Production of cholera toxin-like toxin by Vibrio mimicus and non-O1 Vibrio cholerae: batch culture conditions for optimum yields and isolation of hypertoxigenic lincomycin-resistant mutants

Abstract

Vibrio mimicus 61892, isolated in 1977 from a case of watery diarrhea in Bangladesh, produces an enterotoxin which possesses activity in Y-1 mouse adrenal cells and in rabbit ileal loops which is identical to the prototype cholera toxin (CT) produced by Vibrio cholerae 569B. The neutralization of the adrenal cell activity of 61892 toxin and 569B CT by homologous and heterologous antisera generates parallel titration curves which show complete neutralization in all cases. Paired titrations in the ganglioside GM1 enzyme-linked immunosorbent assay (using either CT or Escherichia coli heat-labile toxin antitoxin) of both toxins indicates that 61892 toxin is antigenically indistinguishable from 569B CT. The specific activity of the two toxins in the rabbit ileal loop is virtually identical. Batch culture production of CT-like toxin and CT by isolates of V. mimicus and different biotypes of V. cholerae was found to be highest in shake flask cultures of Casamino Acids-yeast extract broth grown at 27 degrees C with vigorous aeration. Incorporation of lincomycin into the growth medium at a concentration of 50 micrograms/ml increased yields from wild-type strains. Dramatically higher yields were obtained when a spontaneous resistance mutant of strain 61892 was grown in the presence of 200 to 300 micrograms of lincomycin per ml. Under these conditions, yields of CT-like toxin were increased by 300- to 500-fold, and the highest yields reached more than 100 micrograms/ml after 44 h of culture. This is substantially higher than that reported in the literature for CT production by any strain of V. cholerae, including hypertoxigenic strain 569B.