Vibrio cholerae non-O1 and V. mimicus in diarrhoeal disease : a study of virulence factors

Vibrio cholerae non-01 and V. mimicus, isolated from clinical and environmental sources, were examined for factors related to virulence. The aim was to identify factors which would distinguish pathogenic from non-pathogenic strains and to establish a correlation, if any, with serogroup. The lipopolysaccharide (LPS) of epidemic V. cholerae belonging to serogroups 01 and 0139 are regarded as'virulence factors. In this study certain other serogroups, such as 02,05 and 09 were associated with diarrhoeal disease. LPS of these organisms may have a role in adhesion. Although 90% of V. cholerae non-01 and V. mimicus colonised tissue culture cells, neither smooth LPS, the presence of flagella nor the possession of the toxin coregulated pilus gene (tcpA) were essential for adhesion. Cholera toxin (CT) is the factor responsible for the dramatic symptoms of epidemic cholera and is produced by V. cholerae serogroups 01 and 0139. However, less than 1% of V. cholerae non-01, non-0139 and V. mimicus possessed the gene for CT. Southern blot analysis of the CT genes revealed that most strains carried one CT gene except for serogroups 023 and 0139 which had two hybridising fragments. The variations in fragment size within the 0139 serogroup suggested that this serogroup was not strictly clonal. In addition to CT both V. cholerae non-01 and V. mimicus expressed several other toxins, often concurrently, which made detection of specific toxins difficult. Over 80% of strains produced haemolysin(s) and cytotoxin(s). The cytotoxic effects on tissue culture cells masked cytotonic effects, such as that caused by CT. V. cholerae and V. mimicus did not produce verocytotoxin (VT) and did not possess the genes for VT. The heat-stable enterotoxin (NAG-ST) gene was found in 3% of V. cholerae non-01 and 12% of V. mimicus strains. All V. cholerae strains belonging to serogroup 014, were NAG-ST positive and Southern blot analysis of the NAG-ST genes suggested that this serogroup represented a single clone. In addition to the established toxins two novel factors, which may contribute to the virulence of V. cholerae non-01 and V. mimicus, were found. A novel cytotoxin, produced by 16% of strains, was expressed on Vero cells causing vacuolation of cell cytoplasm. Another putative "toxin" found in 19% of strains was demonstrated in a GMI-ELISA. This "toxin" had the ability to bind ganglioside GMI and shared antibody binding sites with CT. V. cholerae non-01 and V. mimicus also expressed species specific high affinity iron chelating molecules; 74% of V. cholerae non-01 produced enterochelin, whereas 90% of V. mimicus produced aerobactin. Aerobactin production is usually associated with invasive organisms and is unusual among Vibrio spp. None of the virulence factors, except CT, was more prevalent in clinical than in environmental strains. This is perhaps not surprising as vibrio-associated disease is often linked with exposure to the aquatic environment or consumption of seafood. Within V. cholerae the 01 and 0139 serogroups were linked with CT and 014 with NAG-ST, in future, other serogroup - virulence factor associations may be found. For V. mimicus the main virulence factors expressed were aerobactin and NAG-ST. Therefore the pathogenicity of V. cholerae and V. mimicus appears to be multifactorial and it is likely that, as with diarrhoeagenic, E. coli, a heterogeneous pattern of virulence will be found.