JOURNAL OF CLINICAL MICROBIOLOGY, July 1979, p. 85-90 0095-1137/79/07-0085/06$02.00/0
Vol. 10, No. 1
Serotyping of Non-Cholera Vibrios HARRY L. SMITH, JR. Department ofMicrobiology, Jefferson Medical College of Thomas Jefferson University, Philadelphia,
Pennsylvania 19107
Received for publication 30 April 1979
The serotyping system for non-cholera vibrios reported by us in 1965 has been re-evaluated and extended. Results from serotyping 2,624 cultures are presented. These isolates were accumulated over a period of 20 years and came from clinical and environmental samples in many parts of the world. Included is a discussion of the use of terms "Vibrio cholerae" and "non-cholera vibrios" for clinical reports. In 1965 we reported (13) a serotyping system for those vibrios that were not halophiles and which were not agglutinated by the 0 group I serum described by Gardner and Venkatraman (3). This was begun because of difficulty in characterizing the many strains of vibrios isolated by our laboratory people or by other workers from environmental samples and specimens from patients. The results with 0 group I serum and with biochemical tests such as the Heiberg fermentation scheme (4) gave a limited number of groups of different kinds of vibrios. The serotyping system allowed the finer distinction among the organisms that was sought for the purpose of obtaining data regarding the epidemiology and ecology of this group of organisms. Since the time of our first report, there appears to have been an increased awareness of the roles of non-cholera vibrios in causing human diseases (7). Some attention has been di,ected to the distribution of these organisms in nature (1). Many of the isolates were sent to us for serotyping. As more strains were processed it became evident that the original serotyping system needed to be re-evaluated. The definition of the genus Vibrio by Hugh and Sakazaki (6) provided an objective basis for inclusion or exclusion of strains, new and old, in the study. The typing sera were re-examined, and some were found to be partially or totally identical to others in the system. For those vibrio cultures not typed with any of the sera, the process of establishing new serotypes was begun. This report covers the current status of the serotyping system and gives data on the distribution of serotypes of strains in the non-cholera vibrio culture collection of our laboratory. MATERIALS AND METHODS
grounds. A more precise and less subjective method for defining Vibrio was reported by Hugh and Sakazaki (5). Using their scheme, the 2,624 strains employed in this study fit the minimal number of characters for the identification of Vibrio species, with the exception that the guanine-plus-cytosine ratio determinations were not made. In addition, none of these strains was agglutinated by serum prepared in our laboratory which corresponds to the 0 group I serum of Gardner and Venkatraman (3). A list of the cultures used for the preparation of the sera for typing and absorption is given in Table 1. The strains were maintained as lyophilized preparations and on slants of Trypticase-1% sodium chloride (TMNO) agar under sterile liquid paraffin at room temperature. The stock cultures were transferred minimally, usually twice, after their receipt by the laboratory. Sera preparation. The method for preparation of a serum for typing has been previously described (13). Briefly, the selected strain was grown on a 3-inch (ca. 76.2-mm) T,N, agar slant at 37°C overnight. The growth from the slant was suspended in 6 ml of sterile saline. The suspension contained approximately 107 organisms per ml. A 5-ml volume of this suspension was injected subcutaneously into the shaved side of a rabbit. After 10 to 14 days, the animal was exsanguinated by cardiac puncture. Not all animals survived to this point in time. Many that did, developed ulcerations which extended to dependent areas. The serum, after inactivation (56°C for 30 min), was titrated by the slide agglutination method. Drops of dilutions of the serum were placed on a slide, and living vibrios of the strain used for injection of the rabbit were suspended in the drops. The slide was rotated, and clumping was noted within 30 s. The reagent for use in further testing contained twice the amount of serum needed to give strong and complete agglutination, e.g., if a 1:10 dilution gave a rapid fourplus reaction, the reagent was prepared at 1:5. The diluted serum was then tested against all strains previously used for serum preparation by the slide agglutination method. If cross-reactions occurred, absorption studies were initiated to determine Strains. The criteria for including strains in this whether the serum was identical to or shared antibodstudy had been based primarily upon morphological ies with other reagent sera. Live cultures were used 85
J. CLIN. MICROBIOL.
SMITH
86
TABLE 1. Vibrio strains used to prepare typing sera Absorbed, if
Absorbed, if
Sero-
type
neces-
Cul-
uSource
no.
11 12 13 14 15 16
6305 5162 5064 5008 6707 5096
17 18 19 20 21 22
5047 7977 6701 5042 5487 5103
23 24
5072 5066
25 27 28 29 30 31 32
5163 6345 6541 6696 5128 5410 7888
33 37
7920 5078
38 40
5052 5079
42 43 44 45 48
5411 5165 5037 5009 5069
50
5152
Diarrhea, Dacca, 1961 Diarrhea, Dacca, 1961 Nanking 32/106, 0 group IV Water, Chicago, 1953 Night soil, Hong Kong, 1958 Felsenfeld Thai Series, In-28, 1960 Water, Thailand, 1959 Diarrhea, Dacca, 1965 Water, Hong Kong, 1958 Water, Thailand, 1959 Water, Dacca, 1962 Post-mortem gut, Thailand, 1959 Diarrhea, Dacca Felsenfeld Thai Series, N-1, 1960 Diarrhea, Dacca Unknown, Dacca Water, Bangladesh, 1965 Night soil, Hong Kong Diarrhea, Dacca, 1962 Diarrhea, Dacca, 1961 Felsenfeld Thai Series, diarrhea 1-4 Contact, Japan, 1968 Water, Felsenfeld Thai Series, NAG-3" Water, Thailand, 1959 Water, Felsenfeld Thai Series, NAG-4 Diarrhea, Dacca, 1961 Diarrhea, Dacca, 1961 Water, Thailand, 1959 Diarrhea, unknown Contact, Felsenfeld Thai Series, N-4 Water, Chesapeake Bay, Md.,
56 57
5053 6355
Water, Thailand, 1959 Diarrhea, Dacca
Serotype
Cul-
1 59
5092
68 48
60 61 62
6326 5029 5059
75
64 68 69
7449 5028 5086
74 75 76 77 79 83 94 102 106 107 110 111 113 115 148 160 175 176 201 308 309 312 320 332 340 342 343 345 346 347 348 349
7900 5681 6335 5694
sary, with serotype culture no.
321
176, 115
321 115 18 45 45
115
68, 107
321 32
1960
50
no.
5444 5697 5051 7647 5803 6306 7555 7556 6312 7902 5722 6358
9009 5180 7165 7349 6313 6970 7995 8536 8585 8462 8497 9115 9166 9183 9211 9248
neces-
sary, with serotype culture no. 32 Contact, Felsenfeld Thai Series, NAG-24 Cow dung, Dacca, 1962 Water, Thailand, 1959 A.M.S. 20-A-59, Van Rooyen, Q67 Unknown, Philippines, 1962 32 Water, Thailand, 1959 Food, Felsenfeld Thai Series, 326 NAG-15 13, 22 Unknown, Philippines, 1962 Contact, Dacca, 1962 Goat, Dacca, 1962 Water, Dacca, 1962 Water, Dacca, 1962 45 Water, Dacca, 1962 68 Water, Thailand, 1959 107 Water, U.S.A. 326 Water, Philippines, 1964 321 Diarrhea, Dacca, 1961 Unknown, Dacca, 1962 Unknown, Philippines, 1962 Diarrhea, Dacca, 1962 Diarrhea, Dacca, 1962 32 Diarrhea, Philippines, 1964 32 Source unknown Water, Hungary, 1976 Calcutta, NAG group II, 1963 Water, Dacca, 1966 Diarrhea, Dacca, 1966 Diarrhea, Dacca, 1962 Diarrhea, Dacca, 1966 Latrine, unknown, 1968 Muic. 145, sewage, Yugoslavia 321 Human feces, Iraq, 1966 Diarrhea, Iran, 1972 Septic tank, U.S.A., 1973 Nonenteric, Philippines, 1976 Diarrhea, Dacca, 1977 Unknown, Guam, 1977 Water, Maryland, 1977 Water, Maryland, 1977
Source
a NAG, Non-agglutinable vibrio designation.
for absorption. Table 1 lists the sera that were absorbed and the strains used for the absorption. The serum was tested with strains not previously typed. Those cultures that could not be typed by this and other sera similarly prepared were possible candidates for preparation of new sera. Processing of strains received. Each culture was streaked on a brain heart infusion agar plate and inoculated into TIN, broth, and incubated at 370C. The broth culture was used 4 to 6 h later to inoculate glucose broth and amino acid media (12). All were incubated overnight at 37°C. The plate culture was examined macroscopically for contamination or colonial variation. If either situation occurred, colonies were picked from the plate and were tested individually. If the plate culture was acceptable, the oxidase
and the String (11) tests were performed. Both had to give positive results. In addition, the following results had to be obtained before proceeding: glucose fermentation without gas; lysine and ornithine decarboxylated but arginine not affected. Stock cultures were prepared. A portion of the growth on the plate was suspended in sterile skim milk and lyophilized. In addition, two T1NI agar slants were inoculated from the confluent area of the plate. After overnight incubation, sterile liquid paraffin was added to each tube so that the entire slant was covered. The remainder of the plate culture was used as the source of the organisms for the slide agglutination test with the typing sera. The initial test was performed with cholera grouping serum which corresponded to 0 group I of Gardner and Venkatraman (3). A system of
serum pools was devised to facilitate serotyping. Each culture was tested in each pool. When agglutination was observed, the sera which constituted that pool(s) were tested. Additional biochemical tests such as fermentations, salt tolerance, etc. were conducted for further data useful in separating vibrios (13).
with the Heiberg classification it was possible to recognize 160 different kinds of vibrios (Table 3). It should be noted from this table that there does not appear to be any correlation between serotype and Heiberg fermentation pattern. The TABLE 2. The Heiberg classification of vibrios and the frequency of types Fennentation of % of Heiberg
RESULTS When the strains which fit the minimal characteristic for vibrios (6) were inoculated into separate media containing sucrose, arabinose, or mannose as suggested by Heiberg (4), they could be subdivided into four groups (Table 2). Although most were Heiberg II, the frequencies given in this table may represent the effects of other factors, which will be discussed. The serotyping system that was developed currently consists of 72 sera raised against nonO group I vibrios (Table 1). By combining this
Serotype 11 12
13 14 15 16 17 18 19 20 21 22 23 24 25 27 28 29 30 31 32 33. 37 38 40 42 43 44 45 48 50 56 57 59 60 61 62
87
SEROTYPING OF NON-CHOLERA VIBRIOS
VOL. 10, 1979
group
I II III IV V VI VII VIII
Sucrose
Arabi-
+ + + +
-
-
-
nose
+ +
Mannose
strains
+ + +
25 66 0 0 6
-
-
-
0
-
+ +
+
3 0
-
TABLE 3. Representatives of combination of Heiberg and serotype systems Heiberg Heiberg Serotype I
II
+ + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + +
V
I
VII
64 68 69 74 75 76 77 79 83 94 102 106 107 110
+
++ + + +
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
I
113 115 148 160 175 176 201 308 309 312 320 332 340 342 343 345 346 347 348 349 998 999
II
V
+
+ +
+ + +
+
111
+
+
+ + + + + + + + + + + + +
+ +
+ +
+
+
+
+
+
+
+
+ +
+
+ +
+ +
+
+
+
+ +
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
VII
88
SMITH
strains designated "998" (spontaneous agglutination) and "999" (untypable) represent over 20% of the cultures in our collection and are being investigated as to the possibility of new serotypes.
The results obtained with the sera tested against cultures isolated by ourselves or sent by others are presented in Table 4. The different sources are listed and, for the most part, are selfexplanatory. In the "non-G.I. illness" column are listed strains from wounds, otitis media, bacteremia, etc., of hospitalized patients. "Healthy individuals" represents strains isolated primarily from contacts of cholera patients; "Water" includes fresh-, brackish-, and seawater isolates; "Food" includes fish and shellfish isolates; and "Miscellaneous" represents isolates from insects, soil, plants, and other infrequently sampled sources. DISCUSSION This paper is a summary of work to date on the serology of vibrios, which has been in progress for a score of years. Some background may be helpful in understanding the results presented. Vibrios, as a group, are rather homogeneous as regards biochemical reactions. The Heiberg scheme allows some separation. It was noted early in this work that the Heiberg groupings did not give sufficient identity to strains for epidemiological purposes. Serotyping was begun. Those organisms not agglutinated by the 0 group I serum of Gardner and Venkatraman (3) were used. The numbering system was begun at 11 to leave the first 10 numbers available for "cholera vibrios." The criterion initially used for calling an organism "Vibrio" was morphology. When Hugh and Sakazaki (6) defined the minimal number of characters for the identification of Vibrio species, these were applied to all cultures in our collection. Many cultures, including some used to prepare typing sera, were not vibrios. The serotype was not discarded until all strains were rechecked biochemically and serologically. Also, as a part of the re-evaluation, the remaining sera were tested again against all type culures. Some sera were identical based upon reactivity and absorption studies. As a result, 72 serotypes are now recognized, as compared to 187 reported in 1965. The missing numbers for serotypes in the tables are explained on one of these bases. A new numbering system is being considered. Also, with approximately 20% of strains untyped, the development of new serotypes continues. It has been possible, using the serotyping system, to characterize vibrios more precisely than
J. CLIN. MICROBIOL.
if the Heiberg grouping were used by itself (Table 2). Combining both to describe strains, it was possible in one instance to distinguish 10 different kinds of vibrios isolated from a single specimen. As previously indicated, there does not appear to be any correlation between serotype and Heiberg group (Table 3). From the onset of this work one did not know if certain serotypes were associated with human disease or particular environmental samples. The data in Table 4 would indicate that no such relationship exists. However, one must exercise some restraint in drawing conclusions from this table as well as Table 2. The strains reported came from laboratories in various parts of the world over a 20-year period. The specimen from which the culture was isolated was provided by the sender, but the procedure(s) for isolation are not known. The culture collection would best be described as random isolates. Systematic studies on the ecology of these organisms are needed before an understanding of the epidemiology of human vibriotic disease is achieved. The naming of vibrios has been a concern for us. Early in the work, the term non-cholera vibrio was recommended to describe the cultures studied. This was to replace "non-agglutinable" vibrios, since the serotyping system was based upon agglutination of these vibrios. In 1972, the International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Vibrios (5) decided to use the designation Vibrio cholerae for both true cholera, and non-cholera vibrios. One cannot argue with the fact that, on scientific grounds, the subcommittee is correct. However, as Cowan (2) points out, "this was a committee decision taken without regard for the consequences in medicine." Clinicians assume, if the laboratory isolates V. cholerae from a patient, that the person has cholera. It has been suggested that the phrases "type 1" or "not type 1" be appended to the name so that the physician will be better informed. The question is whether this will clarify the situation for the clinician. It might be preferable and practical to be scientifically incorrect and call these organisms non-cholera vibrios rather than to create confusion while being a taxonomic purist. Another serotyping system for vibrios was proposed by Sakazaki et al. (9) in 1970, with further elaboration recently (8, 10). Briefly, rabbits were given multiple injections of heat-killed organisms. The sera were absorbed with a rough strain of a "classic" cholera vibrio and, apparently, other strains. The culture to be typed was heat killed and incubated with sera at 56°C overnight. In contrast, and as a summary of our procedure, rabbits were given a single subcutaneous injection of live organisms. The sera were
SEROTYPING OF NON-CHOLERA VIBRIOS
VOL. 10, 1979
89
TABLE 4. Distribution of serotypes among strains of vibrios isolated from various specimens Source Serotype
11 12
13 14 15 16 17 18 19 20 21 22 23 24 25 27 28 29 30 31
32 33 37 38 40 42 43 44
45 48 50 56 57 59 60 61 62 64 68 69 74 75 76 77 79 83 94 102
106 107 110 111
113 115 148 160
175 176
Source unknown
12 31 49 8 6 8 16 3 3 22 29 9 20 9 33 2 6 5
G.I.a ness
18 103 80 26 8 4 34 3 6 9 4 28 74 3 13 26
3 8 4 2
NonG.I. illness
2 1 2
5 2 2 4
5 9 3 2 4 1
1 5 2 4 1 3 1 2 1
2
2
Feces or night soil
11 138 28 19 1 8 4 5 2 9 26 7 14
3 30 9 6 7 3 15
3 2
4 11
1
1 2 4 1 1
1 3 15 2 4 2 4
Healthy individuals
1
2 5 3 2 1 1
10 2 10 6 3
10 3 1 5
3 1 1
4
2
1 1
1
1 2
2
1
3 2 1
1 1
2 2
1 1 8 7 11 3 1 3 1 2 1 1 2
1 1 14 15
3 2
8 3 7
Sewage
19 7 8
1 2
29 7 15 7 3 13 17 13 7 8 4
4 2 1 5
4 14 5 1 7 5
10 2 1
Food
1 1 1
3 2 2 2 2 1
Animal feces 6 1 2 5
Misce
laneous
Total
3 1 7 3 2
70 318 179 107 34 44
2 1
4 3
1
1
3 1 4 1
1 6
8 1
3 4
2
1
15 1
4 1
1
1
1
2 1 1 2
Water
1 1 1
10
1
2
2
5
4
2 2 1 5 12 6 14 14 2 11 2 4 4 5 1 3 5
1 8
1
1
1
1 3 1
1
4 1 1
1 2 1
1
1
1
2
9 1 1
*
1 5 4 1 3 7 3 17 11 1 5 9
3 8
1 1 1
1 1 1
2 3
4
1 1 2
1 21
1 1
3
3 2
89 27 29 74 89 62 126 9 38 104 5 12
35 31 4 4 3 1 31 55 14 22 7 27 4 19 9 13 14 8 9 4 27 3 5 9 14 8 5 5 7 17 60 46 4 1 15 10 47 2
7 4
90
J. CLIN. MICROBIOL.
SMITH
TABLE 4-continued Source Serotype
Source un-
201 308 309 312 320 332 340 342 343
known 1 1
Q. ness
NonG.I. illness
4 1
2 1
Healthy Feces individ- or night uals soil 1 2 3 1
2 1
5
1
Mlscel-
998b 54
4 152
19
48
19
1 8 160
Total 15 21 3 3 7 1 2 1
1
2
3 1 1 1 1
1 1
348 349
c
7 10 2 1 1 1 1
Animal feces
1
347
b
Food
2
1 1
346
999C
Sewage
1
345
a
Water
6 37
1 8
8
16
1 19 521
G.I., Gastrointestinal. 998, Spontaneous agglutination. 999, Untypable.
absorbed if necessary. Live organisms from cultures to be typed were suspended in drops of sera on slides and were observed for clumping within 1 min. There are obvious differences in the details of the two serotyping systems, but the most important point is whether the results obtained by either are comparable. Studies seeking an answer to this are in progress in several laboratories in Japan and the United States. ACKNOWLEDGMENTS This work was supported by Public Health Service contract NO1-AI-3,2502 under the U.S.-Japan Cooperative Medical
Program, administered by the National Institute of Allergy and Infectious Diseases. I wish to thank Janet LaMartina for her excellent technical assistance.
LITERATURE CITED 1. Colwell, R. R., J. Kaper, and S. W. Joseph. 1977. Vibrio cholerae, Vibrio parahaemolyticus, and other vibrios: occurrence and distribution in Chesapeake Bay. Science 198:394-396. 2. Cowan, S. T. 1973. The vibrio and vibrio-like group, p. 97-102. In Cowan and Steel's manual for the identification of medical bacteria, 2nd ed. Cambridge University Press, London. 3. Gardner, A. D., and K. V. Venkatraman. 1935-36. The antigens of the cholera group of vibrios. J. Hyg. 35-36: 262-282.
4. Heiberg, B. 1934. Des reactions de fermentation chez les vibrions. C. R. Soc. Biol. (Paris) 115:984. 5. Hugh, R., and J. C. Feeley. 1972. Report (1966-1970) of the Subcommittee on Systematic Bacteriology Subcommittee on Taxonomy of Vibrio to the International Committee on Nomenclature of Bacteria. Int. J. Syst.
Bacteriol. 22:123. 6. Hugh, R., and R. Sakazaki. 1972. Minimal number of characters for the identification of Vibrio species, Vibrio cholerae, and Vibrio parahaemolyticus. J. Conf. Public Health Lab. Directors 30:133-137. 7. Hughes, J. N., D. G. Hollis, E. J. Gangarosa, and R. E. Weaver. 1978. Non-cholera vibrio infections in the United States: clinical, epidemiologic and laboratory features. Ann. Int. Med. 88:602-606. 8. Sakazaki, R., and T. Shimada. 1977. Report: serovars of Vibrio cholerae identified during 1970-1975. Jpn. J. Med. Sci. Biol. 30:279-282. 9. Sakazaki, R., K. Tamura, C. Z. Gomez, and R. Sen. 1970. Serological studies on the cholera group of vibrios. Jpn. J. Med. Sci. Biol. 23:13-20. 10. Shimada, T., and R. Sakazaki. 1977. Additional serovars and inter-O antigenic relationships of Vibrio cholerae. Jpn. J. Med. Sci. Biol. 30:275-277. 11. Smith, H. L., Jr. 1970. A presumptive test for vibrios: the "String" test. Bull. W.HO. 42:817-818. 12. Smith, H. L., Jr., and P. Bhat-Fernandes. 1973. Modified decarboxylase-dihydrolase medium. Appl. Microbiol. 26:620-621. 13. Smith, H. L., Jr., and K. Goodner. On the classification of vibrios, p. 4-8. In 0. A. Bushnell and C. S. Brookhyser (ed.), Proceedings of the Cholera Research Symposium, Honolulu, Hawaii, January 24-29, 1965. U. S. Government Printing Office, Washington, D.C.
Vol. 10, No. 1
Serotyping of Non-Cholera Vibrios HARRY L. SMITH, JR. Department ofMicrobiology, Jefferson Medical College of Thomas Jefferson University, Philadelphia,
Pennsylvania 19107
Received for publication 30 April 1979
The serotyping system for non-cholera vibrios reported by us in 1965 has been re-evaluated and extended. Results from serotyping 2,624 cultures are presented. These isolates were accumulated over a period of 20 years and came from clinical and environmental samples in many parts of the world. Included is a discussion of the use of terms "Vibrio cholerae" and "non-cholera vibrios" for clinical reports. In 1965 we reported (13) a serotyping system for those vibrios that were not halophiles and which were not agglutinated by the 0 group I serum described by Gardner and Venkatraman (3). This was begun because of difficulty in characterizing the many strains of vibrios isolated by our laboratory people or by other workers from environmental samples and specimens from patients. The results with 0 group I serum and with biochemical tests such as the Heiberg fermentation scheme (4) gave a limited number of groups of different kinds of vibrios. The serotyping system allowed the finer distinction among the organisms that was sought for the purpose of obtaining data regarding the epidemiology and ecology of this group of organisms. Since the time of our first report, there appears to have been an increased awareness of the roles of non-cholera vibrios in causing human diseases (7). Some attention has been di,ected to the distribution of these organisms in nature (1). Many of the isolates were sent to us for serotyping. As more strains were processed it became evident that the original serotyping system needed to be re-evaluated. The definition of the genus Vibrio by Hugh and Sakazaki (6) provided an objective basis for inclusion or exclusion of strains, new and old, in the study. The typing sera were re-examined, and some were found to be partially or totally identical to others in the system. For those vibrio cultures not typed with any of the sera, the process of establishing new serotypes was begun. This report covers the current status of the serotyping system and gives data on the distribution of serotypes of strains in the non-cholera vibrio culture collection of our laboratory. MATERIALS AND METHODS
grounds. A more precise and less subjective method for defining Vibrio was reported by Hugh and Sakazaki (5). Using their scheme, the 2,624 strains employed in this study fit the minimal number of characters for the identification of Vibrio species, with the exception that the guanine-plus-cytosine ratio determinations were not made. In addition, none of these strains was agglutinated by serum prepared in our laboratory which corresponds to the 0 group I serum of Gardner and Venkatraman (3). A list of the cultures used for the preparation of the sera for typing and absorption is given in Table 1. The strains were maintained as lyophilized preparations and on slants of Trypticase-1% sodium chloride (TMNO) agar under sterile liquid paraffin at room temperature. The stock cultures were transferred minimally, usually twice, after their receipt by the laboratory. Sera preparation. The method for preparation of a serum for typing has been previously described (13). Briefly, the selected strain was grown on a 3-inch (ca. 76.2-mm) T,N, agar slant at 37°C overnight. The growth from the slant was suspended in 6 ml of sterile saline. The suspension contained approximately 107 organisms per ml. A 5-ml volume of this suspension was injected subcutaneously into the shaved side of a rabbit. After 10 to 14 days, the animal was exsanguinated by cardiac puncture. Not all animals survived to this point in time. Many that did, developed ulcerations which extended to dependent areas. The serum, after inactivation (56°C for 30 min), was titrated by the slide agglutination method. Drops of dilutions of the serum were placed on a slide, and living vibrios of the strain used for injection of the rabbit were suspended in the drops. The slide was rotated, and clumping was noted within 30 s. The reagent for use in further testing contained twice the amount of serum needed to give strong and complete agglutination, e.g., if a 1:10 dilution gave a rapid fourplus reaction, the reagent was prepared at 1:5. The diluted serum was then tested against all strains previously used for serum preparation by the slide agglutination method. If cross-reactions occurred, absorption studies were initiated to determine Strains. The criteria for including strains in this whether the serum was identical to or shared antibodstudy had been based primarily upon morphological ies with other reagent sera. Live cultures were used 85
J. CLIN. MICROBIOL.
SMITH
86
TABLE 1. Vibrio strains used to prepare typing sera Absorbed, if
Absorbed, if
Sero-
type
neces-
Cul-
uSource
no.
11 12 13 14 15 16
6305 5162 5064 5008 6707 5096
17 18 19 20 21 22
5047 7977 6701 5042 5487 5103
23 24
5072 5066
25 27 28 29 30 31 32
5163 6345 6541 6696 5128 5410 7888
33 37
7920 5078
38 40
5052 5079
42 43 44 45 48
5411 5165 5037 5009 5069
50
5152
Diarrhea, Dacca, 1961 Diarrhea, Dacca, 1961 Nanking 32/106, 0 group IV Water, Chicago, 1953 Night soil, Hong Kong, 1958 Felsenfeld Thai Series, In-28, 1960 Water, Thailand, 1959 Diarrhea, Dacca, 1965 Water, Hong Kong, 1958 Water, Thailand, 1959 Water, Dacca, 1962 Post-mortem gut, Thailand, 1959 Diarrhea, Dacca Felsenfeld Thai Series, N-1, 1960 Diarrhea, Dacca Unknown, Dacca Water, Bangladesh, 1965 Night soil, Hong Kong Diarrhea, Dacca, 1962 Diarrhea, Dacca, 1961 Felsenfeld Thai Series, diarrhea 1-4 Contact, Japan, 1968 Water, Felsenfeld Thai Series, NAG-3" Water, Thailand, 1959 Water, Felsenfeld Thai Series, NAG-4 Diarrhea, Dacca, 1961 Diarrhea, Dacca, 1961 Water, Thailand, 1959 Diarrhea, unknown Contact, Felsenfeld Thai Series, N-4 Water, Chesapeake Bay, Md.,
56 57
5053 6355
Water, Thailand, 1959 Diarrhea, Dacca
Serotype
Cul-
1 59
5092
68 48
60 61 62
6326 5029 5059
75
64 68 69
7449 5028 5086
74 75 76 77 79 83 94 102 106 107 110 111 113 115 148 160 175 176 201 308 309 312 320 332 340 342 343 345 346 347 348 349
7900 5681 6335 5694
sary, with serotype culture no.
321
176, 115
321 115 18 45 45
115
68, 107
321 32
1960
50
no.
5444 5697 5051 7647 5803 6306 7555 7556 6312 7902 5722 6358
9009 5180 7165 7349 6313 6970 7995 8536 8585 8462 8497 9115 9166 9183 9211 9248
neces-
sary, with serotype culture no. 32 Contact, Felsenfeld Thai Series, NAG-24 Cow dung, Dacca, 1962 Water, Thailand, 1959 A.M.S. 20-A-59, Van Rooyen, Q67 Unknown, Philippines, 1962 32 Water, Thailand, 1959 Food, Felsenfeld Thai Series, 326 NAG-15 13, 22 Unknown, Philippines, 1962 Contact, Dacca, 1962 Goat, Dacca, 1962 Water, Dacca, 1962 Water, Dacca, 1962 45 Water, Dacca, 1962 68 Water, Thailand, 1959 107 Water, U.S.A. 326 Water, Philippines, 1964 321 Diarrhea, Dacca, 1961 Unknown, Dacca, 1962 Unknown, Philippines, 1962 Diarrhea, Dacca, 1962 Diarrhea, Dacca, 1962 32 Diarrhea, Philippines, 1964 32 Source unknown Water, Hungary, 1976 Calcutta, NAG group II, 1963 Water, Dacca, 1966 Diarrhea, Dacca, 1966 Diarrhea, Dacca, 1962 Diarrhea, Dacca, 1966 Latrine, unknown, 1968 Muic. 145, sewage, Yugoslavia 321 Human feces, Iraq, 1966 Diarrhea, Iran, 1972 Septic tank, U.S.A., 1973 Nonenteric, Philippines, 1976 Diarrhea, Dacca, 1977 Unknown, Guam, 1977 Water, Maryland, 1977 Water, Maryland, 1977
Source
a NAG, Non-agglutinable vibrio designation.
for absorption. Table 1 lists the sera that were absorbed and the strains used for the absorption. The serum was tested with strains not previously typed. Those cultures that could not be typed by this and other sera similarly prepared were possible candidates for preparation of new sera. Processing of strains received. Each culture was streaked on a brain heart infusion agar plate and inoculated into TIN, broth, and incubated at 370C. The broth culture was used 4 to 6 h later to inoculate glucose broth and amino acid media (12). All were incubated overnight at 37°C. The plate culture was examined macroscopically for contamination or colonial variation. If either situation occurred, colonies were picked from the plate and were tested individually. If the plate culture was acceptable, the oxidase
and the String (11) tests were performed. Both had to give positive results. In addition, the following results had to be obtained before proceeding: glucose fermentation without gas; lysine and ornithine decarboxylated but arginine not affected. Stock cultures were prepared. A portion of the growth on the plate was suspended in sterile skim milk and lyophilized. In addition, two T1NI agar slants were inoculated from the confluent area of the plate. After overnight incubation, sterile liquid paraffin was added to each tube so that the entire slant was covered. The remainder of the plate culture was used as the source of the organisms for the slide agglutination test with the typing sera. The initial test was performed with cholera grouping serum which corresponded to 0 group I of Gardner and Venkatraman (3). A system of
serum pools was devised to facilitate serotyping. Each culture was tested in each pool. When agglutination was observed, the sera which constituted that pool(s) were tested. Additional biochemical tests such as fermentations, salt tolerance, etc. were conducted for further data useful in separating vibrios (13).
with the Heiberg classification it was possible to recognize 160 different kinds of vibrios (Table 3). It should be noted from this table that there does not appear to be any correlation between serotype and Heiberg fermentation pattern. The TABLE 2. The Heiberg classification of vibrios and the frequency of types Fennentation of % of Heiberg
RESULTS When the strains which fit the minimal characteristic for vibrios (6) were inoculated into separate media containing sucrose, arabinose, or mannose as suggested by Heiberg (4), they could be subdivided into four groups (Table 2). Although most were Heiberg II, the frequencies given in this table may represent the effects of other factors, which will be discussed. The serotyping system that was developed currently consists of 72 sera raised against nonO group I vibrios (Table 1). By combining this
Serotype 11 12
13 14 15 16 17 18 19 20 21 22 23 24 25 27 28 29 30 31 32 33. 37 38 40 42 43 44 45 48 50 56 57 59 60 61 62
87
SEROTYPING OF NON-CHOLERA VIBRIOS
VOL. 10, 1979
group
I II III IV V VI VII VIII
Sucrose
Arabi-
+ + + +
-
-
-
nose
+ +
Mannose
strains
+ + +
25 66 0 0 6
-
-
-
0
-
+ +
+
3 0
-
TABLE 3. Representatives of combination of Heiberg and serotype systems Heiberg Heiberg Serotype I
II
+ + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + +
V
I
VII
64 68 69 74 75 76 77 79 83 94 102 106 107 110
+
++ + + +
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
I
113 115 148 160 175 176 201 308 309 312 320 332 340 342 343 345 346 347 348 349 998 999
II
V
+
+ +
+ + +
+
111
+
+
+ + + + + + + + + + + + +
+ +
+ +
+
+
+
+
+
+
+
+ +
+
+ +
+ +
+
+
+
+ +
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
VII
88
SMITH
strains designated "998" (spontaneous agglutination) and "999" (untypable) represent over 20% of the cultures in our collection and are being investigated as to the possibility of new serotypes.
The results obtained with the sera tested against cultures isolated by ourselves or sent by others are presented in Table 4. The different sources are listed and, for the most part, are selfexplanatory. In the "non-G.I. illness" column are listed strains from wounds, otitis media, bacteremia, etc., of hospitalized patients. "Healthy individuals" represents strains isolated primarily from contacts of cholera patients; "Water" includes fresh-, brackish-, and seawater isolates; "Food" includes fish and shellfish isolates; and "Miscellaneous" represents isolates from insects, soil, plants, and other infrequently sampled sources. DISCUSSION This paper is a summary of work to date on the serology of vibrios, which has been in progress for a score of years. Some background may be helpful in understanding the results presented. Vibrios, as a group, are rather homogeneous as regards biochemical reactions. The Heiberg scheme allows some separation. It was noted early in this work that the Heiberg groupings did not give sufficient identity to strains for epidemiological purposes. Serotyping was begun. Those organisms not agglutinated by the 0 group I serum of Gardner and Venkatraman (3) were used. The numbering system was begun at 11 to leave the first 10 numbers available for "cholera vibrios." The criterion initially used for calling an organism "Vibrio" was morphology. When Hugh and Sakazaki (6) defined the minimal number of characters for the identification of Vibrio species, these were applied to all cultures in our collection. Many cultures, including some used to prepare typing sera, were not vibrios. The serotype was not discarded until all strains were rechecked biochemically and serologically. Also, as a part of the re-evaluation, the remaining sera were tested again against all type culures. Some sera were identical based upon reactivity and absorption studies. As a result, 72 serotypes are now recognized, as compared to 187 reported in 1965. The missing numbers for serotypes in the tables are explained on one of these bases. A new numbering system is being considered. Also, with approximately 20% of strains untyped, the development of new serotypes continues. It has been possible, using the serotyping system, to characterize vibrios more precisely than
J. CLIN. MICROBIOL.
if the Heiberg grouping were used by itself (Table 2). Combining both to describe strains, it was possible in one instance to distinguish 10 different kinds of vibrios isolated from a single specimen. As previously indicated, there does not appear to be any correlation between serotype and Heiberg group (Table 3). From the onset of this work one did not know if certain serotypes were associated with human disease or particular environmental samples. The data in Table 4 would indicate that no such relationship exists. However, one must exercise some restraint in drawing conclusions from this table as well as Table 2. The strains reported came from laboratories in various parts of the world over a 20-year period. The specimen from which the culture was isolated was provided by the sender, but the procedure(s) for isolation are not known. The culture collection would best be described as random isolates. Systematic studies on the ecology of these organisms are needed before an understanding of the epidemiology of human vibriotic disease is achieved. The naming of vibrios has been a concern for us. Early in the work, the term non-cholera vibrio was recommended to describe the cultures studied. This was to replace "non-agglutinable" vibrios, since the serotyping system was based upon agglutination of these vibrios. In 1972, the International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Vibrios (5) decided to use the designation Vibrio cholerae for both true cholera, and non-cholera vibrios. One cannot argue with the fact that, on scientific grounds, the subcommittee is correct. However, as Cowan (2) points out, "this was a committee decision taken without regard for the consequences in medicine." Clinicians assume, if the laboratory isolates V. cholerae from a patient, that the person has cholera. It has been suggested that the phrases "type 1" or "not type 1" be appended to the name so that the physician will be better informed. The question is whether this will clarify the situation for the clinician. It might be preferable and practical to be scientifically incorrect and call these organisms non-cholera vibrios rather than to create confusion while being a taxonomic purist. Another serotyping system for vibrios was proposed by Sakazaki et al. (9) in 1970, with further elaboration recently (8, 10). Briefly, rabbits were given multiple injections of heat-killed organisms. The sera were absorbed with a rough strain of a "classic" cholera vibrio and, apparently, other strains. The culture to be typed was heat killed and incubated with sera at 56°C overnight. In contrast, and as a summary of our procedure, rabbits were given a single subcutaneous injection of live organisms. The sera were
SEROTYPING OF NON-CHOLERA VIBRIOS
VOL. 10, 1979
89
TABLE 4. Distribution of serotypes among strains of vibrios isolated from various specimens Source Serotype
11 12
13 14 15 16 17 18 19 20 21 22 23 24 25 27 28 29 30 31
32 33 37 38 40 42 43 44
45 48 50 56 57 59 60 61 62 64 68 69 74 75 76 77 79 83 94 102
106 107 110 111
113 115 148 160
175 176
Source unknown
12 31 49 8 6 8 16 3 3 22 29 9 20 9 33 2 6 5
G.I.a ness
18 103 80 26 8 4 34 3 6 9 4 28 74 3 13 26
3 8 4 2
NonG.I. illness
2 1 2
5 2 2 4
5 9 3 2 4 1
1 5 2 4 1 3 1 2 1
2
2
Feces or night soil
11 138 28 19 1 8 4 5 2 9 26 7 14
3 30 9 6 7 3 15
3 2
4 11
1
1 2 4 1 1
1 3 15 2 4 2 4
Healthy individuals
1
2 5 3 2 1 1
10 2 10 6 3
10 3 1 5
3 1 1
4
2
1 1
1
1 2
2
1
3 2 1
1 1
2 2
1 1 8 7 11 3 1 3 1 2 1 1 2
1 1 14 15
3 2
8 3 7
Sewage
19 7 8
1 2
29 7 15 7 3 13 17 13 7 8 4
4 2 1 5
4 14 5 1 7 5
10 2 1
Food
1 1 1
3 2 2 2 2 1
Animal feces 6 1 2 5
Misce
laneous
Total
3 1 7 3 2
70 318 179 107 34 44
2 1
4 3
1
1
3 1 4 1
1 6
8 1
3 4
2
1
15 1
4 1
1
1
1
2 1 1 2
Water
1 1 1
10
1
2
2
5
4
2 2 1 5 12 6 14 14 2 11 2 4 4 5 1 3 5
1 8
1
1
1
1 3 1
1
4 1 1
1 2 1
1
1
1
2
9 1 1
*
1 5 4 1 3 7 3 17 11 1 5 9
3 8
1 1 1
1 1 1
2 3
4
1 1 2
1 21
1 1
3
3 2
89 27 29 74 89 62 126 9 38 104 5 12
35 31 4 4 3 1 31 55 14 22 7 27 4 19 9 13 14 8 9 4 27 3 5 9 14 8 5 5 7 17 60 46 4 1 15 10 47 2
7 4
90
J. CLIN. MICROBIOL.
SMITH
TABLE 4-continued Source Serotype
Source un-
201 308 309 312 320 332 340 342 343
known 1 1
Q. ness
NonG.I. illness
4 1
2 1
Healthy Feces individ- or night uals soil 1 2 3 1
2 1
5
1
Mlscel-
998b 54
4 152
19
48
19
1 8 160
Total 15 21 3 3 7 1 2 1
1
2
3 1 1 1 1
1 1
348 349
c
7 10 2 1 1 1 1
Animal feces
1
347
b
Food
2
1 1
346
999C
Sewage
1
345
a
Water
6 37
1 8
8
16
1 19 521
G.I., Gastrointestinal. 998, Spontaneous agglutination. 999, Untypable.
absorbed if necessary. Live organisms from cultures to be typed were suspended in drops of sera on slides and were observed for clumping within 1 min. There are obvious differences in the details of the two serotyping systems, but the most important point is whether the results obtained by either are comparable. Studies seeking an answer to this are in progress in several laboratories in Japan and the United States. ACKNOWLEDGMENTS This work was supported by Public Health Service contract NO1-AI-3,2502 under the U.S.-Japan Cooperative Medical
Program, administered by the National Institute of Allergy and Infectious Diseases. I wish to thank Janet LaMartina for her excellent technical assistance.
LITERATURE CITED 1. Colwell, R. R., J. Kaper, and S. W. Joseph. 1977. Vibrio cholerae, Vibrio parahaemolyticus, and other vibrios: occurrence and distribution in Chesapeake Bay. Science 198:394-396. 2. Cowan, S. T. 1973. The vibrio and vibrio-like group, p. 97-102. In Cowan and Steel's manual for the identification of medical bacteria, 2nd ed. Cambridge University Press, London. 3. Gardner, A. D., and K. V. Venkatraman. 1935-36. The antigens of the cholera group of vibrios. J. Hyg. 35-36: 262-282.
4. Heiberg, B. 1934. Des reactions de fermentation chez les vibrions. C. R. Soc. Biol. (Paris) 115:984. 5. Hugh, R., and J. C. Feeley. 1972. Report (1966-1970) of the Subcommittee on Systematic Bacteriology Subcommittee on Taxonomy of Vibrio to the International Committee on Nomenclature of Bacteria. Int. J. Syst.
Bacteriol. 22:123. 6. Hugh, R., and R. Sakazaki. 1972. Minimal number of characters for the identification of Vibrio species, Vibrio cholerae, and Vibrio parahaemolyticus. J. Conf. Public Health Lab. Directors 30:133-137. 7. Hughes, J. N., D. G. Hollis, E. J. Gangarosa, and R. E. Weaver. 1978. Non-cholera vibrio infections in the United States: clinical, epidemiologic and laboratory features. Ann. Int. Med. 88:602-606. 8. Sakazaki, R., and T. Shimada. 1977. Report: serovars of Vibrio cholerae identified during 1970-1975. Jpn. J. Med. Sci. Biol. 30:279-282. 9. Sakazaki, R., K. Tamura, C. Z. Gomez, and R. Sen. 1970. Serological studies on the cholera group of vibrios. Jpn. J. Med. Sci. Biol. 23:13-20. 10. Shimada, T., and R. Sakazaki. 1977. Additional serovars and inter-O antigenic relationships of Vibrio cholerae. Jpn. J. Med. Sci. Biol. 30:275-277. 11. Smith, H. L., Jr. 1970. A presumptive test for vibrios: the "String" test. Bull. W.HO. 42:817-818. 12. Smith, H. L., Jr., and P. Bhat-Fernandes. 1973. Modified decarboxylase-dihydrolase medium. Appl. Microbiol. 26:620-621. 13. Smith, H. L., Jr., and K. Goodner. On the classification of vibrios, p. 4-8. In 0. A. Bushnell and C. S. Brookhyser (ed.), Proceedings of the Cholera Research Symposium, Honolulu, Hawaii, January 24-29, 1965. U. S. Government Printing Office, Washington, D.C.
