Mannitol Egg Yolk Polymyxin Agar Research Articles

Effects of spore purity on the wet heat resistance of Clostridium perfringens, Bacillus cereus and Bacillus subtilis spores

Heat resistance of spores of Clostridium perfringens 8238 (Hobbs Serotype 2), Bacillus cereus NCTC 11143 (4810/72), and Bacillus subtilis PS533, an isogenic derivative of strain PS832 (a 168 strain) was determined in ground beef at 95 °C. Spore purification was by centrifugation and washing with sterile distilled water (dH2O), followed by sonication and then Histodenz centrifugation for B. subtilis and C. perfringens, and centrifugation and washing with sterile dH2O followed by Histodenz centrifugation for B. cereus. Bags containing inoculated beef samples were submerged in a temperature-controlled water bath and held at 95 °C for predetermined lengths of time. Surviving spore populations were enumerated by plating on mannitol egg yolk polymyxin agar (MYP) agar plates for B. cereus and B. subtilis, and on tryptose-sulfite-cycloserine agar (TSC) agar plates for C. perfringens. Survivor curves were fitted to linear, linear with tail, and Weibull models using the USDA Integrated Pathogen Modeling Program (IPMP) 2013 software. The Weibull model provided a relatively better fit to the data since the root mean square error (RMSE), mean square error (MSE), sum of squared errors (SSE), and Akaike information criterion (AIC) values were lower than the values obtained using the linear or the linear with tail models. Additionally, the Weibull model accurately predicted the observed D-values at 95 °C for the three spore-formers since the accuracy factor (Af) values ranged from 1.03 to 1.08 and the bias factor (Bf) values were either 1.00 or 1.01. Times at 95 °C to achieve a 3-log reduction decreased from 206 min for C. perfringens spores purified with water washes alone to 191 min with water washes followed by sonication and Histodenz centrifugation, from 7.9 min for B. cereus spores purified with water washes alone to 1.4 min with water washes followed by Histodenz centrifugation, and from 20.6 min for B. subtilis spores purified with water washes alone to 6.7 min for water washes followed by sonication and Histodenz centrifugation. Thermal-death-time values reported in this study will assist food processors to design thermal processes to guard against bacterial spores in cooked foods. In addition, clearly spore purity is an additional factor in spore wet heat resistance, although the cause of this effect is not clear.

Characterization of Protease Enzyme Produced from Locally Isolated Bacillus sp.

This study aimed to characterization of protease enzyme purified from locally isolated Bacillus sp.. A total of 50 soil samples were taken for this purpose. Only 35 (or 70%) of the sample were found to have given rise to bacterial growth. As the first phase in the cultivation of bacteria, the samples were grown on mannitol egg yolk polymyxin agar and nutrition agar. More than 20 bacterial isolates were discovered, according to the findings. The results of the cultural, microscopic, and biochemical examinations used to identify these isolates revealed that 15 of them were Bacillus spp. Additionally, the use of the Vitek 2 technique ensured that each of the 15 isolates belonged to this species. Examining these isolates' capacity to create proteases revealed that all 15 of the Bacillus sp. isolates were capable of doing so, albeit to varying degrees. The isolate with the number S9 was the best at producing proteases, with a specific activity value of 40.19 U/mg proteins. On a casein hydrolyzed medium plate with a diameter of 3.0 mm and 2.2 mm, respectively, they demonstrated a clean zone. After 42 hours and 72 hours of incubation at 37°C, respectively, the strain 9 produces 1.01 units of proteolytic enzyme per milliliter. Proteolytic activity decreases with longer incubation times. It has potential for industrial use because the isolated Bacillus sp. was generating protease.

Detection of Bacillus cereus as a causative agent of emetic food poisoning by an unconventional culture procedure

Bacillus cereus is known to cause two types of food poisoning: emetic and diarrhoeal. Both diseases are usually self-limiting; however, severe cases have been reported, presenting with acute liver failure and encephalopathy, including rarely fatal cases of vomiting. Clinical laboratories do not routinely test for B. cereus in patients with gastrointestinal disease. Therefore, B. cereus causing food poisoning goes undetected. We report a successful isolation of emetic B. cereus from a patient with food poisoning who presented with severe vomiting, fulminant hepatic failure, and acute encephalopathy, by a non-conventional method. Initially, stool specimens from the patients were routinely cultured to identify the causative organisms of food poisoning. No foodborne pathogens were detected in this study. In contrast, additional clinical and epidemiological information strongly suggested food poisoning by emetic B. cereus. Consequently, we allowed Drigalski agar medium smeared with patient stool specimens to stand at room temperature (approximately 25 °C) for 9 days. After 9 days, mixed bacteria grown on the medium were inoculated onto mannitol egg yolk polymyxin (MYP) agar plates, a selective medium for B. cereus. Typical colonies of B. cereus developed on MYP agar plates. The isolated B. cereus had a cereulide-producing genetic locus (ces) gene encoding the emetic toxin cereulide. The method used in this case study was unique. This method is easy to apply after obtaining an additional clinical and epidemiological information, and this method will improve the diagnostic rate of severe B. cereus food poisoning. This will contribute to the advancement of therapeutics in the future.

A Novel, Selective, Ready-to-Use Culture Medium for the Enumeration of Bacillus cereus

Bacillus cereus is a Gram-positive, sporulating microorganism that causes food poisoning and / or spoilage. Hence, it is important to detect and enumerate B. cereus; this typically involves culturing with mannitol egg yolk polymyxin agar and NaCl glycine Kim Goepfert agar, both of which require the addition of aseptic egg yolk. To eliminate this unstable and variable step, we developed and evaluated a novel B. cereus group-specific ready-to-use dry medium, CompactDry BC. Inclusivity study results revealed that CompactDry BC showed growth comparable to classical media. Exclusivity study results revealed no typical blue colonies on CompactDry BC. Relative trueness study results revealed that CompactDry BC showed a linear association and almost no negative bias compared to the classical media. Furthermore, we also report the comparison studies of CompactDry BC with CompactDry X-BC, which detects the B. cereus group. Throughout the experiments, CompactDry BC showed higher growth of the B. cereus group than CompactDry X-BC.

Activities of Macrolides in Combination with Vitamin D against Bacillus cereus Isolated from Soil of a Lanfill Site in Nsukka South East Nigeria

Background: The fast moving world of antibiotics resistance is now a big concern, since our antibiotics are in danger of extinction. This is a concern to us because; an empty ‘antibiotic pipeline’ leads to devastating future.
 Aim: The aim of this study was to isolate Bacillus cereus from soil contaminated with various toxic and domestic wastes and investigate its sensitivity to macrolide antibiotics (erythromycin ERY and azithromycin AZM) in combination with vitamin D supplement.
 Methodology: Five soil samples were collected from a landfill site and serially diluted up to 10ˉ6. B. cereus was isolated from the soil sample using Mannitol egg Yolk Polymyxin agar medium (MYP), a selective nutrient medium for B. cereus isolation. The sensitivity of B. cereus to erythromycin and azithromycin was screened in Mueller-Hinton agar (MHA) containing varying concentrations (5, 10, 20 mg/ml) of vitamin D. Agar without vitamin D (0.00mg/ml) served as the control. The activities of ERY and AZM in combination with vitamin D were determined by measuring the zones of inhibition.
 Results: In vitro antibiotic susceptibility tests on our isolate showed maximum sensitivity to ERY and maximum resistance to AZM with 0.00 mg/ml (no combination) of vitamin D. Contrarily, ERY was found to be least active, with the zones of inhibitions decreasing from 28mm to 16mm, with increasing concentration of vitamin D. On the other hand, AZM achieved maximum sensitivity from 12mm to 25mm, with increasing concentration of vitamin D.
 Conclusion: The activities appeared to be important but dose-dependent. So, the systematic use of Vitamin D in combination with macrolide antibiotics may be efficient in treating food and soil infections caused B. cereus. However, in vivo evaluation of these activities is needed.

DETECTION OF HBLD TOXIN GENE BY BACILLUS CEREUS ISOLATED FROM MEAT CURRY FOOD SAMPLES IN MALAYSIAN RESTAURANTS

Bacillus cereus is a ubiquitous foodborne pathogen, can cause food poisoning, leading to infections, have two major types of food poisoning emetic and diarrheal. Foods rich in protein such as meat are associated with foodborne outbreaks of diarrhea caused by B. cereus. The aim of this study is to isolate and identify B. cereus from ready to eat (RTE) meat curry from restaurants in Malaysia and to detect hblD pathogenic gene of B. cereus isolates. Mannitol egg yolk polymyxin agar was used as a selective isolation medium. Commercially available kits and boiling methods were used for DNA extraction, samples acquired from restaurants were examined for the presence of Hemolysin BL gene by polymerase chain reaction (PCR). Among all isolates, twenty-four of B. cereus isolates detected for HBL enterotoxin production by the discontinuous pattern on HBL sheep blood agar then confirmed by biochemical tests. More than 58.33 % of the isolate showed discontinuous hemolysis pattern on HBl blood agar and 29.16% of the samples were shown positive for hblD gene that can cause diarrhea with the size of 807bp on gel. This study demonstrated that RTE meat curry was a potential source for entero-toxigenic B. cereus and the presence of the hblD toxin genes for the HBL complex in the isolates tested were highly associated. Therefore, these meat curry isolates should be regarded as potential toxin producers.

Thermal inactivation of Bacillus cereus spores during cooking of rice to ensure later safety of boudin

The heat resistance of a four-stain cocktail of Bacillus cereus was determined in rice. Bags containing inoculated rice samples with hot water were submerged in a temperature-controlled water bath and held at 90.5, 95, or 99 °C for predetermined lengths of time. Surviving spore population were enumerated by plating on mannitol egg yolk polymyxin agar. Survivor curves were fitted to linear and Weibull models using the USDA Integrated Pathogen Modeling Program (IPMP) 2013 software. While both models provided good fit to the data, Weibull model was relatively better since root mean square error values were lower (<0.32). The time durations to achieve a 4-log reduction were 256.26, 138.81 and 44.38 min at 90.5 °C, 95 °C, and 99 °C, respectively. Thermal-death-time values reported in this study will assist food processors to design lethality processes for the manufacture of foods incorporating cooked rice, such as boudin.

Deciphering rhizosphere microbiome for the development of novel bacterial consortium and its evaluation for salt stress management in solanaceous crops in India

Eighty bacteria were isolated from the salt affected solanaceous crops (potato, tomato, chilli and brinjal) rhizosphere and fallow land in Mau, Faizabad, Ballia, Gazipur and Varanasi districts of Uttar Pradesh, India. All the bacterial isolates showed morphological variation on Nutrient agar, Jenson’s medium, Modified Czapek-Dox medium, Mannitol egg yolk polymyxin agar, Pseudomonas isolation agar medium, Luria Bertni agar, Tryptic soya agar, King B agar and Nutrient broth yeast extract. Majority of the isolates showed creamy colony (32.5%), spherical (66.25%), shiny (88.7%), raised (62.25%), translucent (71.25%) and entire type of surface margin (78.75%). The gram staining, catalase test, glucose fermentation, IAA test, H2S production, starch hydrolysis, tween 80, gelatin hydrolysis, casein hydrolysis, P solubilization, Zn solubilization and ammonia production tests revealed that 26, 28, 13, 3, 11, 10, 18, 10, 17, 24, 23 and 16 isolates were positive for these tests respectively. The molecular characterization of bacterial isolates Brevibacillus fluminis, Brevibacillus agri, Bacillus paralicheniformis, and Microbacterium arborescens revealed that these are highly salt tolerant (18%) produced 37 alleles using box primer. The number of bands (400–3190 bp) varied from 1 to 10. A cluster analysis with unweighted pair-group method with arithmetic means (UPGMA) based on Jaccard’s similarity coefficient divided the bacterial isolates into two major and three minor clusters with coefficient of similarities ranging from 1 to 23%. Phylogenetic analysis of salt tolerant bacterial isolates showed that out of 22 isolates, 17 (77.27%) were found to be phylogenetically closely related to Bacillus, with 99–100% similarity in their 16S rDNA sequences, making Bacillus the most dominant genus among salt tolerant bacteria in the study.

Predictive Model for Growth of Bacillus cereus at Temperatures Applicable to Cooling of Cooked Pasta.

A model was developed to predict the growth of Bacillus cereus from spores during cooling of cooked pasta. Cooked pasta was inoculated with a cocktail of four strains of heat-shocked (80 °C/10 min) B. cereus spores to obtain a final spore concentration of approximately 2 log CFU/g. Thereafter, growth was determined at isothermal temperatures starting at 10 °C and every three degrees up to 49 °C. Samples were removed periodically and plated on mannitol egg yolk polymyxin agar. The plates were incubated for 24 hr at 30 °C. Baranyi, Huang, and modified Gompertz primary growth models were used to fit growth data. The modified Ratkowsky secondary model was used to fit growth rates determined by the primary growth models with respect to temperature. All three primary models fitted the growth data well. The modified Ratkowsky secondary model adequately fit growth rates generated by the three primary models (R2 values ranging from 0.96 to 0.98). After acceptable prediction zone (APZ) validation and goodness of fit statistical analyses, it was determined that the Baranyi primary growth model was best suited for these data. For both single-rate exponential cooling and biphasic linear cooling model validation, all Baranyi model predictions (n = 24 and 28, respectively) fell within the APZ (-1.0 to 0.5 log CFU/g). The model will assist institutional food service settings to determine the safety of cooked pasta subjected to longer cooling times or stored at improper temperatures. PRACTICAL APPLICATION: Predictive model can be used to estimate extent of microbial growth during cooling of cooked pasta and in designing HACCP program and setting of critical control levels. Retail food industry would need fewer challenge studies to validate the safety of their products. The model will provide regulatory agencies and food industry with an objective means of assessing the microbial risk and ensuring that the public is not at risk of acquiring food poisoning.

Determination of enterotoxin genes in bacillus cereus from raw milk using multiplex polymerase chain reaction technique

A Gram-positive bacterium, which can usually be found in cultivating plants and soil, causing it commonly universal is regarded as Bacillus cereus. It is said that most strains present in B. cereus is specifically called the primary cause of food poisoning illnesses which is characterised by either emetic or diarrheal syndrome, the most essential purpose of the current research was, therefore, to isolate and identify the enterotoxin genes in B. cereus through using a multiplex polymerase chain reaction (mPCR)technique. To achieve this aim, thus B.cereus was isolated from raw milk then cultured on mannitol egg yolk polymyxin agar and chromogenic B. cereus agar. The samples were then examined using biochemical investigation then established using partial sequencing of 16SrDNA. Among 40 samples, 20 of them were found to contain hblC and hblD, a hemolytic enterotoxin (HBL) complex encoding genes. The total mean of contamination samples in the B. cereus was proved to be higher (1.4×105 – 3.2×105 CFU/mL) than the acceptable detection level of B. cereus in dairy products ~103 CFU/mL. The isolates (10%) were shown to express hblC gene whereas the isolates (20%) were shown to express the hblD gene. The findings suggested that the detected enterotoxin genes of B. cereus which were found in raw milk may create a potential danger to the health of the public.

Predictive model for growth of Bacillus cereus during cooling of cooked rice

Bacillus cereus is frequently implicated in foodborne outbreaks associated with the consumption of cooked rice. The main contributing factors leading to outbreaks is rice cooked in large quantities and subsequently, inadequately chilled or stored at room temperatures for a prolonged period of time prior to consumption. Bacillus cereus growth in cooked rice inoculated with approximately 2 log CFU/g of heat-shocked (80 °C/10 min) spores at several isothermal conditions (between 10 and 49 °C) was quantified. B. cereus populations were determined by plating on mannitol egg yolk polymyxin agar and incubating at 30 °C for 24 h. Primary growth models, namely Baranyi, Huang, modified Gompertz, and logistic models were fitted to growth data. Specific growth rates from all four primary models were used to fit the modified Ratkowsky square-root model with respect to temperature. All four primary models were well fitted by the modified Ratkowsky model (R2 values from 0.90–0.99). Based on the goodness of fit secondary model statistics (R2, SSE, RMSE), the Baranyi model performed the best and was chosen for tertiary modeling. Acceptable prediction zone (APZ) analysis was performed for validation of the Baranyi model predictions during single rate exponential and biphasic linear cooling temperature profiles. For single rate cooling, 23 of the 24 predictions fell within the APZ (−1.0 to 0.5 log CFU/g). For biphasic linear cooling, 26 of the 28 predictions fell within the APZ. The developed dynamic model can be used to predict potential B. cereus growth from spores in cooked rice during chilling and thus, support the disposition of product subject to cooling deviations.

Influence of Cooling Rate on Growth of Bacillus cereus from Spore Inocula in Cooked Rice, Beans, Pasta, and Combination Products Containing Meat or Poultry

The objective of this study was to assess the ability of Bacillus cereus spores to germinate and grow in order to determine a safe cooling rate for cooked rice, beans, and pasta, rice-chicken (4:1), rice-chicken-vegetables (3:1:1), rice-beef (4:1), and rice-beef-vegetables (3:1:1). Samples were inoculated with a cocktail of four strains of heat-shocked (80°C for 10 min) B. cereus spores (NCTC 11143, 935A/74, Brad 1, and Mac 1) to obtain a final spore concentration of approximately 2 log CFU/g. Thereafter, samples were exponentially cooled through the temperature range of 54.5 to 7.2°C in 6, 9, 12, 15, 18, and 21 h. At the end of the cooling period, samples were removed and plated on mannitol egg yolk polymyxin agar. The plates were incubated at 30°C for 24 h. The net B. cereus growth from spores in beans was <1 log after 9 h of cooling, but the pathogen grew faster in rice and pasta. In combination products, the net growth was as follows: 3.05, 3.89, and 4.91 log CFU/g in rice-chicken; 3.49, 4.28, and 4.96 log CFU/g in rice-beef; 3.50, 4.20, and 5.32 CFU/g in rice-chicken-mixed vegetables; and 3.68, 4.44, and 5.25 CFU/g in rice-beef-mixed vegetables after 15, 18, and 21 h of cooling, respectively. This study suggests safe cooling rates for cooling cooked rice, beans, pasta, rice-chicken, rice-chicken-vegetables, rice-beef, and rice-beef-vegetables to guard against the hazards associated with B. cereus.

Dynamic Predictive Model for Growth of Bacillus cereus from Spores in Cooked Beans

Kinetic growth data for Bacillus cereus grown from spores were collected in cooked beans under several isothermal conditions (10 to 49°C). Samples were inoculated with approximately 2 log CFU/g heat-shocked (80°C for 10 min) spores and stored at isothermal temperatures. B. cereus populations were determined at appropriate intervals by plating on mannitol-egg yolk-polymyxin agar and incubating at 30°C for 24 h. Data were fitted into Baranyi, Huang, modified Gompertz, and three-phase linear primary growth models. All four models were fitted to the experimental growth data collected at 13 to 46°C. Performances of these models were evaluated based on accuracy and bias factors, the coefficient of determination ( R2), and the root mean square error. Based on these criteria, the Baranyi model best described the growth data, followed by the Huang, modified Gompertz, and three-phase linear models. The maximum growth rates of each primary model were fitted as a function of temperature using the modified Ratkowsky model. The high R2 values (0.95 to 0.98) indicate that the modified Ratkowsky model can be used to describe the effect of temperature on the growth rates for all four primary models. The acceptable prediction zone (APZ) approach also was used for validation of the model with observed data collected during single and two-step dynamic cooling temperature protocols. When the predictions using the Baranyi model were compared with the observed data using the APZ analysis, all 24 observations for the exponential single rate cooling were within the APZ, which was set between -0.5 and 1 log CFU/g; 26 of 28 predictions for the two-step cooling profiles also were within the APZ limits. The developed dynamic model can be used to predict potential B. cereus growth from spores in beans under various temperature conditions or during extended chilling of cooked beans.

Detection of presumptive Bacillus cereus in the Irish dairy farm environment

AbstractThe objective of the study was to isolate potentialBacillus cereussensu lato (B.cereus s.l.)from a range of farm environments. Samples of tap water, milking equipment rinse water, milk sediment filter, grass, soil and bulk tank milk were collected from 63 farms. In addition, milk liners were swabbed at the start and the end of milking, and swabs were taken from cows’ teats prior to milking. The samples were plated on mannitol egg yolk polymyxin agar (MYP) and presumptiveB. cereus s.l. colonies were isolated and stored in nutrient broth with 20% glycerol and frozen at -80 °C. These isolates were then plated on chromogenic medium (BACARA) and colonies identified as presumptiveB. cereus s.l. on this medium were subjected to 16S ribosomal RNA (rRNA) sequencing. Of the 507 isolates presumed to beB. cereus s.l. on the basis of growth on MYP, only 177 showed growth typical ofB. cereus s.l. on BACARA agar. The use of 16S rRNA sequencing to identify isolates that grew on BACARA confirmed that the majority of isolates belonged toB. cereus s.l. A total of 81 of the 98 isolates sequenced were tentatively identified as presumptiveB. cereus s.l. Pulsed-field gel electrophoresis was carried out on milk and soil isolates from seven farms that were identified as having presumptiveB. cereus s.l. No pulsotype was shared by isolates from soil and milk on the same farm. PresumptiveB. cereus s.l. was widely distributed within the dairy farm environment.

Diagnostic properties of three conventional selective plating media for selection of Bacillus cereus, B. thuringiensis and B. weihenstephanensis

The aim of this study was to assess the diagnostic properties of the two selective plating media and a chromogenic medium for identification of Bacillus cereus. The 324 isolates were B. cereus (37%), Bacillus weihenstephanensis (45%) or Bacillus thuringiensis (18%), as identified by a new combination of techniques. All isolates were growing on mannitol-egg yolk-polymyxin agar (MYP), and they did not form acid from mannitol. However, a significant lower number of B. thuringiensis isolates did not show lecithinase activity. All isolates were also growing on polymyxin-egg yolk-mannitol-bromothymol blue agar (PEMBA); however, 11% isolates indicated that they did produce acid from mannitol, and 15% isolates did not show any lecithinase activity. Five of the isolates did not grow at all on the chromogenic agar, and 14 of the growing isolates were β-glucosidase negative. It is concluded that the two recommended selective plating media MYP and PEMBA for detection of B. cereus group bacteria both have their limitations for identification of some B. cereus, B. weihenstephanensis or B. thuringiensis. However, MYP is preferable compared to PEMBA. The chromogenic medium has its own advantages and limitations, and some of the limitations seem to be solved by incubation at 30°C instead of the recommended 37°C.

Prevalence of Bacillus cereus in Rabbit Meat Consumed in Burdur-Turkey, Its Enterotoxin Producing Ability and Antibiotic Susceptibility

Summary This study was undertaken to determine the existence, enterotoxin producing ability and antibiotic susceptibility of Bacillus cereus at rabbit meat. B. cereus was enumerated by the surface plating method with mannitol egg yolk polymyxin agar. The BCET-RPLA test was used for detecting of diarrheal type-enterotoxin. The disk diff usion method was used for antimicrobial sensitivity test. B. cereus was found in 18 (36%) of 50 samples of rabbit meat, with the mean contamination level of 2.89x10 3 cfu/g in positive samples. The 8 (44.4%) of the total isolates of B. cereus was able to produce enterotoxin. While antibiotic resistance of B. cereus isolates was found to be 100% penicillin, 94.4% ampicillin, 27.7% streptomycin, 22.2% gentamicin and erythromycin, no resistance was detected to chloramphenicol and vancomycin.

Occurrence of Bacillus sporothermodurans and other aerobic spore-forming species in feed concentrate for dairy cattle.

To determine the aerobic spore composition and presence of Bacillus sporothermodurans spores in feed concentrate for dairy cattle. Six feed concentrate samples from five different farms were analysed. High levels of spores (up to 10(6) spores g(-1)) were found. Identification of 100 selected isolates was obtained by a combination of fatty acid methyl esters analysis, amplified ribosomal DNA restriction analysis and 16S rDNA sequencing. Ninety-seven isolates could be identified to the species level or assigned to a phylogenetic species group. Most of the isolates obtained after a heat treatment of 10 min at 80 degrees C were identified as members of the B. subtilis group (32 isolates), B. pumilus (25 isolates), B. clausii (eight isolates) and B. licheniformis (eight isolates). The isolates with very heat-resistant spores, obtained after a heat treatment of 30 min at 100 degrees C, were identified as members of the B. subtilis group (five isolates), B. sporothermodurans (three isolates), B. amyloliquefaciens (one isolate), B. oleronius (one isolate) and B. pallidus (one isolate). Bacillus cereus was present in each feed concentrate sample and was isolated using a selective mannitol egg yolk polymyxin agar medium. Feed concentrate for dairy cattle contains known as well as as yet unknown species of Bacillus and related genera with properties relevant to the dairy sector. The results formulate the hypothesis that feed concentrate can be a contamination source of spores, including those of B. sporothermodurans, for raw milk at the farm level.

Isolation and enumeration of Bacillus cereus from foods on a novel chromogenic plating medium

Bacillus cereus is an aerobic sporeformer commonly found in raw and processed foods. Foodborne illnesses associated with this pathogen are caused primarily by consumption of cooked foods with inadequate refrigeration. Mannitol-egg yolk-polymyxin (MYP) agar is widely used for isolation and enumeration of the pathogen. In the present study, a new selective medium, which contains a chromogenic substrate (5-bromo-4-chloro-3-indoxyl- myo -inositol-1-phosphate) for the detection of phosphatidylinositol-specific phospholipase C in B. cereus/B. thuringiensis organisms, was evaluated and compared to MYP. Twenty B. cereus strains, four B. thuringiensis strains, 95 other Gram-positive and 14 Gram-negative organisms were examined on BCM®B. cereus/B. thuringiensis (BioSynth) and MYP agar. Only B. cereus and B. thuringiensis formed large (2–7 mm), turquoise, flat colonies±turquoise halos on BCM®after incubation for 24 h at 35°C, while other Bacillus spp. formed white, 1–2 mm colonies or were inhibited. Some Listeria strains formed <1-mm colonies, white or turquoise, on the medium. Of the Gram-positive non- B. cereus/B. thuringiensis organisms tested, growth of >50% was inhibited on BCM®, compared to <1% on MYP. No significant difference (P>0·05) was observed on the two media in the recovery of B. cereus strains from foods inoculated or naturally contaminated with the organisms. However, colony enumeration and isolation were easier on BCM®than on MYP because of higher selectivity and formation of discrete, non-coalescing colonies. Ribotyping (Riboprinter®Microbial Identification System, Qualicon) of five randomly selected food isolates identified three B. cereus and two B. thuringiensis strains. Agar plates were stable for >3 months at 2–5°C.

Rapid Identification of Bacillus cereus Based on the Detection of a 28.5-Kilodalton Cell Surface Antigen

Conventional procedures for the identification of suspect Bacillus cereus isolated on mannitol-egg yolk-polymyxin(MYP) agar may need several days. To facilitate the identification of the bacterium, an enzyme-linked immunosorbent assay (ELISA) was developed. The assay was based on the detection of a 28.5-kDa cell surface antigen of B. cereus. Bacterial colonies grown on MYP agar or nutrient agar were suspended in phosphate-buffered saline (pH 7.2) containing 0.1% Teepol. The cell suspensions were heated at 100°C for 5 min and added to the microtiter plates coated with antibodies against the 28.5-kDa antigen. After washing, the same antibodies labeled with horseradish peroxidase were used as secondary antibodies to reveal the signal of antigen-antibody reaction. For 38 strains of B. cereus and 127 strains of non-B. cereus bacteria (including 79 isolates of Bacillus spp.) tested, the sensitivity and specificity of the ELISA were 100 and 88.2%, respectively. Strains producing false-positive results were members of the B. cereus group (i.e., Bacillus anthracis, Bacillus mycoides, and Bacillus thuringiensis), which are genetically and biochemically similar to B. cereus. Similar ELISA results were obtained by using antibodies against another cell surface antigen with a molecular mass of 20 kDa. If members of the B. cereus group were recognized as a single species, the sensitivity and specificity of the ELISA were 100 and 99.1%, respectively. The ELISA could be used as a rapid method for presumptive identification of B. cereus grown on MYP agar.

Evaluation of the ISO 7932 standard for the enumeration of Bacillus cereus in foods

Twenty, mostly European laboratories took part in a collaborative study to validate the general 1993 ISO 7932 standard for the enumeration of Bacillus cereus in foods (Anonymous, ISO 7932, Microbiology – General Guidance for the Enumeration of Bacillus cereus – Colony-count Technique at 30°C. International Organisation for Standardization, Geneva, Switzerland, 1993). The objective was to determine the precision data in terms of repeatability ( r) and reproducibility ( R) of the method using three different food types at various inoculum levels. The results are intended for publication in Comité Européen de Normalisation (CEN) and ISO standards. The method was challenged with three types of food product: fresh cheese, minced beef and potato powder. Each participant received eight samples for each food type: blind duplicates at four inoculum levels (target values of B. cereus of 0, 10 3, 10 4 and 10 5–10 6 colony forming particles (cfp)/g). In addition, two reference materials (RMs, capsules containing milk powder, artificially contaminated with B. cereus) were included in the study. All test materials were tested extensively for homogeneity and stability prior to the collaborative trial. In addition to determining the precision parameters of the ISO method, polymyxin pyruvate egg yolk bromothymol blue agar (PEMBA, incubation at 37°C) was included in the study to evaluate possible differences in performance compared to mannitol egg yolk polymyxin agar (MEYP, incubation at 30°C) which is prescribed in the ISO standard. In this study no difference in performance between MEYP and PEMBA medium was observed. Results from the glucose fermentation confirmation test indicated that generally 48 h was needed to obtain a yellow colour throughout the whole test tube. A high number of false negative results with the Voges Proskauer (VP) reaction, even after 48 h of incubation of the tubes, was observed in some laboratories. Values for r and R were therefore calculated without VP test results. Further studies have been initiated by the ISO technical committee in order to improve the performance of the confirmation tests. The precision of the test method appeared to be unaffected by the type of food or the concentration of B. cereus present in the test sample. The overall repeatability value found for the food samples was 0.29 log 10 units and the overall reproducibility value was 0.42 log 10 units. For the reference material, the repeatability was 0.11 log 10 units and the reproducibility 0.22 log 10 units. It was recommended to CEN and ISO to include these values into the revised horizontal standard for the enumeration of Bacillus cereus.