Virulotyping of <Emphasis Type="Italic">Salmonella enterica</Emphasis> subsp. <Emphasis Type="Italic">enterica</Emphasis> isolated from indigenous vegetables and poultry meat in Malaysia using multiplex-PCR

The increased occurrence of Salmonella occurrence in local indigenous vegetables and poultry meat can be a potential health hazards. This study is aimed to detect the prevalence of twenty different virulence factors among Salmonella enterica strains isolated from poultry and local indigenous vegetables in Malaysia via an optimized, rapid and specific multiplex PCR assay. The assay encompasses a total of 19 Salmonella pathogenicity islands genes and a quorum sensing gene (sdiA) in three multiplex reaction sets. A total of 114 Salmonella enterica isolates belonging to 38 different serovars were tested. Each isolate in under this study was found to possess up to 70% of the virulence genes tested and exhibited variable pathogenicity gene patterns. Reproducibility of the multiplex PCR assay was found to be 100% and the detection limit of the optimized multiplex PCR was tested with lowest detectable concentration of DNA 0.8 pg μl⁻¹. This study demonstrated various Salmonella pathogenicity island virulence gene patterns even within the same serovar. This sets of multiplex PCR system provide a fast and reliable typing approach based on Salmonella pathogenicity islands, thus enabling an effective monitoring of emerging pathogenic Salmonella strains as an additional tool in Salmonella surveillance studies.     

Association Between the <Emphasis Type="Italic">cfx</Emphasis>A Gene and Transposon Tn<Emphasis Type="Italic">4555</Emphasis> in <Emphasis Type="Italic">Bacteroides distasonis</Emphasis> Strains and Other <Emphasis Type="Italic">Bacteroides</Emphasis> Species

The Bacteroides genus, the most prevalent anaerobic bacteria of the intestinal tract, carries a plethora of the mobile elements, such as plasmids and conjugative and mobilizable transposons, which are probably responsible for the spreading of resistance genes. Production of β-lactamases is the most important resistance mechanism including cephalosporin resistance to β-lactam agents in species of the Bacteroides fragilis group. In our previous study, the cfxA gene was detected in B. distasonis species, which encodes a clinically significant broad-spectrum β-lactamase responsible for widespread resistance to cefoxitin and other β-lactams. Such gene has been associated with the mobilizable transposon Tn4555. Therefore, the aim of this study was to detect the association between the cfxA gene and the presence of transposon Tn4555 in 53 Bacteroides strains isolated in Rio de Janeiro, Brazil, by PCR assay. The cfxA gene was detected in 11 strains and the Tn4555 in 15. The transposon sequence revealed similarities of approximately 96% with the B. vulgatus sequence which has been deposited in GenBank. Hybridization assay was performed in attempt to detect the cfxA gene in the transposon. It was possible to associate the cfxA gene in 11 of 15 strains that harbored Tn4555. Among such strains, 9 presented the cfxA gene as well as Tn4555, but in 2 strains the cfxA gene was not detected by PCR assay. Our results confirm the involvement of Tn4555 in spreading the cfxA gene in Bacteroides species.     

Species identification of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> ssp. <Emphasis Type="Italic">jejuni</Emphasis> and <Emphasis Type="Italic">C. coli</Emphasis> by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and PCR

The Campylobacter species strains (n = 42; isolated from clinical samples and deposited in Czech National Collection of Type Cultures, Prague) originally phenotypically (and biochemically) identified as Campylobacter jejuni were re-classified using molecular biological and mass spectrometric methods. Whole-cell MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) separated the isolates into two genetically related strains — C. jejuni (n = 26) and C. coli (n = 16) and, moreover, distinguished the intimate details in the group of tested strains. It also made it possible to create the MALDI-TOF MS dendrogram; similarly, the spectral characteristics were used for the 3D cluster analysis. Polymerase chain reaction (PCR) confirmed the results obtained by mass spectrometry. Both methods (PCR and MALDI-TOF MS) gave the same results which supports their suitability in the rapid and accurate Campylobacter-species determination. Part of this work was presented at the 24th Congress of Czechoslovak Society for Microbiology, Liberec (Czech Republic) 2007.     

Diversity and recombination in <Emphasis Type="Italic">Wolbachia</Emphasis> and <Emphasis Type="Italic">Cardinium</Emphasis> from <Emphasis Type="Italic">Bryobia</Emphasis>spider mites

BACKGROUND: Wolbachia and Cardinium are endosymbiotic bacteria infecting many arthropods and manipulating host reproduction. Although these bacteria are maternally transmitted, incongruencies between phylogenies of host and parasite suggest an additional role for occasional horizontal transmission. Consistent with this view is the strong evidence for recombination in Wolbachia, although it is less clear to what extent recombination drives diversification within single host species and genera. Furthermore, little is known concerning the population structures of other insect endosymbionts which co-infect with Wolbachia, such as Cardinium. Here, we explore Wolbachia and Cardinium strain diversity within nine spider mite species (Tetranychidae) from 38 populations, and quantify the contribution of recombination compared to point mutation in generating Wolbachia diversity. RESULTS: We found a high level of genetic diversity for Wolbachia, with 36 unique strains detected (64 investigated mite individuals). Sequence data from four Wolbachia genes suggest that new alleles are 7.5 to 11 times more likely to be generated by recombination than point mutation. Consistent with previous reports on more diverse host samples, our data did not reveal evidence for co-evolution of Wolbachia with its host. Cardinium was less frequently found in the mites, but also showed a high level of diversity, with eight unique strains detected in 15 individuals on the basis of only two genes. A lack of congruence among host and Cardinium phylogenies was observed. CONCLUSIONS: We found a high rate of recombination for Wolbachia strains obtained from host species of the spider mite family Tetranychidae, comparable to rates found for horizontally transmitted bacteria. This suggests frequent horizontal transmission of Wolbachia and/or frequent horizontal transfer of single genes. Our findings strengthens earlier reports of recombination for Wolbachia, and shows that high recombination rates are also present on strains from a restrictive host range. Cardinium was found co-infecting several spider mite species, and phylogenetic comparisons suggest also horizontal transmission of Cardinium among hosts.     

Incidence of the endosymbionts <Emphasis Type="Italic">Wolbachia</Emphasis>, <Emphasis Type="Italic">Cardinium</Emphasis> and <Emphasis Type="Italic">Spiroplasma</Emphasis> in phytoseiid mites and associated prey

Endosymbiotic bacteria that potentially influence reproduction and other fitness-related traits of their hosts are widespread in insects and mites and their appeal to researchers’ interest is still increasing. We screened 20 strains of 12 agriculturally relevant herbivorous and predatory mite species for infection with Wolbachia, Cardinium and Spiroplasma by the use of PCR. The majority of specimens originated from Austria and were field collected or mass-reared. Eight out of 20 strains (40%) tested, representing seven of 12 mite species (58%), carried at least one of the three bacteria. We found Wolbachia in the herbivorous spider mites Tetranychus urticae and Bryobia rubrioculus, with the former also carrying Spiroplasma and the latter also carrying Cardinium. Cardinium was furthermore found in two populations of the predatory mite Euseius finlandicus and the spider mite Eotetranychus uncatus. Spiroplasma was detected in the predatory mite Neoseiulus californicus. All bacteria positive PCR products were sequenced, submitted to GenBank and analyzed in BLAST queries. We found high similarities to complete identity with bacteria found in the same and different mite species but also with bacteria found in insect species like ladybirds, butterflies and minute pirate bugs, Orius. We discuss the significance of potential (multiple) infections with the investigated bacteria for biological control.     

Biochemical and molecular characterization of <Emphasis Type="Italic">Cronobacter</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> (formerly <Emphasis Type="Italic">Enterobacter sakazakii</Emphasis>) isolated from foods

The aim of this study was to identify and characterize Cronobacter spp. isolated from a range of foods. A total of 71 Cronobacter strains were isolated from 602 foods in our laboratory. The highest contamination was observed in foods of plant origin, e.g. spices, teas, chocolate, nuts, pastries and vegetables. On the basis of genus and species identification performed using genus-specific PCR, 16S rRNA sequencing and AFLP genotyping, most of the strains belonged to Cronobacter sakazakii. Biochemical profiling by the tests included in API 20E, complemented with relevant additional tests, classified the strains into 13 biogroups. AFLP genotyping facilitated discrimination of six main groups at the 70% similarity level and strain grouping correlated clearly with species identification. Our results indicate that molecular typing by AFLP may be applied as a useful tool not only for direct comparison of Cronobacter isolates, providing traceability, but also for the reliable species classification. Moreover, tracing of these bacteria in a wider variety of foods should be important to enhance the knowledge of their transmission.     

The genome of <Emphasis Type="Italic">Streptomyces rimosus</Emphasis> subsp. <Emphasis Type="Italic">rimosus</Emphasis> shows a novel structure compared to other <Emphasis Type="Italic">Streptomyces</Emphasis> using DNA/DNA microarray analysis

DNA/DNA genome microarray analysis together with genome sequencing suggests that the genome of members of the genus Streptomyces would seem to have a common structure including a linear genomic structure, a core of common syntenous Actinomycete genes, the presence of species specific terminal regions and two intermediate group of syntenous genes that seem to be genus specific. We analyzed Streptomyces species using DNA/DNA microarray comparative genome analysis. Only Streptomyces rimosus failed to give a congruent genome pattern for the genes found in Streptomyces coelicolor. We expanded the analysis to include a number of strains related to the type strain of S. rimosus and obtained a similar divergence from the main body of Streptomyces species. These strains showed very close identity to the original strain with no gene deletion or duplication detected. The 16S rRNA sequences of these S. rimosus strains were confirmed as very similar to the S. rimosus sequences available from the Ribosomal Database Project. When the SSU ribosomal RNA phylogeny of S. rimosus is analyzed, the species is positioned at the edge of the Streptomyces clade. We conclude that S. rimosus represents a distinct evolutionary lineage making the species a worthy possibility for genome sequencing.     

PCR screening and sequence analysis of <Emphasis Type="Italic">iol</Emphasis> clusters in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> strains isolated from koumiss

The iol cluster (consisting of genes involved in myo-inositol utilization) was investigated in Lactobacillus casei strains isolated from koumiss. Ten strains were tested for the presence of iol cluster by PCR screening; three strains encoded this cluster. Full-sequencing procedure was conducted; the iol cluster was identical to that of L. casei BL23 (GenBank access. no. FM177140) except for an upstream transposase. The iol cluster is not a common feature for L. casei strains isolated from koumiss.     

Negative Evidence of <Emphasis Type="Italic">Wolbachia</Emphasis> in the Predaceous Mite <Emphasis Type="Italic">Phytoseiulus persimilis</Emphasis>

The cytoplasmically inherited bacterium Wolbachia is widespread in arthropod species and has been repeatedly detected in the predaceous mite Phytoseiulus persimilis. Our original goal was to assess the prevalence of Wolbachia infection in P. persimilis and the potential fitness consequences for this host. To accomplish that goal, seven P. persimilis strains were obtained from Europe, Africa and the USA and reared on the phytophagous mite Tetranychus urticae as prey. After preliminary results showed that the T. urticae used was infected with Wolbachia, the minimum starvation time of the predators to prevent false positive results from undigested prey was determined. We tested DNA samples by PCR (polymerase chain reaction) after starving the predators or feeding them Wolbachia-free T. urticae for various periods. Those experiments showed that Wolbachia could not be detected after 16 h at 25 °C and 48 h at 20 °C. To verify the results of the PCR analyses, we furthermore conducted crossing experiments with antibiotic-treated and untreated individuals. No indications of Wolbachia effects were recorded. Additionally, we screened live eggs of four of the seven strains reared in our laboratory and alcohol samples of 10 other P. persimilis strains for the occurrence of Wolbachia by PCR, none of which tested positive. Synthesis of our study and previous reports suggests that infection of P. persimilis with Wolbachia is extremely rare and of minor importance. We discuss the significance of our findings for future studies on the presence of Wolbachia in predaceous arthropods.     

Epidemiological investigation of <Emphasis Type="Italic">eaeA</Emphasis>-positive <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Escherichia albertii</Emphasis> strains isolated from healthy wild birds

Escherichia coli has commonly been associated with diarrheal illness in humans and animals. Recently, E. albertii has been reported to be a potential pathogen of humans and animals and to be carried by wild birds. In the present study, the prevalence and genetic characteristics of intimin-producing E. coli and E. albertii strains were evaluated in wild birds in Korea. Thirty one of 790 Enterobacteriaceae strains from healthy wild birds were positive for the intimin gene (eaeA) and twenty two of the 31 strains were identified as atypical enteropathogenic E. coli (aEPEC) that did not possess both EAF and bfpA genes. A total of nine lactose non-fermenting coliform bacterial strains were identified as E. albertii by PCR and sequence analysis of housekeeping genes. A total of 28 (90.3%) eaeA-positive strains were isolated from waterfowl. Fifteen aEPEC (68.2%) and two E. albertii (22.2%) strains had a β-intimin subtype and 14 aEPEC strains harboring β-intimin belonged to phylogenetic group B2. AU eaeA-positive E. albertii and 3 aEPEC strains possessed the cytolethal distending toxin gene (cdtB). The eaeA-positive E. coli and E. albertii strains isolated from healthy wild birds need to be recognized as a potential pathogroup that may pose a potential threat to human and animal health. These findings indicate that eaeA-positive E. coli as well as E. albertii can be carried by wild birds, posing a potential threat to human and animal health.     

Application of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">parE</Emphasis> sequence similarity analyses for differentiation of species within the genus <Emphasis Type="Italic">Geobacillus</Emphasis>

The primary structures of the genes encoding the β-subunits of a type II topoisomerase (gyrase, gyrB) and a type IV topoisomerase (parE) were determined for 15 strains of thermophilic bacteria of the genus Geobacillus. The obtained sequences were used for analysis of the phylogenetic similarity between members of this genus. Comparison of the phylogenetic trees of geobacilli constructed on the basis of the 16S rRNA, gyrB, and parE gene sequences demonstrated that the level of genetic distance between the sequences of the genes encoding the β-subunits of type II topoisomerases significantly exceeded the values obtained by comparative analysis of the 16S rRNA gene sequences of Geobacillus strains. It was shown that, unlike the 16S rRNA gene analysis, comparative analysis of the gyrB and parE gene sequences provided a more precise determination of the phylogenetic position of bacteria at the species level. The data obtained suggest the possibility of using the genes encoding the β-subunits of type II topoisomerases as phylogenetic markers for determination of the species structure of geobacilli.     

Identification of iron-reducing<Emphasis Type="Italic"> Thermus</Emphasis> strains as<Emphasis Type="Italic"> Thermus scotoductus</Emphasis>

Thermus strain SA-01, previously isolated from a deep (3.2 km) South African gold mine, is closely related to Thermus strains NMX2 A.1 and VI-7 (previously isolated from thermal springs in New Mexico, USA, and Portugal, respectively). Thermus strains SA-01 and NMX2 A.1 have also been shown previously to grow using nitrate, Fe(III), Mn(IV) or S^O as terminal electron acceptors and to be capable of reducing Cr(VI), U(VI), Co(III), and the quinone-containing compound anthraquinone-2,6-disulfonate. The objectives of this study were to determine the phylogenetic positions of the three known metal-reducing Thermus strains and to determine the phylogenetic significance of metal reduction within the genus Thermus. Phylogenetic analyses of 16S rDNA sequences, BOX PCR genomic fingerprinting, and DNA–DNA reassociation analyses indicated that these strains belong to the previously described genospecies T. scotoductus. The morphologies and lipid fatty acid profiles of these metal-reducing strains are consistent with their identification as T. scotoductus; however, the T. scotoductus strains tested in this study evinced a wide intraspecies variability in some other phenotypic traits, e.g., carbon substrate utilization and pigmentation. Iron reduction occurred in all strains of T. scotoductus tested except the mixotrophic, sulfur-oxidizing strain IT-7254. Thermus strains belonging to other species did not reduce Fe(III) to Fe(II) or reduced it only poorly.Communicated by J. Wiegel     

<Emphasis Type="Italic">IGHV1</Emphasis>,<Emphasis Type="Italic"> IGHV5</Emphasis> and<Emphasis Type="Italic"> IGHV7</Emphasis> subgroup genes in the Rhesus macaque

The diversity of the antibody response is achieved, in part, by rearrangement of different immunoglobulin (Ig) genes. The Ig heavy chain is made up of a variable region (IGHV), a diversity region (IGHD) and a joining region (IGHJ). Human germline IGHV genes have been grouped into seven multigene subgroups. Size and usage of these subgroups is not equal, the IGHV3 subgroup is the most commonly used (36%), followed by IGHV1/7 (26%), then IGHV4, IGHV5, IGHV2, IGHV6 (15%, 12%, 4%, 3% respectively). The rhesus macaque (Macaca mulatta) is a useful non-human primate model for studies of infection and the database of germline Ig genes for the macaque is gradually growing to become a useful tool in the study of B-cell responses. The proportions of IGHV subgroup usage in the macaque are similar to those in man. Representatives from IGHV3 and IGHV4 subgroups for the macaque have been published, as have germline sequences of the IGHD and IGHJ genes. However, to date there have been no sequences published from the second largest IGHV subgroup, IGHV1. We report the isolation and sequencing of a genomic fragment containing an IGHV1 gene from the macaque. Polymerase chain reaction (PCR) primers designed from this sequence enabled us to amplify and sequence 25 new IGHV1 germline genes. We also isolated two IGHV7 genes, using the same primers, and two IGHV5 genes, using human IGHV5 primers.     

A <Emphasis Type="Italic">gyrB</Emphasis>-targeted PCR for Rapid Identification of <Emphasis Type="Italic">Salmonella</Emphasis>

Salmonella causes the majority of infections in humans and homeothermic animals. This article describes a specific polymerase chain reaction (PCR) method developed for a rapid identification of Salmonella. A gyrB-targeted species-specific primer pair, S-P-for (5′-GGT GGT TTC CGT AAA AGT A-3′) and S-P-rev (5′-GAA TCG CCT GGT TCT TGC-3′), was successfully designed. PCR with all the Salmonella strains produced a 366- bp DNA fragment that was absent from all the non-Salmonella strains tested. The detection limit of the PCR was 0.01 ng with genomic DNA or 3.2 cells per assay. Good specificity was also demonstrated by fecal samples, from which only the gyrB gene of Salmonella was amplified. Using the culture-PCR method, 27 isolates on Salmonella-Shigella (SS) medium were rapidly identified as Salmonella, which was confirmed by the sequencing of the gyrB gene.     

Diversity of Arsenate Reductase Genes (<Emphasis Type="Italic">arsC</Emphasis> Genes) from Arsenic-Resistant Environmental Isolates of <Emphasis Type="Italic">E. coli</Emphasis>

A polymerase chain reaction (PCR) approach was used to assess the occurrence and diversity of arsenate reductase gene (arsC gene) in arsenic-resistant environmental E. coli strains. For this purpose, two different sets of primers were designed for the specific amplification of approximately 370-bp fragments from the arsC gene. These primers were used to screen a collection of 25 environmental arsenic-resistant strains isolated from different geographical regions of India, as well as Bangladesh. The PCR results showed that 17 out of the 25 environmental isolates (68%) contained a gene related to the arsC family. Phylogenetic analysis of the protein sequences deduced from the amplicons indicated a prevalence of arsC genes in the isolated strains. A significant divergence in the DNA sequence was found in the arsC genes among As-resistant environmental E. coli strains from this study, and arsenic resistance, a genetic character, arose from a common ancestral background.     

Molecular analysis of the genus <Emphasis Type="Italic">Anoxybacillus</Emphasis> based on sequence similarity of the genes <Emphasis Type="Italic">recN</Emphasis>, <Emphasis Type="Italic">flaA</Emphasis>, and <Emphasis Type="Italic">ftsY</Emphasis>

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies. We analyzed three genes, recN, flaA, and ftsY, for determining if these genes are useful tools for systematic analyses in the genus Anoxybacillus. The genes encoding a DNA repair and genetic recombination protein (recN), the flagellin protein (flaA), and GTPase signal docking protein (ftsY) were sequenced for ten Anoxybacillus species. The sequence comparisons revealed that recN sequence similarities range between 61% and 99% in the genus Anoxybacillus. Comparisons to other bacterial recN genes indicated that levels of similarity did not differ from the levels within genus Anoxybacillus. These data showed that recN is not a useful marker for the genus Anoxybacillus. A 550–600-bp region of the flagellin gene was amplified for all Anoxybacillus strains except for Anoxybacillus contaminans. The sequence similarity of flaA gene varies between 61% and 76%. Comparisons to other bacterial flagellin genes obtained from GenBank (Bacillus, Pectinatus, Proteus, and Vibrio) indicated that the levels of similarity were lower (3–42%). Based on these data, we concluded that the variability in this single gene makes it a particularly useful marker. Another housekeeping gene ftsY suggested to reflect the G+C (mol/mol) content of whole genome was analyzed for Anoxybacillus strains. A mean difference of 1.4% was observed between the G+C content of the gene ftsY and the G+C content of the whole genome. These results showed that the gene ftsY can be used to represent whole G+C content of the Anoxybacillus species.     

Isolation and characterization of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> strains in China

Important staple foods (peanuts, maize and rice) are susceptible to contamination by aflatoxin (AF)-producing fungi such as Aspergillus flavus. The objective of this study was to explore non-aflatoxin-producing (atoxigenic) A. flavus strains as biocontrol agents for the control of AFs. In the current study, a total of 724 A. flavus strains were isolated from different regions of China. Polyphasic approaches were utilized for species identification. Non-aflatoxin and non-cyclopiazonic acid (CPA)-producing strains were further screened for aflatoxin B₁ (AFB₁) biosynthesis pathway gene clusters using a PCR assay. Strains lacking an amplicon for the regulatory gene aflR were then analyzed for the presence of the other 28 biosynthetic genes. Only 229 (32%) of the A. flavus strains were found to be atoxigenic. Smaller (S) sclerotial phenotypes were dominant (51%) compared to large (L, 34%) and non-sclerotial (NS, 15%) phenotypes. Among the atoxigenic strains, 24 strains were PCR-negative for the fas-1 and aflJ genes. Sixteen (67%) atoxigenic A. flavus strains were PCRnegative for 10 or more of the biosynthetic genes. Altogether, 18 new PCR product patterns were observed, indicating great diversity in the AFB₁ biosynthesis pathway. The current study demonstrates that many atoxigenic A. flavus strains can be isolated from different regions of China. In the future laboratory as well as field based studies are recommended to test these atoxigenic strains as biocontrol agents for aflatoxin contamination.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Infections of the Whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

We report the first systematic survey for the presence of Wolbachia endosymbionts in aphids and whiteflies, particularly different populations and biotypes of Bemisia tabaci. Additional agriculturally important species included were predator species, leafhoppers, and lepidopterans. We used a polymerase chain reaction (PCR)-based detection assay with ribosomal 16S rDNA and Wolbachia cell surface protein (wsp) gene primers. Wolbachia were detected in a number of whitefly populations and species, whitefly predators, and one leafhopper species; however, none of the aphid species tested were found infected. Single, double, and triple infections were detected in some of the B. tabaci populations. PCR and phylogenetic analysis of wsp gene sequences indicated that all Wolbachia strains found belong to group B. Topologies of the optimal tree derived by maximum likelihood (ML) and a ML tree in which Wolbachia sequences from B. tabaci are constrained to be monophyletic are significantly different. Our results indicate that there have been at least four independent Wolbachia infection events in B. tabaci. The importance of the presence of Wolbachia infections in B. tabaci is discussed. RID= ID= <E5>Correspondence to: </E5>K. Bourtzis; <E5>email:</E5> kbourtz&commat;cc.uoi.gr Received: 9 September 2002 / Accepted: 25 September 2002     

Determination of Toxigenic Potentials of <Emphasis Type="Italic">Bacillus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Okpehe</Emphasis>, a Nigerian Fermented Condiment

The toxigenic potential of Bacillus species isolated from the traditional fermented condiment okpehe was determined; this is aimed at selection of non-toxigenic bacilli as starter cultures to bring about production of safe product. B. subtilis and B. cereus strains isolated from okpehe were evaluated for their possible possession of virulence characteristics. Fifty isolates were screened for their ability to produce diarrhoea enterotoxin by reversed passive latex agglutination (BCET-RPLA) test kit; the result showed that 40% of the B. cereus strains were toxigenic. The ability of the selected isolates to compete in situ and in vitro toxin production during the fermentation was also determined. The enterotoxin was not detected using BCET-RPLA kit in the spontaneously fermented samples of okpehe, but the toxin was detected in the okpehe samples fermented using B. cereus enterotoxin producer in mixed starter culture fermentation. The PCR amplification of virulence genes revealed that Bacillus cereus and B. licheniformis, a strain from the B. subtilis group, contained DNA sequences encoding the haemolysin BL (hblD) enterotoxin complex. The growth ability of B. cereus strains to high population during the fermentation and the presence of detectable diarroheagenic genes in B. cereus and B. licheniformis showed that strains carrying virulence characteristics cannot be totally ruled out in traditionally fermented okpehe.