EFFECT OF ISOLEUCINE ON TOXIN PRODUCTION BY CLOSTRIDIUM-DIFFICILE IN A DEFINED MEDIUM

Supplementation of a carbohydrate-free minimal medium with a high leve l (100 mM) of histidine, methionine, valine, isoleucine, proline and l eucine, in particular isoleucine, markedly increased toxin production by Clostridium difficile WI 10463. The effect of isoleucine was furthe r examined. Increasing the concentration of isoleucine from 20 to 100 mM remarkably increased toxin production, while bacterial growth decre ased gradually. Amino acid analysis of the culture revealed that, at 1 00 mM isoleucine, consumption of isoleucine was remarkably increased. During the incubation period when toxin titers increased markedly but bacterial growth was declining, isoleucine, leucine and cysteine were taken up preferentially and alanine and cystathionine, which were not found at 1 mM isoleucine, were produced in large quantities. These fin dings suggest that isoleucine may play an important role in toxin prod uction by C. difficile and that alanine and cystathionine production m ay be co-regulated with the toxin production in the absence of ferment able carbohydrates.