Abstract
A collection of 400 Vibrio-like organisms, isolated from clinical and environmental sources in different geographical locations, was assembled at the Cholera Research Laboratory, Dacca, Bangladesh. From this collection 222 isolates were selected for further study. The method of selection favoured the inclusion of freshly isolated human associated non cholera vibrios (NCVs) although a representative number of V. cholerae and other strains were also included. The following models were used to determine the spectrum of biological activity among isolates : infant rabbit; adult rabbit ligated ileal loop; rabbit skin permeability assay; the adult and suckling mouse and the CHO and adrenal tissue culture assay. The observed pattern of biological activity was separated into 4 categories. Isolates were characterised as producing: a cholera-like toxin; a low molecular weight, heat stable toxin; enteritis without demonstrable extracellular enterotoxin and no activity in these models. Culture filtrates from the cholera-like toxin producing strains elicited fluid accumulation in ligated rabbit ileal loops, a blueing response in rabbit’s backs, morphological changes in adrenal and CHO cell cultures; this toxin was completely neutralised by antiserum against purified choleragen. The heat stable toxin was similar in behaviour and nature to E. coli ST enterotoxin. Enteritis producing strains were characterised by producing a positive infant rabbit response and fluid accumulation in the ileal loop when whole cultures were used but sterile culture filtrates lacked biological activity. Out of 140 NCVs examined 23 (16. 4%) produced a CT-like toxin; 2 (1. 4%) produced a ST toxin; 90 (64. 3%) produced an enteritis reaction and 25 (17. 9%) were inactive. Human associated NCVs possessed a greater pathogenic potential than their environmental counterparts. No enterotoxic factors were detected in strains of V. parahaemolvticus: V. fluvialis or A. hydrophila although enteritis type reactions were noted. Of the 222 isolates screened for biological activity 195 isolates were compared over 121 characterisation tests and the results were subjected to a numerical taxonomic analysis. From the dendrograms 5 phenons which corresponded to descriptions of cholerae 01 and non 01; V. metschnikovii: A. hydrophila; V. fluvialis and parahaemolvticus and 4 miscellaneous phenons were distinguished. The mean %GC composition of representative strains of cholerae. A. hvdrophila. V. parahaemolvticus V. fluvialis and V. metschnikovii was 46. 9; 59. 6; 45. 6; 50. 4 and 43. 3 respectively. The V. cholerae phenon contained strains of the 01 and non 01 serovars and was separated into 3 biotypes: the 01 serovars; non 01 serovars of Heiberg group I and II and non 01 serovars of Heiberg group V. Each biotype was distinguished by 2 characterisation tests and contained strains which elaborated a cholera-like toxin. All strains within the A. hydrophila phenon were anaerogenic; minor variation was noted in sucrose fermentation and ampicillin sensitivity. Minor variation among V. parahaemolyticus strains was noted for arabinose fermentation and the enteritis response in rabbits was not correlated with a positive Kanagawa test reaction. Two environmental isolates did not correspond to descriptions of any current species, they were most similar to the V. metschnikovii phenon but differed by: resistance to 10ug of 0129, ornithine decarboxylase and arginine dihydrolase activity, positive oxidase and amylase activity and inositol fermentation; they possessed a %GC of 43. 4. Another isolate, sensitive to 0129, oxidase positive, failed to reduce nitrates and possessed a %GC content of 44 was also not identifiable to a current Vibrio species. A computer derived diagnostic scheme for the recognition of these phenons was prepared. The frequency of multiple antibiotic resistance in non 01 V. cholerae strains was low: most strains were sensitive to 7 of 9 test antibiotics. Higher resistance frequencies were noted for strains of V. parahaemolyticus. V. fluvialis and A. hydrophila. Attempts to transfer antibiotic resistance from the resistant strains were unsuccessful. Plasmids were found in 13 strains although their function remained cryptic. The vibriostatic agent 0129 was found to be of intra and intergeneric value. However 0129 sensitivity was adversely affected by high sodium chloride levels. The fate of 4 V. cholerae strains in Ringer’s solution and under glucose limitation in the chemostat was determined. Two environmental nonpathogenic V. cholerae isolates survived longer in the simple salts solution and were more competitive under glucose limitation in the chemostat than 2 human associated toxigenic V. cholerae. Cholera-like toxin production in non 01 V. cholerae (and thus pathogenicity) was independent of serotype and Heiberg fermentation pattern and was not significantly correlated with any other bacteriological character or the presence of extrachromosomal DNA. It is concluded that enterotoxin production has to be tested directly : there is no bacteriological proxy.