J. Med. Microbiol. - Vol. 33 (1990), 107-1 14 0 1990 The Pathological Society of Great Britain and Ireland 0022-261 5/90/00334107/$10.00 Plasmids and factors associated with virulence in environmental isolates of Vibrio cholerae non-0 I in Bangladesh J. L. BARJA, YSABEL SANTOS, I. HUQ", RITA R. COLWELLt and ALICIA E. TORANZO Departamento de Microbiologia y Parasitologia, Facultad de Biologia, Universidad de Santiago de Compostela 15706, Spain, "International Centre for Diarrhoea1 Disease Research, Dacca, Bangladesh and t Department of Microbiology, University of Maryland, College Park 20742, USA Summary. Plasmid profiles and factors associated with toxigenicity in 51 strains of Vibrio cholerae non-01 isolated from water samples collected in Bangladesh were analysed. Eleven (21.5%) strains were found to harbour at least one plasmid of 1.7- 115 Mda; seven of these strains shared a 115-Mda plasmid. Six of 13 strains tested gave positive cytotoxic and enterotoxic responses. However, two non-cytotoxicstrains were enterotoxigenic. Only three of the six cytotoxic and enterotoxic strains caused haemagglutination of human erythrocytes which indicated that toxin production and haemagglutinating activity were unrelated in these V. cholerae non-0 1 strains. Conjugal transfer assays demonstrated that the 1 15-Mdaplasmid harboured by some of the toxigenic V. cholerae non-01 strains carried genes coding for antibiotic resistance and cytotoxin production but not for enterotoxin production. However, this plasmid was also carried by non-toxigenic strains. Some other strains carrying no plasmids or only small-mo1.-wt plasmids, were found to be toxigenic. Therefore, toxin production is not plasmid-mediated in all V. cholerae non-01 strains. Regardless of their pathogenic potential, V. cholerae non-01 strains possessed the capacity to grow in conditions of iron limitation and, under these conditions, synthesis of at least two new outer-membrane proteins was induced. Introduction At present there is little doubt about the autochthonous aquatic nature of the non-01 serovars of Vibrio cholerae. V. cholerae non-01 strains are widely distributed in marine environ- ments, especially bays, estuaries and other brackish waters, and in shellfish. seem to indicate that V. cholerae non-01 strains are not associated with sewage pollution, being more abundant in the natural environment where they are associated frequently with the micro- and macro-flora and fauna of fresh and coastal The number of reports of diarrhoea1 illness in different countries caused by V. cholerae non-01 strains has increased over the past decade as a result of the recognition of their importance as pathogens for man*-" and anirna1s.j' ''? l2 Al- though it has been reported'j that V. cholerae non- 0 1 strains can produce more than one type of Ecological studies' Received 2 March 1990; accepted 19 April 1990. cholera-li ke enterotoxin, the pat hophysiological mechanism underlying V. cholerae non-01 induced diarrhoea remains largely unknown because many strains do not produce cholera toxin (CT).l9 2y 12* l4 Furthermore, whether clinical or environmental isolates possess the higher enteropathogenic poten- tial is controversial. 14-16 In the work reported here, several factors associated with the toxigenicity of environmental V. cholerae non-01 isolates from waters in Bangla- desh were studied and the drug resistance patterns and plasmid profiles of these strains were analysed. Materials and methods Bacterial strains and antimicrobial susceptibility tests In this study, 51 I/. cholerae non-01 strains isolated from water samples collected at three different sites in Bangladesh-Dacca (D), Matlab (M) and Teknaf (TK)- were examined. Strains were cultured routinely at 25°C in Trypticase Soy Broth (TSB; Oxoid, CM129) supple- mented with NaCl 1%, and maintained on Trypticase Soy Agar (TSA; Oxoid, CM131). Strains were identified by the procedures and criteria of West and Colwell.' 107