Multiple-reaction monitoring for multiplex detection of three bacterial toxins using liquid chromatography-tandem mass spectrometry.

Clostridium perfringens epsilon toxin, staphylococcal enterotoxin B and shiga toxin are implicated in a number of diseases and food-borne intoxications and are considered potential agents for bioterrorism and warfare. Artificially generated aerosol is the likely mode of delivery of these for nefarious uses, potentially capable of causing mass destruction to human and animal health by inhalation of toxic bioaerosol. Multiplex and unambiguous detection of these agents is of paramount importance for emergency response in a biothreat scenario and for food safety. Multiple-reaction monitoring (MRM) assay for simultaneous monitoring of the three toxins is reported here using reverse-phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Three different peptides with two fragment ions each were considered for quantification and confirmation. One of the three MRM transitions from each toxin, which exhibited the best sensitivity, was selected for multiplexing of the assay. Simulating a biothreat scenario wherein the bioaerosol is collected in 10 ml of buffer, the multiplex assay was tested with blind samples with one or more of the three toxins even in the presence of interfering Escherichia coli lysate proteins.

Highly Expressed Recombinant SEB for Antibody Production and Development of Immunodetection System.

Staphylococcal enterotoxins (SEs) are the second most common causal agents of food poisoning throughout the world. Staphylococcal enterotoxin B (SEB) is one of the most potent and a listed biological warfare agent. Therefore, its quick, accurate and sensitive detection is of paramount importance. But availability of sensitive and specific antibodies against SEB is the major bottleneck in the development of an immunodetection system. Therefore, in the present study seb gene was cloned and expressed in a heterologous host resulting in a yield of 92 mg pure toxin per litre of culture broth after Ni-NTA affinity purification. Antibodies raised against the recombinant toxin did not cross react with related enterotoxins and organisms that can gain access in the food. Further, a sandwich ELISA was developed to detect SEB after extraction from artificially spiked food samples like milk, orange juice, skim milk and khoya. The sandwich ELISA was able to detect SEB in the range of 0.25 to 0.49 ng/ml or g of food. The detection system developed in the present study is at least as specific and sensitive as other commercially available kits which use monoclonal antibodies.

Isolation, phenotypic characterization and comparative genomic analysis of 2019SD1, a polyvalent enterobacteria phage.

Shigella has the remarkable capability to acquire antibiotic resistance rapidly thereby posing a significant public health challenge for the effective treatment of dysentery (Shigellosis). The phage therapy has been proven as an effective alternative strategy for controlling Shigella infections. In this study, we illustrate the isolation and detailed characterization of a polyvalent phage 2019SD1, which demonstrates lytic activity against Shigella dysenteriae, Escherichia coli, Vibrio cholerae, Enterococcus saccharolyticus and Enterococcus faecium. The newly isolated phage 2019SD1 shows adsorption time < 6 min, a latent period of 20 min and burst size of 151 PFU per bacterial cell. 2019SD1 exhibits considerable stability in a wide pH range and survives an hour at 50 °C. Under transmission electron microscope, 2019SD1 shows an icosahedral capsid (60 nm dia) and a 140 nm long tail. Further, detailed bioinformatic analyses of whole genome sequence data obtained through Oxford Nanopore platform revealed that 2019SD1 belongs to genus Hanrivervirus of subfamily Tempevirinae under the family Drexlerviridae. The concatenated protein phylogeny of 2019SD1 with the members of Drexlerviridae taking four genes (DNA Primase, ATP Dependent DNA Helicase, Large Terminase Protein, and Portal Protein) using the maximum parsimony method also suggested that 2019SD1 formed a distinct clade with the closest match of the taxa belonging to the genus Hanrivervirus. The genome analysis data indicate the occurrence of putative tail fiber proteins and DNA methylation mechanism. In addition, 2019SD1 has a well-established anti-host defence system as suggested through identification of putative anti-CRISPR and anti-restriction endonuclease systems thereby also indicating its biocontrol potential.

Role of nodD gene product and flavonoid interactions in induction of nodulation genes in Mesorhizobium ciceri.

Mesorhizobium ciceri is a host specific bacterium which nodulates the genus, Cicer. Host specificity is regulated at first step by induction of nodulation (nod) genes in the presence of NodD protein and inducers (flavonoids) of plant origin. The inducer specificity of M. ciceri nodD gene was studied in NodD-mutant strain HN-9 carrying heterologous nodD genes and nodAlacZ fusion. The induction profile of nod promoter in M. ciceri revealed that nodD gene product of M. ciceri is specifically activated by chickpea root exudates only. M. ciceri HN-9 (nodA-lacZ) containing heterologous nodD genes from Rhizobium leguminosarum bv. viciae, R. leguminosarum bv. trifolii and Sinorhizobium meliloti was induced in presence of a number of flavonoids. On the other hand, induction profile of nod promoter showed that heterologous nodD gene products were activated to different levels in NodD(-) mutant of M. ciceri in presence of root exudates from homologous as well as heterologous legume hosts. The transfer of FITA (Flavonoid independent transcription activation) nodD gene in NodD(-) mutant, M. ciceri HN-9, was able to break the inducer specificity barrier and nod promoter was induced to maximum level irrespective of the presence or absence of inducer. It is concluded from the results that host specificity in M. ciceri - chickpea (Cicer arietinum) symbiosis is regulated at first step by the host specific interaction of nodD gene product of M. ciceri and inducers present in the root exudates of chickpea.

A large cholera outbreak due to a new cholera toxin variant of the Vibrio cholerae O1 El Tor biotype in Orissa, Eastern India.

A total of 32 Vibrio cholerae isolates were collected during a recent large cholera outbreak in Eastern India. Biochemical and serological studies revealed that all of the isolates belonged to serogroup O1, biotype El Tor, serotype Ogawa. Two multiplex PCR assays confirmed the presence of various toxigenic and pathogenic genes - ace, ctxAB, hlyA, ompU, ompW, rfbO1, rtx, tcp, toxR and zot - in all of the isolates. Sequencing of the ctxB gene from the isolates revealed a novel mutation in the gene. Sequencing also confirmed the presence of altered cholera toxin B of the classical biotype in all of the El Tor isolates, suggesting infection of isolates by classical CTXPhi. The molecular diversity of V. cholerae isolates studied by enterobacterial repetitive intergenic consensus sequence PCR, BOX-PCR and randomly amplified polymorphic DNA analysis uniformly showed the clonal relationship among the outbreak V. cholerae O1 isolates. The results of this study suggest that cholera-causing V. cholerae strains are constantly evolving in epidemic areas, highlighting the potential of the emergence of more virulent strains.

Single multiplex polymerase chain reaction for environmental surveillance of toxigenic-pathogenic O1 and non-O1 Vibrio cholerae.

A multiplex PCR assay was developed for the detection of toxigenic and pathogenic V. cholerae from direct water sources using specific primers targeting diverse genes, viz. outer membrane protein (ompW), cholera toxin (ctxB), ORF specific for O1 (rfbG), zonula occludens (zot) and toxin co-regulated pilus (tcpB); among these genes, ompW acts as internal control for V. cholerae, the ctx gene as a marker for toxigenicity and tcp for pathogenicity. The sensitivity of multiplex PCR was 5 x 10(4) V. cholerae cells per reaction. The procedure was simplified as direct bacterial cells were used as template and there was no need for DNA extraction. The assay was specific as no amplification occurred with the other bacteria used. Toxigenic V. cholerae were artificially spiked in different water samples, filtered through a 0.45 microm membrane, and the filters containing bacteria were enriched in APW for 6 h. PCR following filtration and enrichment could detect as little as 8 V. cholerae cells per mL in different spiked water samples. Various environmental potable water samples were screened for the presence of V. cholerae using this assay procedure. The proposed method is rapid, sensitive and specific for environmental surveillance for the presence of toxigenic-pathogenic and nonpathogenic V. cholerae.

In-Vivo Neutralization of Botulinum Neurotoxin Serotype E Using Rabbit Polyclonal Antibody Developed against BoNT/E Light Chain.

BACKGROUND: Clostridium botulinum is an obligate anaerobic, Gram positive bacterium that secretes extremely toxic substances known as botulinum neurotoxins (BoNTs) that cause serious paralytic illness called botulism. Based upon the serological properties, these neurotoxin have been classified into seven serotypes designated from A to G. Due to extreme toxicity of BoNTs, these neurotoxins have been designated as category A biowarfare agents. There is no commercial neutralizing antibody available for the treatment of botulism. Hence there is an urgent need to develop therapeutic intervention for prevention and cure of botulism within short period. BoNT antiserum injection is still the effective treatment. METHOD: In the present study, the recombinant light chain of BoNT/E was successfully purified in soluble form. The purified rBoNT/E LC was used for the generation of polyclonal antibody in rabbit. In order to find out the neutralizing capacity of generated antisera, rabbit antiserum was incubated with 20 LD50 of botulinum neurotoxin type E for 1 hour at 37°C and then injected intraperitoneally (IP) into mice. Further in another set of experiments antiserum was administered in different ways that included administration of - antiserum and BoNT/E toxin simultaneously without preincubation, one after another at the same and different time points for its therapeutic ability. To find out cross neutralization capacity, rBoNT/E LC antiserum was pre-incubated with 5 LD50 of BoNT/A, BoNT/B, BoNT/F and then injected (IP) into mice. In all the cases mice were observed continuously for 96 hours. RESULT: The results clearly indicate that developed polyclonal rabbit antiserum showed serotype specific neutralization of BoNT/E toxin only but not of BoNT/A, BoNT/B and BoNT/F. CONCLUSION: The developed antibodies will be used for preventive and therapeutic intervention of type 'E' botulism.

Construction of Recombinant Single Chain Variable Fragment (ScFv) Antibody Against Superantigen for Immunodetection Using Antibody Phage Display Technology.

Superantigens are a class of antigens that bind to the major histocompatibility complex class (MHC) II and T-cell receptor (TCR) and cause the nonspecific activation of T cells, resulting in a massive release of pro-inflammatory mediators. They are produced by the gram-positive organisms Staphylococcus aureus and Streptococcus pyogenes, and by a variety of other microbes such as viruses and mycoplasma, and cause toxic shock syndrome (TSS) and even death in some cases. The immunodetection of superantigens is difficult due to the polyclonal activation of T-cells leading to nonspecific antibody production. The production of recombinant monoclonal antibodies against superantigens can solve this problem and are far better than polyclonal antibodies in terms of detection. Here, we describe the construction of recombinant single chain variable fragments (ScFv) antibodies against superantigens with specific reference to SEB (staphylococcal enterotoxin B) using antibody phage display technology.

Direct immunofluorescence assay for rapid environmental detection of Vibrio cholerae O1.

An immunofluorescence assay for direct detection of V. cholerae O1 was developed using polyclonal antibodies raised against outer membrane proteins (OMPs) of V. cholerae O1. Production of OMPs varied with growth media used; maximum production was found in tryptic soy broth. The detection system was specific because no cross-reactivity was observed with other bacteria including V. cholerae O139, E. coli, S. dysenteriae and Salmonella enterica subsp. enterica serovar Typhi. The technique was able to detect 240 CFU/mL of V. cholerae O1 suspended in phosphate-buffered saline. The assay coupled with bacterial enrichment in APW for 6 h detected as few as 5 CFU of V. cholerae in spiked samples. Moreover, a 2-h incubation of enriched bacterial cells in 0.1% yeast extract with 10 ppm nalidixic acid enhanced the bacterial size and helped in morphological identification of V. cholerae. Among 32 potable water samples from afflicted hand pumps and wells collected from a cholera-plagued area 12 were found to be contaminated with V. cholerae by immunofluorescence assay as well as by conventional culture methods. The proposed method could thus be employed in environmental surveillance of V. cholerae O1.

Preincubation of Mesorhizobium ciceri with flavonoids improves its nodule occupancy.

Strains of M. ciceri, symbionts of chickpea (Cicer arietinum) were incubated with the flavonoids naringenin, daidzein and quercetin which have earlier been reported as inducers and inhibitors of nodABC-lacZ fusion of M. ciceri. Preincubation of M. ciceri with naringenin and daidzein (100 nmol/L) for 1 d improved the competitive ability of the inoculated strain while preincubation with quercetin decreased the nodule occupancy of inoculated strain under sterile conditions. Under non-sterile conditions induced strains of Rcd 301 and HT-6 formed by 23 and 18% more nodules, respectively, than untreated control. Quercetin-treated strains showed by 13-20% fewer nodules than untreated controls. Therefore, it is possible to regulate the competitive ability of inoculated strains by flavonoid treatment.

Multidrug resistant Vibrio cholerae O1 El Tor carrying classical ctxB allele involved in a cholera outbreak in South Western India.

Cholera is a fatal diarrheal disease characterized with enormous fluid loss through stools. A total of 41 Vibrio cholerae isolates collected from a recent cholera outbreak in Solapur, South Western India were found to belong to serogroup O1, biotype El Tor and serotype Ogawa. Molecular analysis revealed the prevalence of different toxigenic and pathogenic genes in the isolates. All the isolates harboured rstR(El)(Tor) allele indicating the presence of CTXΦ(El)(Tor). However, cholera toxin (ctxB) gene sequencing and a ctxB allele specific PCR of the isolates confirmed the presence of ctxB of classical biotype. The antibiogram profile revealed the resistance for several antibiotics including nalidixic acid, polymyxin B, streptomycin, sulfamethoxazole, trimethoprim, rifampicin and vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). All the isolates were PCR positive for class 1 integron and SXT elements also. Fingerprinting analysis revealed the clonal relationship among the outbreak isolates. The results suggested the involvement of multidrug resistant V. cholerae El Tor biotype isolates having ctxB gene of classical biotype in the cholera outbreak.

Virulence profile and clonal relationship among the Vibrio cholerae isolates from ground and surface water in a cholera endemic area during rainy season.

All the V. cholerae non-O1, non-O139 isolates from ground and surface water samples collected during the rainy season (rainfall contributes significantly in the spread of cholera) contained ompW and a regulatory toxR gene, while many others possessed accessory cholera toxin (ace), hemolysin (hlyA) and outer membrane protein (ompU) genes. All the isolates lacked ctxAB, tcp, zot, rfbO1 and rfbO139 genes. The strains could be grouped into two main clusters colligating the isolates from ground water and surface water samples. The results suggest that surface water harbors various virulent V. cholerae strains that contaminate the ground water due to rain or poor hygienic practices, and result in the emergence of new toxigenic strains for cholera.