Hemolysin-destructive factor of Vibrio cholerae (Vibrio comma).

Feeley and Pittman (Bull. World Health Organ. 28:347, 1963) speculated that a hemolysin (HL) destructive enzyme produced by El Tor vibrio at a late stage of growth lowers the HL titer. We proved the production of an HL-destructive factor (HDF) by strains of classical cholera and El Tor vibrio and established an in vitro method for quantitative titration of HDF. HL was prepared by growing a hemolytic El Tor strain in heart infusion broth (Nihon Eiyo) or Syncase (Finkelstein et al., J. Infect. Diseases 114:203, 1964) at 37 C for 20 hr and by centrifuging the culture in the cold. Filtration of the supernatant liquid through Millipore filters (type PH) decreased the HL titer. Titration of HL was performed by the method of Feeley and Pittman. HDF was prepared by the method used by Finkelstein et al. for preparation of choleragen. An HDF-producing strain was grown in 100 ml of brain heart infusion broth or Syncase in a 500ml flask at 37 C for 18 hr with shaking at 112 strokes per minute. The supernatant fluid (pH 8.0 to 8.6), obtained by centrifugation, was filtered. The Millipore filtrate contained a reduced HDF titer. HL and HDF were kept frozen until ilse. The method for titration of HDF is given in Table 1. HDF was detected at the 4th hour of cultivation, although the titer varied depending upon each strain. No HDF was detected when a rugose single colony was used as the inoculum. A high HDF titer was detected in rice water stools from cholera patients. The foregoing results may be an indication that HDF is the choleragenic factor itself. HDF is thermolabile, is resistant to 0.2% formalin, precipitates at 60% saturation of ammonium sulfate, and is active in a pH range of 6.90 to 8.60. HL of Ubon-type El Tor and that of a strain of NAG (NCTC 4715) was inhibited by HDF from a strain of classical cholera (Ogawa type).