Functional characterization and evaluation of protective efficacy of EA752-862 monoclonal antibody against B. anthracis vegetative cell and spores.

The most promising means of controlling anthrax, a lethal zoonotic disease during the early infection stages, entail restricting the resilient infectious form, i.e., the spores from proliferating to replicating bacilli in the host. The extractible antigen (EA1), a major S-layer protein present on the vegetative cells and spores of Bacillus anthracis, is highly immunogenic and protects mice against lethal challenge upon immunization. In the present study, mice were immunized with r-EA1C, the C terminal crystallization domain of EA1, to generate a neutralizing monoclonal antibody EA752-862, that was evaluated for its anti-spore and anti-bacterial properties. The monoclonal antibody EA752-862 had a minimum inhibitory concentration of 0.08 mg/ml, was bactericidal at a concentration of 0.1 mg/ml and resulted in 100% survival of mice against challenge with B. anthracis vegetative cells. Bacterial cell lysis as observed by scanning electron microscopy and nucleic acid leakage assay could be attributed as a possible mechanism for the bactericidal property. The association of mAb EA752-862 with spores inhibits their subsequent germination to vegetative cells in vitro, enhances phagocytosis of the spores and killing of the vegetative cells within the macrophage, and subsequently resulted in 90% survival of mice upon B. anthracis Ames spore challenge. Therefore, owing to its anti-spore and bactericidal properties, the present study demonstrates mAb EA752-862 as an efficient neutralizing antibody that hinders the establishment of early infection before massive multiplication and toxin release takes place.

Development and evaluation of a novel combinatorial selective enrichment and multiplex PCR technique for molecular detection of major virulence-associated genes of enterotoxigenic Staphylococcus aureus in food samples.

AIMS: To develop a multiplex PCR assay coupled with selective enrichment step to detect major virulence-associated genes of enterotoxigenic Staphylococcus aureus and evaluate the same directly on contaminated food samples. METHODS AND RESULTS: The most important virulence-associated genes of Staph. aureus, which are commonly related to food safety issues, are targeted in this study. They include five major enterotoxigenic genes-sea, seb, sec, seg and sei, tst-which encodes TSST-1, mecA-which confer methicillin resistance and coa-for the enzyme coagulase along with an internal amplification control (IAC) to rule out false-negative result. A modified mannitol salt broth (MSB) supplemented with sodium pyruvate was used for selective enrichment of Staph. aureus from food samples prior to PCR. Evaluation of efficiency of different media revealed that enrichment of samples in modified MSB followed by PCR resulted in specific, sensitive and effective amplification of the targeted genes in comparison with other enrichment media. Incorporation of bovine serum albumin (BSA) as PCR enhancer improved the intensity of amplicons. The standardized multiplex PCR (mPCR) format was able to detect all the target genes at a bacterial load of 10(6) CFU ml(-1) in any sample. The PCR results were unequivocally correlated with the conventional methods when the mPCR format was assessed on a total of 91 Staph. aureus isolates. The entire assay was found to be effectual when evaluated on naturally contaminated food samples. CONCLUSIONS: The combinatorial approach involving selective enrichment followed by mPCR developed in this study was found to be effective for the detection of toxigenic Staph. aureus directly from various food sources. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed format would find a promising application in early detection of food contaminations as well as in the diagnosis of food poisoning due to Staph. aureus.

Application of r-PFE hyperimmune sera for concurrent detection of Bacillus anthracis, Yersinia pestis and staphylococcal enterotoxin B.

AIM: To evaluate the potential of an intergeneric multidomain recombinant chimeric protein for the simultaneous detection of Bacillus anthracis, Yersinia pestis and Staphylococcal enterotoxin B. METHODS AND RESULTS: Truncated portions of protective antigen (pag) of B. anthracis, fraction 1 capsular antigen (F1) of Y. pestis and enterotoxin B (entB) of Staphylococcus aureus were PCR amplified and linked each other using ligation-dependent cloning. The fusion gene was codon-optimized for expression in Escherichia coli and encoded a 55 kDa recombinant PFE protein (rPFE). Hyperimmune antiserum raised against rPFE specifically reacted individually with the native PA of B. anthracis, F1 antigen of Y. pestis and SEB of S. aureus on Western blot analysis as well as in enzyme-linked immunosorbent assay (ELISA). For simultaneous detection of these three antigens from culture supernatants, common media consisting of BHI broth supplemented with 0·2% xylose were used. To assess the detection capability, a known number of these organisms (10(8) -10(2) CFU ml(-1)) were experimentally spiked on to the meat and blood samples, the polyclonal antibodies were again clearly able to identify all three target proteins up to a dilution of 10(5) CFU ml(-1). CONCLUSIONS: This recombinant chimeric protein-based immunodetection approach may eventually provide advantages over PCR formats during onsite investigations of biological emergencies or even during routine testing by laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: The trivalent recombinant PFE protein could be a novel intervention for possible diagnosis/detection of potential biological agents simultaneously in environmental and clinical samples to reduce the responding time and minimize the impact of the bioattack.

Development of ISSR-derived SCAR marker-targeted PCR for identification of Aspergillus section Flavi members.

UNLABELLED: Aspergillus section Flavi is a heterogeneous fungal cluster including some of the most economically important Aspergillus species. The section is comprised of toxigenic and nontoxigenic aspergilli that are phenotypically undistinguishable. The aim of this study was to develop a genetic marker specific to Aspergillus section Flavi on the whole. Based on inter-simple sequence repeat (ISSR) fingerprinting profiles of major Aspergillus section Flavi members, a sequence-characterized amplified region (SCAR) marker was identified. Primers were designed in the conserved regions of the SCAR marker and were utilized in a PCR for concurrent identification of major members of the section. The detection level of the SCAR-PCR was found to be 0·1 ng purified DNA, and when applied to 45 naturally contaminated food samples, 28 samples were found infected with Aspergillus section Flavi members. The present SCAR-PCR is rapid and less cumbersome unlike conventional identification techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of Aspergillus section Flavi members is important owing to their impact on human health and economy. The ISSR-based SCAR-PCR developed in this study is superior over the other existing Aspergillus section Flavi detection systems due to its simplicity and minimal requirement of sample handling. This PCR could be a supplementary strategy to time-consuming and rather ambiguous conventional polyphasic detection techniques and a reliable tool for high-throughput sample analysis.

Nisin based stabilization of novel fruit and vegetable functional juices containing bacterial cellulose at ambient temperature.

The current study reports the preparation and stabilization of novel functional drinks based on fruit and vegetable juices incorporating bacterial cellulose from Acetobacter xylinum. Pineapple, musk melon, carrot, tomato, beet root and a blend juice containing 20 % each of carrot and tomato juice with 60 % beet root juice has been studied. These juices have been stabilized over a storage period of 90 days at 28 °C, by the use of nisin and maintaining a low pH circumventing the need for any chemical preservatives or refrigeration. Instrumental color values have been correlated with the pigment concentrations present in the fresh as well as stored juices. There was 36, 72 and 60 % loss of total carotenoids in the case of carrot, pineapple and musk melon juices respectively while the lycopene content remained unchanged after 90 days of storage. The betanin content decreased 37 % in the case of beetroot juice and 25 % in the case of beetroot juice blended with carrot and tomato juices. Sensory analysis has revealed a clear preference for the beetroot blended mixed juice.

Development and validation of an immunochromatographic assay for rapid detection of fumonisin B1 from cereal samples.

Fumonisins are one of the most agriculturally significant environmental toxins produced by Fusarium and Aspergillus species that grow on agricultural commodities in the field or during storage. Cereals contaminated with fumonisins causes serious loss to agricultural produce leads to health problems in humans and other farm animals. In the present study, polyclonal hyperimmune sera was raised against FB1 in rabbits immunized with FB1-keyhole limpet haemocyanin (KLH). Purified antibodies were used to establish a sensitive gold nanoparticle based immunochromatographic strip (ICG) for detecting FB1 levels in cereal grains. Effective on-site detection of FB1 was achieved by developing a rapid and sensitive pAb based ICG strip. This strip had a detection limit of 5 ng mL(-1) for FB1 in cereal samples and it could be completed within 3 min. Close examination of 150 cereal samples by ICG strip method revealed that 77 were fumonisin-positive. Results obtained by the developed method was further validated with well standardized HPLC method and results of strip method was correlated well with those obtained by HPLC method. In conclusion, the developed method was a better alternative for onsite detection of FB1 in cereal samples intended for human consumption to reduce risk of humans and other farm animals. The high level of FB1 concentrations recorded in present study warrants the need to develop an awareness creation programme to the farmers of India for safe handling of cereal grains at the time of harvesting and storage of grains.

Evaluation of a multiplex PCR assay for concurrent detection of four major mycotoxigenic fungi from foods.

AIM: To develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of four major mycotoxin metabolic pathway genes, viz. nor1 (aflatoxin), Tri6 (trichothecene), FUM13 (fumonisin) and otanps (ochratoxin A). METHODS AND RESULTS: A mPCR assay with competitive internal amplification control, employing specific primers for each of the aforementioned four genes, was optimized and validated using 10 reference strains and 60 pure culture isolates. The standardized mPCR assay detected all four mycotoxin metabolic genes in artificially contaminated maize samples with a sensitivity of 2 × 10(3) CFU g(-1) for nor1-positive Aspergillus strains, Tri6 and FUM13-positive Fusarium strains and 2 × 10(4) CFU g(-1) for otanps-positive Penicillium strains. When the developed mPCR assay was applied to 40 natural foods, 35% (14 of 40) of the samples were contaminated with either one or more mycotoxins. The mPCR results were further evaluated with high-performance liquid chromatography (HPLC), and in general, both the methods provided unequivocal results. CONCLUSION: The current mPCR assay is a rapid and reliable tool for simultaneous specific and sensitive detection of aflatoxigenic Aspergillus strains, trichothecene- and fumonisin-producing Fusarium strains, and ochratoxigenic Penicillium species from naturally contaminated foods. SIGNIFICANCE AND IMPACT OF THE STUDY: This mPCR assay could be a supplementary strategy to current conventional mycotoxin analytical techniques such as thin-layer chromatography (TLC), high performance thin layer chromatography, HPLC, etc., and a reliable tool for high-throughput monitoring of major mycotoxin-producing fungi during the processing steps of food and feed commodities.

Corrigendum: Development of sandwich dot-ELISA for specific detection of Ochratoxin A and its application on to contaminated cereal grains originating from India.

[This corrects the article DOI: 10.3389/fmicb.2015.00511.].

Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications.

AIM: To evaluate an inter-generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. METHODS AND RESULTS: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap-extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r-αCS specifically detected 42-kDa and 33-kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild-type toxins in vitro. CONCLUSIONS: The r-αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild-type toxins in vitro. SIGNIFICANCE OF THE STUDY: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co-infections like gangrenous ischaemia, gangrenous mastitis, etc.

Development of a diagnostic system for Burkholderia pseudomallei infections.

Monoclonal antibodies were generated against whole cell lysate of Burkholderia pseudomallei. Two out of 6 monoclonal antibodies were found specific and exhibited high affinity against B. pseudomallei, one of which, was utilized to develop sandwich ELISA for detection of specific B. pseudomallei antigen. Immunoassays were found to be specific as no reaction was observed with closely related Burkholderia and Pseudomonas species. Blood samples from experimentally infected mice were found positive for isolation till 4 days post infection (DPI) and ELISA till 10 DPI. One out of 40 sick animal serum samples tested in Thailand was found positive by sandwich ELISA that was earlier confirmed by isolation of B. pseudomallei. The results indicate the potentiality of the assay for its applicability in specific diagnosis of septicaemic melioidosis.

Specific identification of pathogenic Yersinia enterocolitica by monoclonal antibodies generated against recombinant attachment invasion locus (rAil) protein.

Classical pathogenic strains of Yersinia enterocolitica produce a 17 kDa outer membrane protein, Ail (attachment-invasion locus), which mediates bacterial attachment to some cultures epithelial cell lines and invasion of others. In the present study, hybridomas were developed for the production of monoclonal antibodies (MAbs) against Ail protein of Y. enterocolitica. A set of five stabilized hybridoma cell lines were generated, of which, two MAbs, YEA 302 and YEA 303, exhibited specific reaction to the native Ail protein (17 kDa) present in whole cell lysate of Y. enterocolitica strains beside having reaction with rAil. The other three MAbs, YEA 5, 17 and 32, had some cross reactions with proteins other than Ail. Two out of five MAbs were IgG1, two were IgG2b and one in IgM in nature. MAbs (YEA 302 and YEA 303) did not show any cross-reaction with whole cell lysate of Brucella abortus, Vibrio cholerae, Salmonella typhimurium and Escherichia coli and other species of Enterobacteriaceae including Y. pestis in ELISA and Western blot analysis. The presence of Ail protein among the strains recovered from pork and milk samples was evaluated by these sets of MAbs and the results were compared with the duplex PCR. Collectively, the data suggest that these MAbs may have the potential for their use in the detection of pathogenic Y. enterocolitica reliably, rapidly and at a relatively low cost.

Application of a Chimeric Protein Construct having Enterotoxin B and Toxic Shock Syndrome Toxin Domains of S. aureus in Immunodiagnostics.

Staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 are the super antigens responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome. At low serum concentrations, SEB can trigger toxic shock, profound hypotension and multi organ failure and hence is recognized as biowarfare molecule. In this study, a multidomain fusion protein (r-TE) was generated with specificity for SEB and toxic shock syndrome toxin (Tsst-1). The fusion gene comprising the conserved regions of seb and the tsst genes was codon-optimized for expression in Escherichia coli and encoded a 26 kDa recombinant multidomain chimeric protein (r-TE). Hyperimmune antiserum raised against r-TE specifically reacted with SEB (~28 kDa) and Tsst-1 (~22 kDa) components during Western blot analysis and by plate ELISA in confirmed toxin producing strains of S. aureus. The antigenicity of the SEB component of the r-TE protein was also confirmed using TECRA kit. The described procedure of creating a single protein molecule carrying components of two different toxins whilst still retaining the original antigenic determinants of individual toxins proved highly advantageous in the development of rapid, reliable and cost effective immunoassays and may also have the potential to serve as candidate molecule for vaccine studies.

Cloning, expression and characterization of attachment-invasion locus protein (Ail) of Yersinia enterocolitica and its utilization in rapid detection by immunoassays.

AIMS: Rapid detection of pathogenic Yersinia enterocolitica isolates by using antisera raised against recombinant attachment-invasion locus (Ail) protein. METHODS AND RESULTS: The complete gene (471 bp) encoding for the Ail protein was amplified by PCR and cloned in pQE 30 UA vector. The recombinant clones were selected by polymerase chain reaction (PCR). Recombinant protein was expressed using induction with 1 mmol l(-1) final concentration of isopropylthiogalactoside (IPTG). Polyclonal antibodies were raised in mice against this purified recombinant protein. An indirect plate ELISA was standardized based on rAil protein for the detection of Y. enterocolitica. Western blot analysis with the sera raised against recombinant Ail protein exhibited reaction at 17 kDa region of the native Ail protein present in pathogenic Y. enterocolitica standard strains and strains isolated from pork samples suggesting that the antigenicity of recombinant Ail protein was similar to that of native Ail protein. Nonpathogenic Y. enterocolitica and the other species of Yersinia, namely, Y. pseudotuberculosis, Y. intermedia, Y. kristenseni, Y. fredrickseni and also the Enterobacteriaceae organisms tested were not found reacting to polyclonal antisera against this recombinant Ail protein. CONCLUSION: The antibodies raised against recombinant Ail protein could specifically identify pathogenic Y. enterocolitica strains both by indirect plate ELISA and Western blot immunoassay. SIGNIFICANCE AND IMPACT OF THE STUDY: The method developed in this study may find application in the detection of pathogenic Y. enterocolitica not only from food and environmental samples but also from clinical samples.

Detection of toxigenic strains of Aeromonas species in foods by a multiplex PCR assay.

Aeromonas hydrophila and other aeromonads are ubiquitous organisms found in meat, vegetables, drinking water and various other food items. They cause diarrhea and extra-intestinal infections in normal and immunocompromised patients. The aim of the study was to develop a multiplex PCR assay for the detection of virulence-associated genes of Aeromonas including hemolysin (hlyA), aerolysin (aerA), glycerophospholipid-cholesterol acyl transferase (GCAT) alongwith a 16S rRNA gene. Internal amplification control (IAC), which was coamplified with aerA primers, was also included in this study. The results showed that all cultures of Aeromonas were accurately identified by the assay without showing non-specificity. A. hydrophila could be detected at a range of 10-50 CFU ml(-1) from experimentally spiked fish, chicken and milk samples following overnight enrichment in alkaline peptone water supplemented with 10 µg/ml ampicillin (APW-A) by this multiplex PCR (mPCR). When evaluated on a total of 74 naturally occurring food samples, four samples were identified to contain Aeromonas by mPCR. All these results were compared to the conventional culture, isolation and biochemical identification procedures. The high throughput and cost-effective mPCR method developed in this study could provide a powerful tool for detection of pathogenic Aeromonas spp. from food and environmental samples and in addition, the method has advantages in terms of specificity, sensitivity and ease of use compared to other reported PCR methods and DNA hybridization assays.

Multiplex PCR assay for the detection of enterotoxic Bacillus cereus group strains and its application in food matrices.

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are the major concerns for the food safety in terms of frequency and/or seriousness of the disease. Being members of the same group and sharing DNA homology to a larger extent, they do create problems when their specific detection/identification is attempted from different food and environmental sources. Numerous individual polymerase chain reaction (PCR) and few multiplex PCR (mPCR) methods have been employed to detect these organisms by targeting toxin genes but with lack of internal amplification control (IAC). Therefore, we attempted a mPCR with IAC for the detection of enterotoxic B. cereus group strains by selecting hbl A, nhe A and cyt K genes from B. cereus, indicative of the diarrheal potential and cry I A and pag genes, the plasmid borne phenotypic markers specific to B. thuringiensis and B. anthracis strains, respectively. Multiplex PCR assay validation was performed by simultaneous comparison with the results of single-target PCR assays and correlated to the classical conventional and biochemical identification of the organisms. The mPCR was able to detect as low as 10(1)-10(2) organisms per ml following overnight enrichment of spiked food samples (vegetable biriyani and milk) in buffered peptone water (BPW). The presence of these organisms could also be detected by mPCR in naturally contaminated samples of rice based dishes and milk. The high throughput and cost-effective mPCR method described could provide a powerful tool for simultaneous, rapid and reliable detection of enterotoxic B. cereus group organisms.

Molecular characterization of lactic acid bacteria recovered from natural fermentation of beet root and carrot Kanji.

The lactic acid bacteria (LAB) play an important role in the fermentation of vegetables to improve nutritive value, palatability, acceptability, microbial quality and shelf life of the fermented produce. The LAB associated with beetroot and carrot fermentation were identified and characterized using different molecular tools. Amplified ribosomal DNA restriction analysis (ARDRA) provided similar DNA profile for the 16 LAB strains isolated from beetroot and carrot fermentation while repetitive extragenic palindromic PCR (rep-PCR) genotyping could differentiate the LAB strains into eight genotypes. Thirteen strains represented by five genotypes could be clustered in five distinct groups while three LAB strains exhibiting distinct genotypes remained ungrouped. These genotypes could be identified to be belonging to L. plantarum group by 16S rDNA sequencing. The recAnested multiplex PCR employing species-specific primers for the L. plantarum group members identified the LAB strains of six genotypes to be L. paraplantarum and the other two genotypes to be L. pentosus. Three genotypes of L. paraplantarum were consistently found on the third and sixth day of beetroot fermentation whereas a distinct genotype of L. paraplantarum and L. pentosus appeared predominant on the tenth day. From carrot Kanji two distinct genotypes of L. paraplantarum and one genotype of L. pentosus were identified. REP-PCR DNA fingerprinting coupled with 16S rDNA sequencing and recA-nested multiplex PCR could clearly identify as well as differentiate the diverse L. plantarum group strains involved in the fermentation.

Construction of a recombinant intergenus multidomain chimeric protein for simultaneous expression of haemolysin BL of Bacillus cereus, listeriolysin O of Listeria monocytogenes and enterotoxin B of Staphylococcus aureus.

Haemolysin BL (HBL) of Bacillus cereus, listeriolysin O (LLO) of Listeria monocytogenes and enterotoxin B (SEB) of Staphylococcus aureus are among the major toxin components contributing to the pathogenicity of these organisms in foodborne illnesses. In this study, an intergenus non-toxic multidomain fusion protein (r-HLE) was generated with specificity for HBL, LLO and SEB. The fusion gene (r-hle) comprising the conserved regions of hblD and the hly and entB genes was codon-optimized for expression in Escherichia coli and encoded a 50 kDa recombinant multidomain chimeric protein (r-HLE). Hyperimmune antiserum raised against r-HLE specifically reacted with the L1 (38 kDa) component of the HBL complex of B. cereus, LLO (58 kDa) of L. monocytogenes and SEB (28 kDa) of S. aureus during Western blot analysis when tested on standard strains. During testing on isolates, the antiserum again identified the appropriate toxin molecules and was highly specific to the relevant bacterial species. The antigenicity of the SEB component of the r-HLE protein was also confirmed using a commercially available TECRA kit. The described procedure of creating a single antigenic molecule carrying components of three different toxins whilst still retaining the original antigenic determinants of individual toxins will be highly advantageous in the development of rapid, reliable and cost-effective immunoassays.

Th1-type immune response to infection by pYV-cured phoP-phoQ null mutant of Yersinia pseudotuberculosis is defective in mouse model.

The PhoP-PhoQ two-component system of Yersinia pseudotuberculosis, a Gram-negative enteric pathogen which causes a variety of gastrointestinal and extraintestinal infections in humans, has been shown to be necessary for virulence. A phoP-phoQ null mutant of a strain of Y. pseudotuberculosis cured of its native plasmid pYV was obtained and studied for generation of immune response in mouse model following intravenous inoculation. The phoP-phoQ null mutant elicited much weaker IgG antibody response to whole cell sonicated (WCS) antigen, in particular that of IgG2a isotype. Interferon-gamma levels were also significantly reduced in cultured splenocytes of mice immunized with phoP-phoQ null mutant. The null mutant was found to be about 72-fold less virulent than the parent isogenic strain of Y. pseudotuberculosis. Average counts in spleen of mice inoculated with the null mutant were observed to reduce by at least four logs when compared with the counts in the spleen of mice inoculated with parent isogenic strain. We can thus suggest that the Th1-type immune response of the phoP-phoQ null mutant of Y. pseudotuberculosis is diminished in mice.

Simultaneous detection of pathogenic B. cereus, S. aureus and L. monocytogenes by multiplex PCR.

Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus are of major concern for food safety in terms of frequency and seriousness of the disease. The occurrence these three important pathogens and their coexistence in food matrices are predominant. Moreover, symptoms associated with B. cereus and S. aureus food poisoning not only closely resembles each other but can also be overlapping with other foodborne infections. In this context, detection of these three pathogens simultaneously in food samples by a single multiplex PCR (mPCR) would have advantages in terms of rapidity and cost saving, when compared with single organism specific PCRs. mPCR has been standardized by targeting three major diarrheal enterotoxin genes hbl A, cyt K and nhe A of B. cereus, virulence associated nuc and Ent B genes of S. aureus and virulence associated hly and iap genes of L. monocytogenes along with internal amplification control (IAC). The results showed that mPCR accurately identified all the three organisms individually or in combination without non-specificity. The mPCR was able to detect as low as 10 to 100 organisms per ml of growth following overnight enrichment of spiked food samples (vegetable biriyani and milk) and their presence in naturally contaminated samples also. The high throughput and cost effective multiplex PCR method developed in this study could provide a powerful tool for simultaneous, rapid and reliable detection of B. cereus, S. aureus and L. monocytogenes in food samples.

Antimicrobial susceptibility and molecular characterization of Vibrio cholerae from cholera outbreaks in Chennai.

The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002-2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002-2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.