Preselection of hemolytic variants of el tor vibrios.

Historically, hemolytic activity in tube tests was regarded as the sine qua non in the differentiation of El Tor from classical cholera vibrios. However, in the recent outbreaks of cholera in Southeast Asia, many strains of agglutinable vibrios have been isolated which share a large number of properties ascribed to El Tor vibrios (and not the classical variety), but which lack hemolytic activity. These are now generally regarded as nonhemolytic El Tor vibrios, although there is some disagreement as to how they should properly be classified. J. C. Feeley (J. Bacteriol. 89:665, 1965) suggested reclassification of the choleragenic vibrios into five "types" of a single species on the basis of their reactions in tube and plate hemolysis tests, phage IV sensitivity, the direct slide hemagglutination test with chicken erythrocytes, and the Voges-Proskauer (VP) reaction; he selected only these tests from the many (O. Felsenfeld, Bacteriol. Rev. 28:72, 1964), including virulence for chick embryos (R. A. Finkelstein, Nature 202:609, 1964), which have been used to differentiate El Tor from classical vibrios. The present communication is concerned with observations on strains received (courtesy of A. S. Benenson) from the recent outbreak in Teheran. These strains contained colonial variants, recognizable on meat extract-agar (MEA) with oblique illumination (R. A. Finkelstein and C. Z. Gomez, Bull. World Health Organ. 28:327, 1963), which gave variable reactions on bloodcontaining media. The hemolytic activity could be predicted from the colonial form on MEA. Thus, if we were to employ Feeley's terminology, type 4 variants can be selected from initially type 5 strains. The picture is complicated by the observation that some tube tests resulted in "partial" hemolysis. This phenomenon may be related to the presence of the hemolysin-destructive factor of A. Wake and M. Yamamoto (J. Bacteriol. 91:461, 1966). Each of the 21 strains examined was of the Ogawa serotype, and each was hemagglutinative in the direct slide test of R. A. Finkelstein and S. Mukerjee (Proc. Soc. Exptl. Biol. Med. 112: 355, 1963) confirming their identity as El Tor vibrios. All of the strains and colonial variants tested exhibited enhanced virulence for chick embryos (R. A. Finkelstein, Nature 202:609, 1964), and each was resistant to polymyxin (50 units) in the disc technique. In fact, there has been complete correlation in the results of these three tests with reaction to Mukerjee's group IV phage, which also differentiates El Tor from classical cholera vibrios (Bull. World Health Organ. 28:333, 1963), with all strains tested on primary isolation by the author in cholera epidemics in the Philippines, India, Thailand, and Viet Nam. Undoubtedly, exceptions occur in some stock cultures (J. C. Feeley, J. Bacteriol. 89:665, 1965), and even in fairly recent isolates after a delay in testing coupled with transfer or maintenance in some artificial media (S. Rizvi, M. I. Huq, and A. S. Benenson, J. Bacteriol. 89:910, 1965). With the opportunity for genetic interchange between classical and El Tor vibrios now simultaneously present in India, more exceptions may be expected in the future. Eight pairs of colonial forms, most easily described as opaque (0) and translucent (T), were selected from eight strains for testing of hemolytic activity. Each of the paired substrains gave characteristic reactions on 5% sheep blood-agar, the translucent variety producing zones of hemolysis around isolated colonies, whereas the opaque forms manifested only hemodigestive action in crowded areas of the plates. With the exception of the isolated translucent variants, 17 of the 21 strains were nonhemolytic when received by us. Thus, it appears most likely that the translucent hemolytic variant arises in an initially nonhemolytic opaque population. This was subsequently borne out when cultures freshly isolated by the author in an El Tor Inaba outbreak in Korat, Thailand, were examined. All of the strains were of the opaque type and were nonhemolytic when first tested, but subsequently translucent hemolytic variants could be isolated from laboratorymaintained cultures. Testing of other cholera strains on blood-agar revealed that classical varieties were either totally nonhemolytic or produced hemodigestion in crowded areas, whereas El Tor strains from various sources ranged from markedly hemolytic, more so than the variants noted above, to nonhemolytic, usually with hemodigestion.