to the laboratory, one

gram of each sample was ground in a sterile motar and resuspended in 5

ml of sterile distilled water. Bacillus cereus strains were isolated on Bacillus

cereus selective medium by inoculating 0.1 ml of the food suspension onto

Bacillus cereus agar and incubating for 24 h at 37oC. Resulting colonies

were characterized in terms of Gram stain, spore production, catalase

reaction, haemolysis, gelatin liquefaction, starch hydrolysis and plasmid

profile. Presumed B. cereus colonies were stored in nutrient agar slants at

4oC for further use.

54

3.4 Identification Tests

3.4.1 Motility

SIM (sulphide, indole and motility) medium was used for the

motility test. This demonstrates the ability of the isolates to move away

from the point of inoculation. To 50 ml of distilled water, 1.8 g of SIM

medium was added and heated for complete dissolution. The medium was

dispersed into clean test tubes in 10 ml aliquots and sterilized by

autoclaving at 121oC for 15 min. The tubes were allowed to cool after

which media were stab-inoculated with the isolates and incubated at 37oC

for 48 h. Positive motility test is shown by diffuse growth or turbidity away

from the line of inoculation while growth only along the line of stabbing

indicates a negative result.

3.4.2 Catalase

This test demonstrates the ability of bacteria to produce the

enzyme catalase that breaks down hydrogen peroxide (usually toxic to

microorganism) to water and oxygen (Cheesbrough, 2000).

2H2O2 2H2O + O2

For each isolate, a drop of 3% (w/v) hydrogen peroxide was placed

on a clear grease-free slide. A little quantity of the isolate was then

emulsified on the slide. Positive test or reaction was indicated by

effervescence.

55

3.4.3 Hemolysis

To 100 ml of distilled water, 2.8 g of nutrient agar was added and

heated for complete dissolution. The mixture was sterilized by autoclaving

at 121oC for 15 min. It was allowed to cool to 45oC and 5 ml of human

blood was added aseptically and swirled immediately. It was then poured

into sterile plates and incubated for sterility after which they were

inoculated with the isolates and incubated for 24 h at 37oC. Following

incubation, the plates were examined for haemolysis.

3.4.4 Gelatin Liquefaction

The test is used for the detection of proteolysis (elaboration of

gelatinilytic enzyme) as shown by the liquefaction of gelatin.

The gelatin liquefaction medium was prepared according to Cowan

and Steel, (1966), by adding 1.2 g of gelatin and 2.8 g of nutrient agar to

100 ml of distilled water and heating for complete dissolution. It was then

sterilized at 121oC for 15 min. The solution was poured into plates and

incubated for 24 h at 37oC for sterility. The isolates were streaked onto the

plates and incubated for 24 h at 37oC. After the incubation, the surface

was flooded with acid mercuric chloride solution (Mercuric chloride, 12 g,

distilled water, 50 ml concentrated hydrochloric acid, 16 ml). A zone of

clearing around the growth was indicative of gelatin liquefaction (positive

result), while no zone of clearing indicated negative result.

56

3.4.5 Starch Hydrolysis

This test demonstrates the production of amylase by the isolates.

One gram of starch was added to 100 ml of nutrient agar and sterilized by

autoclaving at 121oC for 15 min. The medium was allowed to cool to about

60oC before dispensing into sterile Petri dishes. The Petri dishes were

incubated for 24 h at 37oC for sterility. The Petri dishes were then

inoculated with the isolates and incubated for 24 h at 37oC. Starch

hydrolysis was determined by flooding the culture plates with Gram’s

iodine solution. A zone of clearing shown by the starch hydrolyzing

bacteria after flooding detects the starch hydrolysis. Blue-black colour

around bacterial growth indicates a negative result (Ogbulie et al., 1998).

3.4.6 Spore Staining

This test is to demonstrate the production of spores by the isolates.

A smear of a 24 h culture of each isolate was prepared on a clean, grease

free slides, air-dried and heat fixed. The smear was covered with

malachite green solution and heated over a beaker of boiling water for 5

seconds or until it steamed. Then the slide was rinsed with water. The

smear was counter stained with safranin for about 30 seconds, washed off

with water, drained, air-dried, and examined under the light microscope

using the oil immersion objective lens. The spores stained green and the

vegetative cells stained red (Ogbulie et al., 1998).

57

3.5 Standardization of inoculum

A small quantity of B. cereus was inoculated into 10 ml of nutrient

broth and incubated at 37oC for 24 h. Following incubation, hundred fold

serial dilutions were made of the 24 h culture in sterile tubes up to 10-12

dilution. Thereafter, 0.1 ml of each dilution was spread-plated onto

duplicate agar plates and incubated at 37oC for 24 h for viable counts. The

absorbance of each tube was also taken in the spectrophotometer at 580

nm. The dilution yielding 1.0 to 1.5 x 1010 cfu/ml was taken as the

standard and its optical density (OD) of 0.2 was noted. Subsequently,

standard inocula were prepared by adding or removing B. cereus cells

from tubes of nutrient broth until the OD of 0.2 was attained.

3.6 Determination of the Effect of Temperature on the

Vegetative Growth and Spore Germination

Three isolates (BC7, BC9, NBC) and a typed strain (NRRL-B14724)

were used in this study. These isolates were tested for the effect of

temperature on the vegetative growth and spore germination according to

Finlay et al., (2000). Three flasks containing 100 ml of nutrient broth,

each, were inoculated with 0.1 ml of standard inoculum containing 1.0 x

109 cells and incubated at 37oC, 28oC and 4oC respectively. Samples of 1

ml were taken at intervals of 24 h from each incubation temperature.

From this 1 ml, 100 µl was taken to ascertain the vegetative growth by

plating out on duplicate agar plates. The remaining 900 µl of each sample

was placed in a water bath at 80oC for 10 min to kill vegetative cells.

58

Thereafter, 100 µl of this heat-treated sample was taken to provide spore

germination by plating out on duplicate nutrient agar plates.

3.7 Determination of the Effect of pH on the Growth of Bacillus

cereus

Six flasks of Nutrient broth (100 ml per flask) were adjusted to pH

4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 respectively. They were later sterilized at

121oC for 15 min. Then 0.1 ml of standard inoculum prepared as

previously described was added to each of the flasks. They were then

incubated at 37oC and monitored for seven days. On each day, 0.1 ml

from each flask was serially diluted and plated out in duplicate nutrient

agar plates to determine the viable cell numbers.

3.8 Evaluation of Heat Resistance of the Isolates

The four isolates were tested for heat resistance within the

temperature range of 40oC – 100oC. The test isolates were grown in

nutrient broth for 24 h at 37oC after which they were adjusted to 0.2

optical density (580nm) containing 1.0 x 1010 cells/ml. For each test, 1 ml

of the diluted culture was added to 9 ml sterile nutrient broth in a bijou

bottle maintained in a water bath at the given test temperature. Each test

was performed in duplicates. The bijou bottles were held in the water bath

with the surface of the broth cultures 2 to 3 mm below the surface of the

water in the bath and swirled intermittently by hand so as to promote

59

equal temperature distribution in the broth. Care was taken to prevent

contamination of the tops of the bijou bottles.

At set times (20-min intervals for 40 to 80oC and 5-min intervals for

90 to 100oC), the bottles were taken out and loopfuls were withdrawn and

inoculated onto duplicate plates of nutrient agar and incubated at 37oC

overnight to determine viable counts. Decimal reduction times (D-values)

were calculated using this formula:

D-value = t1 – t0

Log N0 – log N1

where t1 = time after heating

t0 = Time at start

N0 = Start population

N1 = Population after heating

3.9 Determination of Effect of Carbon source on the growth of

Bacillus cereus.

The isolates were evaluated for the effect of carbon source by

growing the cells on glucose at different concentrations or rice as sole

carbon source.

To evaluate the effect of glucose, according to Spira et al., (1979),

a growth medium comprising (g/100 ml) of (NH4)2 PO4, 0.1 g; Na2HPO4,

2.5 g; 1 ml of mixture of 5% MgSO4, 0.5% MnSO4, 0.5% iron citrate and

0.02 g yeast extract, was supplemented with 1%, 5% or 10% glucose

60

(w/v) separately in 250 ml Erlenmeyer flasks. The pH was adjusted to 7 in

all the flasks before sterilizing in the autoclave at 121oC for 15 min.

The flasks were each inoculated with 0.1 ml of the standard

inoculum. The cultures were incubated at 37oC and monitored for seven

days. Every 24 hours, 0.1 ml from each flask was serially diluted and

plated out in duplicate nutrient agar plates for viable counts.

On the effect of complex carbohydrate, rice culture was used. Five

grams of boiled rice (wet weight) was cooked and dried and 4.3 g (dry

weight) was added to 100 ml of distilled water in a flask. This medium was

adjusted to pH 7 and sterilized. The medium was inoculated with 0.1 ml of

standard inoculum which contains 1.0 x 109 cells and incubated at 37oC for

seven days. On every 24 h, 0.1 ml from each flask was serially diluted and

plated out in duplicate nutrient agar plates for viable counts.

3.10 Plasmid DNA Profiling

Eleven Bacillus cereus strains, a Bacillus thuringiensis (Bt) strain

and a non- Bacillus cereus strain were examined to know their plasmid

DNA profiles, according to the method of Zhou et al., (1990). These

strains were inoculated into different sterile test tubes containing nutrient

broth and incubated for 72 h at 37oC. After the incubation, the cells were

spinned at 10,000 rpm for 5 min in a microcentrifuge to pellet cells. The

supernatants were gently decanted leaving the cell pellets. Lysis buffer

(300 µl) comprising 10 mM Tris-HCl, 1 mM EDTA, 0.1 N NaOH and 0.5%

sodium dodecyl sulphate, at pH 8.0, was added to the cell pellet and

61

vortexed at high speed for 2 – 5 seconds until the mixture became sticky.

Then 150 µl of 3.0 M sodium acetate (pH 5.2) was added. The mixture

was again centrifuged for 5 min in microcentrifuge to pellet cell debris and

chromosomal DNA

The supernatant was then transferred to a fresh tube and mixed

well with 1.0 ml of 100% ethanol, which was pre-cooled to -20oC. This

was then spinned for 5 min to pellet plasmid DNA. At this juncture, pellet

was observed. The supernatant was discarded and pellet was rinsed twice

with 1 ml of 70% ethanol and the pellet was dried by evaporation. The

pellet was re-suspended in 20 – 40 µl of TE buffer (Tris-HCl and EDTA) for

further use.

To each 6 µl DNA sample, 3 µl of loading dye was added and mixed

by vortexing the mixture. A 1.3% agarose gel was prepared by dissolving

1.3 g of agarose in 100 ml of Tris-Borate buffer (pH 7.6) and heating to

dissolution. After cooling, the solution was then poured into the gel

column and left to solidify. The samples were loaded into the gel wells

(along with Hind III digest of lambda DNA (Sigma chemicals) used as

molecular weight standard) and subjected to electrophoresis at 60 V for 1

½ h. Following electrophoresis, bands were viewed under UV light in a

transilluminator. The graph of the standard was plotted and from this

graph, the molecular weights of the plasmids were extrapolated.

62

3.11 Data Analysis.

Data from all the experiments were analyzed by one-way ANOVA

and Least Significant Difference (LSD) at the 95% confidence limit.

63

CHAPTER FOUR

RESULTS

4.1 Isolation and Characterization of Bacillus cereus

4.1.1 Incidence of Bacillus cereus in different food samples

A total of 325 food samples were examined comprising 115

(35.39%) rice, 103 (31.69%) meat and 107 (32.93%) fish samples. All

samples (100%) yielded Bacillus cereus as determined by growth on

Bacillus cereus selective medium. A total of 350 strains of B. cereus were

isolated made up of 123 strains (35.14%) from rice, 111 (31.71%) from

meat and 116 (33.14%) from fish. These results are shown in Table 1.The

table also shows the bacterial load of the different food samples with

highest load of up to106 cfu/g obtained from fish samples.

4.1.2 Morphological and Metabolic Characterization of

Isolates and Reference Strains

A summary of morphological and metabolic characteristics of the isolates

are shown in Table 2. A total of 259 (74%) of the strains were haemolytic,

58 (16.57%) hydrolyzed starch, 252 (72.00%) isolates liquefied gelatine

and 97 (27.71%) did not ferment mannitol. All isolates were motile,

catalase positive and contained endospores. The morphological and

metabolic characteristics of the reference strains are shown in Table

3.They were all Gram positive short rods, spore formers, motile, catalase

positive and liquefied gelatine.

64

Table 1. Incidence of Bacillus cereus in different food samples

Food

samples

Number of

samples

examined

(percentage)

Number positive

for Bacillus

cereus*

(percentage)

Number of

Bacillus cereus

isolates

(percentage)

Bacterial

load

(Range)

(cfu/g)

Rice 115 (35.39%) 115 (100%) 123 (35.14%) 103 – 105

Meat 103 (31.69%) 103 (100%) 111 (31.71%) 104 – 105

Fish 107 (32.93%) 107 (100%) 116 (33.14%) 102 – 106

Total 325 325 350

* Samples yielding growth on Bacillus cereus selective medium were taken

as positive

65

Table 2: Summary of Morphological and Metabolic Characteristics

of the Isolates

Characterization

Number of isolates

positive

(percentage)

Number of isolates

negative

(percentage)

Hemolysis 259 (74%) 91 (26%)

Starch hydrolysis 58 (16.57%) 292 (83.43%)

Gelatin liquefaction 252 (72%) 98 (28%)

Mannitol fermentation 253 (72.29%) 97 (27.71%)

Gram reaction 350 (100%) 0 (0%)

Spore stain 350 (100%) 0 (0%)

Motility 350 (100%) 0 (0%)

Catalase 350 (100%) 0 (0%)

66

Table 3: Morphological and Metabolic Characterization of Reference

Strains

Isolates

Characterization on

Bacillus cereus

selectve medium

Hemolysis

on blood

agar

Starch

hydrolysis

Gram

reaction

Spore

strain

Gelatine

liquefaction

Motility

Catalase

NRRL-B14724 No distinct

colonies, there is

zone of clearing

around the

growth, the

growth is flat

with creanated

edges. It does

not ferment

mannitol i.e. the

purple colour still

retains. The

growth is milky in

colour.

β-

Hemolytic

greenish

colour

without

zone of

clearing.

The

colonies

are flat

with

creanated

or rough

edges

Negative.

There is

large grey

white flat

colonies

with no

zone of

clearing.

Gram

positive

short rods

in chains.

+ Positive

with wide

zone of

clearing.

+ +

NRRL-B14725

No distinct

colonies, there is

zone of clearing

around the

growth, the

growth is flat

with creanated

edges. It does

not ferment

mannitol i.e. the

purple colour still

retains. The

growth is milky in

Non-

hemolytic

with flat

white

growth

Positive

before

addition of

iodine,

there is

zone of

clearing

around the

growth.

After

addition of

iodine,

there is

Gram

positive

short rode

in chains.

+

Positive

with small

zone of

clearing

+

+

67

colour. zone of

clearing.

Bt (GSC 3) No growth β-

Hemolytic

Greenish

colour

without

zone of

clearing.

The

colonies

are flat

with

creanated

edges

Negative

There is

large grey

white flat

colonies

with blue-

black

colour

around the

growth

+ + Negative + +

68

4.1.3 Selection of Classical Bacillus cereus Isolates

A total of 10 isolates were selected based on their morphological

and metabolic similarity to the reference strains. These strains were

designated BC 1 to BC10. Another isolate which differed markedly from

the reference strains (ferments mannitol and has smooth edges) was

designated as Non- Bacillus cereus (NBC) and was used as negative

control for the rest of the study. The morphological and metabolic

characteristics of these isolates are shown in Table 4.

4.1.4 Plasmid profile of Bacillus cereus strains isolated from foods

The ten selected isolates, the reference strains and the negative control

were characterized by plasmid profiling. The results showed that the

strains contained multiple plasmid bands. Most had three observable

plasmid bands with molecular weights of approximately 5 kb, 21 kb and a

large plasmid >35 kb, respectively. Two strains had profiles that were

different; BC10 (lane 13) and Bacillus thuringiensis (Bt, lane 15). The

BC10 strain contained the large plasmid in addition to two others of

molecular weights 33 kb and 26 kb. The Bt contained only the 21 kb

plasmid. These results are shown in Figure 1.

Table 4: Morphological and Metabolic Characteristics of classical B. cereus isolates

Isolates

(Source)

Characterization on Bacillus

cereus selective medium

Hemolysis on blood

agar

Starch hydrolysis

Gram reaction

Spore

strain

Gelatine

liquefaction

Motility

Catalase

BC1

(Meat)

Non-mannitol

fermenting. The

purple colour still

retains. The growth

is milky in colour with

zone of clearing. The

growth is flat with

creanated edges.

β-

Hemolytic

Greenish

colour without

zone of

clearing.

+

There is large

grey white flat

colonies with

zone of

clearing.

Gram positive

short rods in

chains.

+ + + +

BC2

(Rice)

The purple colour still

retains. The growth



growth is flat with

creanated edges.

β-

Hemolytic

Greenish

colour without

zone of

clearing.

+

There is large

grey white flat

colonies with

zone of

clearing.

Gram positive

short rods in

chains.

+ + + +

52

BC3

(Fish)


retains. The growth



growth is flat with

creanated edges.

β-

Hemolytic

Greenish

colour without

zone of

clearing.

Negative

There is large

grey white flat

colonies with

blue-black

colour around

the growth.

Gram positive

short rods in

chains.

+ + + +

BC4

(Rice)


retains. The growth



growth is flat with

creanated edges.

β-

Hemolytic

Greenish

colour without

zone of

clearing.

Negative

There is large

grey white flat

colonies with

blue-black

colour around

the growth.

Gram positive

short rods in

chains.

+ + + +

Table 4 Continued

53

BC5

(Fish)


retains. The growth



growth is flat with

creanated edges.

β-

Hemolytic

Greenish

colour without

zone of

clearing.

Negative

There is large

grey white flat

colonies with

blue-black

colour around

the growth.

Gram positive

short rods in

chains.

+ + + +

BC6

(Meat)


retains. The growth is

milky in colour with


growth is flat with

creanated edges.

β-

Hemolytic

Greenish colour

without zone of

clearing.

+

There is large

grey white flat

colonies with

zone of

clearing.

Gram positive

short rods in

chains.

+ + + +

BC7

(Meat)


retains. The growth is

milky in colour with


growth is flat with

creanated edges.

β-

Hemolytic

Greenish colour

without zone of

clearing.

Negative There

is large grey

white flat

colonies with

blue-black

colour around

the growth.

Gram positive

short rods in

chains.

+ + + +

Table 4 Continued

54

BC8

(Rice)


retains. The growth



growth is flat with

creanated edges.

β-

Hemolytic

Greenish

colour without

zone of

clearing.

+

There is large

grey white flat

colonies with

zone of

clearing.

Gram positive

short rods in

chains.

+ + + +

BC9

(Rice)


retains. The growth



growth is flat with

creanated edges.

β-

Hemolytic

Greenish

colour without

zone of

clearing.

Negative

There is large

grey white flat

colonies with

blue-black

colour around

the growth

Gram positive

short rods in

chains.

+ + + +

BC10

(Rice)

The purple colour

changes to yellow.

The growth is milky

in colour without


colony is raised with

Non-

hemolytic.

Negative

There is large

grey white flat

colonies with

blue-black

colour around

Gram positive

short rods in

chains.

+ - + +

Table 4 Continued

55

smooth edges. The

growth is flat with

creanated edges.

the growth

NBC/Non

Bacillus

cereus

(fish)

The colonies change

the purple colour of

the indicator to

yellow. That is, the

organism ferments

mannitol. The colony

is raised with smooth

edges.

β-

Hemolytic

Greenish

colour without

zone of

clearing.

+

There is large

grey white flat

colonies with

zone of

clearing.

Gram positive

short rods

+ + + +

Table 4 Continued

56

Figure 1. Plasmid profile of Bacillus cereus strains isolated from

foods. The molecular weight standard (Hind III digest of λ DNA)

was loaded in Lane 1; Lanes 3 – 13, plasmid DNA from food

isolates; Lane 14, plasmid DNA from refernce strain NRRL-

B14724; Lane 15, plasmid DNA from Bacillus thuringiensis (Bt)

and Lane 16, plasmid DNA from the negative control NBC. Lanes

2 and 11 were left empty. Note the multiple plasmid bands in most

strains. Also, note the similar profile in most strains, except BC10

(Lane 13) and Bt (Lane 15).

1 2 3 4 5 6 7 8 9 10 11

1 12 13 14 15 16

23,130 bp

9,146 bp

6,556 bp

4,321 bp

2,322 bp

2,027 bp

>35 kb

33 kb

26 kb

21 kb

5 kb

57

4.2 Effect of Temperature on the Vegetative Growth of Bacillus

cereus Strains

The Bacillus cereus strains were grown at 37oC, 28oC and 4oC

respectively to ascertain their vegetative growth. At 37oC, all the strains grew

well with NRRL-B14724 strain having the highest growth (1.1x1010 – 7.0x1015

cfu/ml) (Figure 2).

At 28oC, all strains grew again with NRRL-B14724 having the highest

growth (9.0x109 – 5.0x1012 cfu/ml). There was, however, an increased lag at

this temperature (Figure 3).

At 4oC, there was no growth. It was observed, however, that after the

7th day, when the plates were brought out from 4oC and kept at 28oC

overnight, growth occurred.

4.3 Effect of Temperature on the Spore Germination of Bacillus

cereus strains

The germination of spores followed the pattern of vegetative growth

(Figure 4 and 5). However, a rapid loss of viability was recorded after 6 days

for the type strain NRRI-B14724, at both 37oC, and 28oC.

At 4oC, none of the spores germinated. However, that after the 7th

day, when the plates were brought out from 4oC and kept at 28oC overnight,

the spores germinated.

58

Fig.2: Vegetative growth of Bacillus cereus strains at 370C

59

Time (Days)

Fig.3: Vegetative growth of Bacillus cereus strains at 280C

60

Time (Days)

Fig.4: Spore Germination of Bacillus cereus strains at 370C

61

Time (Days)

Fig.5: Spore Germination of Bacillus cereus strains at 280C

62

4.4 Effect of pH on the growth of Bacillus cereus strains

Isolates BC9, BC10, NBC and reference strain NRRL-B14724 were

grown at different pH values (pH 4 – pH 9). There was significant (p < 0.05)

growth of all strains from pH 6 – pH 8 and no growth at pH 4 (Figure 6 – 10).

At pH 5, there was a prolonged lag for all strains except the negative control

NBC, which did not grow at all (Figure 9). These differences were significant

at the 95% confidence level. One of the test strains, BC10, lost viability after

six days at pH 9 (Figure 10).

63

Fig.6: Effect of pH on the Growth of NRRL-B14724 strains of Bacillus cereus

64

Fig.7: Effect of pH on the Growth of BC7 strain of Bacillus cereus

65

Fig.8: Effect of pH on the Growth of BC9 strain of Bacillus cereus

66

Fig.9: Effect of pH on the Growth of NBC strain of Bacillus cereus

67

Fig. 10: Effect of pH on the growth of BC10 strain of Bacillus cereus

68

4.5 Heat Resistance

All strains were resistant to heat up to 60oC. However, there was

variability in heat resistance of the spores at higher temperatures. The heat

resistance of the tested strains were in the order NRRL-B14724 > BC7 > BC9

> NRRL-B14725 > NBC. These results are shown in Figures 11 – 17 and

Table 5

69

Fig.11: Heat Resistance of isolates at 40 degrees

70


71


72


73


74


75


76

Table 5: The decimal reduction time (D-value) for the test strains.

Strains D80oc-values

(min)

D90oc-values

(min)

D100oc-values

(min)

BC7 208 64 17

BC9 383 110 16

NBC 23 0 0

NRRL-B14724 250 134 21

NRRL-B14725 192 15 0

77

4.6 Effect of carbon source on the growth of Bacillus cereus strains

The growth of three strains BC7, BC9 and NBC and a reference strain

NRRL-B14724 were compared on simple sugar (glucose) and complex

carbohydrate (rice).

At 1%, 5% and 10% concentrations of glucose, there was no

significant (p > 0.05) growth of BC7, BC9 and NRRI-14724 strains, while NBC

grew up to day 4. That is, the cell numbers increased from 4.17x1011 –

2.0x1013 cfu/ml (1%), 4.27x1011 – 3.3x1012 cfu/ml (5%) and 2.14x1011 –

2.0x1015 cfu/ml (10%) but lost its viability at day 5 (Figures 18 – 21).

On complex carbohydrate (rice), all strains grew well except the

reference strain NRRL-B14724 (Figure 21). The cell numbers increased from

1.66x1011 – 6.6x1015 cfu/ml (BC7), 1.37x1011 – 1.9x1016 cfu/ml (BC9) and

3.82x1011 – 1.73x107 cfu/ml (NBC).

78

Fig.18: Growth of Bacillus cereus strains on 1% glucose as the sole carbon source

79


80


81

Fig.21: Growth of Bacillus cereus strains on Rice as the sole carbon source

82

CHAPTER FIVE

DISCUSSION

Bacillus cereus is the causative agent of two forms of food poisoning; a

diarrhoeal form, which is mild and resembles Clostridium perfringens food

poisoning and an emetic form, which resembles staphylococcal food

intoxication and may become fulminant and fatal (Mahler et al., 1997;

Andersson et al., 1998; Finlay et al., 2000; Dierick et al., 2005). The ubiquity

of the organism and the ability of its spores to survive dried storage means

that most raw and ready to eat foods may contin B. cereus. The results from

this study showed that all meat, fish and rice samples examined were

contaminated with B. cereus and the bacterial loads of the samples were

within the range of 102 – 106 cfu/g (Table 1). These results are in line with

reports from other researchers, which state that B. cereus spores occur

widely in foods and all kinds of foods have been implicated in B. cereus food

poisoning (Haggblom et al., 2002; EFSA, 2005). The levels of B. cereus

reported in food poisonings range from 102 to 108 cfu/g and it is generally

believed that any food exceeding 104 to 105 cells or spores per gram may not

be safe for consumption (Notermans and Batt, 1998; Beattie and Williams,

2000; Finlay et al., 2000). The levels of B. cereus recorded from the samples

in this study are therefore a cause for concern.

Studies have also shown, however, that although the number of cells

recovered from foods falls frequently within ranges that may be considered

83

unsafe, illness from these occurrences are relatively rare (Notermans et al.,

1997). This has been attributed mainly to the wide variation of pathogenic

potential and overall diversity among B. cereus strains (Helgason et al.,

2000). For instance, B. cereus strains may differ morphologically,

metabolically, in genetic arrangement as well as in the physical and chemical

conditions required for growth and toxin production (Parry and Gilbert 1980;

Shinagawa et al., 1992; Carlson and Kolso, 1993; Granum and Lund, 1997;

Graham et al., 2006). In this study, a total of 350 different isolates recovered

on B. cereus selective medium were characterized. The results showed that

there was indeed metabolic diversity among the strains in terms of

haemolytic reaction, starch hydrolysis, gelatin liquefaction and mannitol

fermentation (Table 2). By comparison with reference B. cereus strains, ten

isolates (BC1 to BC10) were selected as classical strains. The ten strains were

all non-mannitol-fermenting and β-haemolytic, but differed in terms of starch

hydrolysis and gelatin liquefaction. The results on starch hydrolysis and

gelatin liquefaction are particularly important, since some authors have

suggested that these two properties may be significant for separating emetic

B. cereus strains from other B. cereus; emetic strains not being able to

hydrolyze starch and giving wide zones of clearing on gelatin-liquefaction

tests (Christiansson et al., 1989). Of the ten classical strains, six (BC3, BC4,

BC5, BC7, BC9 and BC10) did not hydrolyze starch, suggesting that these

may be emetic strains and only BC10 did not liquefy gelatin. The plasmid

84

profiles also showed BC10 to be the only strain with a different profile (Figure

1). It was interesting, however, to observe that the strain designated NBC

(non-Bacillus cereus) had a profile similar to the classical and reference

strains. This would suggest that the strain, though atypical, is also a B.

cereus strain and again emphasizes the diversity among the strains.

Studies on the effect of temperature on growth showed that all strains

grew well at 28oC and 37oC but not at 4oC (Figures 2 and 3). Also, the strains

showed significant (P < 0.05) growth from pH 6 to pH 8. Outside of this pH

range, however, there was variability in the strains. There was no growth of

any of the strains at pH 4 while at pH 5, there was a prolonged lag for the

strains that grew, specifically, BC7, BC9 and NRRL-B14724 (Figure 6-8). The

atypical NBC strain did not grow at all at pH 5. It also had a prolonged lag at

pH 9 (Figure 9). These observations are in line with reports of other

researchers, which show that refrigeration significantly reduces growth of B.

cereus by increasing the generation time as well as lag time and that B.

cereus is not particularly acid tolerant (Choma et al., 2000; Valero et al.,

2000; Clavel et al., 2004).

In terms of heat resistance, the results showed that all the strains

were resistant to heat up to 60oC, but there was variability in the heat

resistance of the spores at higher temperatures (Table 5). A broad spread of

heat resistance of B. cereus spores has been reported in literature. For

instance, among a set of strains from various origins, D90oC-values varied from

85

a few min to >100 min (Dufrenne et al., 1994), strains from foodborne

outbreaks had D values at 100oC ranging from 6 to 27 min (Rajkowski and

Mikolajeik, 1987) and spores isolated from vegetables had D values at 105oC

around 0.63 min (Fernandez et al., 1999). It has been suggested that the

most heat-resistant strains belong to serotype 1 of the species and this

serotype is also the most frequently isolated from emetic outbreaks (Parry

and Gilbert, 1980; Shinagawa et al., 1992; Graham and Paul, 2006). They

also suggested that the preparation of rice might select for this serotype and

therefore explain why this serotype is most commonly implicated in

foodborne outbreaks. For comparative purposes, some of the strains in this

study, specifically BC7 and BC9, which have previously been suggested to be

possibly emetic, also had D100oC-values of 17 and 16 min respectively (Table

5). Again, this suggests emetic nature of the strains.

Results from the evaluation of effect of carbon source showed that

growth occurred highest on rice with no significant growth on glucose as sole

carbon source. This is in accord with epidemiological findings where fried or

cooked rice has been implicated in approximately 95% of cases of emetic B.

cereus poisoning, suggesting that there is a relationship between substrate

and emetic toxin production (Graham and Paul, 2006).

From the results of this study, it is evident that most raw and cooked

foods available to consumers are highly contaminated with B. cereus spores,

including the dangerous emetic strains. The high prevalence and highly heat-

86

resistant nature of these strains represent a significant health risk to

consumers. In addition, the great variability in the biological properties of the

B. cereus strains would present a difficulty in terms of food monitoring and

assessment of risk based on strains. Therefore, any food containing B. cereus

poses a potential risk. However, the results of this study have also shown

that the risks may be minimized by maintaining foods, particularly rice dishes

under adequate refrigeration or any conditions which might prevent growth of

the organism.

87

Conclusion

From the results of this work, it is concluded that:

• There is a high incidence of contamination of foods with Bacillus

cereus and this has associated health risks.

• The inability of the strains to grow at 4oC indicates that storage

temperature is critical for controlling growth of Bacillus cereus in food

samples

• There is a high degree of variability in morphological and metabolic

characteristics of Bacillus cereus strains and this could pose a problem

in terms of proper screening and monitoring of foods for Bacillus

cereus.

• Inhibition of growth of Bacillus cereus can be achieved by reducing pH

and/or temperatures .

88

Recommendations

From the results of this work, it is recommended that:

• To avoid high numbers of Bacillus cereus at the stage of consumption,

cooked foods should be eaten soon after cooking, or kept above 63oC or

cooked rapidly and kept below 7 – 8oC (ideally below 4oC).

• Temperature below 10oC greatly slows multiplication and temperatures

below 4oC prevent it. Rapid cooling of heat treated foods through the

temperature range supporting growth will also minimize multiplication

before storage.

• Food vendors and restaurants should be monitored to avoid the

multiplication since all the samples collected were contaminated.

• All food samples should be taken care of since these organisms are highly

resistant and heating to inactivate the growth of the organisms may alter

the food quality.

• All foods containing rice should be handled properly since B.cereus strains

grow well in rice and can produce toxins.

89

REFERENCES

Adams, M. R., and Moss, M.O. (1995). Food Microbiology. The Royal Society of Chemistry, Cambridge. pp 160 – 163.

Agaisse, H., Gominet, M., Okstad, O. A., Kolsto, A, B., mand lereclus, D.

(1999). PLCR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Molecular Microbiology 32: (5), 1043 – 1053.

Agata, N., Mori, M. Ohta, M. Suwan, S., Ohtani, and Isobe, M., (1994). A

Novel dodecadepepetide, cereulide, isolated from Bacillus cereus causes vacuole formation in HEP – 2 cells. FEMS Microbiol Lett 121: 31 – 4.

Agata, N., Ohta, M., Arakawa, Y., and Mori, M. (1995a). The bceT gene of

Bacillus cereus encodes on enterotoxic Protein. Microbiology 141: (4), 983 – 988.

Agata, N., Ohta, M., Mori M. and Isobe M. (1995b). A novel

Dodecadepsipeptide, cereulide, is an Emetic Toxin of Bacillus cereus. FEMS Microbiology letters 129: 129 – 19.

Andersen – Borge, G. L., Skeie, M., Sorhaug, T., Langsrud, T. and Granum, P.

E. (2001). Growth and Toxin Profiles of Bacillus cereus isolated from different food sources. International Journal of Food Microbiology 69: 237 – 246.

Andersson, A., Ganum P. E. and Ronner, U. (1998). The adhesion of Bacillus

cereus spores to epithelial cells might be an additional virulence mechanism. International Journal of Food Microbiology 39: 93 – 99.

Andersson, A., Ronner, U. and Granum P. E. (1995). What Problems does the

food Industry have with the spore-forming Pathogens Bacillus cereus and Clostridium perfringens? Int J food Microbiol 28: 145 – 155.

Ash, C., Farrow, J. A. E., Wallbanks, S. and Collins, M. D. (1991).

Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small submit-ribosomal RNA sequences. Letters in Applied Microbiology 13: 202 – 206.

90

Ash,C. and Collins, M. D. (1992). Comparative Analysis of 235 ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR Direct sequencing. FEMS Microbiology letters 73: 78 – 80.

Beattie, S. H. and Williams, A. G. (2000). Detection of toxins. In: R. K.

Robinson, C. A. Batt, and P.D. Patel (ed), Encyclopedia of Food Microbiology, Vol. 1. Academic Press San Diego, Calif. pp 141 – 158.

Beecher, D. J. and Macmillan, J. D. (1990). A novel bicomponent hemolysin

from Bacillus cereus. Infect. Immun 58: 2220 – 2227. Beecher, D. J. and Macmillan, J. D. (1991). Characterization of the

components of hemolysin BL from Bacillus cereus. Infection and Immunity 59: (5), 1778 – 1784.

Beecher, D. J. and Wong, A. C. L. . (1994). Identification of hemolysin BL-

Producing Bacillus cereus by a discontinuous hemolytic haemolytic pattern in blood agar. Appl Environ Microbiolo 60: 1646 – 51.

Beecher, D. J., Schoeni, J. L., and Lee Wong A. C. (1995). Entero toxic

activity of hemolysin BL from Bacillus cereus. Infection and Immunity 63: (11), 4423 – 4428.

Bennett, R. W., and Harmon, S. M. (1988). Bacillus cereus food poisoning. In:

Laboratory Diagnosis of Infections Diseases: Principles and Practice, A. Balows, W. J. Hausler, Jr., M. Ohashi, an A. Turano (eds). Springer – Verlag, New York Vol. 1: pp 88 – 93.

Beuchat, L. R., Ma – lin, C. F. A., Carpenter, J. A. (1980). Growth of Bacillus

cereus in media containing plant seed materials and ingredients used in Chinese cooker Y. J. Appl Bacteriol., 48: 397 – 404.

Bradshaw, J. G., Peeler J. T. and Twedt, R. M. (1975). Heat resistance of

illeal loop reactive Bacillus cereus strains isolated from commercially canned food. Applied Microbiology 30: (6) 943 – 945.

91

Carlson C. R. and Kolsto, A. B. (1993). A complete physical map of a Bacillus thuringiensis Chromosome. J. Bacteriology 175: 1053 – 1060.

Cheesbrough,M. (2000). District laboratory practice in tropical countries.

University press Cambridge, United Kingdom – volume II. Pp 38 – 64. Choma, C., Guinebretiere, M. H., Carlin, F., Schmitt, P., Velge, P., Granum, P.

E. and Nguyen – the C. (2000). Prevalence, Characterization and growth of Bacillus cereus in commercial cooked chilled foods containing vegetables. Journal of Applied Microbiology 88: 617 – 625.

Christiansson, A., Naidu, A. S., Nilssm, I., Wadstrom, T. and Pettersson, H.

(1989). Toxin production by Bacillus cereus Dairy isolates in milk at low temperatures. Applied and Environmental Microbiology 55 (10): 2595 – 2000.

Chrisitiansson, A., Bertilsson J. and Svensson, B. (1999). Bacillus cereus

spores in raw milk: Factors affecting the contamination of milk during the grazing period. Journal of Dairy Science 82: 305 – 314.

Claus, D. and Berkeley, R. C. W. (1986). Genus Bacillus Cohn 1987, 174 In:

Sneath, P. H. A; Mair, N. S., Sharpe M. E. and Holt, J. G. (eds), Bergey’s manual of Systematic Bacteriology, William & Wilkins. pp 1105 – 1139.

Clavel, T. Carlin, F., Lairon D., Nguyen – The, C., Schmitt, P. (2004). Survival

of Bacillus cereus spores and vegetative cells in acid media stimulating human stomach. Journal of Applied Microbiology. 97: (1): 214 – 219.

Contrepois, M. (1993). Adherence bacterienne et colonization des muqueuses

digestives. In: Rambaud, J. C. and Rmapal, P. (eds). Diarrhees aigues infectiveness. Doin, Paris. Pp 11 – 19.

Cowan, S. T. and Steel, K. J. (1966). Manual for the Identification of Medical

Bacteria. Cambridge University press, New York pg. 116 – 156. Dierick. K. Van Coillie, E., Swiecicka, 1 & 7 other authors (2005). A fatal

family outbreak of Bacillus cereus food poisoning. J. Clin Microbiol (in press).

Dufrenne, J., Soentoro, P., Tatini, s., Day, T. and Notermans, S. (1994).

Characteristics of Bacillus cereus related to safe food production. International Journal of food Microbiology 23: 99 – 109.

92

European Food and Safety Agency (EFSA) (2005). Bacillus cereus and other Bacillus spp in foodstuffs. The EFSA Journal 175:1-48.

Eneroth, A., Svensson, B., Molin G and Christiansson, A. (2001).

Contamination of Pasteurized milk by Bacillus cereus in the filling machine. Journal of Dairy Research 68: 189 – 196.

Fernandez, A., Collado, J., Cunha, L. M., Ocio, M. J. and Martinez, A. (2002).

Empirical model building based on Weibull distribution to describe the joint effect of pH and temperature on the thermal resistance of Bacillus cereus in vegetable substrate. International Journal of Food Microbiology 77: 147 – 153.

Fernandez, A., Collado, J., Cunha, L. M., Ocio, M. J., Fernandez, P. S.,

Rodrigo, M. and Martinez, A. (1999). Application of non-linear depression analysis to the estimation of kinetic parameters for two enterotoxigenic strains of Bacillus cereus spores. Food Microbiology 16: 607 – 613.

Finlay, W. J. J., Logan N. A. and Sutherland, A. D. (2000). Bacillus cereus

produces most emetic toxin at lower temperatures. Letters in Applied Microbiology 31: 335 – 339.

Francis, K. P., Mayr, R., Von Stetten, F., Stewart, G. and Scherer, S. (1998).

Discrimination of Psychrotophic and Mesoplilic strains of the Bacillus cereus group by PCR targeting of major old shock protein genes. Applied and Environmental Microbiology 64: 3525 – 3529.

Gilbert, R. J., Stringer, M. F., and Peace, T. C. (1974). The survival and

growth of Bacillus cereus in boiled and fried rice in relation to outbreaks of food poisoning. Journal of Hygiene, Cambridge 73: 433.

Graham, B. and Paul, H. (2006). Development of Improved Molecular

Detection methods for Bacillus cereus toxins. A report for the rural industries research and development corporation. Pg 1 – 60.

Granum, P. E. (1994). Bacillus cereus and its toxins. Symposium series

society for Applied Bacteriology. 61s – 66s. Granum, P. E. (1997), Bacillus cereus Dissection of toxins. In: R. K. Robinson,

C. A. Batt and P. D. Patel (ed.), Encyclopedia of Food Microbiology, Academic press, San Diego, Calif vol. 1: pp 327 – 336.

93

Granum, P. E., Naestvold, A. and Gunderby, K. N. (1995). An outbreak of Bacillus cereus food poisoning in the junior Norwegian ski championships. Norsk veterinaertidsskrift 107: 945 – 948.

Granum, F. E., Andersson, A., Gayther, C., te Giffel, M., Larsen, H., Lund, T.

and O’ Sullivan, K. (1996). Evidence for a further enterotoxin complex produced by Bacillus cereus. FEMS Microbiol Lett 141: 145 – 149.

Granum P. E. and Lund, T. (1997). Bacillus cereus and its food poisoning

toxins. FEMS Microbiology letters 157: (2), 223 – 228. Granum, P.E. O’ Sullivan, K., and Lund, T. (1999). The sequence of the non-

haemolytic enterotoxin operon from Bacillus cereus. FEMS Microbiology letters 177: (2), 225 – 229.

Guinebretiere, M. H. and Nguyen –the, C. (2003). Sources of Bacillus cereus

contamination in a pasteurized Zucchini puree processing plant, differentiated by two PCR – based methods. FEMS Microbiology Ecology 43: 207 – 215.

Guinebretiere, M. H. and Sanchis, V. (2003). Bacillus cereus Sensu lato.

Bulletin de la societe francaise de microbiologie 18: 95 – 103. Halverson, L. J., Clayton, M. K. and Handelsmen, J. (1993). Population

biology of Bacillus cereus UW 85 in rhizosphere of field grown soy beans. Soil Biological Biochemistry 25: 485 – 493.

Hardy, S. P., Lund, T., Granum, P. E. (2001). Cyt K toxin of Bacillus cereus

forms pores in planar lipid bilayers and is cytotoxic to intestinal epithelia. FEMS Microbiology letters 197: 47 – 51.

Hauge, (1950). Food Poisoning caused by Bacillus cereus preliminary report

(In Norwegian). Nord. Hyg. T. 31: 189 – 206. Heganson, E., Caugant, D. A., Olsen, I. And Kolsoto, A. (2000). Genetic

structure of population of Bacillus cereus and Bacillus thuringensis isolates associated with periodontitis and other human infections. J. Clin Microbiol 38: 1615 – 1622.

94

Helgason, E., Okstad, O. A., Caugent D. A., Johansen, H. A., Fouet, A., Moik, M., Hegna, 1.8 Kolsto, A. A., (2000). Bacillus antracis, Bacillus cereus and Bacillus thuringiensis. One species on the basis of genetic evidence. Appl Environ Microbiol 66: 2627 – 2630.

Helgason, E., Tourasse, N. J., Meisal, R., Caugant, D. A. and Kolston, A. B.

(2004). Multilocus sequence typing Scheme for bacteria of the Bacillus cereus group. Microbiology Applied and Environmental 70: 191 – 201.

Hendriksen, N. B. and Hensen, B. M. (2002). Long-term survival and

germination of Bacillus thuringiensis var. Kurstak in a field trial. Canadian Journal of Microbiology 48: 256 – 261.

Hsieh, Y. M., Sheu S. J., Chen Y. L. and Tsen, H. Y. (1999). Enterotoxigenic

profiles and polymerase chain reaction detection of Bacillus cereus group and Bacillus cereus strains from foods and food borne outbreaks. J. Appl Microbiol 87: 481 – 490.

http://mic.sgmijounals.org. Husmark, U. and Ronner, U. (1990). Forces involved in adhesion of Bacillus

cereus spores to solid surfaces under different environmental conditions. Journal of Applied Bacteriology 69: 557 – 562.

Husmark, U. (1993). Adhesion mechanisms of bacterial spores to solid

surface. Ph. D. thesis, Department of Food Science, Chalmers University of Technology and sik, the Swedish Institute for Food Research Got

ICMSF (International Commission in Microbiological Specification for foods

(2003). Microbial theology of food commodities, 2nd edition, Kluwer Academic/Plenum Publishers, New York (in press).

Johnson, K.M., Nelson, C. L., Busta F. F. (1983). Influence of temperature on

germination and growth of spores of emetic and diarhoeal stran of Bacillus cereus in a broth medium and in rice. J. Food Sci. 48: 286 – 287.

Kramer, J. M. and Gilbert, R. J. (1989). Bacillus cereus and other Bacillus

species. Food borne bacterial pathogens. Marcel Dekkar Inc New York, USA.

95

Lechner, S., Mayr, R. Francis, K. P., Pruss, B. M., Kaplan, T., Wiessner. Gunkel, E., Stewartz, G. and Scherer, S. (1998). Bacillus weihenstephanensis sp. Nov. in a new psychrotolerant species of the Bacillus cereus group. International Journal of Systematic Bacteriology 48: 1373 – 1382.

Logan, N. A. and Berkeley, R. C. (1984). Identification of Bacillus strains using

the API system. Journal of General Microbiology 130: 1871 – 1882. Lubenau. C. (1906). Bacillus peptonificans als Erreger emer Gastroenteritis –

Epidemic. 2bl. Bakt. 1. Orig; 40 431 – 437. Lund, B. M. (1990). Food – borne disease due to Bacillus and Clostridium

species. Lancet 336: 982 – 986. Lund, T., DeBuyser, M. L., Granum, P. E. (2000). A new cytotoxin from

Bacillus cereus that may cause necrotic enteritis. Moleculer Microbiology 38: 254 – 261.

Lund T. and Granum, P. E. (1996). Characterization of a non-heamolytic

enterotoxin complex from Bacillus cereus isolated after a food borne outbreak. FEMS Microbiology letters 141: (213), 151 – 156.

Lund, T. and Granum P. E. (1997). Comparison of biological effect of the two

different enterotoxin complexes isolated from three different strains of Bacillus cereus. Microbiology 143: 3329 – 3336.

Lundsay, D., Brozel, V. s., Mostert, J. f. and Von Holy, A. (2000). Physiology

of dairy associated Bacillus Spp over a wide pH range. Int. J. Food Microbilogy 54: 49 – 62.

Mahler, H., Pasi, A., Kramer, J. M., Schulte, P., Scoging, A. C., Bar, W. and

Krahen buhl, S. (1997). Fulminant liver failure in association with the emetic toxin of Bacillus cereus. New England Journal of medicine 336: 1142 – 1148.

Mantynen, V. and Lindstrom, K. (1998). A rapid PCR-based DNA test for

enterotoxic Bacillus cereus. Appl. Environ Microbiol, 64: 15334 – 15339.

96

Max, M. H., Apetroaie, C., Andersson, M. A. and Salkinoja – Salonen, M. S. (2002). Quantitative Analysis of Cereulide, the Emetic Toxin of Bacillus cereus produced under various conditions. Appl Environ Microbiol. 68: 2479 – 2483.

Mazas, M., Lopez, M., Gonzalez, I., Gonzalez, J., Bernardo, A and Martin, R.

(1998). Effects of the heating medium pH on heat resistance of Bacillus cereus spores. Journal of food safety 18: 25 – 36.

Mikkola, R., Saris N. E., Grigoirer, P. A., Andersson, M. A. and Salkinoja –

Salonen, M. s. (1999). Ionophoretic Properties and Mitochondrial effects of cereulide: the emetic toxin of Bacillus cereus. Eur J. Biochem 26: 112 –117.

Mossel, D. A. A., Koopman, M. J. and Jongerius, E. (1967). Enumeration of

Bacillus cereus in foods. Applied Microbiology 15: 650 – 653. Nakamura, L. K. (1998). Bacillus pseudomycoides Sp. Nov. International

Journal of Systematic Bacteriology 18: 1031 – 1035. Nguyen –the, C. and Carlin, f. (2003). Bacillus cereus et securite des

aliments. Bulletin de la societe Francaise de Microbiologie 18: 104 – 112.

Notermans, S. and Batt, C. A. (1998). A risk assessment approach for food –

borne Bacillus cereus and its toxins. Symposium, series – society for Applied Bacteriology 84: 515 – 615.

Notermans, S., Dufernne, J., Teunis, P., Beumer, R., Giffel, M. T., Peeters

Weem, P., (1997). A risk assessment study of Bacillus cereus present in pasteurized milk. Food Microbiology 14: (2) 143 – 151.

Ogbulie, J. N. Uwaezuoke, J. C., and Ogiehor, S. I. (1998). Introductory

Microbiology practical Springfield publishers, Nigeria. pg. 32. Parry, J. M. and Gilbert, R. J. (1980). Studies on the heat resistance of

Bacillus cereus spores and growth of the organism in boiled rice. Journal of Hygiene, Cambridge 84: 77 – 82.

Periago, P. M. Moezelaar, P. (2001). The combined effect of nisin and

carvacol at different pH and temperature levels on the viability of different strains of Bacillus cereus. International Journal of Food Microbiology 68: 141 – 148.

97

Peters, A. C., Thomas, L. and Winpenney, J. W. T.(1991). Effects of salt concentration on bacterial growth on plates with gradients of pH and temperature. FEMS Microbiological letters 77: 309 – 314.

Pirttijarvi, T. S., Andersson, M. A. and Salknoja – Salonen, M. S. (2000).

Properties of Bacillus cereus and other bacilli contaminating biomaterial. Journal of Food Microbiology 60: 231 – 239.

Pol, I. And Smith, E. J. (1999). Combined action of nisin and Carvacrol on

Bacillus cereus and Listeria monocytogenes. Letters in Applied Microbiology 29: 166 – 170.

Quintavalla, S., Parolari, G. (1993). Effects of temperature aw and pH on the

growth of Bacillus cereus. International Journal of Food Microbiology 19: 207 – 216.

Raevuon, M. T. and Genigeorgis, C. (1975). Effects of pH and sodium on

growth of Bacillus cereus in laboratory media and certain foods. Applied Microbiology 29: 68 – 73.

Raevuori, M., T. Klutamo, and A. Niskaneo (1977) Comparative studies of

Bacillus cereus strains isolated from various foods and food poisoning outbreaks. Acta Vet. Scan. 18: 397 -407.

Rajkowski, K. T. and Mikolajcik, E. M. (1987). Characteristics of selected

strains of Bacillus cereus. Journal of food protection 50: 199 – 205. Ranald, M. A. (1996). Handbook of Microbiological Media. Parka,L.C. (eds.).

Robert Stern, USA. pp 137-138. Ray, B. (1992). Nisin of Lactococcus lactis sp lactus as a food biopreservative.

In: B. Ray and M. A. Daeschel (ed). Food Bio preservatives of Natural origin. C R C Press BoCa Raton Fla. pp. 207 – 264.

Runner, U. Husmark, U., Henriksson, A. (1990). Adhesion of Bacillus spores

in relation to hydrophobicity. J. Appl. Bacteriol. 69: 550 – 556. Salamitou, S., Ramisee, F., Brhelin, M., bouguet, D., Gilois, N., Gominent, M.,

Hermandez, E. and Lereclus, D. (2000). The pclR regulon is involed in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and Insects. Microbiology 146: 2825 – 2832.

98

Salkinoja – Salonen, M. S., Vuorio, R. Andersson, M. A., Kampfer, P., Andersson, M. C., Honkanen – Buzalski, T. and Scoging, A. C. (1999). Toxigenic strains of Bacillus licheniformis Related to food poisoning. Appl. Environ Microbiol, 65: 4637 – 4645.

Schraft, H. and Griffiths, M. W. (1995). Specific Oligonucletide primers for

detection of Lecithinase positive Bacillus cereus spp by PCR. Appl Environ Microbiol 61: 469 – 474.

Setlow, P. and Johnson, E. a. (1997). Spores and their significance. In M. P.

Doyle, L. R. Beuchat, and T. L. Montuille (ed.), Food Microbiology: Fundamental and frontiers. ASM press, Washington, D. C. pp 30 – 64.

Svensson, B., Eneroth, A., Brendehaug, J. and Christiansson, A. (1999).

Investigation of Bacillus cereus contamination sites in a dairy plant with RAPD-PCR. International Dairy Journal 9: 903 – 912.

Shinagawa, K., N. Matsusaka, H. Konuma, and H. Kurate. (1985). The

relation between the diarrheal and other biological activities of Bacillus cereus involved in food poisoning outbreaks. J. Vet. Sc. 47: 557 – 565.

Shinagawa, K., Takechi, T., Matasusaka, N. and Shugii, S. (1992). Purification

of an enterotoxin produced by Bacillus cereus by immuno affinity chromatography using a monoclonal antibody. Canadian journal of Microbiology 38 (2): 153 – 156.

Shinagawa, K., Otake, S., Matsuasaka, N. and Sugii, S. (1992a).Production of

the vacillation factor of Bacillus cereus isolated from vomiting – type food poisoning J. Vet. Med. Sci. 54: 443 – 446.

Shinagawa, K., Takechi, T., Matasusaka, N. and Shugii, S. (1992b).

Purification of an enterotoxin produced by Bacillus cereus by immuno affinity chromatography using a monoclonal antibody. Canadian Journal of Microbiology 38 (2): 153 – 156.

Spira, W. M. and Silverman, G. J. (1979). Effects of Glucose, pH and

Dissolved – Oxygen Tension on Bacillus cereus growth and permeability factor production in Batch culture. Applied and Environmental Microbiology 37: 109 – 116.

99

Svensson, B., Ekelund, K., Ogura, H. and Chrgtiansson, A. (2004). Characterization of Bacillus cereus isolated from milk silo tanks at eight different diary plants. International Diary Journal 14: 17 – 27.

Te Giffel, M. C., Beumer, R. R., Bonestroo, M. H. and Rombouts, F. M.

(1996). Incidence and characterization of Bacillus cereus in two dairy processing plant. Netherlands milk and Dairy Journal 50: 479 – 492.

Te Giffel, M. C., Beumer, R. P., Slaghuis, B. A. and Rombouts, F. M. (1995).

Occurrence and Characterization of Psychrotrophic Bacillus cereus on farms in the Netherlands. Netherlands milk & Dairy Journal 49: 125 – 138.

Te Giffel, M. C., Beumer, R. R., Granum, P. E. and Rombouts, F. M. (1997).

Isolation and characterization of Bacillus cereus from pasteurized milk in household refrigerators in the Netherlands. International Journal of Food Microbiology 34: 307 – 318.

Thompson, N. E., Ketterhagen, M. J., Bergdoll, M. S. and Schantz, E. J.

(1984). Isolation and some properties of an enterotoxin produced by Bacillus cereus. Infect Immun 43: 887 – 894.

Valero M., Leontidis S., Fernandez P., Martinez A. and Salmeron C. (2000).

Growth of Bacillus cereus in natural and acidified carrot substrates over the temperature range 5 – 30OC. Food Microbiology 17: 605 – 612.

Van Netten, P., Van de Moosdjik, A., Van Hoensel, P., Mossel, D. A. A. and

Perales, I. (1990). Psychrotrophic strains of Bacillus cereus producing enterotoxin. Journal of Applied Bacteriology 69: 73 – 79.

Von Stetten, F., Mayr, R. and Scherer, S. (1999). Climatic Influence on

Mesophilic Bacillus cereus and psychrotolerant Bacillus wihenstephansis populations in tropical temperate and alpine soil. Environmental Microbiology 1: 505 – 515.

Wiencek, K. M., Hapes, N. A. and Foegeding, P. M. (1990). Hydrophobicity of

Bacillus and Clostridium spores. Applied and Environmental Microbiology. 65: 470 – 475.

Willardsen, R., Busta, F., Allen, C., Smith L. (1978). Growth and survival of

Clostridium perfringens during constantly rising temperatures. J. Food Sci. 43: 470 – 475.

100

Yamada, A., Tsukagoshi, N., Udaka, S., Sasaki, t., Makino, S., Nakamura, S.,

Little, C., Tomita, M. and Ike Zawa, H. (1988). Nucleotide sequence and expression in Escherichia coli of the gene Coding for Sphingomyelinase of Bacillus cereus. Eur J. Biochem. 175: 213 – 20.

Zhou, C., Yang, Y. Y., Jong, A. Y. (1990). Mini prep Plasmid DNA in Ten

minutes. Biotechniques 11: (2) 217.

101

APPENDIX 1 Electrophoretic Mobility of Standard Molecular weight Markers in Figure1.

Electrophoretic mobility (mm) Molecular weight (bp)

17 23130

21 9416

24

26

6557

4321

37 2322

41 2027

102

APPENDIX II

0

5000

10000

15000

20000

25000

30000

35000

40000

0 10 20 30 40 50

Electrophoretic mobility (mm)

Mo

lecu

lar

wei

gh

t (b

p)

Reference graph of the molecular weight versus

electrophoretic mobility of the standard molecular weight

marker.

103

APPENDIX III

The eletrophoretic mobilities and calculated molecular weights of plasmid

bands in Figure 1.

Bands No of Bands

Electrophoretic mobilities

Molecular weights

Lane 3 3 1 mm, 18 mm, 28 mm

>35 Kbp, 21 Kbp, 5 Kbp

Lane 4 3 1 mm, 18 mm, 28 mm


Lane 5 3 1 mm, 18 mm, 28 mm


Lane 6 3 1 mm, 18 mm, 28 mm


Lane 7 3 1 mm, 18 mm, 28 mm


Lane 8 3 1 mm, 18 mm, 28 mm


Lane 9 3 1 mm, 18 mm, 28 mm


Lane 10 3 1 mm, 18 mm, 28 mm


Lane 11 - No sample loaded No sample loaded

Lane 12 3 1 mm, 18 mm, 28 mm


Lane 13 3 1 mm, 5 mm, 12 mm >35 Kbp, 33 Kbp, 26 Kbp

Lane 14 3 1 mm, 18 mm, 28 mm


Lane 15 1 18 mm 21 Kbp

Lane 16 3 1 mm, 18 mm, 28 mm


104

APPENDIX IV

***** Analysis of variance for Effects Temperature *****

Variate: NRRL_BC

Source of variation d.f. s.s. m.s. v.r. F pr.

Temp_oC 2 96.260 48.130 12.71 <.001

Residual 18 68.138 3.785

Total 20 164.399

***** Tables of means *****

Variate: NRRL_BC

Grand mean 12.79

Temperature (oC) 4.00 28.00 37.00

10.00 13.17 15.20

*** Least significant differences of means (5% level) ***

Table Temp_oC

rep. 7

d.f. 18

l.s.d. 2.185

Least Significant Difference Table

Treatments

Means

Difference between treatment

means (i-j)

4oC – 28

oC

-37oC

28oC – 4

oC

-37oC

37oC – 4

oC

-28oC

-3.17*

-5.2*

3.17*

-2.03

5.2*

2.03

* Significance

105

APPENDIX V

***** Analysis of variance for Growth of BC7 at different

Temperatures

Variate: BC7_Gth


Temp_oC 2 47.693 23.847 11.15 <.001

Residual 18 38.493 2.139

Total 20 86.186


Variate: BC7_Gth

Grand mean 11.85

Temperature(oC) 4.00 28.00 37.00

10.00 11.85 13.69


Table Temp_oC

rep. 7

d.f. 18

l.s.d. 1.642


Treatments

Means


means (i-j)

4oC – 28

oC

-37oC

28oC – 4

oC

-37oC

37oC – 4

oC

-28oC

-1.85*

-3.69*

1.85*

-1.84*

3.69*

1.84*

* Significance

106

APPENDIX VI

***** Analysis of variance *****

Variate: BC9_Gth


Temp_oC 2 46.282 23.141 9.60 0.001

Residual 18 43.383 2.410

Total 20 89.665


Variate: BC9_Gth

Grand mean 11.85

Temp_oC 4.00 28.00 37.00

10.00 11.92 13.63


Table Temp_oC

rep. 7

d.f. 18

l.s.d. 1.743


Treatments

Means


means (i-j)

4oC – 28

oC

-37oC

28oC – 4

oC

-37oC

37oC – 4

oC

-28oC

-1.92*

-3.63*

1.92*-1.71

3.63*

1.71

* Significance

107

ugwu, celestina chibuzo - university of nigeria … · mrs. ugwu, celestina chibuzo a postgraduate...

Documents