Dev Vrat Kamboj

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-O1vibrio cholerae

A multiplex PCR assay was developed for the detection of toxigenic and pathogenicV. 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 forV. cholerae, thectx gene as a marker for toxigenicity andtcp for pathogenicity. The sensitivity of multiplex PCR was 5 × 104V. 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. ToxigenicV. cholerae were artificially spiked in different water samples, filtered through a 0.45 µm membrane, and the filters containing bacteria were enriched in APW for 6 h. PCR following filtration and enrichment could detect as little as 8V. cholerae cells per mL in different spiked water samples. Various environmental potable water samples were screened for the presence ofV. cholerae using this assay procedure. The proposed method is rapid, sensitive and specific for environmental surveillance for the presence of toxigenicpathogenic and nonpathogenicV. cholerae.

Construction of a single-chain variable-fragment antibody against the superantigen Staphylococcal enterotoxin B.

ABSTRACT Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detection of SEB is of the utmost importance. High-affinity and specific monoclonal antibodies are being used for detection of SEB, but hybridoma clones tend to lose their antibody-secreting ability over time. This problem can be overcome by the use of recombinant antibodies produced in a bacterial system. In the present investigation, genes from a hybridoma clone encoding monoclonal antibody against SEB were immortalized using antibody phage display technology. A murine phage display library containing single-chain variable-fragment (ScFv) antibody genes was constructed in a pCANTAB 5E phagemid vector. Phage particles displaying ScFv were rescued by reinfection of helper phage followed by four rounds of biopanning for selection of SEB binding ScFv antibody fragments by using phage enzyme-linked immunosorbent assay (ELISA). Soluble SEB-ScFv antibodies were characterized from one of the clones showing high affinity for SEB. The anti-SEB ScFv antibody was highly specific, and its affinity constant was 3.16 nM as determined by surface plasmon resonance (SPR). These results demonstrate that the recombinant antibody constructed by immortalizing the antibody genes from a hybridoma clone is useful for immunodetection of SEB.

Surveillance methodology for Vibrio cholerae in environmental samples

Abstract The purpose of this study was to examine the prevalence of Vibrio cholerae in environmental water samples by using a series of biochemical tests. A total of 223 V. cholerae-like bacteria were isolated from TCBS agar after spreading the alkaline peptone water enriched sewer (n = 21) and water (n = 16) samples. All oxidase positive isolates were subjected to confirmation for V. cholerae by seven other biochemical tests and polymerase chain reaction. Only 74.2% isolates were found to be V. cholerae by PCR using primers against an outer membrane protein (ompW) gene, out of which only 2 isolates were positive for cholera toxin (ctxAB) gene. Among the various biochemical tests studied, arginine hydrolysis, arabinose fermentation and string test showed 92 – 100% sensitivity and 42 – 67% specificity. Eight isolates including the toxigenic ones, showed agglutination with V. cholerae O1 antiserum. The present study showed that no biochemical test is 100% specific for V. cholerae. However, a few tests, if performed in a sequence after growing the alkaline peptone water enriched samples onto TCBS media can be used for screening of V. cholerae from the environmental samples. This study also showed that most of the environmental isolates are non-O1/non-O139 and the chances of presence of toxigenic V. cholerae are very rare in the environment.

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.

Effect of propionate toxicity on methanogenesis of night soil at phychrophilic temperature.

The effect of propionate concentrations on biodegradation of human waste (night soil) was studied at 10 degrees C. Propionate was toxic for the biomethanation at all the pH tested (6.0, 7.0 and 8.0). The maximum reduction in biogas production in presence of 200 mM propionate was observed at pH 7.0 followed by 8.0. The methane content in biogas also followed a similar trend and at pH 7.0 an 11.5% decrease was observed. Propionate caused the reduction of methanogenic count by an approximately 2log value. Total volatile fatty acids increased with the increase in propionate concentration and particularly accumulation of propionate was observed. The results were also compared with the 30 degrees C fermentation.

Direct immunofluorescence assay for rapid environmental detection ofVibrio cholerae O1

An immunofluorescence assay for direct detection ofV. cholerae O1 was developed using polyclonal antibodies raised against outer membrane proteins (OMPs) ofV. chloerae 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 includingV. cholerae O139,E. coli, S. dysenteriae andSalmonella enterica subsp.enterica serovar Typhi. The technique was able to detect 240 CFU/mL ofV. cholerae O1 suspended in phosphate-buffered saline. The assay coupled with bacterial enrichment in APW for 6 h detected as few as 5 CFU ofV. 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 ofV. cholerae. Among 32 potable water samples from afflicted hand pumps and wells collected from a cholera-plagued area 12 were found to be contaminated withV. cholerae by immunofluorescence assay as well as by conventional culture methods. The proposed method could thus be employed in environmental surveillance ofV. cholerae O1.

Relative efficiency of zinc sulfide (ZnS) quantum dots (QDs) based electrochemical and fluorescence immunoassay for the detection of Staphylococcal enterotoxin B (SEB)

In this paper an attempt was made to detect Staphylococcal enterotoxin B (SEB) both by electrochemical and fluorescence immunoassay methods using zinc sulphide (ZnS) QDs. Wet-chemical method was adopted for the preparation of fluorescent ZnS QDs (diameter ∼ 5–10 nm). These QDs were bioconjugated with monoclonal antibodies and then characterized by various method. A detection limit of 0.02 ng mL−1 by fluorescence assay and 1.0 ng mL−1 by electrochemical assay for SEB was achieved. While by sandwich ELISA it is possible to detect 0.24 ng mL−1 only. The sensitivity of all techniques is very good, since the LD50 of SEB is 20 ng kg−1. Electrochemical assay is faster, need low-cost instrument, independent to the size of QDs and found to be one of the best alternative methods as compared to the other existing methods studied herein. The presented method could be expanded to the development of electrochemical and fluorescence biosensors for various agents for field and laboratory use.

Sensitive detection of staphylococcal enterotoxin B (SEB) using quantum dots by various methods with special emphasis on an electrochemical immunoassay approach

Staphylococcal enterotoxin B (SEB) is a potent foodborne pathogen and categorized as a class B type of biological warfare agent. In this research work, SEB is detected by various sensitive analytical methods such as enzyme linked immunosorbent assay (ELISA), quantum dots-based fluorescence linked immunosorbent assay (QDs-FLISA) and square-wave voltammetry (SWV). The obtained results were compared in terms of sensitivity, ease of experimentation and analysis time. For the QD-based detection, fluorescent lead sulfide (PbS) QDs were prepared by a bottom-up approach and characterized by various techniques. Highly specific antibodies against SEB were conjugated with the prepared PbS QDs and were used as revealing antibodies. For the electrochemical detection of SEB, rabbit anti-SEB polyclonal antibodies (primary antibodies) were immobilized on screen-printed electrodes (SPEs) followed by the addition of various concentrations of SEB antigen. These electrodes were further incubated with revealing antibodies. Finally, 1 M HCl solution was added to the SPE to dissolve the PbS QDs which were captured in a sandwiched immunoassay, and resulting Pb2+ ions were determined by the SWV method using a glassy-carbon electrode. The obtained peak current is proportional to the amount of Pb2+ ions which indirectly depends on the SEB concentration. Linearity was observed in the concentration range of 1 ng mL−1 to 1 μg mL−1 of SEB antigen. The limit of detection was found to be 0.01 ng mL−1 for SEB. The results reveal that electrochemical SWV sensing is much easier, faster and provides high sensitivity as compared to the other methods. It is found that the detection limits achieved for sandwich ELISA and QDs-FLISA were 0.24 ng mL−1 and 0.03 ng mL−1 respectively. In addition, the developed SWV method can be implemented for the on-site detection of SEB particularly for civil and defense applications where security is of prime importance.

Characterization of Vibrio cholerae from deep ground water in a cholera endemic area in Central India.

A total of 8 out of 11 deep ground water samples collected from different villages in Central India were found contaminated with Vibrio cholerae non O1, non O139. In a multiplex PCR, isolates were found positive for ompW gene but negative for ctxAB and rfbO1 genes. However, isolates from two places were positive for tcp and zot genes, indicating their intestinal colonization and toxigenic potential. Antibiotic susceptibility studies revealed that all isolates were multidrug resistant. Although, none of the isolates was found PCR positive for the mobile genetic elements, class 1 integrons and SXT constins. The results of this study corroborated that deep ground water can also be an important reservoir of V. cholerae in plane endemic areas, suggesting a continuous monitoring of water samples for timely prevention of the disease.