Cloning and Nucleotide Sequence Analysis of gyrB of Bacillus cereus, B. thuringiensis, B. mycoides, and B. anthracis and Their Application to the Detection of B. cereus in Rice (original) (raw)
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International Journal of PharmTech Research, 2015
The aim of this study has been an investigation of the presence of Bacillus cereus and detection of enterotoxigenic genes in cooked rice samples through utilizing a PCR technique. In this study the providence of B.cereus was carried out to cooked rice samples and the B.cereus isolates were investigated for enterotoxigenic gene. The cooked rice samples were purchased from several restaurants in the area of (Bangi, Kajang and UKM) Selangor, Malaysia. A total of 70 samples have been analyzed. B. cereus contamination has been formed between 1.2 × 10 4 to 1.6 × 10 6 cfu/g cooked of 110 colonies of tentative B. cereus have been tested onto mannitol egg yolk polymyxin agar and Chromogenic Bacillus cereus Agar, and 35 colonies have been detected as B. cereus using biochemical test and partial sequence of 16s r DNA sequences analysis. The B. cereus isolates that are BC1 to BC35 have been distinguished for hemolytic enterotoxin (HBL complex encoding gene (hblD), and ematic (ces) gene toxin. 12 isolates have been reported to be positive towards hblD, None of the B. cereus isolates have been found positive towards ematic(ces) gene. Therefore, the presence of B. cereus and their enterotoxigenic genes in cooked rice samples can be regarded as a potential risk for public health.
Detection of toxigenic Bacillus cereus and Bacillus thuringiensis spores in U.S. rice
International Journal of Food Microbiology, 2009
Bacillus cereus is a gram-positive, endospore forming pathogenic bacterium that is ubiquitous in the environment and is frequently associated with emetic and diarrheal types of foodborne illness. In this study, 178 samples of raw rice from retail food stores were analyzed for the presence of B. cereus spores. Spores of Bacillus species were found in 94 (52.8%) of the rice samples with an average concentration of 32.6 CFU/g (3.6-460 CFU/g for B. cereus and 3.6-23 CFU/g for Bacillus thuringiensis). Eighty three of the 94 isolates were identified as B. cereus and 11 were identified as B. thuringiensis. Bacillus mycoides (240 CFU/g) was the predominant isolate in one rice sample. Using PCR the isolates were checked for the presence of the cereulide synthetase gene (ces), the hblA and hblD genes of the hemolysin BL (HBL) complex and the nheA and nheB genes of the nonhemolytic (NHE) enterotoxin complex. The ces gene was not identified in any of the isolates. By contrast 47 (56.6%) B. cereus isolates possessed the hblA and hblD genes and 74 (89.1%) isolates possessed the nheA and nheB genes. As determined by commercial assay kits, forty four (53.0%) of the 83 B. cereus isolates produced both NHE and HBL enterotoxins whereas 78 (93.9%) were positive for either one or the other. Protein toxin crystals were detected visually in the 11 B. thuringiensis isolates. PCR analysis revealed 10 (90.9%) of those 11 isolates carried the cry gene. All the B. thuringiensis isolates were positive for NHE and HBL enterotoxins. Our results suggest that foodborne illness in the U.S. due to B. cereus with rice as the vehicle would be most likely associated with the diarrheal-type syndrome.
Enumeration, isolation and characterization of Bacillus cereus strains from Spanish raw rice
Food Microbiology, 2002
Bacillus cereus was present in 61 samples of raw rice analysed representing unhusked, husked and commercial origins. B. cereus in husked and white rice samples did not reach 10 2 cfu g À1 , while in the unhusked rice B. cereus densities exceeded 10 3 cfu g À1 . Processing steps such as drying, husking and polishing reduced the number of B. cereus in the ¢nal product. Eight strains with typical morphology of B. cereus on Polymyxin^Mannitol^EggYolk^Phenol Red Agar (PMYPA) were isolated. According to ISO con¢rmatory tests, the API System tests and supplementary tests of motility, oxidase activity and enterotoxin production, these isolates were characterized and identi¢ed as B. cereus. All strains were motile, oxidase-negative and produced diarrheal enterotoxin inTSB. D and z-values were used to characterize heat resistance of spores obtained from the eight strains of B. cereus characterized. A large diversity in heat resistance was observed among the isolates. At 901C, D-values ranged from 2?23 to 23?26 min, with ¢ve groups of D-value means signi¢cantly di¡erent at the 95% con¢dence level. D 95and D 100 values calculated for the eight strains ranged from 0?69 to 5?17 min and from 0?43 to 1?09 min, respectively. Statistical analysis revealed that there was signi¢cant di¡erence between the D-value means obtained for the strains at each temperature.The z-values for the eight strains of B. cereus tested in this study ranged from 7?421C to 8?201C with an average of 7?71C.
Bacillus cereus and Bacillus thuringiensis in ready-to-eat cooked rice in Malaysia
Bacillus cereus (B. cereus) isolates are toxigenic and can cause food poisoning. Cooked rice is a potentially hazardous food, especially in tropical countries. The aim of this study was to determine the prevalence of B. cereus and B. thuringiensis in raw and cooked rice marketed in Selangor, Malaysia. A combination of Most Probable Number-Polymerase Chain Reaction (MPN-PCR) method was used to detect gyrB gene in B. cereus and B. thuringiensis. Five local varieties of raw rice samples were negative for B. thuringiensis but all (100%) were positive for B. cereus. A total of 115 cooked rice samples (nasi lemak, nasi briyani, nasi ayam and nasi putih) were studied for the presence of B. cereus and B. thuringiensis. Nasi ayam was found to have the highest prevalence (100%) of B. cereus compared to nasi putih (76.2%) and nasi lemak (70.4%). Nasi briyani had the lowest prevalence (50%) of B. cereus. The frequencies of B. thuringiensis were found to be 10, 30 and 35.2 % in nasi putih and nasi ayam, nasi briyani and nasi lemak, respectively. Occurrence of B. cereus and B. thuringiensis in the samples ranged from < 3 to 1100 MPN/g in different samples. Maximum number of B. cereus was observed in nasi lemak, nasi briyani and nasi putih (> 1100 MPN/g) while nasi ayam showed less contamination (460 MPN/g) with B. cereus which was significantly different (P < 0.05) from others. The number of B. thuringiensis in nasi lemak, nasi briyani, nasi putih and nasi ayam were found to be >1100, 93, 9.2 and 3.6 MPN/g, respectively.
Current Journal of Applied Science and Technology
Aims: Experiments were conducted to evaluate the specificity and rapidity of the application of conventional, immunoassay and direct Polymerase Chain Reaction (dPCR) techniques in the detection of Bacillus cereus and its toxins in contaminated rice. Place and Duration of Study: Department of Life Sciences, Glasgow Caledonian University, UK, between May 2015 and December 2015. Methodology: Conventional testing for the presence of B. cereus and associated toxins was achieved using Polymixin Egg-yolk Mannitol Agar (PEMBA) culture plates while immunoassays were conducted using a commercially available Reverse Passive Latex Agglutination (TD0950 BCET-RPLA, Oxoid, UK) kit. Direct PCR was used to detect the HBL-E gene in the food samples and the PCR amplicons were visualised after separation by gel electrophoresis. Results: Total Mean B. cereus count was recorded as 5.3 ×107 cfu / g of the rice sample from the PEMBA plate culture. The PEMBA method was the least in terms of rapidity of assa...
International Journal of Food Microbiology, 2003
A method was developed to differentiate between Bacillus cereus, Bacillus mycoides and Bacillus thuringiensis using the polymerase chain reaction combined with a restriction endonuclease (PCR-RE) technique. This fast and simple protocol, applied to pure culture strains, was developed using the gyrB DNA sequence, as previously proposed by other authors. Strains from international collections were used to optimize the method which was then applied to the identification of strains isolated from food samples. Amplifications were specific for the B. cereus group. Only Staphylococcus aureus gave the same size PCR product, but it was easily differentiated from strains in the B. cereus group by using restriction analysis, based on digestion with the RsaI, Sau3AI and EcoRI endonucleases. Specific amplifications and good differentiations were obtained using pure strains, suggesting the possibility of using the method described to identify the B. cereus group directly in food samples. D
Food Control, 2010
A centrifugation-plating method was developed, using amylase and Tween 80 pre-treatment, for detection and enumeration of Bacillus spp. in rice products. The high sensitivity of this method improved detection of a variety of Bacillus species in rice products compared to the spread-plate method. Bacillus spp. were detected in 33 out of 35 raw rice samples with the centrifugation-plating method, but only 13 samples using the spread-plating method, even though 1 mL of a 10 À1 sample dilution was used, to increase sensitivity of the method. Known toxigenic or potentially toxigenic species (Bacillus cereus/Bacillus thuringiensis, Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus) were those most frequently found in both raw and cooked rice.
A Review of the development in the multiplex PCR technique for the detection of Bacillus cereus
Authorea (Authorea), 2023
Bacillus cereus is a Gram positive rod-shape bacterium, that causes severe food poisoning. Bacillus form spores that enable it to resist the environmental stresses, such as drought, heat, pH changes. Spores can remain dormant for many years, and back to the vegetative cells when suitable conditions available for bacterial growth. Several food poisoning outbreaks in cereal products showed that B. cereus was the main causative agent. Recently, the multiplex polymerase chain reaction (m-PCR) has provide a rapid and highly sensitive method for the detection of specific pathogenic microorganisms in the aquatic environment. To date, most m-PCR assays for pathogen detection have focused on only one, two or three different types of organisms. The lack of knowledge in the development of a multiplex PCR assay for the specific detection of Bacillus cereus inspire us to spot the light on the development of the method for Bacillus cereus detection in one rapid multiplex PCR assay and the potential application of Response Surface Methodology (RSM) for the simultaneous detection of other multi-pathogen systems.
Bangladesh Journal of Microbiology, 2023
The 'Bacillus cereus group' is one of the largest and most ubiquitous microbes of high economic, medical and biodefense importance, comprised of at least eight phylogenetically very closely related Bacillus species. These are B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, B. weihenstephanensis, B. cytotoxicus, and B. toyonensis 1. With the exception of B. cytotoxicus, which is the most divergent of the group with a chromosome of 4.085 Mb, the genomes of member species are highly conserved, with sizes of 5.2 to 5.5 Mb having very similar 16S rRNA gene sequences 2. The most studied members of the group, Bacillus anthracis, B. cereus, and B. thuringiensis are well known and possess substantial pathogenic potential. While B. anthracis is the causative agent of anthrax, few members of B. cereus group are commonly recognized as food poisoning agents, some can also cause localized wound and eye infections as well as systemic diseases 2. Certain B. thuringiensis strains are entomopathogens and are being used commercially as biopesticides, however, some strains have been reported to cause infections in the immunocompromised individuals 2. Despite the presence of high degree phylogenetic relatedness that prompted several researchers to consider the members of the Bacillus cereus group as a unique species, the members demonstrated differences in plasmid content and expression of key regulatory genes. Several approaches were employed to differentiate B. cereus group members including whole-genome DNA hybridization 8 , sequence analysis of the 16S-23S operons, the gyrB-gyrA intergenic spacer region, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis analysis 4 , amplified fragment length polymorphism 6 , virulence factors, arbitrary PCR, multiplex PCR and PCR-restriction fragment length polymorphism 6. However, the need to adopt a rapid and simpler method remained an issue to ponder.. HiCrome TM Bacillus Agar is a selective and differential media which is recommended for the isolation and differentiation amongst various species of Bacillus. This medium is based on the formulation of MYP (Mannitol, egg yolk, polymyxin) agar where different Bacillus sp. show different colored colonies 9. However, culture-based method of identification does not always