Pathogenic potential of Aeromonas hydrophila isolated from surface waters in Kolkata, India.

Members of the genus Aeromonas (family Aeromonadaceae) are medically important, Gram-negative, rod-shaped micro-organisms and are ubiquitous in aquatic environments. Aeromonas species are increasingly recognized as enteric pathogens; they possess several virulence factors associated with human disease, and represent a serious public health concern. In the present study, putative virulence traits of Aeromonas hydrophila isolates collected from different natural surface waters of Kolkata, India, were compared with a group of clinical isolates from the same geographical area using tissue culture and PCR assays. Enteropathogenic potential was investigated in the mouse model. Of the 21 environmental isolates tested, the majority showed cytotoxicity to HeLa cells (81 %), haemolysin production (71 %) and serum resistance properties (90 %), and they all exhibited multi-drug resistance. Some of the isolates induced fluid accumulation (FA ratio>or=100), damage to the gut and an inflammatory reaction in the mouse intestine; these effects were comparable to those of clinical strains of A. hydrophila and toxigenic Vibrio cholerae. Interestingly, two of the isolates evoked a cell vacuolation effect in HeLa cells, and were also able to induce FA. These findings demonstrate the presence of potentially pathogenic and multi-drug-resistant A. hydrophila in the surface waters, thereby indicating a significant risk to public health. Continuous monitoring of surface waters is important to identify potential water-borne pathogens and to reduce the health risk caused by the genus Aeromonas.

Putative virulence traits and pathogenicity of Vibrio cholerae Non-O1, Non-O139 isolates from surface waters in Kolkata, India.

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR, and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin (ctxA), heat-stable toxin (est), heat-labile toxin (elt), and Shiga toxin variants (stx) of Escherichia coli. Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin (hly). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (>/=100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae. Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.

Development of a simple latex agglutination assay for detection of shiga toxin-producing Escherichia coli (STEC) by using polyclonal antibody against STEC.

Rabbit antiserum raised against the whole-cell antigen of Shiga toxin-producing Escherichia coli (STEC) strain VT3 (stx1+ stx2+ eae+) was repeatedly adsorbed with heat-killed cells of different non-STEC strains and other enteric bacteria. Thus, the antiserum obtained was designated VT3 antiserum. VT3 antiserum reacted with intimin type gamma. We assessed the reactivity of VT3 antiserum to whole-cell lysates of 87 strains of E. coli and other enteric bacteria by immunoblotting. The antiserum recognized the 97-kDa protein in whole-cell lysate from strain VT3, and 36 (83.7%) of the 43 STEC strains were positive for the STEC antigen. None of the non-STEC strains or strains of other species examined tested positive by immunoblotting. Based on this result, we developed a latex agglutination assay for the detection of STEC strains. Thirty-five (81.4%) of the 43 STEC strains tested positive for the STEC antigen by the latex agglutination assay. One (3.3%) of the 30 non-STEC strains and none of the strains of the other enteric bacteria included in this study tested positive by the latex agglutination assay. The corresponding specificity of the latex agglutination assay was approximately 98%. Results of this study showed the production of STEC antiserum and the generation of a simple, cost-effective, sensitive, and specific latex agglutination assay for establishing an etiological diagnosis of STEC.

Antibacterial, antisecretory and antihemorrhagic activity of Azadirachta indica used to treat cholera and diarrhea in India.

Indigenous uses of Azadirachta indica A. juss (Maliaceae) (locally known as neem) leaves in different parts of India for curing gastrointestinal disorder such as diarrhea and cholera is wide spread. The objective of the present study was to evaluate the antibacterial and antisecretory activity of neem extract against Vibrio cholerae, a causative agent of watery diarrhea such as cholera. The methanol extract of neem leaf was tested for its antibacterial, antisecretory and antihemorrhagic activity against Vibrio cholerae. Azadirachta indica extract had significant antibacterial activity against the multi-drug-resistant Vibrio cholerae of serotypes O1, O139 and non-O1, non-O139. The minimum inhibitory concentration reached by 50% (MIC50) and 90% (MIC90), and minimum bactericidal concentration for the extract were 2.5, > 5, and 10 mg/ml, respectively. Neem extract showed antisecretory activity on Vibrio cholerae induced fluid secretion in mouse intestine with inhibition values of 27.7%, 41.1%, 43.3%, 57.0%, and 77.9% at doses of 100, 200, 300, 450 and 1800 mg/kg, respectively. Oral administration of the extract inhibited hemorrhage induced by Vibrio cholerae in mouse intestine at a dose > or = 300 mg/kg. The results obtained in this study give some scientific support to the uses of neem employed by the indigenous people in India employed for the treatment of diarrhea and dreadful disease cholera.