Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27.5 degrees C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10(4) spores/g whereas with 800 g loaves survival occurred with doughs containing 5.0 X 10(3) spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0.2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

Incidence of Staphylococcus aureus, coliforms and antibiotic-resistant strains of Escherichia coli in rural water supplies in Port Harcourt

The bacteriological quality of some rural water supplies in Port Harcourt was monitored over a 3 month period. The supplies were unsatisfactory as judged by standard plate counts (10(3)/ml) and the presence of presumptive and faecal coliforms and Staphylococcus aureus. The recovery of potentially pathogenic bacteria (e.g. Pseudomonas aeruginosa) further substantiated the existence of health hazards. The most frequently isolated coliforms were Escherichia coli, Enterobacter aerogenes and Klebsiella pneumoniae. Coliform contamination was greater in well water than in river or stream water samples. An antibiotic sensitivity test revealed that 17.5-27.2% of E. coli strains were resistant to three or more antibiotics. Escherichia coli isolated from well water samples exhibited the greatest degree of multiple resistance. Some strains were resistant to all the six antibiotics tested. The danger of an epidemic of waterborne diseases in the communities as a result of drinking water from these non-potable sources is noted.     

Incidence of Staphylococcus aureus, coliforms and antibiotic-resistant strains of Escherichia coli in rural water supplies in Port Harcourt

The bacteriological quality of some rural water supplies in Port Harcourt was monitored over a 3 month period. The supplies were unsatisfactory as judged by standard plate counts (10³/ml) and the presence of presumptive and faecal coliforms and Staphylococcus aureus. The recovery of potentially pathogenic bacteria (e.g. Pseudomonas aeruginosa ) further substantiated the existence of health hazards. The most frequently isolated coliforms were Escherichia coli, Enterobacter aerogenes and Klebsiella pneumoniae. Coliform contamination was greater in well water than in river or stream water samples. An antibiotic sensitivity test revealed that 17.5–27.2% of E. coli strains were resistant to three or more antibiotics. Escherichia coli isolated from well water samples exhibited the greatest degree of multiple resistance. Some strains were resistant to all the six antibiotics tested. The danger of an epidemic of waterborne diseases in the communities as a result of drinking water from these non-potable sources is noted.     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27·5°C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10⁴ spores/g whereas with 800 g loaves survival occurred with doughs containing 5·0 times 10³ spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0·2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

Characterization of lactic acid bacteria isolated from the one humped camel milk produced in Morocco

One hundred and twenty (120) strains of lactic acid bacteria (LAB) were enumerated and isolated from raw dromedary milk in Morocco using various cultured media. Strains isolated were characterized by phenotypic, physiological and biochemical properties. Results showed that high counts of LAB were found. Presumptive lactobacilli counts ranged from 2.5x10(2) to 6x10(7)cfu/ml, presumptive lactococci levels varied from 5x10(2) to 6x10(7)cfu/ml, presumptive streptococci counts varied from 4.2x10(2) to 8x10(7)cfu/ml, presumptive leuconostoc levels ranged from 5.4x10(2) to 5.4x10(7)cfu/ml. Results showed also that Lactobacillus and Lactococcus were the predominant genera with 37.5% and 25.8%, respectively. The dominated species found were Lactococcus lactis subsp. lactis (17.5%), Lactobacillus helveticus (10%), Streptococcus salivarius subsp. thermophilus (9.20%), Lactobacillus casei subsp. casei (5.80%) and Lactobacillus plantarum (5%). This is the first report on the characterization of LAB strains isolated from the one humped camel milk produced in Morocco.     

Identification of hemolysin BL-producing Bacillus cereus isolates by a discontinuous hemolytic pattern in blood agar.

Bacillus cereus causes distinct exotoxin-mediated diarrheal and emetic food poisoning syndromes and a variety of nongastrointestinal infections. Evidence is accumulating that hemolysin BL is a major B. cereus virulence factor. We describe two methods for detection of hemolysin BL in crude samples and on primary culture media. In the first method, the highly unusual discontinuous hemolysis pattern that is characteristic of pure hemolysin BL was produced in sheep and calf blood agar around wells filled with crude culture supernatant from hemolysin BL-producing strains. In the second method, the pattern was formed surrounding colonies of hemolysin BL-producing strains grown on media consisting of nutrient agar, 0.15 M NaCl, 2% calf serum, and sheep or calf blood. Hemolysin BL production was detected with these methods in 41 of 62 (66%) previously identified B. cereus isolates and in 46 of 136 (34%) presumptive B. cereus isolates from soil. All nine isolates tested that were associated with diarrhea or nongastrointestinal illness were positive for hemolysin BL. The methods presented here are specific, simple, inexpensive, and applicable to the screening of large numbers of samples or isolates.     

Occurrence and significance of Bacillus cereus and Bacillus thuringiensis in ready-to-eat food

Among 48,901 samples of ready-to-eat food products at the Danish retail market, 0.5% had counts of Bacillus cereus-like bacteria above 10(4) cfu g(-1). The high counts were most frequently found in starchy, cooked products, but also in fresh cucumbers and tomatoes. Forty randomly selected strains had at least one gene or component involved in human diarrhoeal disease, while emetic toxin was related to only one B. cereus strain. A new observation was that 31 out of the 40 randomly selected B. cereus-like strains could be classified as Bacillus thuringiensis due to crystal production and/or content of cry genes. Thus, a large proportion of the B. cereus-like organisms present in food may belong to B. thuringiensis.     

Potential application of a HEp-2 cell assay in the investigation of Bacillus cereus emetic-syndrome food poisoning

Abstract When grown for 15 h in rice culture, 13 out of 15 Bacillus cereus strains associated with emetic-syndrome food poisoning (87%) caused vacuoles to appear in HEp-2 cells, compared with 5 out of 11 B. cereus strains from other sources (45%). No other Bacillus species tested gave rise to this response under these conditions. Six out of eight rice samples involved in incidents of B. cereus emetic illness produced vacuoles in HEp-2 cells, whereas control rice samples and foods from vomiting episodes caused by other Bacillus spp. failed to do so. This vacuole response may have application as a simple in vitro assay for organisms and foods implicated in B. cereus emetic-syndrome food poisoning.     

Enumeration and Identification of Bacillus cereus in Foods: I. 24-Hour Presumptive Test Medium

An egg yolk-polymyxin medium (KG) for rapid enumeration of Bacillus cereus is described. The test is presumptive in that differentiation of B. cereus (and closely related organisms) from other species is based on the formation of turbidity in the agar surrounding the colonies of the cereus group organisms. The medium is formulated to encourage sporulation and release of free spores for serological confirmatory tests within the 24-hr incubation period. The production of turbidity in egg yolk and free-spore production by 25 strains of B. cereus on KG agar were measured. The recovery of food poisoning strains of B. cereus inoculated into nonsterile food slurries was assessed. A comparison of KG agar and mannitol-egg yolk-polymyxin-agar indicated that the two media were comparable in their abilities to recover low levels of B. cereus from naturally contaminated foods. Since KG agar enhances spore formation by B. cereus, thus permitting early serological testing, its use in screening food products is advocated.     

Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B. cereus strains from foods and food-borne outbreaks

Bacillus cereus is one of the important food pathogens. Since B. cereus group cells, such as B. cereus, B. thuringiensis, B. anthracis and B. mycoides, share many phenotypical properties and a high level of chromosomal sequence similarity, it is interesting to investigate the virulence profiles for B. cereus group cells, including B. cereus strains isolated from foods and samples associated with food-poisoning outbreaks. For this investigation, the presence of enterotoxin genes, such as those of haemolysin BL, B. cereus enterotoxin T and enterotoxin FM, were assayed by polymerase chain reaction (PCR) methods. Meanwhile, their enterotoxin activities were assayed using the BCET-RPLA kit, haemolytic patterns on sheep blood agar and their cytotoxicity to Chinese hamster ovary (CHO) cells. Results showed that there were 12 enterotoxigenic profiles for the 98 B. cereus group strains collected. In addition, if any of the three types of enterotoxins was present in the B. cereus group cells, these cells were shown to be cytotoxic to the CHO cells. Similar enterotoxigenic profiles could be found among strains of B. cereus, B. mycoides and B. thuringiensis. Thus, all B. cereus group strains may be potentially toxigenic and the detection of these cells in foods is important. We thus designed PCR primers, termed Ph1/Ph2, from the sphingomyelinase gene of B. cereus cells. These primers were specific for all B. cereus group strains and could be used for the detection of B. cereus cells contaminated in food samples.     

Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens . It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus . It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43°C-45°C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10–20 μg/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens .
The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfringens could be obtained by pre-incubation at 37°C of inoculated media for 2–4 h followed by overnight incubation at 43°C-45°C. Tryptose-sulphite-cyclo-serine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens . This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus . Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clos-tridia on BCP is presented.     

The Occurrence of Bacillus cereus in some Dried Foods Including Pulses and Cereals

In a survey of 39 dried food samples which represented 12 different pulses and cereals. 22 (56%) were found to be contaminated with Bacillus cereus. The numbers varied between 1 times 102 and 6 times 10⁴ organisms/g. During normal cooking procedures and on storage at room temperature, the B. cereus resident on red lentils and kidney beans increased to a level at which enterotoxin production could become significant. Some physiological characteristics including deoxyribonuc-lease (DNase) and ribonuclease (RNase) secretion and salicin fermentation did not correlate with the ability or otherwise of a strain to cause food poisoning. Nevertheless, serotype 8 strains were prevalent on these foods and these have been implicated in both the diarrhoeal and vomiting-type food poisoning syndromes.     

Microbiological survey of prepackaged pâté and ham in New Zealand

AIMS: To gauge the effectiveness of paté and ham manufacturers' management of the microbial safety and quality of their products. METHODS AND RESULTS: A survey of 60 batches of prepackaged paté showed that 41.7% of the batches had aerobic plate counts (APC) exceeding 10(5) CFU g(-1), one of paté sample contained a Bacillus cereus count of >5000 CFU g(-1) and another contained 1700 CFU g(-1) of Listeria monocytogenes. No other pathogens were isolated from any of the samples. The survey of prepackaged ham showed that only 1% (1/104) of the ham samples were positive for L. monocytogenes (50 CFU g(-1)). CONCLUSIONS: The presence of microbial hazards in these foods has generally declined since the early 1990s in New Zealand. Noncomplying APC levels may be due to an over-estimation of product shelf life or poor food handling practices during manufacture. SIGNIFICANCE AND IMPACT OF THE STUDY: Few of the samples tested contained pathogens at significant levels. The prevalences of L. monocytogenes in paté and ham were low. The presence of 1700 CFU g(-1) of L. monocytogenes in a paté sample indicates that occasionally, the population can be exposed to levels of L. monocytogenes above the zero tolerance level set in New Zealand.     

Air-liquid interface biofilms of Bacillus cereus: formation, sporulation, and dispersion

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.     

Staphylococcal enterotoxin production in the presence of non-enterotoxigenic staphylococci.

Enterotoxigenic Staphylococcus aureus strains were grown with a non-enterotoxigenic strain in laboratory medium, in milk, and in ham. Differences in pigmentation were used to differentiate the enterotoxigenic strains from the non-enterotoxigenic ones. Enterotoxin was detectable in milk when the colony counts of the non-enterotoxigenic strain were 15 to 20 times greater than those of the enterotoxigenic ones and in ham when the ratio was 60 to 77:1. Enterotoxin was detectable in milk when the enterotoxigenic strains reached counts of 10(7) colony-forming units per ml and in ham when the counts reached 10(8) colony-forming units per ml. It may be necessary in some food poisoning outbreaks to examine many isolates (up to 50 or 60) for enterotoxin production to be able to detect the enterotoxigenic staphylococci.     

Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method

Despite the emergence of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, E. coli serotype O157 is still the most commonly identified STEC in the world. It causes high morbidity and mortality and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain the gold standard. Here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157. Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. The method then was optimized for detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1 × 10⁻² to 1 × 10² CFU per 25 g or ml of food. Samples were then preenriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrices with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% confidence interval [CI], 82.83 to 100), a sensitivity of 97.22% (95% CI, 83.79 to 99.85%), and an accuracy of 98.33% (CI 95%, 83.41 to 99.91%). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day.     

Prevalence and characterization of Clostridium perfringens from spices in Argentina

Spices can present high microbial counts and Clostridium perfringens, Bacillus cereus, Salmonella and Shigella, among others have been isolated from spices. C. perfringens is an important pathogen agent causing, among other diseases, enteritis in humans caused by C. perfringens enterotoxin (CPE) which causes human food poisoning and enterotoxemia in domestic animals. The aims of the present work were (i) to establish the hygienic sanitary quality of some spices in San Luis, Argentina; (ii) to determine the presence of C. perfringens in these spices by means of the most probable number (MPN) and count on plate methods; (iii) to characterize the enterotoxigenic strains of C. perfringens by PCR and immunological methods such as reverse passive latex agglutination (RPLA) and (iv) to type by PCR C. perfringens strains isolated. A total of 115 samples of spices, 67 of which were purchased in local retail stores and 48 domestically collected were analysed. Total aerobe counts on tryptone glucose yeast extract agar medium of the 115 samples were between <10 and 10(6) CFU/g. The colifecal counts using Mac Conkey broth of the 115 samples were <4-10(3)CFU/g, with 28 samples (24.34%) exceeding the limit established by the Spanish Alimentary Code (10 CFU/g) while 2 samples (1.73%) had a sulfite reducing anaerobe load above standard limits. A total of 14 C. perfringens strains (12.17%) were isolated and characterized from 115 samples by the standard biochemical tests. Four of which (28.60%) turned out to be enterotoxigenic by PCR and RPLA. In order to type C. perfringens strains based on their main toxins, the 14 strains were analysed by PCR. All strains belonged to type A. All RPLA positive strains were cpe(+) by PCR. The percentage of enterotoxigenic strains was more elevated that those reported in other studies for this type of sample. These results indicate that sanitary conditions in different production stages of species must be improved to reduce health hazards. The high percentage (24.34%) of samples with colifecal values above standard limits is an indication of deficient sanitary conditions. These results suggest the need to provide legislation on the sanitary and hygienic quality of spices in our country.     

Quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, produced under various conditions

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 microg ml(-1). The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 microg of cereulide g(-1) (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 microg of cereulide ml(-1) in liquid medium at room temperature (21 +/- 1 degrees C) in 1 to 3 days, during the stationary growth phase when cell density was 2 x 10(8) to 6 x 10(8) CFU ml(-1). Cereulide production at temperatures at and below 8 degrees C or at 40 degrees C was minimal.     

Improved cytotoxicity assay for Bacillus cereus diarrhoeal enterotoxin

An improved McCoy cell cytotoxicity assay for Bacillus cereus diarrhoeal toxin, which includes a staining procedure in addition to visual examination, was developed and the method was compared with two commercially available kits (Oxoid BCET-RPLA and Tecra BDE-VIA). A total of 71 strains of 15 different Bacillus, Brevibacillus and Paenibacillus species, including 16 strains of B. cereus from outbreaks of food-borne illness, were evaluated for toxin production. Eleven of the outbreak strains exhibited cytotoxicity, including all six B. cereus strains associated with diarrhoeal illness. Several other isolates of B. cereus, and its relatives B. anthracis, B. mycoides and B. thuringiensis, exhibited similar cytotoxicity. The other species showed no cytotoxicity, with the exception of certain B. subtilis strains. The cytotoxicity assay was more sensitive than the Oxoid kit and unlike the Tecra kit, did not give false positive results with supernatant fluids heat-treated to destroy the toxin.