JOURNAL OF CLINICAL MICROBIOLOGY, May 1997, p. 1151–1156 0095-1137/97/$04.0010 Copyright q 1997, American Society for Microbiology
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Molecular Evolution of Vibrio cholerae O1 Strains Isolated in Lima, Peru, from 1991 to 1995 A. DALSGAARD,1* M. N. SKOV,1† O. SERICHANTALERGS,2 P. ECHEVERRIA,2 R. MEZA,3 AND D. N. TAYLOR3 Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Frederiksberg, Denmark1; Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand2; and Departments of Microbiology and Medical Ecology, United States Naval Medical Research Institute Detachment, Lima, Peru3 Received 7 October 1996/Returned for modification 19 December 1996/Accepted 29 January 1997
Following the emergence of cholera in Lima, Peru, in 1991, isolates of Vibrio cholerae O1 biotype El Tor recovered from patients in various parts of Lima were selected and characterized. Ribotyping and pulsed-field gel electrophoresis (PFGE) revealed four BglI ribotypes and eight NotI PFGE types among 50 V. cholerae O1 strains recovered from patients with cholera in Lima from 1991 to 1995, with certain genotypes appearing to cluster geographically. While differences in ribotype and PFGE type patterns suggest that genetic changes are occurring in the strain responsible for the Latin American cholera epidemic, more frequently than previously reported, 40 (80%) O1 strains showed an identical ribotype pattern and 41 (82%) strains showed closely related PFGE types, types 1, 2, or 3, that differed by less than three restriction fragments. All strains were susceptible to nine antibacterial agents studied. In 1991, more than 95% of the clinical V. cholerae O1 strains were serotype Inaba, whereas from 1992, serotype Ogawa began to predominate, with more than 90% of the isolates being of the Ogawa serotype in 1995. The small differences in genotypes of V. cholerae O1 is remarkable because cholera is highly seasonal in coastal areas of Peru and support the hypothesis that the epidemic strain reemerges from an environmental source. However, the relative high rate of genetic changes within V. cholerae O1 as shown by ribotyping and PFGE should be taken into consideration when typing patterns of V. cholerae O1 associated with cholera in Latin America are evaluated. 1991, the Latin American strain appears to have undergone genetic changes, although the rate of changes remains to be determined (9). Whereas the Latin American epidemic appears to be associated with a single strain of V. cholerae O1, distinct O1 strains were reported to be associated with cholera in Mexico and Brazil (4, 9). The ability of these and other possibly introduced strains to compete with the Latin American epidemic strain is unknown. Within this epidemic setting, we studied the evolution of V. cholerae O1 biotype El Tor isolates recovered in Lima, Peru, from 1991 to 1995. A collection of 50 isolates were characterized by antibiotic susceptibility testing, plasmid profiling, ribotyping, restriction fragment length polymorphism analysis of cholera toxin (CT) genes, and PFGE typing. Molecular typing showed that the isolate initially introduced into Peru in 1991 has undergone several genetic changes. Furthermore, a distinct new V. cholerae O1 serovar Ogawa strain isolated in 1994 was identified.
In January 1991, cholera appeared in several cities along the coast in Peru, including the capital Lima, and spread rapidly to other countries in Latin America (14). The outbreak of cholera marked the first epidemic in Latin America in this century. Among Latin American countries, Peru has reported the highest number of cholera cases, with more than 600,000 cases reported from 1991 to 1993 (14). From 1993, cholera has been endemic in most areas of Peru, a pattern often observed in countries affected by significant morbidity (14). A variety of phenotypic and molecular typing methods, such as multilocus enzyme electrophoresis, ribotyping, and pulsedfield gel electrophoresis (PFGE), have been used to study the epidemiology of Vibrio cholerae O1 isolates from Latin America (32). Previous studies of V. cholerae O1 strains isolated from 1991 to 1993 found that all isolates were essentially identical, as shown by ribotyping and PFGE (1, 21, 31). Popovic et al. (21) found that all isolates related to the Latin American epidemic in 1991 and 1992 had an identical ribotype, which was also shown by some V. cholerae O1 isolates associated with the seventh pandemic in other parts of the world. Cameron et al. (1) found that all isolates related to the current epidemic in Latin America were identical by PFGE with the restriction enzyme NotI. However, in a recent study of the diversity of electrophoretic types of V. cholerae O1, two isolates from Peru showed a PFGE pattern which differed by a single band from those of all isolates previously associated with the Latin American cholera epidemic (9). Thus, following its introduction in
MATERIALS AND METHODS Bacteria. A total of 50 V. cholerae O1 biotype El Tor strains recovered from patients from 1991 to 1995 in various parts of Lima, Peru, were included in the study (Table 1). Five strains each which agglutinated with antisera to serotype Ogawa and to serotype Inaba were selected from each year. Strains with designations CHO originated from Callao, the port of Lima or north of Lima, at a military training area; BAB strains were isolated at city hospitals; strains with the designations VIG, HMA, and ECC were recovered in south Lima during a cholera surveillance to evaluate the efficacy of a V. cholerae vaccine (5, 25); strains with number designations only were isolated in north Lima; and CIT strains were isolated at a military base in Callao. Isolates O51A serotype Ogawa and O51B serotype Inaba were recovered from the same stool sample of a single patient, as were isolates O19A and O19B. Fecal specimens were analyzed for V. cholerae by enrichment for 6 h in alkaline peptone water (pH 8.6) and were then plated onto thiosulfate-citrate-bile saltsucrose agar (Difco, Detroit, Mich.). On the basis of standard biochemical reactions (24), all suspected V. cholerae isolates were tested by agglutination tests
* Corresponding author. Mailing address: Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Bu ¨lowsvej 13, 1870 Frederiksberg C, Denmark. Phone: 45-35-282720 Fax: 45-35-282757. E-mail:
[email protected]. † Present address: National Veterinary Laboratory, Aarhus, Denmark 1151
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TABLE 1. Plasmid profiles, ribotypes, and PFGE types of 50 clinical V. cholerae O1 strains isolated in Peru from 1991 to 1995 Strain
Date of isolation (day, mo, yr)
Serotype
Plasmid size (kb)
Ribotypea
PFGE typeb
CHO 467 CHO 474 CHO 471 CHO 468 CHO 239 BAB 2305 BAB 2303 BAB 2292 BAB 2307 BAB 2301 CHO 563 CHO 561 CHO 569 CHO 567 O19Ad O19Bd CHO 539 CHO 549 CHO 548 CHO 552 O70 O59 O54 O912 O51Ad O51Bd O58 O24 O61 O880 VIG 707 VIG 705 VIG 795 VIG 753 VIG 1490 VIG 1398 HMA 34 CIT 171 CIT 175 CIT 178 VIG 10917 VIG 10852 VIG 10989 HMA 159 ECC 1091 VIG 9562 VIG 9835 VIG 10179 VIG 10371 VIG 9834
26 Nov 1991 26 Nov 1991 26 Nov 1991 26 Nov 1991 11 June 1991 20 Nov 1991 20 Nov 1991 14 Nov 1991 21 Nov 1991 16 Nov 1991 6 Feb 1992 29 Jan 1992 8 Feb 1992 8 Feb 1992 12 Dec 1992 12 Dec 1992 15 Jan 1992 16 Jan 1992 16 Jan 1992 16 Jan 1992 18 Jan 1993 5 Jan 1993 5 Jan 1993 16 July 1993 5 Jan 1993 5 Jan 1993 5 Jan 1993 2 Jan 1993 7 Jan 1993 9 July 1993 24 Jan 1994 24 Jan 1994 24 Jan 1994 25 Jan 1994 4 Feb 1994 4 Feb 1994 15 Dec 1994 3 Feb 1994 3 Feb 1994 3 Feb 1994 24 Mar 1995 21 Mar 1995 28 Mar 1995 21 Mar 1995 24 Mar 1995 26 Jan 1995 8 Feb 1995 20 Feb 1995 28 Feb 1995 7 Feb 1995
Ogawa Ogawa Ogawa Ogawa Ogawa Inaba Inaba Inaba Inaba Inaba Ogawa Ogawa Ogawa Ogawa Ogawa Inaba Inaba Inaba Inaba Inaba Ogawa Ogawa Ogawa Ogawa Ogawa Inaba Inaba Inaba Inaba Inaba Ogawa Ogawa Ogawa Ogawa Ogawa Inaba Inaba Inaba Inaba Inaba Ogawa Ogawa Ogawa Ogawa Ogawa Inaba Inaba Inaba Inaba Inaba
—c — — — 48 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 37 — — 37 37 37 — — — — 37 — — 37 — —
R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R2 R2 R2 R1 R1 R1 R1 R1 R1 R1 R1 R1 R3 R1 R4 R1 R1 R1 R1 R1 R3 R3 R3 R3 R3 R1 R1 R1 R1 R1
P1 P1 P1 P1 P1 P1 P1 P1 P1 P1 P1 P2 P1 P1 P2 P1 P3 P3 P3 P3 P5 P6 P6 P1 P3 P3 P3 P3 P3 P4 P2 P2 P2 P2 P7 P1 P3 P1 P1 P1 P2 P2 P2 P2 P8 P4 P3 P4 P4 P3
a
Ribotype was established by using the enzyme BglI. PFGE type was established by using the enzyme NotI. —, no plasmids were found. d Isolates O51A and O51B were recovered from the same patient, as were isolates O19A and O19B. b c
with polyvalent O1, monospecific Ogawa and Inaba, and O139 polyclonal goat antisera (Naval Medical Research Institute, Bethesda, Md.). Antibiotic susceptibility testing. The 50 V. cholerae O1 isolates were tested for their susceptibilities to nine antibacterial agents by the disk diffusion method recommended by the National Committee for Clinical Laboratory Standards (18) with disks (Sensi-disc; BBL, Becton Dickinson, Cockeysville, Md.) containing cephalothin at 30 mg/disk, chloramphenicol at 30 mg/disk, doxycycline at 30 mg/disk, furazolidone at 100 mg/disk, gentamicin at 10 mg/disk, kanamycin at 30 mg/disk, streptomycin at 10 mg/disk, sulfisoxazole at 250 mg/disk, and tetracycline at 30 mg/disk. In addition, the O1 isolates were tested for their susceptibilities to
a low level of tetracycline by using disks containing 10 mg/disk and following the instructions of the disk manufacturer (Neo-Sensitabs; Rosco, Taastrup, Denmark). Isolates were recorded as either sensitive or resistant. Isolation of plasmid DNA. Plasmid preparation was carried out by the method of Kado and Liu (11), modified by incubating the cells at elevated pH (pH 12.75) for 30 min at 568C during the lysis step. Following electrophoresis, the plasmids were visualized essentially as described previously (19). V. cholerae O1 V1075/25 containing an approximately 150-kb plasmid was used as the control strain (27). Plasmid sizes were estimated from the migration distance in the agarose gels relative to the migration distance of reference plasmids in Escherichia coli V517 and 39R861 (12, 29) by the method of Rochelle et al. (23). Repeated extraction of plasmid DNA was carried out for all isolates. Ribotyping. Total bacterial DNA was extracted by the method of Murray and Thompson (17). On the basis of previous studies (6, 7, 21), the restriction enzyme BglI was used to digest chromosomal DNA. Ribotyping was performed by the procedure described by Dalsgaard et al. (7) with digoxigenin-labeled 16S and 23S rRNA probes. A 1-kb DNA molecular size standard (GIBCO BRL, Gaithersburg, Md.) was used as a size marker. Each isolate was ribotyped at least twice. Ribotype patterns were considered to be different when there was a difference of one or more bands between isolates. Each ribotype was designated R followed by an arbitrary number. CT genotyping. Restriction fragment length polymorphism analysis of DNA sequences encoding CT genes was performed by hybridization of nylon membranes with BglI-digested DNA with a digoxigenin-labeled probe consisting of a 950-bp fragment of an XbaI-HincII digest of plasmid DNA isolated from E. coli MS 371(pJM17) (20). The hybridization procedures and detection of the probe were performed as described by the manufacturer (Boehringer Mannheim). PFGE. Strains were grown as described previously (26), and DNA was prepared directly in a solid agarose plug (Bio-Rad, Hercules, Calif.) as described by Cameron et al. (1). For restriction endonuclease digestion, thin slices were cut off the agarose plugs, equilibrated in the appropriate nuclease buffer for 1 h, and then digested for 4 h with 20 U of enzyme per plug (8). On the basis of previous PFGE studies of V. cholerae O1, the enzyme NotI (Amersham, Arlington Heights, Ill.) was selected for restriction analysis, allowing for comparison with previously published PFGE types (1, 9, 16). PFGE types were considered to be different if they differed by one or more bands and were designated P followed by an arbitrary number. The samples were loaded as plugs into the wells of 1.0% agarose (Litex LSL 4000; FMC A/S, Vallensbaek Strand, Denmark) gels prepared in 0.53 TBE buffer (103 TBE buffer is 89 mM Tris base, 90 mM boric acid, and 2.5 mM disodium EDTA [pH 8.3]). PFGE was carried out by using a modified contourclamped homogeneous electric field system (3) (Pulsaphor Plus; Pharmacia LKB, Uppsala, Sweden). The running conditions were 12 V/cm at 148C for 22 h. The pulse times were increased as follows: 5 s for 3 h, 9 s for 5 h, 12 s for 5 h, 20 s for 4 h, 25 s for 3 h, and 30 s for 2 h. Multimeric phage lambda (48.5 kb) DNA (Pharmacia LKB) was used as molecular mass standard. Following electrophoresis, the gels were stained for 15 min in ethidium bromide (2 mg/ml in water; Sigma), destained in distilled water for 15 min, and visualized on a UV light box.
RESULTS Specimens and antibiotic susceptibility patterns. Each of the 50 isolates included in the present study all showed biochemical and serological reactions typical of those of V. cholerae and were identified as V. cholerae O1 biotype El Tor and serotype Inaba or Ogawa (Table 1). Most isolates were found to be susceptible to the antibiotics tested, including low-dose tetracycline; exceptions were strains CHO 471, BAB 2292, and V. cholerae strains isolated in 1994 and 1995, which were resistant to kanamycin. Strain VIG 1398 was the only isolate showing resistance to furazolidone and sulfisoxazole. Plasmid analysis. Plasmid analysis revealed that 7 of 50 (14%) strains carried plasmids, including 6 strains that contained a 37-kb plasmid and 1 strain (strain CHO 239) that carried a 48-kb plasmid (Fig. 1; Table 1). Among the 30 V. cholerae strains isolated from 1991 to 1993, plasmids were found only in strain CHO 239, whereas 6 of 20 strains isolated from 1994 to 1995 carried the 37-kb plasmid. Strain CHO 239 was among the first V. cholerae O1 serotype Ogawa strains isolated in Lima. The presence of plasmids did not appear to correlate with antibiotic susceptibility, because isolates with or without plasmids showed similar susceptibility patterns. The control strain showed a plasmid of approximately 150 kb (Fig. 1).
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FIG. 1. Examples of plasmid profiles of V. cholerae O1 isolated in Peru. Lanes: A, E. coli 39R 861 (four plasmids ranging from 147 to 6.9 kb); B, E. coli V517 (eight plasmids ranging from 54 to 2.0 kb); C, strain V1075/25 (control strain); D, strain CHO 239; E, strain VIG 10179.
Ribotyping. Altogether, four BglI ribotypes were observed among the 50 V. cholerae O1 isolates studied (Table 1). An example of the ribosomal banding patterns is presented in Fig. 2. The chromosomal fragments containing rRNA genes ranged in size from 2 to 10 kb. All strains except strain VIG 1490 contained common fragments of 2.3, 4.1, 4.6, 5.9, 6.1, 6.2, 6.7, 7.0, and 9.6 kb and appeared to be closely related. Variations in ribotype patterns were shown by serotype Ogawa strains only. All strains isolated in 1991 and 1992 showed an identical ribotype, ribotype R1. Ribotype R2 was shown only by three Ogawa strains isolated from the same location in the northern part of Lima in 1993. Strain VIG 795 isolated in south Lima in 1994 showed ribotype R3, which demonstrated a 5.6-kb fragment not seen among the other ribotypes. In addition, each of the five Ogawa strains isolated in south Lima in 1995 was ribotype R3. Strain VIG 1490 was isolated in 1994 and showed a unique ribotype, ribotype R4, which differed from the other ribotypes by four to five fragments. R2 isolates showed a 4.3-kb fragment not seen among isolates of the R1, R3, and R4 ribotypes and, furthermore, lacked a 4.0-kb fragment demonstrated by isolates of the other ribotypes (Fig. 2). All V. cholerae serotype Inaba strains belonged to ribotype R1. Repeated studies of all strains showed no variation in ribotype patterns, although differences in band intensity and the degree of background were observed for a few strains. CT genotypes. Southern blot hybridization of BglI-digested genomic DNA with the CT probe revealed an identical pattern for all strains which showed three fragments of approximately 15, 9, and 7 kb. However, variations in the band intensity of the 9- and 7-kb fragments were found for the majority of strains. PFGE. PFGE analysis with the restriction enzyme NotI gave a suitable distribution of fragments, with about 20 fragments being .50 kb. The analysis of 50 V. cholerae O1 isolates showed that the isolates displayed eight different banding patterns (Fig. 3; Table 1). With the exception of strain VIG 1490, which showed a unique type (type P7) differing from the other PFGE types by more than seven fragments, all isolates appeared to be closely related. Strain VIG 1490 was recovered on 4 February 1994 from a 10-year-old boy with diarrhea. The isolate showed an unique ribotype (ribotype R4) and appears
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to represent a distinct O1 strain compared with the strain causing the present epidemic (28). The mother of the 10-yearold boy was also hospitalized for cholera. However, an isolate recovered from the mother’s stool sample showed PFGE type P2 (data not shown). Several different PFGE types were shown among V. cholerae strains isolated from 1991 to 1995. All isolates from 1991 showed an identical PFGE type (type P1). PFGE type P2, which differed from type P1 by having a 400-kb fragment instead of a 375-kb fragment, was represented by Ogawa isolates only and appeared for the first time in 1992. PFGE type P3 lacked the 175-kb fragment shown by type P1 and P2 isolates and was shown for the first time in 1992 by four serotype Inaba isolates. With the exception of isolate O51A, which agglutinated with serotype Ogawa antiserum, all type P3 isolates were of the Inaba serotype. One and three serotype Inaba isolates recovered in 1993 and 1995, respectively, were type P4. Most PFGE type P1, P2, P3, and P4 isolates belonged to ribotype R1, but four type P2 isolates recovered in 1995 belonged to ribotype R3. One and two serotype Ogawa isolates recovered in 1993 were types P5 and P6, respectively. PFGE types P5 and P6 had a 150-kb fragment not seen among the other types, and each of the three isolates was ribotype R2. PFGE type P8, which had an unique 80-kb fragment, was shown by a single isolate, isolate ECC 1091, recovered in 1995. No isolate recovered in 1995 was PFGE type P1. A comparison of the typing results indicated that PFGE typing and ribotyping were in general agreement, with the four ribotypes being further differentiated by PFGE. However, isolates within ribotypes R1 and R3 showed an identical PFGE type (type P2). From 1992, an association appeared between serotype and PFGE types because serotype Ogawa isolates showed PFGE types P1, P2, P5, P6, P7, and P8, whereas serotype Inaba isolates showed types P3 and P4. However, excep-
FIG. 2. Examples of BglI ribotypes of 50 V. cholerae O1 strains isolated in Lima, Peru, from 1991 to 1995. Unless indicated otherwise, the following explanations for the contents of the lanes indicate ribotype, strain designation, and year of isolation. Lanes: a, 1-kb molecular mass standard; b, ribotype R1, CHO 467, 1991; c, type R1, CHO 239, 1991; d, type R1, BAB 2307, 1991; e, type R2, O70, 1993; f, type R2, O59, 1993; g, type R2, O54, 1993; h, type R1, CHO 569, 1992; i, type R1, O19A, 1992; j, type R1, CHO 539, 1992; k, type R1, 912, 1993; l, type R1, O51A, 1993; m, type R1, O24, 1993; n, type R1, VIG 1398, 1994; o, type R1, CIT 175, 1994; p, type R1, VIG 9562, 1995; q, type R3, VIG 795, 1994; r, type R1, VIG 10371, 1995; s, type R4, VIG 1490, 1994; t, 1-kb molecular mass standard.
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among isolates, as shown by one-dimensional electrophoresis. When extracts of plasmids of the two sizes were subjected to PFGE, fragments were located close to the 194-kb fragments of the phage lambda DNA size marker (data not shown). However, since none of the eight different PFGE types had fragments with similar sizes, the presence of plasmids did not influence the interpretation and stability of PFGE typing. DISCUSSION
FIG. 3. PFGE banding patterns of NotI-digested total cellular DNAs from representative V. cholerae strains isolated in Lima, Peru, from 1991 to 1995. Unless indicated otherwise, the following explanations of the contents of the lanes indicate PFGE type, strain designation, and year of isolation. Lanes: A, multimers of phage lambda DNA (48.5 kb) as molecular mass markers; B, PFGE type P1, CHO 467, 1991; C, type P2, CHO 561, 1992; D, type P3, CHO 539, 1992; E, type P4, VIG 9562, 1995; F, type P8, ECC 1091, 1995; G, type P5, O70, 1993; H, type P6, O59, 1993; I, type P7, VIG 1490, 1994; J, multimers of phage lambda DNA (48.5 kb) as molecular mass markers.
tions were found because PFGE type P1 was also demonstrated by serotype Inaba isolates from 1994 and isolate O19B. V. cholerae O1 isolate O51A serotype Ogawa and serotype Inaba isolate O51B were isolated from the same patient and showed an identical PFGE type (type P3). Repeated agglutination tests and PFGE typing confirmed the serotypes and the PFGE types of the two isolates. By using the PC/GENE, version 6.5, software (IntelliGenetics, Mountain View, Calif.), no restriction sites were identified in the rfbT gene for the enzyme NotI used for PFGE typing. The rfbT operon is responsible for serotype conversion in V. cholerae O1, and the rfbT sequences for the Inaba and Ogawa serotypes have been deposited in GenBank (13). With the absence of restriction sites for NotI within the rfbT operon of the two serotypes, identical PFGE types may occur. On the basis of the identical characteristics of strains O51A and O51B, a serotype conversion may have occurred within the patient during the course of infection. V. cholerae O1 strains O19A serotype Ogawa and O19B serotype Inaba were isolated from the same stool samples but showed PFGE types P2 and P1, respectively. Digested plasmids migrate according to the sizes of the fragments in PFGE gels, whereas uncut plasmids have been reported to have aberrant mobilities (15, 22). In the present study plasmids of approximately 37 and 48 kb were found
A total of four BglI ribotypes and eight NotI PFGE types were found among 50 clinical V. cholerae O1 strains isolated in Lima, Peru, from 1991 to 1995. These results indicate that genetic changes are occurring in the strain responsible for the Latin American cholera epidemic more frequently than has previously been reported (1, 9). While differences in ribotype and PFGE type patterns occurred, 40 (80%) O1 strains showed an identical ribotype pattern (type 1) and 41 (82%) strains showed closely related PFGE types (type 1, 2, or 3) that differed by less than three restriction fragments. Furthermore, genotyping with a CT probe revealed an identical pattern for all strains. The small differences in ribotypes and PFGE types are consistent with relatively minor genetic variations within a largely clonal pool from which the epidemic strain arises every year. This is remarkable because cholera is highly seasonal in Lima and other coastal areas of Peru. Most cases occur in the summer months of January to March. Cholera does not occur at all on the coast from June until December. Despite this 6-month hiatus, our data indicate that the nearly identical cholera strain reemerges the next year. Our data support the hypothesis that the epidemic strain reemerges from an environmental source. In 1991, more than 95% of V. cholerae O1 biotype El Tor isolates recovered from patients in the capital, Lima, and the second largest city, Trujillo, were serotype Inaba, whereas from 1992 serotype serotype Ogawa began to predominate, with more than 90% of the isolates being of the Ogawa serotype in 1995 (30). A similar change in serotype was reported for isolates from most other Latin American countries (9). The closely related ribotype patterns and PFGE types found in the present study demonstrate that the two serotypes are derived from the same clone of V. cholerae O1. Among isolates from Lima, certain ribotype and PFGE types appeared to cluster geographically. For example, isolates of ribotype 2 and PFGE types P5 and P6 were only isolated in north Lima. The strains from each year were found at locations where we were conducting surveillance for cholera. Thus, strains from any one year represent clusters of cholera cases or small outbreaks associated with a common location and date of isolation. Our data indicate that within one of these clusters there is marked genetic similarity of V. cholerae O1. However, there were some notable variations. Strain VIG 1490 isolated in 1994 from a boy represents a distinct PFGE type that occurred at the same time that his mother was infected with a standard PFGE type strain. It is possible that strain VIG 1490 underwent genetic variation after being passed through one individual. Similarly, there were two instances (strains O19A and O19B and strains O53A and O53B) in which Ogawa and Inaba strains isolated from the same stool specimen showed nearly identical typing patterns. These data also provide evidence for point mutations in the rfbT operon within the epidemic O1 clone. Our data provide further support for the clonality of the South American cholera strains. PFGE types P1 and P2 from this study appear to be identical to PFGE types 38 and 66, respectively, described by Evins et al. (9) among clinical V.
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cholerae strains isolated from several South American countries from 1991 to 1993. An apparently identical PFGE type (type 38) was shown by O1 strains isolated from seafood and nonpotable water (ballast, bilge, and sewage) (9). Our ribotype R1 pattern shown by all O1 strains isolated in 1991 and 1992 appears to be identical to ribotype 5 of Popovic et al. (21). However, our ribotypes R2, R3, and R4 have not been described previously among South American O1 strains (9, 21). Although more than half of all cholera cases reported in Latin America have been reported in Peru, significant antimicrobial resistance has not occurred (2). In the present study, the majority of V. cholerae strains were susceptible to all antibiotics tested; the exceptions were O1 strains isolated in 1994 and 1995, which were resistant to kanamycin. However, a high prevalence of multiple-antibiotic-resistant V. cholerae O1 strains was found in Ecuador in 1991 (33). In addition, antibiotic resistance to furazolidone, sulfisoxazole, and streptomycin was found among V. cholerae O1 strains in Mexico, Guatemala, and Brazil (9, 21). This resistance pattern was associated with a specific ribotype pattern 6a which we did not find among isolates in Peru (21). These data indicate that antibiotic-resistant V. cholerae O1 strains are involved in the Latin American cholera epidemic and that some O1 strains may develop antimicrobial resistance at a higher rate than others. The spread of antibiotic-resistant O1 strains should be monitored. Although an increase in the number of strains containing plasmids was registered from 1991 to 1995, plasmids did not appear to encode antibiotic resistance. However, a study carried out in Ecuador in 1991 demonstrated a 100-MDa plasmid in four multiple-antibiotic-resistant V. cholerae O1 strains studied (33). The importance of plasmid analysis in the characterization of Latin American V. cholerae O1 isolates remains to be determined. For future comparisons of ribotypes, we suggest that the method originally described by Popovic et al. (21) be used and that ribotype figures of high photographic quality be included in any reports. Several studies have shown the usefulness of the restriction enzyme NotI in PFGE typing of V. cholerae O1 isolates (1, 9, 16). We therefore suggest that NotI be used in PFGE typing of V. cholerae O1 isolates in the future, although the enzyme ApaI was able to differentiate two clinical O1 strains which were indistinguishable by NotI PFGE typing (16). Computer-assisted numerical analysis, including scanning of ribotyping membranes and PFGE gel patterns, is increasingly being used for quantitative assessment of genetic similarities and differences among V. cholerae O1 strains (10). Such assessments look promising, although the typing methods need further standardization because the ribotyping membranes and gels used for analysis are too often of poor quality. In the future, certain laboratories may establish databases of bacterial typing patterns which scientists could access through the Internet for comparison analysis. The results of our study indicate the usefulness of ribotyping and especially PFGE for studying genetic changes within the V. cholerae O1 strain responsible for the epidemic in Latin America. The higher number of different genotypes shown in the present study compared with those shown in previous studies (9, 21) should be taken into consideration when the typing patterns of V. cholerae O1 isolates associated with cholera in Latin America are evaluated. ACKNOWLEDGMENTS We are grateful for the technical assistance provided by Anita Forslund and Marianne Jacobsen at the Royal Veterinary and Agricultural University in Denmark and by Maruja Bernal at the Naval Medical Research Institute Detachment in Peru.
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