Hbl Genes Research Articles

Biocontrol of Bacillus cereus by Lactobacillus plantarum in Kareish cheese and yogurt

This study aims to biocontrol of Bacillus cereus by Lactobacillus plantarum in Kareish cheese and yogurts. The minimum inhibitory concentrations (MICs), antioxidant potentials, total flavonoids content (TFC) and total phenolic content (TPC) of L. plantarum were also estimated. Results showed that incidence of B. cereus in Kareish cheese and yogurt was 16 and 4%, respectively. Four virulence genes were investigated by PCR in B. cereus isolates (n = 10). Two toxin producing genes, cytotoxin K (cytK), and phosphatidylcholine-hydrolyzing phospholipase C (Pc-plc), were detected in all B. cereus, whereas enterotoxigenic (nhe) and hemolysin BL (hbl) genes were detected in 90 and 50%, respectively. All isolates were vulnerable to erythromycin and gentamicin (100%) with intermediate sensitivity to ciprofloxacin and complete resistance to tetracycline (100%). L. plantarum showed antibacterial power against B. cereus EMCC1006 reference strain with MIC at 3.1 mg/mL. From the different concentrations (1.5, 3.1, 6.25, 12.5, 25.0, 50.0, 100.0 mg/mL) of L. plantarum which mixed with Kareish cheese and yogurt samples, the minimum concentrations displayed the excellent sensory parameters. TPC and TFC of L. plantarum CFS were 18.5 (μg GAE/g) and 2.67 (μg QE/g), respectively. Regarding antioxidant activity, IC50 of L. plantarum was 53.84 μg/mL, while IC50 of ascorbic acid was 26.36 μg/mL. In sum, L. plantarum could be used as a promising antibacterial and antioxidant agent for biocontrol of B. cereus to ensure dairy safe without negative impact on sensorial attributes.

Newly Emerging MDR B. cereus in Mugil seheli as the First Report Commonly Harbor nhe, hbl, cytK, and pc-plc Virulence Genes and bla1, bla2, tetA, and ermA Resistance Genes.

BackgroundBacillus cereus is a common food poisoning pathogen in humans. This study aimed to investigate the prevalence, molecular typing, antibiogram profile, pathogenicity, dissemination of virulence and antibiotic resistance genes associated with natural B. cereus infection among Mugil seheli.MethodsConsequently, 120 M. seheli (40 healthy and 80 diseased) were obtained from private fish farms in Port-said Governorate, Egypt. Afterward, samples were processed for clinical, post-mortem, and bacteriological examinations. The recovered isolates were tested for antimicrobial susceptibility, phenotypic assessment of virulence factors, pathogeneicity, and PCR-based detection of virulence and antibiotic resistance genes.ResultsB. cereus was isolated from 30 (25%) examined fish; the highest prevalence was noticed in the liver (50%). The phylogenetic and sequence analyses of the gyrB gene revealed that the tested B. cereus isolate displayed a high genetic similarity with other B. cereus strains from different origins. All the recovered B. cereus isolates (n =60, 100%) exhibited β-hemolytic and lecithinase activities, while 90% (54/60) of the tested isolates were biofilm producers. Using PCR, the tested B. cereus isolates harbor nhe, hbl, cytK, pc-plc, and ces virulence genes with prevalence rates of 91.6%, 86.6%, 83.4%, 50%, and 33.4%, respectively. Moreover, 40% (24/60) of the tested B. cereus isolates were multidrug-resistant (MDR) to six antimicrobial classes and carried the bla1, bla2, tetA, and ermA genes. The experimentally infected fish with B. cereus showed variable mortality in direct proportion to the inoculated doses.ConclusionAs far as we know, this is the first report that emphasized the existence of MDR B. cereus in M. seheli that reflects a threat to the public health and the aquaculture sector. Newly emerging MDR B. cereus in M. seheli commonly carried virulence genes nhe, hbl, cytK, and pc-plc, as well as resistance genes bla1, bla2, tetA, and ermA.

Detection of Enterotoxin Genes in Bacillus species Isolated from Cassava Processing Environment in Nigeria

Introduction: The health challenges associated with cassava products as a common staple food for approximately 70 % of Africans and part of Asia pose a looming danger due to Bacillus enterotoxins’ presence in the processing environment. Objective: This study investigated the presence of enterotoxigenic genes, namely, Bacillus cereus enterotoxin T (bceT), hemolysin bl (hblC, hblD), and non-haemolytic enterotoxin (nheA, nheB, and nheC) from Bacillus species isolated from soil of cassava processing environment. Methods: Soil samples from 20 cassava processing sites in Ile-Ife and Modakeke, Nigeria, were collected and cultured on nutrient agar at 37ºC for 24 hours. Colonies phenotypically identified as Bacillus were identified using Bacillus-specific 16S rRNA-targeted PCR technique. Screened Bacillus spp were assessed for the presence of enterotoxigenic genes using PCR with previously reported primers. Results: A total of 100 Bacillus isolates were selected from this study, with Bacillus macerans (33 %) showing the highest frequency of occurrence among the identified species, however, 74 isolates were molecularly confirmed as Bacillus. Amongst the 74 molecularly confirmed Bacillus isolates, 28 (37.84 %), 35 (47.30 %), and 37 (50 %) had nhe, hbl, and bceT genes, respectively. Investigation showed that 42 (56.76 %) of the Bacillus species encoded at least one of the screened enterotoxin genes. Conclusion: The presence of these 3 sets of enterotoxin genes in Bacillus isolated from cassava processing sites calls for immediate attention as they could be pivotal in the release of toxins in cassava products, causing lethal effects via consumption. This study demonstrates the possibility of foodborne disease outbreaks in Bacillus toxin-laden cassava products processed under unhygienic conditions.

Aerobic spore-forming bacteria in powdered infant formula: Enumeration, identification by MALDI-TOF mass spectrometry (MS), presence of toxin genes and rpoB gene typing

This study aimed to assess the counts and biodiversity characterization of aerobic sporeforming bacteria (ASB) in powdered infant formula (PIF). Fifty-four (n = 54) samples of PIF of three brands were analyzed for mesophilic aerobic bacteria, and ASB counts. ASB isolated from PIF were assessed for their ability to produce spoilage enzymes and hemolytic activity and further identified by MALDI-TOF mass spectrometry. Then, the isolates were subjected to rpoB gene typing and assessment of bceT, entFM, nhe (A, B, C), and hbl (A, B, C) toxin genes. The main species isolated were B. licheniformis (54%), followed by B. cereus (33%) and B. subtilis (5%). The ASB counts ranged from 1 to 4 log CFU/g, and the mean was 2.9 log CFU/g for mesophilic aerobic sporeforming bacteria (MSC) and 2.5 log CFU/g for thermophilic aerobic sporeforming bacteria (TSC). Most PIF samples presented MSC and TSC counts between 2 and 3 log CFU/g. A total of 13%, 50%, and 37% of the samples presented MSC counts from <2 log CFU/g, between 2 and 3 log CFU/g and between 3 and 4 log CFU/g, respectively. Among the ASB isolates, 97% had protease, 84% hydrolyzed starch, 66% had hemolytic activity, and 61% had lecithinase activity. A total of 44 out of 120 isolates harbored at least one toxin gene; 56% for B. cereus, 34% for B licheniformis, and less than 5% for B. subtilis, B pumilus, and L. sphaericus. All B. cereus isolates harbored the nhe gene, 60% entFM, 44% cytK, 32% bceT, and 28% hbl genes. Besides, 17% of B. licheniformis harbored nhe. A small proportion of B. subtilis, B. pumilus, and L. sphaericus carried toxin genes. The rpoB based phylogenetic tree provided high resolution among Bacillus species. The findings of this study provide insights into the phenotypic and genotypic biodiversity of Bacillus present in PIF.

Comprehensive Study about the Identification of Bacillus Cereus Isolated from Chicken Farms

Bacillus cereus is one of the opportunistic foodborne bacteria producing food poisoning producing severe economic loss and representing a high risk to the human. The contamination of poultry feed with Bacillus cereus produces gastric damage and facilitates other bacterial infections. For these reasons, fresh fecal matters were collected from poultry farms located in 3 Egyptian Governorates (Giza, Gharbia, and Dakahlia) to isolate and identify Bacillus cereus, determine their susceptibility to different antimicrobial agents, and examine their virulence and resistance factors. Eleven B. Cereus out of 50 examined samples was isolated with a percentage of (11/50, 22%) and subjected to confirmation using the VITEK2 instrument. Disc diffusion tests showed that all isolates were resistant to trimethoprim-sulfamethoxazole, imipenem, cefotaxime, ampicillin, amoxicillin/clavulanic acid, and penicillin G and sensitive to chloramphenicol, gentamicin, and ciprofloxacin. The molecular characterizations of virulence and resistance factors revealed the detection of targeting the groEL gene in all isolates (100%). The toxin gene profiling showed the existence of the nhe gene (90.9%) and the hbl genes (18.18%). The ces and cytk genes were not detected in the examined isolates. Two antimicrobial resistance genes were detected in the examined isolates with (33.33%) for the tetA gene and (45.45%) for bla gene. Therefore, it was concluded that the isolation of B. cereus carrying genes responsible for antimicrobial resistance and toxins production in broiler farms, represents major threats for human health and poultry industry. So that control programs of biosecurity should be implemented in poultry farms to mitigate the losses of B. cereus infection.

Psychrotrophic properties, toxigenic characteristics, and PFGE profiles of Bacillus cereus isolated from different foods and spices

ABSTRACT: Bacillus cereus is an aerobic and facultatively anaerobic, spore-forming bacterium, and it is found naturally in soil and poses a risk factor for the contamination of food and foodstuffs including cereals, vegetables, spices, ready-to-eat (RTE) foods, meats, milk, and dairy products. This study determined the prevalence of B. cereus in raw poultry meat, raw cow’s milk, cheese, spices, and RTE foods in Hatay province. The study also analysed the psychrotrophic properties, toxigenic characteristics, and pulsed-field gel electrophoresis (PFGE) profiles of the isolates. The levels of contamination with B. cereus determined for cheese, raw milk, RTE foods, spices, and raw poultry meat were 16.6%, 34.2%, 42.8%, 49%, and 55.5%, respectively. B. cereus was isolated from 84 (42%) of the 200 samples analysed and the 84 isolates were verified by PCR analysis targeting the haemolysin gene specific for B. cereus. Of the total isolates, 64 (76.1%) were psychrotrophic. The toxin gene profiling of B. cereus isolates was determined by amplifying the four genes nhe, hbl, cytK, and ces. The nhe and cytK genes were most frequently detected in the isolates, while the hbl and ces genes were not found. In addition, a high genetic relationship between the isolates was detected at a 92% similarity level by PFGE analysis. In conclusion, the occurrence of both psychrotrophic and toxigenic B. cereus strains in this study indicated a potential risk for food spoilage and food poisoning.

Bacillus cereus in Packaging Material: Molecular and Phenotypical Diversity Revealed.

The Bacillus cereus group has been isolated from soils, water, plants and numerous food products. These species can produce a variety of toxins including several enterotoxins [non-hemolytic enterotoxin (Nhe), hemolysin BL (Hbl), cytotoxin K, and enterotoxin FM], the emetic toxin cereulide and insecticidal Bt toxins. This is the first study evaluating the presence of B. cereus in packaging material. Among 75 different isolates, four phylogenetic groups were detected (II, III, IV, and VI), of which the groups III and IV were the most abundant with 46.7 and 41.3%, respectively. One isolate was affiliated to psychrotolerant group VI. Growth experiments showed a mesophilic predominance. Based on PCR analysis, nhe genes were detectable in 100% of the isolates, while hbl genes were only found in 50.7%. The cereulide encoding gene was found in four out of 75 isolates, no isolate carried a crystal toxin gene. In total, thirteen different toxin gene profiles were identified. We showed that a variety of B. cereus group strains can be found in packaging material. Here, this variety lies in the presence of four phylogenetic groups, thirteen toxin gene profiles, and different growth temperatures. The results suggest that packaging material does not contain significant amounts of highly virulent strains, and the low number of cereulide producing strains is in accordance with other results.

Multiplex Polymerase Chain Reaction for Detection of Toxin Genes of Bacillus cereus Group Isolated from Meat and Chicken Products

Bacillus cereus sensu lato (B. cereus s. l.) is a significant cause of food spoilage issue owing to the activity of certain hydrolytic enzymes. This study aimed to detect the incidence and contamination level of meat and chicken products with B. cereus group in Sharkia Governorate, Egypt, with reference to their enterotoxin genes` profiles. Overall, 43 out of 200 (21.5%) examined samples were contaminated by B. cereus group, with identification of only one species, B. cereus. B. cereus group isolates were frequent in chicken samples (25.71%), with the highest incidence in chicken meat (30%) followed by chicken sausage and chicken luncheon (25% each). Meanwhile, they were isolated from 19.23% of examined meat products, which predominated in meat burger (25%), followed by each of meat kofta, shawarma, and luncheon (20% each), minced meat (17.14%) and meat sausage (15%). Of interest, the highest B. cereus count (>104 colony forming units (CFU)/ g) was found in 2% of positive samples, with a higher percent in meat sausage (33.33%). Whereas 15.5% of positive samples harbored B. cereus with counts ranging from >1x103-104 CFU/g. Molecular analysis of B. cereus enterotoxin genes using multiplex polymerase chain reaction (PCR) revealed that both ces and nhe genes were detected in 100% of the examined isolates, while cytk and hbl genes were present only in 9.52% and 23.8% of analyzed isolates, respectively. These findings, involving a higher occurrence of B. cereus and their toxin genes in meat and chicken products represent a serious public health concern in Egypt.

Simultaneous detection of food pathogens, Staphylococcus aureus, Salmonella Enterica, Bacillus cereus and Vibrio parahaemolyticus by multiplex real-time polymerase chain reaction

The study established a multiplex real-time polymerase chain reaction (PCR) for simultaneous detection and quantitation of Staphylococcus aureus, Salmonella Enterica, Bacillus cereus and Vibrio parahaemolyticus in the same reaction at one time, thus reducing the amounts of reagents and cutting down on the labor and time. Four sets of genetic marker specific primers corresponding to SAOUHSC-02297, invA, hbl and tlhA gene, and probes were labeled with FAM, HEX, TEXAS RED and Cy5 respectively. Multiplex real-time PCR was carried out with an iQ™ Multiplex Powermix Kit (BIO-RAD) Sequence Detection System. The result showed that the correlation coefficient between multiplex real-time PCR estimates and plate counts of 10-serial dilutions in specific bacteria was above 0.98, independent of 101-4-fold numbers of three other pathogenic strains. With optimized conditions, the lowest detection concentrations of four pathogens were 102.5 CFU/mL for Sta. aureus, S. Enterica and V. parahaemolyticus and 103.5 CFU/mL for B. cereus. The duration of the entire experiment from DNA isolation and purification to PCR amplification was less than 12 h.

Enterotoxigenic Profiles of Bacillus Cereus Isolated from Manufacturing Steps of Traditional Soft Cheese Plant

Microbial contamination of soft cheese may originate from various sources such as contaminated equipment or from the workers themselves during cheese production. The study was aimed to characterize Bacillus cereus using culture-based and PCR techniques among 285 samples along Damietta and Kareish cheese producing line in a small cheese plant in Alexandria governorate including raw milk, cheese, whey, rennet, water, and swabs from all contacts surfaces. Overall of 10.90 % and 10 % among raw milk, cheese, whey, rennet, water, and swabs from all contacts surfaces were positive for Bacillus species in Damietta and Kareish cheese producing line. The incidence of Bacillus cereus in receiving raw milk, curd, whey, fresh cheese samples were 6.66, 13.33 and 13.33%, respectively for traditional Damietta cheese plant. Bacillus cereus is found in only 6.66 % in receiving raw milk, pasteurized milk and fresh cheese samples for traditional Kareish cheese plant. Hbl, Nhe and Cytk genes were detected in Bacillus cereus isolated from curd and Damietta cheese. Nhe gene was detected in Bacillus cereus isolated from raw milk, pasteurized milk and Kareish cheese While, Ces gene was detected only in Bacillus cereus isolated from fresh Kareish cheese samples. The prevalence of Eneterotoxigenic virulent B. cereus poses public health threats to consumers. HACCP should be implemented for all steps of manufacture in order to produce a safe and good quality dairy product.

Multifaceted toxin profile of Bacillus probiotic in newly isolated Bacillus spp. from soil rhizosphere

Probiotic bacteria mainly Lactic Acid Bacteria (LAB) demonstrate to equilibrate the intestinal microflora. Strains of the Bacillus species have been extensively used as probiotics for human beings, animal feed as well as plant promoting bacteria. Attempts were made to characterize and assess the safety of Bacillus strains for their putative virulence factors. The present study includes screening the isolates by hemolysis on blood agar, DNase, lecithinase and cytotoxic activity as well as detection of toxins in the culture medium. PCR technique was used to detect genes encoding enterotoxin T (bceT), cytotoxin K (CytK), enterotoxin FM (EntFM), non-hemolytic enterotoxin (NheA, NheB), haemolysin BL (HblA, HblC, HblD) and emetic toxin (ces). Among seven Bacillus isolates, only isolates B. cereus sp. M14, B. subtilis sp. M12 and B. methylotrophicus sp. M8 showed weak hemolysis on blood agar. DNase and lecithinase activity were not observed for all isolates. However, isolates B. subtilis sp. M12 and B. cereus sp. M14 indicated low cytotoxic effects on HepG2 cell line. Using two commercial immunoassay kits, no enterotoxin was detected for all isolates. Nhe and Hbl genes were detected in isolates B. methylotrophicus sp. M8 and B. cereus sp. M14, respectively. In contrast, none of the Bacillus isolates harbored bceT, entFM and ces genes in their chromosomes. Taking together, among the evaluated Bacillus isolates, strains B. pumilus sp. M17 and B. pumilus sp. S10 found to be the most promising candidates to fulfill the safety assessments of putative virulence factors suitable for human consumption.

Enterotoxigenic structures of Bacillus cereus strains isolated from ice creams

AbstractThis study was conducted to investigate the presence of Bacillus cereus in ice cream samples and to identify associated toxin genes by mPCR. 125 ice cream samples were used as material. A total of 38 samples were found to be positive for B. cereus. It was found that 31.9% of the isolates had three enterotoxic HBL complex encoding genes, 10.6% had two hbl genes and 6.3% contained one hbl gene. On the other hand, 15.9% of the isolates contained three NHE complex encoding genes, 31.9% had two nhe genes and 20.2% contained one nhe gene. Also 7.4% of isolates were found to contain both NHE and HBL complexes while ctyK1 was not detected from any isolate. The presence of B. cereus and their enterotoxigenic genes in ice creams may be a potential risk for public health.Practical applicationsThe presence of the B.cereus in high numbers and the toxins in foods pose a potential risk in terms of health and food spoilage. In food poisoning cases, hbl, nhe, cytK, and the effect of emetic toxin are especially notable. The resistance of spores against pasteurization and psychrotolerant feature enable the explanation of the existence of B. cereus in ice‐cream.

Detection of toxin genes and randomly amplified polymorphic DNA typing of Bacillus cereus isolates from infant milk formulas

AbstractBacillus cereus, a ubiquitous human foodborne pathogen, can persist in different environment, including dry conditions. In this study, we explored the genetic diversity of B. cereus isolates from infant milk formulas. Out of 83 samples, B. cereus was confirmed in 26 (31.3%). Isolates were resistant to penicillin G and ampicillin (100%), trimethoprim‐sulfamethoxazole (84.6%), and erythromycin (3.8%). Ent, hbl, and cer genes were detected in 21 (80.8%), 2 (7.7%), and 11 (42.3%) isolates, respectively. Randomly amplified polymorphic DNA (RAPD) analysis, using set of three primers, provided an overall genetic distance of 95% between B. cereus isolates. A group of seven emetic strains and another two enterotoxic strains were indistinguishable by RAPD, revealing their high clonality. In conclusion, we detected a high proportion of pathogenic B. cereus isolates in infant milk formulas. The majority of isolates harboring both cer and ent genes showed high clonality, pointing food processing environment as probable contamination source.Practical applicationsDetection of Bacillus cereus in powdered infant milk formulas, the prevalence of emetic and diarrhegenic genes, and especially the simultaneous presence of both types of genes in the same isolate, confirm the importance of B. cereus as foodborne pathogen. The consumption of contaminated formula may cause serious health problems in babies, which emphasizes the need to improve B. cereus monitoring and characterization. The clonal relationship between strains, revealed by RAPD analysis, may point to the source of infection and the corresponding prevention measures to improve food safety. Moreover, the identical RAPD profile of cer positive strains obtained with AG15 primer enables the application of this analysis in screening for emetic B. cereus isolates.

Prevalence, molecular identification and characterization of Bacillus cereus isolated from beef burgers

AbstractThe 3‐component hemolysin BL (Hbl) and non‐hemolytic enterotoxin (Nhe) are probably associated with Bacillus cereus diarrheal outbreaks. In this study, toxin transcription of 18 biochemically positive isolates isolated from 80 beef burgers containing different percentage of meat (30, 60, and 90%) were evaluated using PCR method. Bacterial growth curves were further obtained by Bioscreen C to predict B. cereus population of uncooked and fried burgers kept at refrigerator and at room temperatures. The results showed that 22.5% of beef burgers (18 of 80 burgers) were contaminated with B. cereus where 14/18 and 5/18 of the samples contaminated to B. cereus with Nhe and Hbl genes, respectively. No significant growth was observed in burgers stored at 4 °C, although after 8 hr the uncooked burgers and within 10 hr the fried burgers stored at 20 °C were considered as spoiled by reaching B. cereus counts of &gt;106 cfu/g.Practical applicationsBacillus cereus is recognized as a causative agent of emetic and diarrheal foodborne poisoning. To differentiate the encoding chromosomal genes of Nhe (i.e., nheA, nheB, and nheC) and Hbl (i.e., hblC, hblD, and hblA), multiplex PCR was quite selective to perform.

Prevalence, virulence factor genes and antibiotic resistance of Bacillus cereus sensu lato isolated from dairy farms and traditional dairy products

BackgroundB. cereus are of particular interest in food safety and public health because of their capacity to cause food spoilage and disease through the production of various toxins. The aim of this study was to determine the prevalence, virulence factor genes and antibiotic resistance profile of B. cereus sensu lato isolated from cattle grazing soils and dairy products in Ghana. A total of 114 samples made up of 25 soil collected from cattle grazing farm land, 30 raw milk, 28 nunu (yoghurt-like product) and 31 woagashie (West African soft cheese). Ninety-six B. cereus sensu lato isolates from 54 positive samples were screened by PCR for the presence of 8 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK and entFM), and one emetic gene (ces). Phenotypic resistance to 15 antibiotics were also determined for 96 B. cereus sensu lato isolates.ResultsAbout 72% (18 of 25 soil), 47% (14 of 30 raw milk), 35% (10 of 28 nunu) and 39% (12 of 31 woagashi) were positive for B. cereus sensu lato with mean counts (log10 cfu/g) of 4.2 ± 1.8, 3.3 ± 2.0, 1.8 ± 1.4 and 2.6 ± 1.8 respectively. The distribution of enterotoxigenic genes revealed that 13% (12/96 isolates) harboured all three gene encoding for haemolytic enterotoxin HBL complex genes (hblA, hblC and hblD), 25% (24/96 isolates) possessed no HBL gene, whereas 63% (60/96 isolates) possessed at least one of the three HBL genes. All three genes encoding for non-haemolytic enterotoxin (nheA, nheB and nheC) were detected in 60% (57/96) isolates, 14% (13/96) harboured only one gene, 19% (18/96) whereas 8% possessed none of the NHE genes. The detection rates of cytk, entFM, and ces genes were 75, 67 and 9% respectively. Bacillus cereus s. l. isolates were generally resistant to β-lactam antibiotics such as ampicillin (98%), oxacillin (92%), penicillin (100%), amoxicillin (100%), and cefepime (100%) but susceptible to other antibiotics tested.ConclusionsBacillus cereus s. l. is prevalent in soil, raw milk and dairy products in Ghana. However, loads are at levels considered to be safe for consumption. Various enterotoxin genes associated with virulence of B. cereus are widespread among the isolates.

Characteristics of enterotoxin distribution, hemolysis, lecithinase, and starch hydrolysis of Bacillus cereus isolated from infant formulas and ready-to-eat foods

Bacillus cereus is a ubiquitous environmental microbe implicated as a main cause of food poisoning with various symptoms, depending on the strain type and the isolation source. In this study, the potential virulence factors and biochemical properties of B. cereus isolated from infant formulas and ready-to-eat (RTE) foods were analyzed and compared. A total of 347 B. cereus strains were isolated and identified from 687 infant food formulas and RTE food samples. All the isolates had one or more enterotoxin genes, and one-half of the strains had all 3 enterotoxin genes (hbl, nhe, and cytK) that are involved in food poisoning in humans. Here, all the 3 genes were detected in 50% of the B. cereus isolates from RTE foods and only 14% of the isolates were identified from infant formulas. The latter harbored low cytK and bceT, and very low hbl genes. Most B. cereus isolates possessed the hemolysis gene, but not the ces gene. The infant formula isolates showed stronger hemolysis activity than the other isolates. In addition, 26% of the total isolates showed low lecithinase activities and 10% showed high lecithinase activities. A greater number of isolates from the infant formula showed high lecithinase activity than those from the RTE foods. Approximately 83% of the isolates were positive and 17% were negative for starch hydrolysis. Over 90% of the RTE food isolates and only 35% of the infant formula isolates were positive for starch hydrolysis. However, all the strains possessed nhe, but their harboring patterns of hbl and cytK were significantly different. Most starch-hydrolyzing strains possessed hbl, but only 23% nonstarch-hydrolyzing isolates possessed this gene. Moreover, very low nonstarch hydrolyzing strains harbored cytK. Most nonstarch-hydrolyzing isolates showed high lecithinase and strong hemolysis activities, and very low hbl and cytK harboring. In summary, most infant formula isolates showed stronger hemolysis and higher lecithinase activities with lower frequency of harboring hbl and cytK and lower starch hydrolysis compared with RTE food isolates.

Comparative analysis of quantitative reverse transcription real-time PCR and commercial enzyme imunoassays for detection of enterotoxigenicBacillus thuringiensisisolates

Entomopathogenic Bacillus thuringiensis is closely related to Bacillus cereus, a human pathogen known to cause emesis and diarrhea. Standard detection methods do not distinguish these bacilli. Hemolysin BL (hbl) and non-hemolytic enterotoxin (nhe) genes that encode, respectively, HBL and NHE enterotoxins, are known to be harbored in both bacterial species, suggesting that differentiation of these bacilli is clinically and epidemiologically relevant. In this study the reliability of quantitative reverse transcription real-time PCR (qRT-PCR) and enzyme immunoassays (EIAs) in detecting hbl and nhe transcripts and corresponding toxins in environmental B. thuringiensis isolates was assessed. At least one enterotoxin gene was present in each isolate, and nhe or hbl genes were found in 85% and 55% of the strains, respectively. Based on statistical analyses, both BCET-RPLA and Duopath detected HBL at similar levels, and TECRA and Duopath can be used interchangeably for the detection of NHE, although TECRA has significantly lower sensitivity than Duopath. Thus, as potential enterotoxic B. thuringiensis strains occur in the natural environment, and EIA results may not correspond with the presence of enterotoxin genes and their expression, we suggest that reliable interpretation will be significantly enhanced by including qRT-PCR to support inferences based on EIAs.

The Bacillus cereus Hbl and Nhe Tripartite Enterotoxin Components Assemble Sequentially on the Surface of Target Cells and Are Not Interchangeable

Bacillus cereus is a spore-forming, Gram-positive bacterium commonly associated with outbreaks of food poisoning. It is also known as an opportunistic pathogen causing clinical infections such as bacteremia, meningitis, pneumonia, and gas gangrene-like cutaneous infections, mostly in immunocompromised patients. B. cereus secretes a plethora of toxins of which four are associated with the symptoms of food poisoning. Two of these, the non-hemolytic enterotoxin Nhe and the hemolysin BL (Hbl) toxin, are predicted to be structurally similar and are unique in that they require the combined action of three toxin proteins to induce cell lysis. Despite their dominant role in disease, the molecular mechanism of their toxic function is still poorly understood. We report here that B. cereus strain ATCC 10876 harbors not only genes encoding Nhe, but also two copies of the hbl genes. We identified Hbl as the major secreted toxin responsible for inducing rapid cell lysis both in cultured cells and in an intraperitoneal mouse toxicity model. Antibody neutralization and deletion of Hbl-encoding genes resulted in significant reductions of cytotoxic activity. Microscopy studies with Chinese Hamster Ovary cells furthermore showed that pore formation by both Hbl and Nhe occurs through a stepwise, sequential binding of toxin components to the cell surface and to each other. This begins with binding of Hbl-B or NheC to the eukaryotic membrane, and is followed by the recruitment of Hbl-L1 or NheB, respectively, followed by the corresponding third protein. Lastly, toxin component complementation studies indicate that although Hbl and Nhe can be expressed simultaneously and are predicted to be structurally similar, they are incompatible and cannot complement each other.

Occurence of potentially enterotoxigenic Bacillus cereus in infant milk powder

Considering that powdered infant milk formula effectively supports the growth of numerous pathogens, this study investigates the prevalence of potentially pathogenic Bacillus cereus in dried milk products by evaluating the occurrence of B. cereus and the presence of virulence-associated genes. The approach consisted of enriching, isolating and biochemical identifying isolates, followed by PCR assays aimed at the hbl (C, D, A, B), nhe (A, B, C) and cytK enterotoxin genes coding HBL complex, NHE complex and cytotoxin K, respectively. Among cytK-positive strains, the discrimination of two different forms for cytotoxin K, cytK-1 and cytK-2 was performed. Bacillus cereus was detected in powdered infant milk formula samples. All the strains harbored at least one gene of the cytK, HBL and NHE enterotoxins. Because of an increasing trend in invasive infections by B. cereus in infants and immunocompromised children, our PCR findings highlight the need to implement an adequate control plan in order to guarantee the health of potentially fragile consumers. From a hygiene point of view, intensive and continuous monitoring of potentially pathogenic B. cereus may be crucial for powdered infant milk formula safety and even recommended in order to assess the infant health risk, as proposed by Commission Regulation (EC) no. 1441/2007 on microbiological criteria for foodstuffs. Furthermore, the detection in this study of B. licheniformis, B. subtilis and B. mycoides strains raises significant health issues regarding Bacillus spp. in powdered infant milk formula.

Molecular identification and safety of Bacillus species involved in the fermentation of African oil beans (Pentaclethra macrophylla Benth) for production of Ugba

Molecular identification of Bacillus spp. involved in the fermentation of African oil bean seeds for production of Ugba, as well as ability of the Bacillus spp. isolated to produce toxins, were investigated. Forty-nine bacteria were isolated from Ugba produced in different areas of South Eastern Nigeria and identified by phenotyping and sequencing of 16S rRNA, gyrB and rpoB genes. Genotypic diversities at interspecies and intraspecies level of the isolates were screened by PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR) and repetitive sequence-based PCR (rep-PCR). The ability of the bacteria to produce toxins was also investigated by detection of genes encoding production of haemolysin BL (HblA, HblC, HblD), non-haemolytic enterotoxin (NheA, NheB, NheC), cytotoxin K (CytK) and emetic toxin (EM1) using PCR with specific primers. Moreover, a Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) was used to screen ability of the isolates to produce haemolysin in broth and during fermentation of African oil bean seeds.The isolates were characterized as motile, rod-shaped, endospore forming, catalase positive, Gram-positive bacteria. They were identified as Bacillus cereus sensu lato (42), Lysinibacillus xylanilyticus (3), Bacillus clausii (1), Bacillus licheniformis (1), Bacillus subtilis (1), and Bacillus safensis (1). B. cereus was the predominant Bacillus species and was present in all samples studied. Using ITS-PCR, interspecies diversity was observed among isolates, with six clusters representing each of the pre-cited species. Rep-PCR was more discriminatory (eight clusters) and allowed further differentiation at intraspecies level for the B. cereus and L. xylanilyticus isolates with two genotypes for each species. Genes encoding production of non-haemolytic enterotoxin (NheA, NheB, NheC) and cytotoxin K (CytK) genes were detected in all B. cereus isolates, while Hbl genes (HblA, HblC, HblD) were detected in only one isolate. The emetic-specific gene fragment was not detected in any of the isolates studied. None of the toxin genes screened was detected in isolates belonging to other Bacillus species. Using RPLA, haemolysin production was detected in one isolate of B. cereus, which showed positive amplicons for Hbl genes, both during cultivation in broth and during fermentation of oil bean seeds.