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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,0066-4804/00/$04.000

May 2000, p. 13151321 Vol. 44, No. 5

Copyright 2000, American Society for Microbiology. All Rights Reserved.

Distribution and Content of Class 1 Integrons in DifferentVibrio cholerae O-Serotype Strains Isolated in Thailand

ANDERS DALSGAARD,1* ANITA FORSLUND,1 ORALAK SERICHANTALERGS,2

AND DORTHE SANDVANG1,3

Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Frederiksberg,DK-1870 Frederiksberg C,1 and Danish Veterinary Laboratory, DK-1790 Copenhagen V,3 and

Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand2

Received 1 June 1999/Returned for modification 26 July 1999/Accepted 8 February 2000

In this study, 176 clinical and environmental Vibrio cholerae strains of different O serotypes isolated inThailand from 1982 to 1995 were selected and studied for the presence of class 1 integrons, a new group ofgenetic elements which carry antibiotic resistance genes. Using PCR and DNA sequencing, we found that 44isolates contained class 1 integrons harboring the aadB, aadA2, blaP1, dfrA1, and dfrA15 gene cassettes, whichencode resistance to gentamicin, kanamycin, and tobramycin; streptomycin and spectinomycin; -lactams; andtrimethoprim, respectively. Each cassette array contained only a single antibiotic resistance gene. Althoughresistance genes in class 1 integrons were found in strains from the same epidemic, as well as in unrelated

non-O1, non-O139 strains isolated from children with diarrhea, they were found to encode only some of theantibiotic resistance expressed by the strains. Serotype O139 strains did not contain class 1 integrons.However, the appearance and disappearance of the O139 serotype in the coastal city Samutsakorn in 1992 and1993 were associated with the emergence of a distinct V. cholerae O1 strain which contained the aadA2resistance gene cassette. A 150-kb self-transmissible plasmid found in three O1 strains isolated in 1982contained the aadB gene cassette. Surprisingly, several strains harbored two integrons containing differentcassettes. Thus, class 1 integrons containing various resistance gene cassettes are distributed among differentV. cholerae O serotypes of mainly clinical origin in Thailand.

In the past, the prevalence of antibiotic resistance of Vibriocholerae was low and routine susceptibility testing was notrecommended (29, 35). However, reports ofV. cholerae strainsresistant to commonly used antibiotics are appearing with in-creasing frequency and susceptibility testing is now recom-

mended for monitoring of resistance among toxigenic V. chol-erae O1 and O139, the two serotypes which cause cholera (4,20, 23, 29, 54).

The importance of non-O1, non-O139 serotypes of V. chol-erae 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 re-ported recently, little is known about the mechanisms of resis-tance (14, 33, 41). Studies of non-O1, non-O139 strains fromThailand showed that strains frequently contained small-sizeplasmids. However, the plasmids did not appear to encodeantibiotic resistance (14, 18).

Antibiotic resistance genes may be acquired and transmittedthrough several mechanisms, of which the acquisition of genes

through mobile genetic elements and dissemination throughhorizontal transfer are of special interest. In addition to plas-mids and conjugative transposons, integrons have also beendescribed as vehicles for the acquisition of resistance genes(24, 44; for reviews, refer to references 25, 38, and 46). Amongthe three classes of integrons that have been identified, class 1integrons are prevalent among clinical isolates. Class 1 inte-grons were originally defined as being composed of two con-served segments, the 5 conserved segment (5-CS) and the 3conserved segment (3-CS), and an internal variable region

which contains gene cassettes encoding antibiotic resistancedeterminants (24, 44). The 5-CS contains the intI1 gene, whichencodes the type 1 integrase. This integrase is responsible forsite-specific insertion and excision of gene cassettes (11). Also,the 5-CS contains the attI1 site, which is responsible for re-combination. The 3-CS contains the qacE1 and sul1 genes,which encode resistance to quaternary ammonium compoundsand to sulfonamides, respectively. However, class 1 integronsdo not always contain the entire 3-CS (38, 46).

Some information is available about the distribution andimportance of class 1 integrons in encoding antibiotic resis-tance in bacterial enteropathogens, and recent studies indicatethat class 1 integrons may be widespread in multiple-drug-resistant clinical isolates (26, 27, 30), e.g., Salmonella entericaTyphimurium (40, 52). Dalsgaard et al. (15) characterized V.cholerae O1 isolated in Vietnam from 1979 to 1996 and foundthat strains isolated after 1990 were resistant to sulfonamidesand streptomycin and harbored class 1 integrons containing anaadA2 gene cassette. A comparison of phenotypic and geno-

typic characteristics of the Vietnamese O1 strains isolated after1990 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 alsocontains class 1 integrons (17). Recently, Falbo et al. (21)found chromosomally located class 1 integrons containing theaadA1 gene cassette among V. cholerae O1 strains isolatedduring a cholera outbreak in Albania and Italy in 1994.

In this background, the objective of the present study was todetermine the distribution and importance of class 1 integronsin encoding antibiotic resistance among a large collection ofdifferent V. cholerae O serotypes isolated from clinical andenvironmental sources in Thailand. As the vast majority ofstrains containing class 1 integrons are resistant to sulfon-amides, we selected strains with different patterns of suscepti-

* Corresponding author. Mailing address: Department of Veteri-nary Microbiology, The Royal Veterinary and Agricultural University,Bulowsvej 13, 1870 Frederiksberg C, Denmark. Phone: 45-35-282720.Fax: 45-35-282757. E-mail: [email protected].

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bility to sulfonamides and tested them for the presence of class1 integrons. Our study showed that integrons were present inV. cholerae O1 and non-O1, non-O139 serotypes of mainlyclinical origin, containing gene cassettes encoding resistance toaminoglycosides, -lactams, and trimethoprim.

MATERIALS AND METHODS

Bacterial strains. A total of 176 V. cholerae strains, mainly isolated in Thai-land, were tested for the presence and contents of class 1 integrons. The straincollection was selected from a large number of V. cholerae strains studied pre-viously for various phenotypic and genotypic characteristics (5, 14, 1619). Themajority of strains were sulfonamide resistant, but strains showing intermediateresistance or susceptibility to sulfonamides were also studied. Further straininformation 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 Nalr) (50) was

used as the recipient in conjugation experiments with the plasmid-containingstrains 1075/25, 1076/25, and 64/26 (49). After mating on nonselective L agarincubated at 37C for 4 to 6 h of growth, exconjugants were harvested andappropriate dilutions were spread on plates of Fluorocult E. coli O157:H7 agar(Merck, Virum, Denmark) supplemented with nalidixic acid at 50 g/ml andadequate concentrations of selected drugs. Phenotypic appearance was used todifferentiate possible spontaneous nalidixic acid-resistant donors from the ex-conjugants.

Plasmid extraction was carried out using the method of Kado and Liu (28), byincubating the cells at elevated pH (12.54) for 30 min at 56C during the lysisstep. Electrophoresis and visualization of plasmids were carried out essentially aspreviously described (36).

PCR amplification and sequencing of integrons. Figure 1 shows the generalstructure 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 1integrons (37, 38). However, as a few class 1 integrons do not contain the qacE1and sul1 genes, these would not be detected using the selected primers. PCR wascarried out by suspension of one bacterial colony in 100 l of sterile distilledH2O, which was then boiled for 15 min at 95C. After lysis, the suspension wascentrifuged at 13,000 rpm (Biofuge 13; Heraeus Sepatech) for 2 min and storedat 20C. For each PCR, 5 l of the lysis suspension was used. The PCRamplification was performed by the method of Aarestrup et al. (1) and optimizedto detect amplicons in the range of 300 to 1,500 bp. The annealing temperature

was set at 58C. Strains yielding a PCR product with the class 1 primers werefurther amplified with the integron primers in-F and in-B (F is forward, and B isbackward), which amplify the region between the 5-CS and the 3-CS, yieldingproducts of various sizes, depending on the number and length of the insertedgene 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 agene cassette encoding resistance to streptomycin and spectinomycin (Table 1)(31). Primers intI1-F and blaP1-B were used to determine if the integron con-tained the -lactam resistance gene cassette using an annealing temperature of52C (55). All of the PCR primers used are listed in Table 1. S. enterica serotypeTyphimurium strain 9720921 and Acinetobactersp. strain R4-96 were included aspositive and negative controls, respectively (40). A 100-bp molecular mass stan-dard (GIBCO BRL, Gaithersburg, Md.) was used as a size marker duringelectrophoresis of PCR products.

Amplified DNA was purified before sequencing using Microspin S-400HRcolumns purchased from Pharmacia Biotech, and the nucleotide sequence wasdetermined by cycle sequencer using the AmplitaqFS dye terminator kit and a373A automatic sequencer (Applied Biosystems/Perkin-Elmer, Foster City, Cal-

if.) (6) or by the Pharmacia Biotech ALF automated DNA sequencing apparatus.To analyze the identity of determined sequences, a comparison was made withgene banks using the BLAST software (3). The DNAsis software (Hitachi SoftwareEngineering Co., Ltd.) was used to identify restriction enzymes for restriction frag-ment length polymorphism (RFLP) analysis of amplicons showing similar sizes.

Nucleotide sequence accession numbers. The nucleotide sequences of theaadA2, dfrA1 and orf, dfrA15, aadB, and blaP1 gene cassettes have been assignedGenBank accession no. AF221903, AF221901, AF221900, AF221902, andAF221899, respectively.

RESULTS

An approximately 800-bp 3-CS PCR product was obtainedfrom 44 of the 176 V. cholerae strains studied by using theprimers qacE1-F and sul1-B. However, the majority of epi-demiologically unrelated clinical strains which showed resis-tance to sulfonamides did not, based on lack of PCR amplifi-cation, contain the sul1 gene. Only a single environmentalstrain amplified this region. The phenotypic characteristics andresistance gene cassettes found in strains containing class 1integrons are further described below (see also Tables 2 and 3).

V. cholerae non-O1, non-O139 strains isolated at the Chil-dren Hospital, Bangkok. Sixty-nine V. cholerae non-O1, non-O139 strains recovered from children as part of a cholerasurveillance conducted from August 1993 to July 1995 at theChildren Hospital in Bangkok were studied (14). These strainsrepresented 37 different O serotypes, of which none containedthe cholera toxin gene. The strains were originally tested for

FIG. 1. Association between integron structure and PCR products (modifiedfrom reference 40). The lines below the integron structure represent amplicons,and the bold line represents the sequenced amplicons. The primers above thePCR products are described in Table 1. attI1 is the attenuation site, and qacE1and sul1 encode resistance to disinfectants and sulfonamides, respectively. Theindividual gene cassettes are drawn to scale. The recombination site (59-bpelement) is shown as a dark circle.

TABLE 1. PCR primers used for identification and characterization of integrons and gene cassettes in

V. cholerae O serotypes isolated in ThailandPrimer Sequence Accession no.a Position of primer Reference

in-Fb (5-CS) GGC ATC CAA GCA GCA AGC U12338 141631433 12in-Bb (3-CS) AAG CAG ACT TGA CCT GAT U12338 483134814 12attI1-F CGG GCA TCC AAG CAG CAA GCG C U12338 141431435 12

qacE1-F ATC GCA ATA GTT GGC GAA GT X15370 2113230 44sul1-B GCA AGG CGG AAA CCC GCG CC X12869 136031341 47

blaP1-Fc CGC TTC CCG TTA ACA AGT AC Z18955 3483367 55blaP1-Bc CTG GTT CAT TTC AGA TAG CG Z18955 7673748 55aadA-B ATT GCC CAG TCG GCA GCG D43625 193331916 31

aAccession numbers are from the published sequences in the GenBank database.b F, forward nucleotide sequence (533); B, backward nucleotide sequence (345).c blaP1 is identical to the pse1 gene (55).

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susceptibility to 12 antibiotics by disk diffusion includingampicillin, chloramphenicol, ciprofloxacin, colistin, gentami-cin, kanamycin, nalidixic acid, neomycin, streptomycin, sul-fisoxazole, tetracycline, and trimethoprim-sulfamethoxazole(14). Of 40 strains that were resistant to colistin, streptomycin,and sulfisoxazole, 28 showed further resistance and were des-ignated multiply resistant (14).

Nine of the V. cholerae non-O1, non-O139 strains, all resis-tant to sulfonamides, contained class 1 integrons (Table 2).A PCR product of approximately 739 bp was obtained fromeach of five strains using the in-F and in-B primers (Fig. 2 andTable 2). DNA sequencing showed that the 739-bp amplicon

of strain VO-556 contained a trimethoprim gene cassette, aswell as 5-CS and 3-CS sequences (GenBank accession no.AF221900). The gene cassette showed 100% identity to thedfrA15 (dhfrXV) gene cassette encoding resistance to tri-methoprim (GenBank accession no. Z83311) (2). RFLP anal-ysis of the 739-bp amplicon using HindIII suggested that eachof the five strains contained the dfrA15 gene cassette (Table 3).Repeated PCR analysis of the five strains with the in-F andblaP1-B primers (Fig. 2) and the blaP1-F and in-B primersyielded 762- and 853-bp products, respectively, correspondingto the conserved regions of the integron, together with the blaP1(pse1) gene cassette, that encode a -lactamase (Table 1) (55).DNA sequencing of the products (GenBank accession no. AF221899) confirmed these findings, showing 100% iden-tity to the blaP1 gene (GenBank accession no. Z18955 and

AF071555) (9, 55). Surprisingly, we did not obtain two dif-ferent-size amplicons when using the in-F and in-B primers. As shown below, the integron containing the blaP1 geneyielded an 1,197-bp amplicon when the in-F and in-B prim-ers were used (Table 2). We cannot explain why only oneamplicon was obtained.

Strain VO-258 contained one integron, and PCR with the

TABLE 2. Phenotypic characteristics and resistance gene cassettes found by PCR in V. cholerae strains containing class 1 integrons

V. choleraestrain(s)

Date of isolation(day/mo/yr or

mo/yr)

O sero-typea

AntibiogrambSize(s) (bp) of PCRproduct(s) obtainedwith in-F and in-B

Aminoglycosideresistance gene

aadA2c

-Lactamresistance

gene blaP1d

Children Hospitalstrainsg

VO-258 27/08/93 O8 COL; STR; SUL; TET 1,009

VO-556 17/09/93 O151 AMP; STR; SUL; TMP 739e

VO-557 17/09/93 O151 AMP; SUL; TMP 739e VO-2339 20/01/94 O62 AMP; SUL; TMP 739e VO-3198 28/03/94 O78 AMP; CHLI; STR; SUL; TMP; TET 1,009, 1,197 VO-3417 22/04/94 O183 AMP; STR; SUL; TMP; TET 739e VO-4967 12/12/94 O110 AMP; COL; KAN; NAL; NEO; STR; SUL; TMP 1,009, 1,197 VO-5398 25/01/95 O13 AMP; COL; NAL; STR; SUL; TMP 1,009, 1,197 VO-6095A 22/03/95 O152 AMP; STR; SUL; TMP 739e

Strains from patientsh

10 strains 05/9006/90 COL; CHL; NEO; STR; SUL; TMP; TET 1,009, 1,237f 292/90 05/9006/90 COL; NEO; STR; SUL; TMP; TET 1,237f

Samutsakorn strainsi

1075/25 15/09/82 O1 AMP; COL; CHL; NEO; GEN; KAN; STR; SUL;TMP; TET

744

1076/25 15/09/82 O1 AMP; COL; CHL; NEO; GEN; KAN; STR; SUL;TMP; TET

744

64/26 11/10/82 O1 AMP; COL; NEO; GEN; KAN; STR; SUL; TMP;TET

744

20 strains 13/11/9209/05/96 O1 COL; STR; SUL 1,009

Seafood strainj SKF-09 02/02/94 AMP; COL; CHL; STR; SUL; TMP 1,009, 1,197

a O serotype according to the scheme of Shimada et al. (43).bAMP, ampicillin; CHL, chloramphenicol; COL, colistin; GEN, gentamicin; KAN, kanamycin; NAL, nalidixic acid; STR, streptomycin; SUL, sulfonmides; TMP,

trimethoprim-sulfamethoxazole; TET, tetracycline; CHLI, intermediate chloramphenicol resistance.c PCR product of 752 bp obtained using primers in-F and aadA-B (GenBank accession no. AF221903).d PCR product of 762 bp obtained using primers in-F and blaP1-B (GenBank accession no. AF221899).e DNA sequencing of the 739 bp showed 100% identity to the dfrA15 trimethoprim resistance gene cassette and part of the conserved segments (GenBank accession

no. AF221900) (2).fDNA sequencing of the 1,237 bp showed 100% identity to the dfrA1 trimethoprim resistance gene cassette and an open reading frame (GenBank accession no.

AF221901) (48).g Non-O1, non-O139 strains isolated at Children Hospital in Bangkok, Thailand (14).h Non-O1, non-O139 strains isolated from patients in Thailand (18).i O1 strains isolated from patients in Samutsakorn, Thailand (17).j

Non-O1, non-O139 strain isolated from seafood in Samutsakorn, Thailand (18).

FIG. 2. Examples of PCR products of V. cholerae O1 strains isolated inThailand 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 (lanesn and o). Lanes: a, 100-bp molecular mass standard; b, strain VO-3417; c, strain1075/25; 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, Acineto-bacter 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, strainVO-557; o, strain 9720921 (positive control); p, PCR mixture, (negative control);q, 100-bp molecular mass standard.

VOL. 44, 2000 CLASS 1 INTEGRONS IN VIBRIO CHOLERAE 1317

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in-F and aadA-B primers confirmed the presence of an aadAaminoglycoside resistance gene cassette (GenBank accessionno. D43625) which confers resistance to streptomycin andspectinomycin (Fig. 2; Tables 1 and 2) (8, 31).

PCR with the in-F and in-B primers yielded two ampliconsof 1,009 and 1,197 bp in strains VO-3198, VO-4967, and VO-5398. PCR with the in-F and aadA-B primers and DNA se-quencing of the 1,009-bp amplicon from strain VO-3198 con-firmed the presence of the aadA2 aminoglycoside resistancegene cassette (GenBank accession no. AF221903) (8, 31) (Fig.2; Tables 1 and 2). RFLP analysis of the 1,009-bp ampliconusing HindIII, which has two restriction sites in the 1,009-bpproduct but none in the 1,197-bp amplicon, suggested thatstrains VO-3198, VO-4967, VO-5398, and VO-258 also con-tained the aadA2 gene cassette (Table 3). PCR with the in-Fand blaP1-B primers yielded a 762-bp product correspondingto the conserved regions of the integron, together with theblaP1 gene cassette, that encode the -lactamase PSE-1 (Fig.

2; Tables 1 and 2) (55). DNA sequencing of the 1,197-bpproduct from strain VO-3198 confirmed these findings, show-ing 100% identity to the blaP1 gene and the 5CS and 3-CSsequences (GenBank accession no. Z18955 and AF071555) (9,55). Digestion with MfeI, which had a single restriction site inthe 1,197-bp fragment, but none in the 1,009-bp amplicon,demonstrated identical fragments for all three of the strains,suggesting that these strains contained the same 764-bp -lac-tamase-encoding blaP1 cassette (Table 3) (40).

V. cholerae non-O1, non-O139 strains recovered from pa-tients in Thailand. Nineteen V. cholerae non-O1, non-O139strains were selected from 41 strains recovered from stool sam-ples from patients with diarrhea (18), including 13 strains isolatedin 1990 during an epidemic of a cholera-like disease amongKhmer refugees in a camp in Aranyaprathet, Thailand (5).

Ten sulfonamide-resistant non-O1, non-O139 strains iso-lated from the Khmer refugees contained class 1 integronseach yielding PCR amplicons of 1,009 and 1,237 bp using thein-F and in-B primers, whereas strain 292/90 yielded a singleproduct of 1,237 bp (Fig. 2 and Table 2). PCR with the in-Fand aadA-B primers confirmed the presence of an aadA genecassette (Fig. 2; Tables 1 and 2). DNA sequencing showed thatthe 1,237-bp amplicon of strain 292/90 showed 100% identityto the dfrA1 (dhfrI) gene encoding resistance to trimethoprim(GenBank accession no. X00926) (22) and an open readingframe downstream of the dfrA1 gene (GenBank accession no.AF221901). This open reading frame was previously noted bySundstrom and Skold (48). Digestion with ClaI, which had asingle restriction site on the 1,237-bp amplicon, suggested thateach strain contained the dfrA1 gene cassette (Table 3). The

remaining strains were intermediately resistant or sensitive tosulfonamides (18).

V. cholerae O1 strains recovered from patients in Samutsa-korn, Thailand. Thirty-one clinical V. cholerae O1 strains wereselected from 70 strains isolated from 1982 to 1996 in Samut-sakorn, a port city 30 km southwest of Bangkok (17). Weshowed in a previous study that the disappearance ofV. chol-

erae O139 from Samutsakorn was associated with distinct phe-notypic and genotypic changes in O1 strains isolated duringand after the O139 epidemic, including the development ofresistance to streptomycin and sulfisoxazole (17).

Twenty V. cholerae O1 strains isolated during and after theO139 epidemic, but also within a 10-months period just beforethe appearance of the O139 serotype, showed resistance tosulfonamides and streptomycin and contained class 1 integrons(Fig. 2; Table 2). PCR and RFLP analysis as described aboveconfirmed the presence of the aadA2 aminoglycoside resis-tance gene cassette (Table 2). One strain isolated in October1991 showed resistance to sulfonamides and streptomycin butdid not contain class 1 integrons.

Strains 1075/25, 1076/25, and 64/26 were isolated by Tab-tieng et al. (49) during a cholera outbreak among children in1982 and found to carry a conjugative 150-kb plasmid confer-ring multiple-drug resistance. Each strain contained class 1integrons, and the use of the in-F and in-B primers yielded aPCR product of 744 bp (GenBank accession no. AF221902)(Table 2). DNA sequencing and RFLP analysis with SphI re-vealed that the 744-bp amplicon of each of the strains con-tained the aadB gene cassette, as well as 5-CS and 3-CS se-quences. This gene cassette showed 100% identity to the aadBgene, which encodes resistance to gentamicin, kanamycin, andtobramycin (GenBank accession no. L06418) (Table 3) (10).Conjugation of the strains with E. coli K-12 strain J53-1 yieldedone type of exconjugant which showed identical antibiogramsand contained the 150-kb plasmid, suggesting that the entireantibiotic resistance pattern was plasmid encoded (Table 2).

Further, PCR of the exconjugants yielded the 744-bp ampli-con, showing that the aadB gene cassette was located and trans-ferred on the 150-kb plasmid.

Clinical V. cholerae O139 strains and environmental non-O1, non-O139 strains isolated from shrimp aquaculture andseafood. Class 1 integrons were not found among 11 represen-tative V. cholerae O139 strains tested which showed resistanceto furazolidone, streptomycin, trimethoprim, and sulfonamidesand included strains isolated from patients in Bangladesh, In-dia, and Thailand from 1992 to 1993 (19). Thus, the antibioticresistances shown by the O139 strains were not associated withthis type of integron.

Of 34 V. cholerae non-O1, non-O139 strains selected among93 strains isolated from water, sediment, and shrimp in ashrimp production area in Thailand (16), none contained class

1 integrons. The strains tested included 23, 6, and 5 strainsshowing resistance (inhibition zone diameters of 19 mm),intermediate resistance (inhibition zone diameters between 20and 22 mm), and susceptibility (inhibition zone diameters of23 mm) to sulfonamides, respectively, when tested in diskdiffusion assays by the Bauer-Kirby method (7, 18).

Twelve V. cholerae non-O1, non-O139 strains were selectedamong 23 strains isolated from seafood samples in Samutsa-korn, Thailand (18). Ten and two strains were intermediateand fully resistant to sulfonamides, respectively (18). One sul-fonamide-resistant strain contained two class 1 integrons con-taining amplicons of 1,009 or 1,197 bp (Table 2). PCR andRFLP analysis suggested that the amplicons found in the sea-food isolate were identical to amplicons found in strains VO-3198, VO-4967, and VO-5398 from the Children Hospital (Ta-

TABLE 3. RFLP analyses of resistance gene cassettes foundin class 1 integrons in V. cholerae

Genecassette

Size (bp)of amplicon

obtained withprimers in-F

and in-B

Restrictionenzyme

used

Restrictionfragmentsizes (bp)

GenBank no.of identical

sequence

Refer-ence

aadA2 1,009 HindIII 216, 275, 517 D43625 31dfrA1 1,237a ClaI 436, 801 X17477 48dfrA15 739 HindIII 315, 424 Z83311b 2aadB 744 SphI 175, 569 L06418 10blaP1 1,197 MfeI 258, 939 AF071555 9

a The amplicon contained both the dfrA1 gene cassette and an open readingframe. See also Fig. 1.

b The sequence with this GenBank no. included some of the aadA genesequence located downstream of the dfrA15 gene cassette.


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bles 2 and 3). Thus, each of these strains seems to contain theaadA2 and blaP1 gene cassettes (Fig. 2; Tables 2 and 3).

DISCUSSION

In the present study, we showed that class 1 integrons con-taining antibiotic resistance gene cassettes were found mainly

in epidemiologically related V. cholerae O serotypes isolatedfrom outbreaks of diarrhea in Thailand. However, several epi-demiologically unrelated V. cholerae non-O1, non-O139 strainsisolated mainly from children with diarrhea also containedclass 1 integrons. Obtained by PCR and DNA sequencing, thefindings of aadB, aadA2, blaP1, dfrA1, and dfrA15 resistancegene cassettes encoding resistances to gentamicin, kanamycin,and tobramycin; streptomycin and spectinomycin; -lactams;and trimethoprim, respectively, represent the first report of thedistribution of class 1 integrons among different V. cholerae Oserotypes. The gene cassettes reported here corroborate pre-vious findings of the content of class 1 integrons in Enterobac-tericeae and Pseudomonas spp., in which antibiotic resistancegene 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, in-cluding determinants of resistance to ampicillin, chloramphen-icol, neomycin, streptomycin, trimethoprim, and tetracycline,were not contained within class 1 integrons. Thus, althoughwidely distributed, resistance genes on class 1 integrons seemto encode only a part of the antibiotic resistance reported inV. cholerae. As our PCR analysis was designed to detect rela-tively smaller-size amplicons, larger arrays of gene cassettesmay have been missed. However, it should be noted that all ofthe strains yielding an amplicon with the primers qacE1-Fand sul1-B also showed a product when the in-F and in-Bprimers were used. Thus, it seems unlikely that larger arrays ofgene cassettes were present.

There was a clear difference in the distribution and fre-

quency of class 1 integrons among clinical and environmentalV. cholerae non-O1, non-O139 strains, as only a single envi-ronmental strain recovered from seafood in Samutsakorn con-tained a class 1 integron. The non-O1, non-O139 strains fromthe shrimp production area in Thailand were isolated fromponds and coastal areas (16) and showed less antibiotic resis-tance than the clinical non-O1, non-O139 strains (unpublishedresults). It is likely that the higher frequency of class 1 inte-grons in clinical isolates is because such strains became dom-inant through the selective pressure caused by the therapeuticuse of antibiotics.

Bagchi et al. (5) reported that during the cholera-like epi-demic among the Khmers in 1982, children and pregnant wom-en 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 resis-tance to trimethoprim-sulfamethoxazole. Thus, it is likely thatthe strains that acquired the dfrA1 gene cassette became pre-dominant through selective pressure. The finding that non-O1,non-O139 strains showing resistance to several antibiotics alsocontained resistance gene cassettes located on integrons is ofconcern. The finding of trimethoprim resistance gene cassettes,including the recently described dfrA15 gene cassette (2), is ofspecial concern, as trimethoprim is often used to treat diarrheaamong children and pregnant women.

The appearance of the O139 serotype in Samutsakorn in1993 and its sudden disappearance in 1994 were associated with the emergence of a sulfonamide-resistant V. cholerae O1strain carrying a class 1 integron which contained the aadA2resistance gene cassette. We reported earlier that this distinct

O1 strain showed unique genotypes, as demonstrated by ribo-typing, PFGE typing, and cholera toxin typing (17). Interest-ingly, Dalsgaard et al. (15) found that O1 strains isolated inVietnam after 1990 also contained class 1 integrons which har-bored the aadA2 gene cassette and showed a ribotype R1 iden-tical to the ribotype seen among O1 strains isolated in Samutsa-korn (17). Thus, it is likely that this distinct O1 strain was

transferred between Thailand and Vietnam and became estab-lished as the main strain causing cholera. It remains to bedetermined how this strain acquired the class 1 integron andthe aadA2 gene cassette and/or if the strain was introducedfrom a third country.

Although the class 1 integrons were first described by Stokesand Hall in 1989 (44), our findings of the aadB resistance geneamong the three O1 strains isolated in 1982 from the choleraoutbreak at the pediatric ward in Samutsakorn show that class1 integrons have been present in V. cholerae in Thailand for anumber of years (49). The demonstration of the class 1 inte-grons being located on a 150-kb self-transmissible plasmid is,to our knowledge, the first report of a plasmid containing in-tegrons in V. cholerae. Tabtieng et al. (49) reported that the150-kb plasmid in each of the three strains belonged to incom-

patibility group C and contained genes coding for type II di-hydrofolate reductase. The genes encoding the type II dihy-drofolate reductase were not found as a class 1 gene cassette.However, after 1982, this plasmid and other plasmids were notfound in O1 strains isolated in Samutsakorn or elsewhere,suggesting that the 150-kb plasmid containing the class 1 inte-grons was lost (17). Other studies have reported multiple-drug-resistant V. cholerae O1 encoded by conjugative incompatibilitygroup C plasmids (51). However, it remains to be shown ifthese or other plasmids in V. cholerae contain class 1 integrons.

Interestingly, class 1 integrons were not found among anyV. cholerae O139 strains. Thus, the 62-kb self-transmissible trans-poson-like SXT element described by Waldor et al. (53) amongO139 strains, some of which were included in the present

study, does not seem to carry class 1 integrons. The V. choleraeO1 El Tor strain which re-emerged in India and Bangladeshafter 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 andBangladesh contain class 1 integrons.

Our findings show that class 1 integrons containing severaldifferent antibiotic resistance gene cassettes were distributedamong different clinical V. cholerae O1 and non-O1, non-O139serotypes in Thailand. Thus, PCR mapping of integrons andDNA sequencing of their genetic contents may be a usefulepidemiological tool with which to study the evolution of mul-tiresistance plasmids and dissemination of antibiotic resistancegenes within V. cholerae. Although the resistance genes within

the integrons encoded only a minor part of the antibiotic re-sistance detected, further studies in Thailand and elsewhereare needed to determine the importance of class 1 integrons inthe horizontal acquisition and dissemination of antibiotic re-sistance genes.

ACKNOWLEDGMENTS

We are grateful for the technical assistance provided by Anne-MettePetersen at the Royal Veterinary and Agricultural University. We alsothank Lorrin Pang and Peter Echeverria at the Armed Forces Re-search Institute of Medical Sciences for critical revision of the manu-script and provision of strains.

Dorthe Sandvang and Anders Dalsgaard were supported by theDanish Agricultural and Veterinary Research Council (grant 9600012)and the Danish Council for Development Research (DANIDA grant90928), respectively.


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