Bacteriocin Typing of Vibrio cholerae

Transcription

1 INFECION AND IMMUN1TY, Mar. 1970, p Vol. 1, No American Society for Microbiology Printed in U.S.A. Bacteriocin Typing of Vibrio cholerae A. N. CHAKRABARTY, SATI ADHYA, JAYANTISRI BASU, AND SUJATA G. DASTIDAR Division of Microbiology, Jadavpur University, Calcutta-32, Ilndia Received for publication 23 October 1969 Bacteriocins of Vibrio cholerae have been demonstrated against enterobacterial and vibrio indicator organisms by conventional techniques. Abundant bacteriocin production took place on casein hydrolysate-yeast extract, tryptic soy, digest broth, proteose-peptone, and neopeptone agars. Essential factors were a citrate-phosphate buffer concentration of 0.5 to 0.7 %, at ph 7.5 to 7.6, and cold shock. Thermal treatment of indicator organisms at 45 C for 12 min increased the percentage of typable strains. The bacteriocins of V. cholerae appeared to be powerful diffusible bactericidal agents. By using 8 indicator strains, 11 bacteriocin types have been recognized among 425 strains, of which 87% are typable at present. Bacteriocinogeny and sensitivity to bacteriocins are stable genetic characters exhibited by many groups of bacteria (19). This relationship has been used to identify types at the infraspecific level, and several typing schemes based on bacteriocins have been successfully applied to epidemiological studieg-of Shigella sonnei, Pseudomonas pyocyanea, and S. flexneri type 6 (2, 7, 10, 16). A bacteriocin-typing method for Vibrio cholerae has not been reported thus far. Although genetic studies have proved the occurrence of bacteriocinogeny among certain Vibrio species (5, 6), its practical demonstration in the population has remained uncertain (4, 22). Farkas-Himsley and Seyfried (12) developed a technique for the detection of vibriocin production by using low Eh medium and streptomycin-resistant V. cholerae as an indicator organism; they also suggested the application of "cold shock" for release of cellbound vibriocins. Wahba, by using a combination of cold shock and preincubation refrigeration of indicator vibrios, described a temporary inhibitory phenomenon present on the 8th hr of incubation as a characteristic of the vibriocin activity (24). Datta and Prescott (11) found this to be unsatisfactory and, by using a modified technique, reported a minimal inhibition of certain enterobacteria by two vibriocinogenic strains. Jayawardene and Farkas-Himsley (20), analyzing the difficulties of a practical demonstration of vibriocin activity, pointed out that failure to detect this substance is due to its presence in minute amounts, which would be overcome by demonstrating it by use of an electron microscope. The present study was undertaken to demonstrate conclusively and characterize the bacteriocins of V. cholerae and to develop a typing method based on this information. MATERIALS AND MEETHODS Cultures. Strains of V. cholerae were identified by the definition proposed by the International Association of Microbiological Societies, Subcommittee on Taxonomy of Vibrios (18); in addition, serologically these strains belonged to 0-subgroup I (14) and biochemically to group I (15). All were of human origin and were isolated between 1961 and A total of 425 strains were studied, of which 48 were isolated by the authors, 263 were received from S. Mukerjee, International Vibrio Reference Center, Calcutta, and 111 were obtained from Ranjit Sen, All India Institute of Hygiene and Public Health, Calcutta. Two strains, ATCC and 14035, representing the neotype strains of V. cholerae (17) and one known bacteriocinogenic strain, NIH35A3 (United States vaccine strain), were also included. Cultures were maintained in stab-slant agar, consisting of 1% peptone, 1% agar, and 0.5% NaCl in distilled water at ph 7.5. The prospective indicator organisms examined consisted of 69 strains of V. cholerae selected at random and 5 strains of Enterobacteriaceae, namely, Escherichia coli Row and S. sonnei 2, 2M, 17, and 56, belonging to the typing set of Abbott and Graham (1). All of these strains were obtained from J. D. Abbott, Manchester, United Kingdom. Additional members of this group consisted of two strains of S. flexneri, 38 (type 6) and 3189 (var. Y), that were discovered by us. The indicator strains finally included in our typing set are given in Table 1. These strains were maintained on Dorset egg medium kept at 4 C. Media. For a comparative study of bacteriocin production, five different basal media known to support such production were tested (7, 21). These were 1.5% each of Difco tryptic soy broth, proteosepeptone, neopeptone and casein hydrolysate-yeast extract in 1.5% agar base. The Difco brand was used in all cases. In addition, Hartley digest broth containing the same concentration of agar was used. For all other studies, the tryptic soy agar base medium was adopted with various modifications. Inhibition of proteolytic enzymes was done by incorporating iodoacetic acid at a final concentration of 10- M. 293

2 294 CHAKRABARTY ET AL. INFEC. IMMUN. TABLE 1. Description of indicator organisms of the typing set Serial Labora - Derived from tory Relationship between _ nno. toiy o. (1)and (2)Decito ~~~~~~~~~~~~~~~~~~~~~Source of Description_Source_of_(2) (2) (1)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~2 1 Row Row Same E. coli J. D. Abbott J. D. Abbott 2 M2/1 2 Mutant selected S. sonnei A. N. Chakra- J. D. Abbott against V. cholerae a 3 M2/2 2 Mutant selected S. sonnei A. N. Chakra- J. D. Abbott against V. cholerae 4 M56 56 Mutant selected S. sonnei A. N. Chakra- J. D. Abbott against V. cholerae Same S. sonnei J. D. Abbott J. D. Abbott Same S. sonnei J. D. Abbott J. D. Abbott 7 38 Wild type S. flexneri 6 A. N. Chakra- A. N. Chak ra Wild type S. flexneri Y A. N. Chakra- A. Vertenyi Wild type V. cholerae A. N. Chakra- Ranjit Sen Wild type V. cholerae A. N. Chakra- Ranjit Sen a Senior author of this paper. The effect of salt concentration in detection of bacteriocins was investigated by the addition of NaCl in 0.2, 0.4, 0.8, 1.5, 3.0, 4.0, and 5.0% concentrations; the effect of ph was studied over a range of 7.1 to 8.0, at intervals of 0.1 adjusted with 0.1 N NaOH. The effects of buffer concentration were examined by the addition of equal amounts of sodium citrate and K2HPO4, ranging from 0.1 to 1.0% at intervals of 0.1%. The media for propagation of the indicator organisms consisted of 0.75% peptone (Difco) and 0.5% NaCl in distilled water at a final ph of 7.5. The bacteriocin-typing medium developed and routinely used by us consisted of 10r M iodoacetic acid in final concentration, 0.6% sodium citrate, and 0.6% K2HPO4 in tryptic soy agar base at ph 7.5. Methods for detection of bacteriocinogeny. Screening tests were performed by the "stab-inoculation" technique (13); confirmatory tests were carried out by the "streak-plate" method (2). When studying bacteriocinogeny by the Wahba technique (24), the method was employed without modification. The indicator organisms were used as 18-hr-old peptone-water cultures either for flooding or for streaking. The concentration of organisms for the enterobacteria varied between 10 X 108 and 14 X 108 per ml, and that for vibrios varied between 12 X 108 and 16 X 108 per ml. Following the analogy of conversion of untypable staphylococci into typable ones (3), culture suspensions heated at 45 C for 12 min were also used. Bacteriocin-typing method. Strains of V. cholerae were grown as streaks 1 cm in width for 54 hr at 37 C on the typing medium and were refrigerated for 18 hr; growth was then scraped off and sterilized with chloroform vapor for 2 hr followed by aeration for an equal period of time. Thereafter, eight indicator strains were applied as parallel lines at right angles to the position of original inoculation, so as to give a heavy confluent growth. For strains giving weak and inconsistent results and for the untypable ones, indicator suspensions were used after heating at 45 C for 12 min. Plates were incubated routinely for 18 hr at 37 C, with extended incubation whenever necessary. Reading of results. Except when studying bacteriocinogeny by the method of Wahba (24), when an additional 8-hr reading was taken, all results were noted after 18 hr of incubation. The inhibition reactions were graded + to when the width of the inhibition zones measured, respectively, 4, 8, 12, and 16 mm or greater. A mere thinning out of growth in the central area was considered to be i reaction. A special form of inhibition was noted with some indicator vibrios; it was characterized by a narrow central band of growth flanked by areas of inhibition together with peripheral growth. This has been designated as a "paradoxical" reaction (Fig. 2). RESULTS Vibriocinogeny against vibrios. Studies on 69 strains of V. cholerae tested against each other in five different basal media, by using the unmodified Wahba technique as well as modifications thereof in tryptic soy agar containing iodoacetic acid and employing heated suspensions of indicator organisms, failed to reveal any bacteriocin activity; the temporary inhibition observed between any two reactant pairs at 8 hr was nonreproducible and absent after 18 hr of incubation. However, we noted a consistently reduced pattern of growth of three strains around the growth of the test organisms. Bacteriocinogeny against indicator enterobacteria. The same strains of V. cholerae were tested against E. coli Row, S. sonnei 17, 56, 2, and 2M, and S. flexneri 38 (type 6) and 3189 (var. Y) on

3 VOL. 1, 1970 BACTERIOCIN TYPING OF V. CHOLERAE 295 TABLE 2. Effects of various factors on bacteriocin production by 69 strains of Vibrio cholerae against one to seven indicator organisms (Escherichia coli Row, Shigella sonnei 2, 2M, 56, and 17, and S. flexneri 38 and 3189) No. of strains producing bacteriocin Test factors NaCi at ph 7.5 Sodium citrate and K2HPO4 ph I % % 3.0% 5.0% 02% 0.3% 0.4% 0-5% 057-1% 0o7% Complete testa Irregular Complete test without cold shock Complete test with 6 hr of Irregular cold shock Complete test without ther Irregular mal treatment Complete test without iodo- 10 I Irregular acetic acid Complete test without pre- 7 2 l l Irregular incubation refrigeration atryptic soy agar, iodoacetic acid, 18 hr of cold shock, thermal treatment of indicator organisms, and preincubation refrigeration. five basal media by the technique of Abbott and Shannon (2). The results were uniformly negative. Detection of bacteriocin production by combined test. This test consisted of (i) tryptic soy agar basal medium at different ph levels and buffer and salt concentrations, (ii) addition of iodoacetic acid, (iii) cold shock of test cultures for 6 and 18 hr, (iv) thermal treatment of indicator organisms, and (v) preincubation refrigeration of plates. The combined test employing all of the five possible determinants showed that different vibrio strains produced bacteriocins, whose spectrum of activity varied from inhibition of one indicator enterobacterial strain at the least to a maximum of seven. As bacteriocin production was detectable by using these five factors, the role of each of the factors was then evaluated by a process of elimination of a single factor at a time from the combined test (Table 2). The essential factors for production and detection of bacteriocin were: citrate-phosphate buffer, at a concentration of 0.5 to 0.7% of each chemical; ph 7.5 to 7.6; and cold shock for 18 hr. Thermal treatment of indicator strains was found to be a useful factor, as this produced uniform results and increased the percentage of typable strains; iodoacetic acid played an occasional and minor role. Preincubation refrigeration did not appear to have any role. The results obtained at the defined optima of test conditions were constant and perfectly reproducible. The pattern of inhibition of growth of the indicator organisms resulting from bacteriocins of V. cholerae showed a number of characteristic features (Fig. 1 to 3). The zones of inhibition of E. coli Row, S. sonnei M2/1, 56, and 17, and S. flexneri 38 had a similar morphology: all of these strains tended to have a diffuse and irregular margin and produced a number of mutant colonies within zones of inhibition. On the other hand, the inhibition zones of S. flexneri 3189 and S. sonnei M2/2 and M56 were rather clear-cut and punched-out with scanty mutants. In Fig. 3, S. flexneri 38 shows a thinning out of growth in the central area which has been designated as a (ht) reaction. The characteristics of inhibition of the vibrio indicator are discussed below. Of 69 strains of V. cholerae, including strains NIH35A3 and 14035, 61 were found to produce detectable bacteriocins. These 61 strains were then examined, under defined test optima, against 69 strains of V. cholerae. The results showed that 15 strains were inhibited by at least one of these bacteriocinogenic strains. The results of a comparison of five different basal media under defined test conditions for supporting bacteriocin production showed that all of the 11 representative types (see below) produced bacteriocins against indicator strain S. flexneri 3189 in all of the media. An assessment of relative amounts of production, indicated by the size of the zones of inhibition, showed that casein hydrolysate-yeast extract, tryptic soy, and digest agars were superior to the other two media. Bacteriocin typing of V. cholerae. The set of indicator organisms that was finally selected consisted of both enterobacteria and vibrios. Selection was based on the maximum distinguishing ca-

4 296 CHAKRABARTY ET AL. INFEC. IMMUN. FiM. 1. Inhibition pattern ofbacteriocin type IB after 18 hr ofincubation. Indicators in descending orderfrom marked end, shown by removal ofagar, are: Escherichia coli Row; Shigella flexneri 3189 (var. Y); S. sonnei M2/1, 56, M2/2, 17, and M56; S. flexneri 38 (type 6); and V. cholerae 541. FIG. 2. Inhibition pattern ofbacteriocin type 2A after 18 hr of incubation. Order of indicators is as in Fig. 1. pacity of each strain and on the stability of reactions. A list of these cultures is given in Table 1. A total of 425 strains were subjected to bacteriocin typing. At present, 11 bacteriocin types are recognized on the basis of their distinctive inhibition patterns. The results of typing are given in Table 3. The cultures in which bacteriocin FiG. 3. Inhibition pattern of bacteriocin type 2B after 18 hr of incubation. Order of indicators is as in Fig. 1. production could not be detected by any of the indicator strains remain as untypable at present; these constitute 13.2% of the total. There are a few strains with weak bacteriocin activity; their typing is facilitated by extending the primary incubation period to 78 hr, other factors remaining the same. There remain a number of strains that give atypical and inconsistent patterns and these have been designated as unclassifiable. They constitute 7.53% of the total. Relationship of bacteriocin types to the origin of strains and other typing methods. All of the strains were isolated from humans; of these, 159 had their origin in Calcutta and represent the years 1961 to 1968; 77 strains originated in Madras during 1967; 140 cultures originated in 11 other different regions of India during During the same year, the 46 cultures which had their origin outside India were as follows: Kuala Lumpur (Malayasia) 32; Nepal 4; and Rangoon (Burma) 10. Two strains belonged to the American Type Culture Collection, and the other one was a United States vaccine strain. Epidemiological history regarding the majority of these strains was lacking. Serologically, 301 strains belonged to the Ogawa serotype, 123 to the Inaba serotype, and only 1 to the Hikojima serotype; 23 of these strains were strongly hemolytic, 8 gave weak positive reactions, and the rest were all nonhemolytic. Information regarding the phage types was available with respect to the 310 strains from S. Mukerjee, Director, International

5 VOL. 1, 1970 BACTERIOCIN TYPING OF V. CHOLERAE 297 TABLE 3. Bacteriocin types and their inhibition patternsa Bacteriocin types Indicator organism A B C A B Shigellaflexneri 3189 Escherichia coli Row S. sonnei M2/1 and 56 S. sonnei M2/2 and V. cholerae 852 S. sonnei 17 S. sonnei M56 S. flexneri 38 V. cholerae 541 No. of strains Per cent of total /+ _ a Symbols: + to , width of inhibition zones measured 4, 8, 12, and 16 mm or greater; ±, thinning out of growth in the central area; -, resistant; -/+, -/4, :1:/+, different strains giving either of the reactions; M, mutants. Vibrio Reference Center, Calcutta: 90 strains were lysable by group IV phage and were sensitive to polymyxin B, whereas the remaining 220 strains were resistant to both of them. The distribution pattern of the different phage types belonging to the first group was: type 1, 54; type 3, 32; type 5, 1; and untypable, 1; the distribution pattern of the second group was: type 1, 4; type 2, 108; type 3, 10; type 4, 74; type 5, 8; type 6, 13; type 8, 1; and untypable, 2. An analysis of the results of distribution of different serotypes, phage types, and hemolytic character did not suggest any correlation among these features, nor could they be correlated to the bacteriocin types. DISCUSSION The difficulties in demonstrating bacteriocins of V. cholerae have produced two hypotheses, that detectable bacteriocins may not exist at all (4, 22) and that bacteriocin production is detectable by the usual methods only as a special phenomenon of transitory inhibition (24) or is demonstrable by electron microscopy (20). It has been postulated that these bacteriocins are produced in minute amounts, and special media, reduced Eh, and mitomycin C, etc., are essential for their production and release. These observations apparently preclude the demonstration of bacteriocins of V. cholerae by conventional tests applicable to other bacteriocins. Repeating the experiment of Wahba (24), we also observed temporary inhibitory effects and reduced growth of some organisms. Since all known bacteriocins are bactericidal in nature (13), the phenomenon of temporary inhibition appears to be a bacteriostatic effect, probably nonspecific in nature l ++++I M+ M+ M Our success in demonstrating and characterizing the bacteriocins of V. cholerae does not support the hypotheses put forth by other workers. We found that these bacteriocins behave very much like other known bacteriocins if the optima for their production, release, and preservation are provided. Common laboratory media are adequate for production of these substances which have been found to be powerful, diffusible agents, bactericidal in nature. The bactericidal character of these agents is supported by the findings that prolonged incubation did not produce any growth in the zones of inhibition, except, occasionally, for a few resistant mutants, and that inoculation of materials from the zones of inhibition on fresh supportive media did not yield any growth. The justification for identifying these inhibitory agents as bacteriocins should be discussed. Each indicator organism had its own characteristic sensitivity. Each of our bacteriocin types exhibited a characteristic inhibitory pattern which remained constant; mutants resistant to one type bred true to this character against the type from which it was selected, with or without an alteration of sensitivity to others which also remained constant. Thus, the indicator organism 2 (Table 1), which was sensitive to all bacteriocin types, produced a mutant resistant to type 7, namely, M2/1, which still retained its sensitivity to the rest of the types. Another resistant mutant of 2, namely, M2/2, selected against type 1C, developed simultaneously resistance against types 5, 6, and 8, whereas its sensitivity to other types remained unchanged. The indicator organism M56, derived from 56 against type 6, bred true to this resistance against type 6, while continu-

6 298 CHAKRABARTY ET AL. INFEC. IMMUN. ing to be sensitive to the other types; in addition, it lost its resistance to type 7. Furthermore, the morphology of the inhibition zones produced by these bacteriocins was indistinguishable from the morphology of the zones produced by colicins. The chances of a phage involvement should also be discussed. It is likely that removal of the primary growth by the scraping technique also removes any phage that it may carry. This, followed by a prolonged exposure to the lethal effects of chloroform vapor, probably leaves little possibility of inhibition of growth of both enterobacteria and vibrios as a result of a phage action. Furthermore, the inhibition zones in most instances extend far beyond the boundaries of the original growth, making such an involvement even less likely. In the course of our studies, we did not observe any plaque suggestive of phage lysis on the streak of growth of the indicator organisms overlaying the area of the original growth. Since V. cholerae is known always to possess catalase which destroys H202, a possible role of the latter as a bacterial inhibitory agent may be excluded. In spite of the basic similarity of action of V. cholerae bacteriocins with those previously known, there are several ways in which they differ. The strict dependence of the bacteriocins of vibrios on a defined concentration of citratephosphate buffers and on cold shock for their activity suggests that these bacteriocins may constitute a special class. It seems possible that cold shock initiates a process of release of cell-bound bacteriocins, whereas an optimum concentration of buffers is essential for their diffusion and preservation. Our studies have shown that the protective action was not due to ph or hypertonicity alone, although hypertonicity at a given ph could produce some bacteriocinogenic effects. In this respect, the buffers seem to play a role similar to that in preservation of serum complement (23). The interesting finding of sensitivity of enterobacterial indicator strains to bacteriocins of vibrios suggests a possible immunochemical relationship of the Enterobacteriaceae with vibrios. Earlier studies (9, 11) indicate that this is possible. That this relationship may be bilateral is supported by our finding of inhibition of some vibrio strains by a colicinogenic S. flexneri 38, type 6 (unpublished data). Our studies have shown that the specificity of the sensitivity reactions of the enterobacteria was constant for the majority of the vibrio strains, and that the same types could be identified by vibrio indicator organisms. It was further observed that, even when an equivalent substitute vibrio indicator organism was available (strain 852), there was no additional advantage in using it; on the contrary, it had certain limitations. We have noted that most indicator vibrios give three types of inhibition reactions in respect to different bacteriocin types: (i) standard inhibition comparable to that of enterobacteria, (ii) paradoxical inhibition, and (iii) partial inhibition in the form of markedly reduced growth. The multiplicity of reactions introduces an element of uncontrollable variability and makes reading and interpretation difficult. It has been reported that some old cultures of V. cholerae may gradually lose their ability to produce bacteriocin (6). We had the opportunity of examining a large number of stab cultures which were 1 to 2 years old and could be tested several times during the last 1 year. We found that the capacity as well as the lack of the capacity to produce bacteriocin did not change among these cultures. The bacteriocin type of each of the 425 cultures had been determined at least three times; the inhibitory pattern of the typable strains with respect to the indicator set used was found to be remarkably constant except for some of those designated as unclassifiable. Likewise, the three mutant indicators belonging to our typing set have been found to be stable. Other strains, namely, E. coli Row, S. sonnei 56, and S. flexneri 3189 and 38 have been used for many years and have not been reported to vary in their reactions (1, 7, 8). Although the indicator organisms used by us were sensitive to the bacteriocins of vibrios per se, thermal treatment proved to be useful in typing some untypable strains. This niy be due to enhanced sensitivity of these indicators to some of the bacteriocins. Some of the bacteriocin types identified by us may undergo further subdivisions when the typing scheme is expanded to involve more strains. Each one of our types consisted of strains isolated from different epidemics in India and abroad occurring between 1961 and 1969, but these strains behaved in an identical way. The so-called unclassifiable organisms represent a collection of miscellaneous types with too few organisms belonging to one type to justify a type recognition. However, with the study of more strains, some of these may form distinct types. Bacteriocin typing of V. cholerae has provided a tool of epidemiological interest. Further studies on the relationship of these types with other methods of subdivision of V. cholerae may provide interesting results. ACKNOWLEDGMENTS We thank S. Mukerjee, Director, International Vibrio Reference Center, Calcutta, Ranjit Sen, All India Institute of Hygiene and Public Health, Calcutta, J. D. Abbott, Manchester, United Kingdom, and A. Vertenyi, Pecs, Hungary, for supplying the various strains and H. C. Guha, Vice-Chancellor, Jadavpur University, for the facilities.

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