ing, serotyping, and bacteriophage typing, but these meth- ods cannot differentiate indigenous strains from imported cases of cholera in Hong Kong. In addition.
JOURNAL OF ClINICAI MICROBIOo(;CY. Aug. 1989. p. 1900-1902
0095-1137/89/081900-03$0('.0)0/0 Copyright ù 1989. American Society l'or
Vol. 27, No. 8
Microbiology
Molecular Epidemiology of Vibrio cholerae in Hong Kong CHEONG
YAMt MARIA LI LUNG,'- KWAN YEE NG.2 AND MUN HON NG1 I)cpartîluîct of Mi(ciobiologv, Uniiîe.rsitv ol Hong Kong,' aIl( Pub/lic Heui/t Luboruortov ,, l Iong Kolng Path/ology IInstitllt(e, M/(Vlie(/l uiI/ Heu/tli !)epu
WING
Received 6 February 1989/Accepted 3 May 1989
We studied restriction fragment length polymorphism of the enterotoxin genes of isolates of Vibrio cholerae El Tor, indistinguishable by bacteriophage typing, which were collected in Hong Kong since 1978. Using this approach, we could distinguish indigenous and exogenous strains obtained from different sources and epidemiological settings.
Cholera toxin is the principal pathogenic attribute of enterotoxigenic Vibrio (hol/rue, the causal agent of cholera (14). V. /hol/crue is conventionally differentiated by biotyping, serotyping, and bacteriophage typing, but these methods cannot differentiate indigenous strains from imported cases of cholera in Hong Kong. In addition. Kaper et aI. (5, 6) and others (4, 7) have used the technique of restriction fragment length polymorphism (RFLP) to examine the molecular epidemiology of V. c/tol/rue Q1 and non-Q1 V. l/olecrue in different settings. We have presently used this technique in an epidemiological study of V. cho/erae in Hong Kong. A total of 52 isolates of' V. c/tol/rue El Tor were obtained in Hong Kong between 1978 and 1987. 36 of which were obtained during one outbreak in 1986. Of the remaining 16 cases, 11 patients had no travel history within 2 weeks before the onset of the disease. 1 was a resident returning from Nepal, 4 were airline hostesses arriving in Hong Kong on the same flight from Manilda and 2 were obtained from Vietnamese refugee children who developed cholera en route to Hong Kong. All of these isolates were identified in the Public Health Laboratory of the Hong Kong government by standard methods (3). The isolates were submitted to the Central Public Health Laboratory. London, England, for phage typing by B. Rowe using the USSR scheme. They were also tested for active toxin production by the rabbit ileal loop assay (2). Standard American Type Culture Collection (ATCC) strains of V. c/ol/crue were purchased from ATCC, Rockville, Md. Chromosomal DNA was extracted by the method of Brenner et al. (1), digested with HindlIl or H/(ill restriction endonuclease (Anglian Biotechnology Ltd.) as described by Maniatis et al. (9). electrophoresed in 1%/, agarose gels. and transferred to nitrocellulose by the method ot`Southern (13). As a probe, the heat-labile toxin (LT) of enterotoxigenic Esc/îeric/îia co/i used. This gene shares sequence homology with cholera toxin (10) and has been used previously in molecular epidemiological studies of V. c/to/lcre (5. 6). We obtained recombinant plasmid EWD299 (LT) from S. Falkow of Stanford University. Stanford. Calif. Although the ct.v gene of V. c/ole/cue has been cloned (8. 12) and used in recent studies (4). we did not have it for these studies. 32P-labeled LT probe was prepared by nick translation as described previously (11, 16). Hybridization of the LTI probe to the Southern blots was performed Linder low-stringencv conditions as described elsewhere (6). After hybridization.
washed filters were air dried and then exposed to X-ray film with Du Pont Lightning-Plus intensifying screens at -70°C. We reviewed 52 cases of cholera occurring in Hong Kong over a period of 10 years since 1978 (Tcable 1). Since cholera is a notifiable disease. this is probably an accurate representation of the extent to which the disease occurred in this city. All except seven of these cases were probably caused by organisms indigenous to Hong Kong, since these patients did not have a recent travel history. Two strains isolated in 1978 and 1981 were serotype Ogawa, while acil of the remaining indigenous strains isolated in the subsequent years were serotype Inaba. Except for one outbreak in August 1986 involving 36 adults working or residing or both in an industrial district of Hong Kong, indigenous cases of cholera otherwise occurred rarely and sporadically during this period. Surprisingly. there have been only seven imported cases of choIera (Table 1) although the disease is endemic in countries neighboring Hong Kong. Ali isolates of V. chlolerue elaborated enterotoxin as TABLE 1. V cho/('rJI El Tor"' isolated in Hong Kong between 1978 and 1987 No. of cases isolated
Date
Serotstpe Ogawa Serotvpe Inaba ____________________________________________________ Indigenotis Exogenotus Indigenous Exogenotis
1978 1981 1984 1985 1986 MaNx Jlune Jtlly Aug
was
Sept 1987
1 1
Nov Nov
2
A B C C
1 1
C
1 36
C C
1
C
4'
Sept
Retrict pattern
D
1
C
1i' 2'
E F
All isolates were phage type 13 (B. Ro\w e. personal communication). Restrictions patteni: type A. 14-kilobaîse (kh) IlindIlI fragment. 2.7-kh 1)ail Ilfragment: type B. t6-kh IlinîdIII fragment. '2.-kb lIplIl firagnment: type C. 22-kh Iii,îlllilfragment. 2.6-kh II1pIl fragment: type D. 14-kh HildIII fragment. 3.7-kb /1palu fragment: t pe E. 16 -kh IlindlII fragment. 2.X-kl Ilp)(ull fragment: type F. 20-klî Hindi Il friament. 2.6-kh /11w(illfragnient. louir airline hostesses arrivînL o n thie saine flight from Manila. Htongy Kon- resident returning fromi NipIl. ]'wo victnanmese relfuee children wx ho developed cholera eni route to
Corresponding authorr.
Hono Kong.
190)(
NOTES
Vot- 27, 1989
A A B C D E F G H I
19(1
B J K L M N O P Q
A B C D E F G H 231M. *
11__
23.1
.l
...
IMu"
_
_
9.4".
6.6"
6. 6i-
FIG. 1. Autoradiogram of Southern blot of V. cholerae El Tor DNA digested with Hindl.1l and probed with LT. (A) V. chluIera El Tor Inaba isolated from sporadic cases between 1984 and 1987 (lIanes A to D). an outbreak in 1986 (lanes E to 1). food and the environment during the 1986 outbreak (lanes J and K). two imported cases fromn Vietnam (lanes L and M). and standard ATCC strains 9459. 25870. 11628, and 14427 (lanes N to Q). (B) V. c/clerae El Tor serotype Ogawa isolated from sporadic case of cholera in 1978 (lane A) and 1981 (lane B). four imported cases from Manila (lines C to F). an imported case from Nepal (lane G). and standard ATCC strain 9458 (lane H). serotype
a
indicated by positive results for the rabbit ileal assay. They aill phage type 13. but they exhibited extensive RFLP of the enterotoxin gene. Figure 1 shows Southern blots obtained from digestion of DNA extracts with HindIII and probed with the LT gene. All serotype Inaba isolates from indigenous cases of cholera gave similar restriction patterns. Representative results are shown in Fig. lA. These include 4 sporadic cases occurring since 1984 (lanes A to D). representatives from 36 cases which occurred during an outbreak in 1986 (lanes E to 1), and isolates from food and the home of a patient during that outbreak (lanes J and K). The serotype Inaba isolates from two Vietnamese children who developed cholera en route to Hong Kong were similar to one another (lanes L and M), but they were different from those obtained with isolates indigenous to Hong Kong (lanes A to K) and standard strains from ATCC (lanes N to Q). Serotype Ogawa isolates were similarly analyzed. The results in Fig. 1B differentiated isolates from two indigenous cholera cases occurring in 1978 (lane A) and 1981 (Jane B). It was also possible to distinguish by this method the indigenous isolates from isolates originating in Manila (lanes C to F). Nepal (lane G), and a standard strain obtained from ATCC (Jane H). In contrast. the four isolates from Manila were indistinguishable from one another by this method. Similar results were aiso obtained when the above strains were subjected to HpbaII digestion (data not shown). RFLP patterns were identified among the isolates (Fig. 1 and Table 1). Our results confirm previous findings that enterotoxin well as genes on the chromosome of V. col/crae El Tor. the plasmid of enterotoxigenic E. coli in Hong Kong (16). exhibit RFLP. This allows us to distinguish indigenous and imported cases of cholera occurring in Hong Kong over 10 years since 1978. Significantly. ail 52 isolates presently investigated have the same phage type. i.e.. phage type 13. which is prevalent in Asia (15). and they are either serotype Inaba or serotype Ogawa. The usefulness of RFLP of the an epidemiological marker further enterotoxin gene evidenced when it found on three separate occasions that the method allows us to relate isolates obtained from different sources under the same epidemiological settings. This includes the restriction pattern of isolates from 36 indigenous cases of cholera outbreak in 1986 (type C). 4 exogenous cases from airline hostesses (type D). and 2 exogenous cases from Vietnamese children (type F). were
as
By this method we distinguished three strains of V. lholerae which are indigenous to Hong Kong. Two were serotype Ogawa with distinguishable RFLP (types A and B). They were isolated from two indigenous cases in 1978 and 1981. respectively. but have not been isolated thereafter. The other strain. which has been prevalent in this community since its first detection in 1984. is serotype Inaba. This strain exhibited one RFLP pattern (type C) and caused an outbreak in 1986 involving 36 cases. The outbreak was confined to a district and subsided within 1 month of its onset. The same or a closely related strain also caused seven sporadic cases of cholera between 1984 and 1987. This situation is similar t1 single strain of V. (Choleirae El Tor serotype Inaba persisting in the U.S. Gulf Coast from 1973 to 1981 (5). Despite its proximity to neighboring countries where cholera is endemic and the frequency of travel to and from these regions, the number of imported cases of cholera in Hong Kong has been surprisingly low. The exogenous strains of V. chlerue isolated on these occasions probably originated from Nepal. Vietnam, and Manila and were readily distinguishable from one another and from the strains indigenous to Hong Kong. There has been no evidence to date. however, to suggest that the exogenous strains have become sufficiently widespread in this community to cause a
disease.
We thank S. Falkow of Stanford University for providing the hacterial strain harboring plasrnid EWD299 (LT) and B. Rowe ofthe Central Public Health L.aboratory for phage typing of our V. c/ol/rac isolates. We also thank W. H. Lau for excellent technical assistance.
LIlERATURE CITED 1. Brenner, D. J., G. R. Fanning, K. E. Johnson, R. V. Citarella, and S. Falkow. 1969. Polynucleotide sequence relationships aniong members of Enicohmicriaccac. J. Bacteriol. 98:637650.
was
as
was
a
2.
Burrows, W., and
G. M.
Musteikis. 1966. Cholera infection and
toxin in the rabbit ileal loop. J. Infect. Dis. 116:183-190.
3. Farmer, J. J., 111 F. W. Hickman-Brenner, and M. T. Kelly. 1985. Vibrio. p. 282-301. ln E. H. Lennette. A. Balows. W. J. Hausler. Jr.. and H. J. Shadorny (ed.). Manual of clinical microbiology. 4th ed. Arnerican Society for Microbiology. Washington. D.C. 4. Hanchalay, S., J. Seriwatana, P. Echeverria, J. Holmgren, C.
19(02
5. 6.
7.
J. CLIN. MICROBIOL.
NOTES
Tirapat, S. L. Moseley, and D. N. Taylor. 1985. Non-Q1 Vi/nio v/Ol/rue in Thailand: homology with cloned choIera toxin genes. J. Clin. Microbiol. 21:288-289. Kaper, J. B., H. B. Bradford, N. C. Roberts, and S. Falkow. 1982. Molecular epidemiology of Vibrio chuol/re in the U.S. Gulf Coast. J. Clin. Microbiol. 16:129-134. Kaper, J. B., S. L. Moseley, and S. Falkow. 1981. Molecular characterization of environmental and nontoxigenic strains of Vibrio chu/oler. Infect. Immun. 32:661-667. Gross, J. Lin, F.-Y. C., J. G. Morris, Jr., J. B. Kaper, Michalski, C. Morrison, J. P. Libonati, and E. Israel. 1986. Persistence of cholera in the United States: isolation of Vi/briu c(/hOl/rc 01 from patient with diarrhea in Maryland. J. Clin. Microbiol. 23:624-626. Lockman, H., and J. B. Kaper. 198-3. Nucleotide sequence analysis of the A2 and B subunits of Vilrio chulcrue enterotoxin. J. Biol. Chem. 258:13722-13726. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Moleculaïr cloning: laboratory manual. p. 97-148. Cold Spring Harbor Laboratory. Cold Spring Harbor. N.Y. Moseley, S. L., and S. Falkow. 1980. Nucleotide sequence homology between the heat-labile enterotoxin gene of Esc/heT.
a
8.
9.
a
10.
11.
12. 13. 14. 15. 16.
richlia coli and Vibrio chu/olvr deoxyribonucleic acid. J. Bacteriol. 144:444-446. Moseley, S. L., I. Huq, A. R. M. A. Alim, M. So, M. SamadpourMotalebi, and S. Falkow. 1980. Detection of enterotoxigenic E.scIwrichia vuli by DNA colony hybridization. J. Infect. Dis. 142:892-898. Pearson, G. D. N., and j. j. Mekalanos. 1982. Molecular cloning of Vibrio chu/crue entertoxin genes in Esclhrichia coli K12. Proc. Nati. Acad. Sci. USA 79:2976-2980. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. World Health Organization. 1980. Cholera and other vibrioassociated diarrhoeas. Bull. W.H .O. 58:353-374. Word Health Organization Scientific Working Group. 1985. Recent advances in cholera research: memorandum from a WHO Meeting. Bull. W.H.O. 63:841-849. Yam, W. C., M. Li Lung, and M. H. Ng. 1988. Clonal origin, restricted natural distribution. and conservation of virulence factors in isolates of enterotoxigenic Eshcrivhia vuoli serogroup 0126. J. Clin. Microbiol. 26:1477-1481.
