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PROJECT TOPIC- ISOLATION AND CHARACTERIZATION OF Bacillus cereus STRAINS FROM VARIOUS FOODS IN NSUKKA

PROJECT TOPIC- ISOLATION AND CHARACTERIZATION OF Bacillus cereus STRAINS FROM VARIOUS FOODS IN NSUKKA

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 Dvalues 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.

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 foodscontaminated 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.
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. 

PROJECT TOPIC- ISOLATION AND CHARACTERIZATION OF Bacillus Cereus STRAINS FROM VARIOUS FOODS IN NSUKKA

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.
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 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 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.

PROJECT TOPIC- ISOLATION AND CHARACTERIZATION OF Bacillus Cereus STRAINS FROM VARIOUS FOODS IN NSUKKA

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