The single bacteriocin typing scheme appeared to be simple and adequate for both groups of organisms, and the producer as well as the indicator bacteria.
Vol. 30, No. 1
INFECTION AND IMMUNITY, Oct. 1980, p. 74-77 0019-9567/80/10-0074/04$02.00/0
Single Bacteriocin Typing Scheme for the Vibrio Group of
Organisms SMITA MITRA,1 T. S. BALGANESH,' SUJATA G. DASTIDAR,' AND A. N. CHAKRABARTY3* Division of Microbiology, Indian Institute of Experimental Medicine'; Division of Microbiology, Jadavpur University2; and Department of Pathology and Bacteriology, University College of Medicine, Calcutta University,3 Calcutta 700 020, India
A total of 743 strains of 0-I "agglutinable" and 293 strains of 0-I "inagglutinable" Vibrio cholerae were subjected to bacteriocin typing based on deferred antagonism of eight indicator bacteria, including two strains of V. cholerae, by the method of Chakrabarty et al. (Infect. Immun. 1:293-299, 1970). A minor modification that was effected in the typing medium was replacement of iodoacetic acid by ammonium chloride (at a final concentration of 0.003%) which appeared to regulate bacteriocin production more accurately and increase the stability of the types. Of the agglutinable strains, 94% were found to be bacteriocinogenic and could be fitted into 11 of the earlier-reported types (Chakrabarty et al., Infect. Immun. 1:293-299, 1970), and 6 newer types were recognized. Likewise, 285 (90.7%) of the inagglutinable strains were found to be bacteriocinogenic and could be accommodated within 10 original and 8 newer types identified for the inagglutinable vibrios. Thirteen types were common to the two groups of vibrios. The single bacteriocin typing scheme appeared to be simple and adequate for both groups of organisms, and the producer as well as the indicator bacteria behaved remarkably stably in this typing scheme over many years. indicator bacteria consisted of 0.75% Bactopeptone (Difco) and 0.5% NaCl (Analar) in distilled water at pH 7.4 to 7.5 The bacteriocin typing medium routinely employed was modified from the medium used earlier (2, 5) and consisted of autoclaved 1.5% agar (Difco) and 1.5% tryptic soy broth (Difco), to which was added a 0.5% amount each of trisodium citrate, K2HPO4, and 0.003% NH4Cl. These were steamed separately for 30 min and mixed thoroughly. Twenty-ml amounts of this medium were poured in 80-mm petri dishes. Technique of bacteriocin typing. The producer strains were grown as 1-cm-wide diametrical streaks for 54 h at 37°C on the typing medium and refrigerated for 18 h. Their growth was scraped off and sterilized with chloroform vapor for 2 h, followed by aeration for an equal period. Thereafter, eight indicator strains were applied as parallel lines by means of a machine (2, 4; S. Mitra, Ph.D. thesis, Calcutta University, Calcutta, 1979) so as to give a heavy confluent growth at right angles to the line of primary (producer) growth. The plates were incubated for 18 h at 37°C, with extended incubation when necessary. Reading of results. The inhibition reactions were graded from + to ++++ when the width of the inhibition zones measured, respectively, up to 4, 8, 12, and 16 mm or more; a - signified resistant growth, and ± indicated a mere thinning out of the indicator bacteria over the area of producer growth.
The bacteriocins of Vibrio cholerae have been described and characterized by conventional methods on the basis of deferred antagonism tests (2). According to these authors, bacteriocins are dependent on citrate phosphate buffers for optimal production, diffusion, and preservation. With a similar procedure, studies on the bacteriocins and bacteriocin typing of inagglutinable vibrios (5) showed that these had a very close similarity to those of V. cholerae (0-I agglutinable). In the present study, we have approached the problem of bacteriocin typing of the vibrio group of organisms by amalgamating the two separate but basically similar organisms, the 0-I agglutinable and 0-I inagglutinable vibrios.
MATERIALS AND METHODS Sources. The bacteria are described in Table 1; 23 strains received from Imdadul Huq were isolated during two different epidemics in Bangladesh, with 9 strains being isolated in 1974 and the remaining 14 strains being isolated in 1975. Fifteen strains isolated from the epidemic among refugees from Bangladesh camping in Dumdum (near Calcutta) were supplied by P. Bhattacharya. All of the strains were of human origin and were isolated between 1957 and 1975. Indicator bacteria. The indicator bacteria used for bacteriocin typing were the same as those described earlier (2, 5). Media. The medium for the propagation of the
RESULTS
Bacteriocin typing V. cholerae (0-I agglutinable). A total of 743 strains subjected to 74
VOL. 30, 1980
BACTERIOCIN TYPING SCHEME
TABLE 1. Sources of V. choleraea studied Strains No. of
belong-
186
Yes
S. C. Pal, Cholera Research Centre, Calcutta.
22
Yes
Authors
3
Yes
J. Sil, WHO International Centre for Vibrio Phage Typing, Calcutta.
23
Yes
Imdadul Huq, Cholera Research Laboratory, Dacca.
15
Yes
P. Bhattacharyya, WHO International Centre for Vibrio Phage Typing, Calcutta.
229
No
R. Sen, All India Institute of Hygiene and Public Health, Calcutta.
26
No
J. Sil, WHO International Centre for Vibrio Phage Typing, Calcutta.
31
No
J. Sil, WHO International Centre for Vibrio Phage Typing, Calcutta.
to 0 Source strains ing subgroup I 494 Yes R. Sen, All India Institute of Hygiene and Public Health, Calcutta.
75
Bacteriocin typing of V. cholerae (O-I inagglutinable). The same eight indicator bacteria could also detect bacteriocin production among the O-I inagglutinable vibrios and could determined their inhibition patterns (Table 2). Of the 293 strains tested, 285 were typable and formed 18 distinct types; 10 bacteriocin types previously reported (5) were detected, and 8 more types were identified. Figure 1 shows the bacteriocin inhibition pattern of a type common to the agglutinable and inagglutinable vibrios. A comparison of the bacteriocin types of the two groups of vibrios shows that types 15 to 19 of the present extended scheme did not contain any agglutinable vibrio strains, whereas types 7, 8, 11, and 14 did not contain any inagglutinable vibrio strains; a total of 13 types were common to both. However, several types had too few strains (Table 2).
DISCUSSION The present study and our earlier studies (2, 5) clearly establish the characteristics of bacteriocins of V. cholerae that constitute a special class among bacteriocins. Our studies have shown that these substances behave very much
7 Authors No Identified on the basis of the Minutes of IAMS Subcommittee on Taxonomy of Vibrios (6). a
bacteriocin typing showed that the 11 types described earlier (2) could all be reproduced in the present medium; 6 more types which were not encountered earlier were identified, thereby decreasing the number of untypable and unclassifiable strains to a minimum of 6% (Table 2). Of 15 strains of V. cholerae isolated in Calcutta from refugees from Bangladesh, 7 strains belonged to bacteriocin type 1B, 5 strains belonged to type 14, and only 1 strain each belonged to types 2B, 8, and 12. A study of vibrios obtained from the 1974 epidemic in Dacca, Bangladesh, showed that three strains each belonged to types 1A, 1B, and 14. In a similar epidemic in 1975 in Dacca, bacteriocin typing of 14 isolates showed that 2 strains of V. cholerae belonged to type 1A, 5 belonged to type 1B, 3 belonged to type 13, and only 1 strain each belonged to types 2A, 4, 10, and 12.
FIG. 1. Inhibition pattern of bacteriocin type 12 after 18 h of incubation. Indicators are (1) Shigella flexneri 3189 (subsp. Y), (2) Escherichia coli ROW, (3) Shigella sonnei 56, (4) S. sonnei M2/2, (5) S. sonnei 17, (6) S. sonnei M56, (7) S. flexneri 38 (type 6), and (8) V. cholerae 541. Indicators 1 to 5 all showed inhibition reactions (++++); 6 to 8 were all resistant (-).
76
INFECT. IMMUN.
MITRA ET AL.
TABLE 2. Single bacteriocin typing scheme for Inhibition reaction in
Indicator strains
1C
1A
1B
Shigella flexneri 3189 Escherichia coli ROW Shigella sonnei 56 S. sonnei M2/2 S. sonnei 17
++++ ++++ ++++ +++ +++
++++ ++++ ++++ ++++ +++
S. sonnei M56
++++ ++++
+++
S. flexneri 38 V. cholerae 541
+++ ++++
+++
No. of AGb vibrios belonging to each type
2A
2B
3
4
5
6
++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ M+ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ +++ -/+ +++ ++++ ++++ ++++ +++ -/± ++++ ++++ ++++ ++++ ++++ -
-1±
-/+
-/+
-
-
+++
+++
+++
-
+++
-
+
+ +
162
±14
1
32
106
20
28
32
16
97 14 1 20 31 4 7 14 No. of NAGb vibrios belonging 4 to each type I I_ I II a +, ++ +++,++++, Width of inhibition zones measuring, respectively, up to 4, 8, 12, and 16 mm or more; mutants. bAG, Agglutinable; NAG, inagglutinable.
The bacteriocin types of both agglutinable like other known bacteriocin if the optimal conditions for their production, release, and pres- and inagglutinable vibrios have been found to be ervation are provided. Ammonium chloride, stable, as is also the behavior of the indicator which replaced iodoacetic acid in the present bacteria. However, information is meager on the medium, seemed to regulate and stabilize bac- stability of these types after in vivo passage of teriocin production more accurately and gave strains. We have noted an accidental swallowing excellent results. The reason for avoiding vibrio of a inagglutinable vibrio by a worker in our indicator bacteria was that these organisms laboratory and its recovery from his feces on his tended to produce central growths within the developing a mild diarrhea when the original zones of inhibition produced by the bacteriocin- bacteriocin type was found to be retained; other ogenic organisms (2, 3) that interfered with de- instances were repeated rabbit leal loop passage velopment of clear-cut reactions and, therefore, of strains 569B and NIH 35A3 which retained with the reading and interpretation of results their original types, namely, types 6 and 3, rewith respect to most vibrio indicator bacteria. spectively. The studies showed that a satisfacHowever, the indicator bacteria V. cholerae 541 torily large number of types could be recognized and 852 were exceptions, and major or frequent among both the 0 serotype I and other serotypes difficulties were not faced in using them, al- of V. cholerae and that there was a large measthough central growths were encountered with ure of overlapping of these types with a fairly even distribution of the strains among the mathem, too. There are various kinds of routinely used me- jority of the types (Table 2); these, therefore, dia that contain citrate (e.g., Simon and Koser served all the major criteria of a typing system citrate, and Desoxycholate-citrate agars), and no intended for use in epidemiology. It was, howinhibitory effect due to citrate has been reported ever, realized that some types had too few thus far. We noted that many prompt citrate- strains, and eventually may have to be excluded utilizing vibrios were non-bacteriocinogenic and from type designation. This was the reason why vice versa. In our study with 47 vibrios and 15 steps to further split the types or recognize adenterobacteria on the effect of carbonates and ditional types were not followed in the present bicarbonates, we found that these chemicals did study, and the supplementary typing scheme not inhibit the growth of these bacteria at a introduced by Chakrabarty et. al. (4) earlier was concentration of 0.5% in tryptic soy broth and not found to be necessary here. The already nutrient agar up to pH 8.0; moreover, the pH of existing combined typing scheme was also apbacteriocin typing medium had never been plied for analysis and investigations of a number found to exceed 7.7. Therefore, the suggestion of of epidemics. It was noted that the strains isoa possible inhibitory effect (1) by carbonates and lated from refugees coming from Bangladesh in bicarbonates (as metabolic end products of cit- 1971 did not belong to a single bacteriocin type, rate) did not appear to be tenable. suggesting that the refugees had probably as-
VOL. 30, 1980
BACTERIOCIN TYPING SCHEME
77
agglutinable and inaggutinable vibrios based on their inhibitory patterns following bacteriocin type': 7
8
9
10
11
12
13
14
15
_
+++ -
++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ -/+
_
_
+++
++ _
+++ ±
+++
++
++++
-
+
+++
++++
+++
+++
-
_
++++ +++ ++ ++/- -/++++ +++/++++ +++ ++ ++++ _ _ ++
M+ M+
++++ +++ ++++
+
++++/-
14
10
24
2
0
0
1
1
-/+
16
17
18
19
-1±
±
M+/±
++++
-+ -
+
++++ ++++ ++++ 7++++
_ ++++ +++/±
_
-1±
_
-
-1±+ -±
-
±1-
-1±
± ± +++_ ±
-
_
_
_
52
34
24
34
0
0
0
0
0
0
4
1
0
63
11
8
3
1
±, thinning of growth in the central area;-, resistant; -/+,
sembled at a particular point from different parts of Bangladesh. Similar multiplicity of the bacteriocin types of the strains was noticed with respect to those isolated in Bangladesh during the 1974 and 1975 epidemics. However, it is difficult to conclude upon, at this stage, an origin of these epidemic strains, in view of their limited number together with an absence of precise epidemiological data. The suitability of the current typing scheme as an epidemiological tool, therefore, can be assessed better when precise epidemiological information becomes available on the
pathogens. LITERATURE CMED 1. Bhaskaran, K., S. S. Iyer, A. W. Khan, and V. C. Vora. 1974. Gross inhibition of enteric bacteria by
2. 3.
4.
5.
6.
++ - _-
+
+
±/+, strains with either of the reactions; M, Vibrio cholerae in nutrient media containing lactate, acetate, or citrate. Antimicrob. Agents Chemother. 6: 375-378. Chakrabarty, A. N., S. Adhya, J. Basu, and S. G. Dastidar. 1970. Bacteriocin typing of Vibrio cholerae. Infect. Immun. 1:293-299. Chakrabarty, A. N., and S. G. Dastidar. 1974. Paradoxical inhibition among bacteria. Characterisation of the phenomenon and nature of the genetic process. J. Gen. Microbiol. 80:339-361. Chakrabarty, A. N., S. G. Dastidar, and S. Adhya. 1971. Bacteriocin typing of Vibrio cholerae-a supplementary typing scheme. Indian J. Med. Res. Supply. 00: 190-193. Chakrabarty, A. N., J. Sil, and S. Mukerjee. 1971. Bacteriocin typing of NAG vibrios. Indian J. Med. Res. Suppl. 00:161-167. Feeley, J. C. 1966. Minutes of IAMS subcommittee on taxonomy of vibrios. Int. J. Syst. Bacteriol. 16.135142.
