INTRODUCTION

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In the past, the prevalence of antibiotic resistance of Vibrio cholerae was low and routine susceptibility testing was not recommended (29, 35). However, reports of V. cholerae strains resistant to commonly used antibiotics are appearing with increasing frequency and susceptibility testing is now recommended for monitoring of resistance among toxigenic V. cholerae O1 and O139, the two serotypes which cause cholera (4, 20, 23, 29, 54).

The importance of non-O1, non-O139 serotypes of V. cholerae as causes of diarrhea are increasingly being recognized (13, 14, 18, 33, 41). Although high prevalences of multiple-antibiotic-resistant non-O1, non-O139 strains have been reported recently, little is known about the mechanisms of resistance (14, 33, 41). Studies of non-O1, non-O139 strains from Thailand showed that strains frequently contained small-size plasmids. However, the plasmids did not appear to encode antibiotic resistance (14, 18).

Antibiotic resistance genes may be acquired and transmitted through several mechanisms, of which the acquisition of genes through mobile genetic elements and dissemination through horizontal transfer are of special interest. In addition to plasmids and conjugative transposons, integrons have also been described as vehicles for the acquisition of resistance genes (24, 44; for reviews, refer to references 25, 38, and 46). Among the three classes of integrons that have been identified, class 1 integrons are prevalent among clinical isolates. Class 1 integrons were originally defined as being composed of two conserved segments, the 5' conserved segment (5'-CS) and the 3' conserved segment (3'-CS), and an internal variable region which contains gene cassettes encoding antibiotic resistance determinants (24, 44). The 5'-CS contains the intI1 gene, which encodes the type 1 integrase. This integrase is responsible for site-specific insertion and excision of gene cassettes (11). Also, the 5'-CS contains the attI1 site, which is responsible for recombination. The 3'-CS contains the qacE1 and sul1 genes, which encode resistance to quaternary ammonium compounds and to sulfonamides, respectively. However, class 1 integrons do not always contain the entire 3'-CS (38, 46).

Some information is available about the distribution and importance of class 1 integrons in encoding antibiotic resistance in bacterial enteropathogens, and recent studies indicate that class 1 integrons may be widespread in multiple-drug-resistant clinical isolates (26, 27, 30), e.g., Salmonella enterica Typhimurium (40, 52). Dalsgaard et al. (15) characterized V. cholerae O1 isolated in Vietnam from 1979 to 1996 and found that strains isolated after 1990 were resistant to sulfonamides and streptomycin and harbored class 1 integrons containing an aadA2 gene cassette. A comparison of phenotypic and genotypic characteristics of the Vietnamese O1 strains isolated after 1990 with a distinct O1 strain recently found in Samutsakorn, Thailand, suggests that they belong to the same clone (15, 17). However, it is unknown if the Thai V. cholerae O1 strain also contains class 1 integrons (17). Recently, Falbo et al. (21) found chromosomally located class 1 integrons containing the aadA1 gene cassette among V. cholerae O1 strains isolated during a cholera outbreak in Albania and Italy in 1994.

In this background, the objective of the present study was to determine the distribution and importance of class 1 integrons in encoding antibiotic resistance among a large collection of different V. cholerae O serotypes isolated from clinical and environmental sources in Thailand. As the vast majority of strains containing class 1 integrons are resistant to sulfonamides, we selected strains with different patterns of susceptibility to sulfonamides and tested them for the presence of class 1 integrons. Our study showed that integrons were present in V. cholerae O1 and non-O1, non-O139 serotypes of mainly clinical origin, containing gene cassettes encoding resistance to aminoglycosides, -lactams, and trimethoprim.

	MATERIALS AND METHODS

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Bacterial strains. A total of 176 V. cholerae strains, mainly isolated in Thailand, were tested for the presence and contents of class 1 integrons. The strain collection was selected from a large number of V. cholerae strains studied previously for various phenotypic and genotypic characteristics (5, 14, 16-19). The majority of strains were sulfonamide resistant, but strains showing intermediate resistance or susceptibility to sulfonamides were also studied. Further strain information is provided in Results.

Transfer of resistance and characterization of plasmids. The nalidixic acid-resistant mutant Escherichia coli K-12 strain J53-1 (lac⁺ pro met Nal^r) (50) was used as the recipient in conjugation experiments with the plasmid-containing strains 1075/25, 1076/25, and 64/26 (49). After mating on nonselective L agar incubated at 37°C for 4 to 6 h of growth, exconjugants were harvested and appropriate dilutions were spread on plates of Fluorocult E. coli O157:H7 agar (Merck, Virum, Denmark) supplemented with nalidixic acid at 50 µg/ml and adequate concentrations of selected drugs. Phenotypic appearance was used to differentiate possible spontaneous nalidixic acid-resistant donors from the exconjugants.

PCR amplification and sequencing of integrons. Figure 1 shows the general structure of class 1 integrons. For PCR amplification, we initially selected a specific class 1 primer set, qacE1 and sul1, directed at the 3'-CS of class 1 integrons (37, 38). However, as a few class 1 integrons do not contain the qacE1 and sul1 genes, these would not be detected using the selected primers. PCR was carried out by suspension of one bacterial colony in 100 µl of sterile distilled H₂O, which was then boiled for 15 min at 95°C. After lysis, the suspension was centrifuged at 13,000 rpm (Biofuge 13; Heraeus Sepatech) for 2 min and stored at 20°C. For each PCR, 5 µl of the lysis suspension was used. The PCR amplification was performed by the method of Aarestrup et al. (1) and optimized to detect amplicons in the range of 300 to 1,500 bp. The annealing temperature was set at 58°C. Strains yielding a PCR product with the class 1 primers were further amplified with the integron primers in-F and in-B (F is forward, and B is backward), which amplify the region between the 5'-CS and the 3'-CS, yielding products of various sizes, depending on the number and length of the inserted gene cassettes (Fig. 1 and Table 1). The in-B primer anneals at the 3'-CS. Primers in-F and addA-B were used to determine if the integron contained a gene cassette encoding resistance to streptomycin and spectinomycin (Table 1) (31). Primers intI1-F and blaP1-B were used to determine if the integron contained the -lactam resistance gene cassette using an annealing temperature of 52°C (55). All of the PCR primers used are listed in Table 1. S. enterica serotype Typhimurium strain 9720921 and Acinetobacter sp. strain R4-96 were included as positive and negative controls, respectively (40). A 100-bp molecular mass standard (GIBCO BRL, Gaithersburg, Md.) was used as a size marker during electrophoresis of PCR products.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Association between integron structure and PCR products (modified from reference 40). The lines below the integron structure represent amplicons, and the bold line represents the sequenced amplicons. The primers above the PCR products are described in Table 1. attI1 is the attenuation site, and qacE1 and sul1 encode resistance to disinfectants and sulfonamides, respectively. The individual gene cassettes are drawn to scale. The recombination site (59-bp element) is shown as a dark circle.

Nucleotide sequence accession numbers. The nucleotide sequences of the aadA2, dfrA1 and orf, dfrA15, aadB, and blaP1 gene cassettes have been assigned GenBank accession no. AF221903, AF221901, AF221900, AF221902, and AF221899, respectively.

	RESULTS

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An approximately 800-bp 3'-CS PCR product was obtained from 44 of the 176 V. cholerae strains studied by using the primers qacE1-F and sul1-B. However, the majority of epidemiologically unrelated clinical strains which showed resistance to sulfonamides did not, based on lack of PCR amplification, contain the sul1 gene. Only a single environmental strain amplified this region. The phenotypic characteristics and resistance gene cassettes found in strains containing class 1 integrons are further described below (see also Tables 2 and 3).

V. cholerae non-O1, non-O139 strains isolated at the Children Hospital, Bangkok. Sixty-nine V. cholerae non-O1, non-O139 strains recovered from children as part of a cholera surveillance conducted from August 1993 to July 1995 at the Children Hospital in Bangkok were studied (14). These strains represented 37 different O serotypes, of which none contained the cholera toxin gene. The strains were originally tested for susceptibility to 12 antibiotics by disk diffusion including ampicillin, chloramphenicol, ciprofloxacin, colistin, gentamicin, kanamycin, nalidixic acid, neomycin, streptomycin, sulfisoxazole, tetracycline, and trimethoprim-sulfamethoxazole (14). Of 40 strains that were resistant to colistin, streptomycin, and sulfisoxazole, 28 showed further resistance and were designated multiply resistant (14).

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Examples of PCR products of V. cholerae O1 strains isolated in Thailand by using the primers in-F and in-B (lanes b to i), in-F and aadA-B (lanes j and k), in-F and blaP1-B (lanes l and m), and qacE1-F and sul1-B (lanes n and o). Lanes: a, 100-bp molecular mass standard; b, strain VO-3417; c, strain 1075/25; d, strain 292/90; e, strain VO-258; f, strain 30/90; g, strain VO-3198; h, S. enterica serotype Typhimurium strain 9720921 (positive control); i, Acinetobacter sp. strain R4-96 (negative control); j, strain 1076/25; k, strain 9720921 (positive control); l, strain SKF-09; m, strain 9720921 (positive control); n, strain VO-557; o, strain 9720921 (positive control); p, PCR mixture, (negative control); q, 100-bp molecular mass standard.

V. cholerae non-O1, non-O139 strains recovered from patients in Thailand. Nineteen V. cholerae non-O1, non-O139 strains were selected from 41 strains recovered from stool samples from patients with diarrhea (18), including 13 strains isolated in 1990 during an epidemic of a cholera-like disease among Khmer refugees in a camp in Aranyaprathet, Thailand (5).

V. cholerae O1 strains recovered from patients in Samutsakorn, Thailand. Thirty-one clinical V. cholerae O1 strains were selected from 70 strains isolated from 1982 to 1996 in Samutsakorn, a port city 30 km southwest of Bangkok (17). We showed in a previous study that the disappearance of V. cholerae O139 from Samutsakorn was associated with distinct phenotypic and genotypic changes in O1 strains isolated during and after the O139 epidemic, including the development of resistance to streptomycin and sulfisoxazole (17).

Clinical V. cholerae O139 strains and environmental non-O1, non-O139 strains isolated from shrimp aquaculture and seafood. Class 1 integrons were not found among 11 representative V. cholerae O139 strains tested which showed resistance to furazolidone, streptomycin, trimethoprim, and sulfonamides and included strains isolated from patients in Bangladesh, India, and Thailand from 1992 to 1993 (19). Thus, the antibiotic resistances shown by the O139 strains were not associated with this type of integron.

	DISCUSSION

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In the present study, we showed that class 1 integrons containing antibiotic resistance gene cassettes were found mainly in epidemiologically related V. cholerae O serotypes isolated from outbreaks of diarrhea in Thailand. However, several epidemiologically unrelated V. cholerae non-O1, non-O139 strains isolated mainly from children with diarrhea also contained class 1 integrons. Obtained by PCR and DNA sequencing, the findings of aadB, aadA2, blaP1, dfrA1, and dfrA15 resistance gene cassettes encoding resistances to gentamicin, kanamycin, and tobramycin; streptomycin and spectinomycin; -lactams; and trimethoprim, respectively, represent the first report of the distribution of class 1 integrons among different V. cholerae O serotypes. The gene cassettes reported here corroborate previous findings of the content of class 1 integrons in Enterobactericeae and Pseudomonas spp., in which antibiotic resistance gene cassettes often encode resistances to aminoglycosides (32, 39, 42), -lactams (40, 45), and trimethoprim (2, 48).

It should be noted that the majority of resistance genes, including determinants of resistance to ampicillin, chloramphenicol, neomycin, streptomycin, trimethoprim, and tetracycline, were not contained within class 1 integrons. Thus, although widely distributed, resistance genes on class 1 integrons seem to encode only a part of the antibiotic resistance reported in V. cholerae. As our PCR analysis was designed to detect relatively smaller-size amplicons, larger arrays of gene cassettes may have been missed. However, it should be noted that all of the strains yielding an amplicon with the primers qacE1-F and sul1-B also showed a product when the in-F and in-B primers were used. Thus, it seems unlikely that larger arrays of gene cassettes were present.

There was a clear difference in the distribution and frequency of class 1 integrons among clinical and environmental V. cholerae non-O1, non-O139 strains, as only a single environmental strain recovered from seafood in Samutsakorn contained a class 1 integron. The non-O1, non-O139 strains from the shrimp production area in Thailand were isolated from ponds and coastal areas (16) and showed less antibiotic resistance than the clinical non-O1, non-O139 strains (unpublished results). It is likely that the higher frequency of class 1 integrons in clinical isolates is because such strains became dominant through the selective pressure caused by the therapeutic use of antibiotics.

Bagchi et al. (5) reported that during the cholera-like epidemic among the Khmers in 1982, children and pregnant women were treated with trimethoprim-sulfamethoxazole. Further, Dalsgaard et al. (18) showed that a high percentage (92%) of the non-O1, non-O139 Khmer outbreak strains showed resistance to trimethoprim-sulfamethoxazole. Thus, it is likely that the strains that acquired the dfrA1 gene cassette became predominant through selective pressure. The finding that non-O1, non-O139 strains showing resistance to several antibiotics also contained resistance gene cassettes located on integrons is of concern. The finding of trimethoprim resistance gene cassettes, including the recently described dfrA15 gene cassette (2), is of special concern, as trimethoprim is often used to treat diarrhea among children and pregnant women.

The appearance of the O139 serotype in Samutsakorn in 1993 and its sudden disappearance in 1994 were associated with the emergence of a sulfonamide-resistant V. cholerae O1 strain carrying a class 1 integron which contained the aadA2 resistance gene cassette. We reported earlier that this distinct O1 strain showed unique genotypes, as demonstrated by ribotyping, PFGE typing, and cholera toxin typing (17). Interestingly, Dalsgaard et al. (15) found that O1 strains isolated in Vietnam after 1990 also contained class 1 integrons which harbored the aadA2 gene cassette and showed a ribotype R1 identical to the ribotype seen among O1 strains isolated in Samutsakorn (17). Thus, it is likely that this distinct O1 strain was transferred between Thailand and Vietnam and became established as the main strain causing cholera. It remains to be determined how this strain acquired the class 1 integron and the aadA2 gene cassette and/or if the strain was introduced from a third country.

Although the class 1 integrons were first described by Stokes and Hall in 1989 (44), our findings of the aadB resistance gene among the three O1 strains isolated in 1982 from the cholera outbreak at the pediatric ward in Samutsakorn show that class 1 integrons have been present in V. cholerae in Thailand for a number of years (49). The demonstration of the class 1 integron's being located on a 150-kb self-transmissible plasmid is, to our knowledge, the first report of a plasmid containing integrons in V. cholerae. Tabtieng et al. (49) reported that the 150-kb plasmid in each of the three strains belonged to incompatibility group C and contained genes coding for type II dihydrofolate reductase. The genes encoding the type II dihydrofolate reductase were not found as a class 1 gene cassette. However, after 1982, this plasmid and other plasmids were not found in O1 strains isolated in Samutsakorn or elsewhere, suggesting that the 150-kb plasmid containing the class 1 integrons was lost (17). Other studies have reported multiple-drug-resistant V. cholerae O1 encoded by conjugative incompatibility group C plasmids (51). However, it remains to be shown if these or other plasmids in V. cholerae contain class 1 integrons.

Interestingly, class 1 integrons were not found among any V. cholerae O139 strains. Thus, the 62-kb self-transmissible transposon-like SXT element described by Waldor et al. (53) among O139 strains, some of which were included in the present study, does not seem to carry class 1 integrons. The V. cholerae O1 El Tor strain which re-emerged in India and Bangladesh after the O139 epidemic were resistant to sulfamethoxazole, trimethoprim, and streptomycin (4, 34). Waldor et al. (53) showed that these O1 strains also contained the SXT element. It remains to be determined if the O1 strains from India and Bangladesh contain class 1 integrons.

Our findings show that class 1 integrons containing several different antibiotic resistance gene cassettes were distributed among different clinical V. cholerae O1 and non-O1, non-O139 serotypes in Thailand. Thus, PCR mapping of integrons and DNA sequencing of their genetic contents may be a useful epidemiological tool with which to study the evolution of multiresistance plasmids and dissemination of antibiotic resistance genes within V. cholerae. Although the resistance genes within the integrons encoded only a minor part of the antibiotic resistance detected, further studies in Thailand and elsewhere are needed to determine the importance of class 1 integrons in the horizontal acquisition and dissemination of antibiotic resistance genes.