ABSTRACT
Changes in the drug susceptibility pattern were observed in Vibrio cholerae O1 isolated in the Lao People's Democratic Republic during 1993 to 2000. In this study, 50 V. cholerae O1 strains were selected during this period for studying the presence of class I integron and SXT constin. Twenty-four streptomycin-resistant strains out of 26 isolated before 1997 contained a class I integron harboring the aadA1 gene cassette. Twenty-four strains isolated after 1997 contained an SXT constin (a large conjugative element). Twenty of the strains were resistant to chloramphenicol, tetracycline, streptomycin, and trimethoprim-sulfamethoxazole, while four strains were susceptible to the antibiotic tested. The resistance genes included in the SXT constins were floR, tetA, strAB, and sulII, which encode resistance to chloramphenicol, tetracycline, streptomycin, and sulfamethoxazole, respectively. The antibiotic resistance gene cluster was found to be deleted in the four susceptible strains. SXTLAOS did not contain dfrA1 or dfr18, which confer resistance to trimethoprim in SXTET and SXTMO10, respectively. A hot spot region of SXTLAOS was sequenced, and we identified two novel open reading frames showing homology to sO24 (exonuclease) and sO23 (helicase) of the genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104. Analysis of SXTLAOS showed that there is a continuous flux of genes among V. cholerae SXT constins which should be carefully monitored.
Cholera, a life-threatening disease with a typical secretory diarrhea, is usually treated with fluid infusion, and antibiotic therapy is not essential. However, antibiotic therapy has been routinely performed, as the duration of diarrhea can be shortened (18). In the Lao People's Democratic Republic (Lao PDR), Vibrio cholerae isolates are collected at the Center for Laboratory and Epidemiology, and their cholera toxin and hemolysin production are studied, as well as their susceptibility to phages and antimicrobials. In the period between 1993 and 2000, although the pathogens were sensitive to the therapeutic antimicrobials as expected until 1996, a change in the drug sensitivity pattern started in 1998 after a period without cholera (13, 17, 20). Strains isolated after 1997 were found to be moderately resistant to tetracycline and chloramphenicol and highly resistant to trimethoprim-sulfamethoxazole (20).
Multiple-antibiotic resistance in V. cholerae has been described, frequently upon the acquisition of R plasmids belonging to the conjugative group C (23). However, no plasmid was detected in 20 randomly selected tetracycline-resistant Laotian strains in 1998 (17). Recently, other genetic elements, such as a class I integron and an SXT constin, have also been reported to be associated with the spread of genetic determinants of resistance to antimicrobial agents (9, 10, 15, 16, 24). Integrons have an integrase gene (intI), an attachment site (attI) into which individual resistance genes are inserted, and a promoter sequence allowing expression of resistance genes (cassette-associated genes), which do not have promoters. The fragment intI-attI is highly conserved in all integrons and is called a 5′ conserved sequence (CS). Integrons have been categorized into nine different classes according to the sequences of their integrases, and those most frequently detected in clinical isolates belong to class I (21). Integrons are not mobile, but they are often found within conjugative plasmids, which assures their mobility (9, 11). Dalsgaard et al. (7) characterized V. cholerae O1 strains isolated in Vietnam from 1979 to 1996 and found that strains isolated after 1990 were resistant to streptomycin and harbored a class I integron containing an aadA2 gene cassette. V. cholerae strains isolated in Thailand and India were also reported to contain a class I integron with various gene cassettes (8, 24). Their presence in V. cholerae strains isolated in the Lao PDR, however, has not been reported.
The SXT constin (a conjugative, self-transmissible integrating element) encodes resistance to sulfamethoxazole, trimethoprim, chloramphenicol, and streptomycin (3). This ∼100-kbp element is always integrated into the 5′ end of the chromosomal gene prfC (14). SXT encodes an integrase in its 5′ end that is required for SXT transfer. This element (SXTMO10) was initially detected in the newly emerged O139 serogroup of V. cholerae in 1992 (28). Since 1994, V. cholerae isolates from India, Bangladesh, and Mozambique have also been reported to contain the SXT constin (2, 10, 16). In SXTMO10, resistance genes are embedded near the 5′ end, in an ∼17.2-kbp composite transposon-like element that interrupts the SXT-encoded rumAB operon. In contrast, in El Tor O1 V. cholerae strains, the resistance genes are located in SXTET, which is closely related but not identical to SXTMO10 (16).
In order to understand the changes in the drug susceptibility patterns in V. cholerae strains isolated in the Lao PDR, in this study, the genetic determinants encoding antibiotic resistance were analyzed, with particular attention directed to the resistance genes in the class I integron and SXT constin.
MATERIALS AND METHODS
Bacterial strains.Among a collection of 284 V. cholerae O1 biotype El Tor strains recovered from cholera patients from 1993 to 2000, 50 strains were included in the present study for further analysis. The various phenotypic characteristics of the strains were studied previously (13, 17, 20, 25). Before PCR analysis, antibiotic resistance was confirmed in these isolates by MIC determination, and the results were compared with the initial MIC results (13, 17, 20). V. cholerae O139 strain MO45 (ATCC 51394), isolated in Madras, India, in 1992, and V. cholerae O1 strain CRC182, isolated in India in 2000, were included for comparison purposes.
PCR amplification and DNA sequencing.All primers used in this study are listed in Table 1. DNA was extracted as previously described (27). Class I integrase (intI1) was detected by PCR using primers inDS-F and inDS-B. DNAs from strains yielding a PCR product with these primers were subsequently amplified with the integron primers in-F and in-B, which amplify the region between the 5′ and 3′ CSs. The primers in-F and aadA-B were used to assess whether the integron contained a gene cassette encoding resistance to streptomycin and spectinomycin (aadA1). To investigate the presence of an SXT constin, primers INT1 and INT2, specific for SXT integrase (intSXT), were used. Antibiotic-resistant genes included in the SXT constin were detected using primers reported by others (16, 22) or designed in this study. The location of the tetA gene within the SXT constin was assessed based upon a strategy of different PCR amplifications, combining several primers based on the sequence of the antibiotic resistance gene element of V. cholerae strain V21 (DDBJ accession number AB114188).
Primers used in this study
Amplified DNA was purified before sequencing using a GFX column (Amersham Pharmacia, Little Chalfont, United Kingdom), and the nucleotide sequence was determined by cycle sequencing with a Big Dye Terminator Cycle Sequencing FS Ready Reaction kit and analyzed with an ABI PRISM 310 Genetic Analyzer or 3730 DNA Analyzer (Applied Biosystems, Foster City, Calif.). The identities of the sequences determined were analyzed by comparison with the gene sequences in databases using BLAST software (1).
Conjugation.The ampicillin-resistant V. cholerae O34 strain 88UDT119/pGV3 (26) was used as the recipient in conjugation experiments with SXT constin-positive strains, and the chloramphenicol-resistant V. cholerae O34 strain AM15 (C. Toma, T. Miyazato, H. Kuroki, Y. Lu, M. Ehara, K. Yamamoto, N. Nakasone, and M. Iwanaga, presented at the 37th Joint Conference of the U.S.-Japan Cooperative Medical Science Program for Cholera and Other Bacterial Enteric Infections Panel, Okinawa, Japan, 2002) was used as the recipient in conjugation experiments with class I integron-positive strains. After mating on nonselective Luria-Bertani agar incubated at 37°C for 16 h, exconjugants were harvested, and appropriate dilutions were spread on Luria-Bertani agar containing 100 μg of ampicillin/ml and 32 μg of trimethoprim/ml to investigate SXT conjugation, and 5 μg of chloramphenicol/ml and 20 μg of streptomycin/ml to investigate the presence of a conjugative plasmid carrying the integron. Exconjugants were analyzed by PCR to exclude possible spontaneous antibiotic-resistant mutants.
Cloning of SXTLAOS-specific fragment.The region between sO73 and traF was amplified by PCR using the primers YL6 and traF-R and DNA from V. cholerae O1 strain 00LA1 as the template. The 5-kbp PCR product was cloned into the pCR 2.1 vector, and the nucleotide sequence of the inserted PCR product was determined by primer walking.
Nucleotide sequence accession number.The nucleotide sequence of the region between sO73 and traF for SXTLAOS has been submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB115497.
RESULTS
Distribution of class I integron and SXT constin.Table 2 shows the characteristics of the V. cholerae strains used in this study. The strains isolated in the Lao PDR can be divided into four groups according to the antibiograms and year of isolation. Group A comprises 24 strains isolated between 1993 and 1996 that were streptomycin resistant. Group B comprises two strains isolated in 1995 that were susceptible to all the antibiotics tested. Group C comprises 20 strains isolated between 1998 and 2000 that were resistant to streptomycin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole. Group D comprises four strains isolated between 1998 and 2000 that were susceptible to all of the antibiotics tested.
Characteristics of V. cholerae strains
PCR with the primers inDS-F and inDS-B yielded a PCR product of ∼800 bp from each of the 24 streptomycin-resistant strains belonging to group A. Strains belonging to groups B, C, and D did not yield an amplicon with these primers, indicating that class I integrons were not present. A PCR product of ∼1,000 bp was obtained from each of the 24 class I integron-positive strains using the in-F and in-B primers. DNA sequencing of the 1,000-bp amplicons from three isolates confirmed the presence of the gene cassette aadA1, which conferred resistance to streptomycin. PCR with the in-F and aadA-B primers yielded an ∼750-bp amplicon from all isolates that yielded a product with the in-F and in-B primers. Streptomycin resistance encoded by the class I integron could not be transferred by conjugation, indicating that the class I integron was not carried in a conjugative plasmid.
PCR for detection of intSXT showed that all strains belonging to groups C and D yielded a 592-bp amplicon of a size identical to those of the positive controls V. cholerae O139 strain MO45 and V. cholerae O1 strain CRC182.
Analysis of SXT constin in group C.Conjugation experiments showed that intsxt was transferable by conjugation. Antimicrobial susceptibility testing of the V. cholerae transconjugants showed that the resistance genes contained in the SXT element were transferred and expressed in each of the transconjugants (Table 3).
MICs and PCR results for V. cholerae strains used in conjugation experiments and transconjugants
PCR assays designed to detect the genes that encode resistance to chloramphenicol (floR), streptomycin (strA and strB), sulfamethoxazole (sulII), and tetracycline (tetA) yielded the expected PCR products of 526 (floR), 383 (strA), 459 (strB), 625 (sulII), and 950 (tetA) bp for each of the 20 strains of group C, while strains in group D failed to react. As tetA was also transferable by conjugation, we investigated the location of tetA within the SXT constin. In PCR analysis, an amplicon of ∼2.3 kbp was obtained with TetA-F and strB-R, and an amplicon of ∼3.0 kbp was obtained with TetA-F and STRA-F (Fig. 1C). The presence of tetA upstream of strB was also confirmed by nucleotide sequence analysis of the 2.3-kbp amplicon obtained with primers TetA-F and strB-R.
(A) Schematic representation of the SXTMO10 constin showing the location of the integrase gene (intSXT), the antibiotic resistance gene cluster, and the three hot spots (triangles). (B) Organization of the region containing the antibiotic resistance genes (solid arrows) in SXTMO10 (3, 16). The locations of primers used for analysis of this region are also shown. A 3.34-kbp fragment that includes dfr18 is missing in SXTLAOS (stippled bar). (C) Primers used in PCR analysis for the location of tetA within SXTLAOS.
Analysis of SXT constin in group D.PCR assays utilizing the primers LEND4 and RUMA (Fig. 1B), which flank the antibiotic resistance genes in SXTMO10, yielded a product of ∼3.3 kbp for each of the four strains in group D. Partial sequencing of these products revealed that there was a deletion of the antibiotic resistance genes as previously reported for V. cholerae O139 strain 2055 by Hochhut et al. (16).
SXTLAOS is different from previously reported SXTs.All strains in group C failed to give an amplicon with primers designed to detect dfr18 and dfrA1, which are the trimethoprim resistance determinants reported for SXTMO10 and SXTET, respectively (16) (Fig. 2). PCR with primers TMP3 and TMP4, which anneal to sequences that flank dfr18 in SXTMO10, gave a PCR product of 1.3 kbp. These primers give a PCR product of 5.35 kbp with V. cholerae O139 DNA as a template (16). The results revealed that a 3.34-kbp fragment which included dfr18 is missing in SXTLAOS as in SXTET (Fig. 1B). However, SXTLAOS is distinct from SXTET in that no PCR product was obtained with primers YL6 and YL3 (Fig. 2). To further characterize SXTLAOS, PCR with primers YL6 and traF-R was performed. An amplicon of ∼5 kbp was obtained for SXTLAOS, as well as SXTMO10 and SXTET. Nucleotide sequencing of this fragment from SXTLAOS revealed the presence of two novel open reading frames (ORFs) (orf1, 1,875 bp, and orf2, 1,605 bp) (Fig. 3). The deduced amino acid sequence of ORF1 had 36% identity and 54% similarity to that of an exonuclease (encoded by sO24) found in the genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104 (5), while the deduced amino acid sequence of ORF2 had 26% identity and 45% similarity to that of a helicase (encoded by sO23) found downstream of the exonuclease in the same genomic island (5). PCR analysis with primers EXO-F and EXO-R showed that only SXTLAOS gave the expected amplicon of 890 bp (Fig. 2).
PCR analysis of SXT constins. Lanes 1 to 3, primers TMP-F and TMP-B; lanes 4 to 6, primers DFR1-F and DFR1-B; lanes 7 to 9, primers YL6 and YL3; lanes 10 to 12, primers EXO-F and EXO-R; lane M, 100-bp DNA ladder. Lanes 1, 4, 7, and 10, strain MO45 (SXTMO10); lanes 2, 5, 8, and 11, strain CRC182 (SXTET); and lanes 3, 6, 9, and 12, strain 00LA1 (SXTLAOS).
Schema of SXT constins around hot spot sO73-traF. The primers used in PCR analysis and to clone this region from V. cholerae strain 00LA1 are indicated. Novel ORFs in SXTLAOS are represented by hatched arrows: orf1 showed homology to sO24 (exonuclease), and orf2 showed homology to sO23 (helicase) of the genomic island conferring multidrug resistance on S. enterica serovar Typhimurium DT104 (5). Common orfs are represented by shaded arrows. The gene mediating trimethoprim resistance in SXTET (dfrA1) is represented by a solid arrow (16).
DISCUSSION
In this paper, we have studied the genetic determinants responsible for the changes in the drug susceptibility pattern of V. cholerae strains isolated in the Lao PDR over the 7-year period between 1993 and 2000. Strains isolated before 1997 (a year without cholera in the Lao PDR) were clearly different from those isolated after 1997. A class I integron with an aadA1 gene cassette was present in pre-1997 strains, while an SXT constin was present in the reemerged (post-1997) El Tor O1 strains. Recently, Amita et al. (2) have reported that a class I integron with an aadA1 gene cassette was widely distributed among the pre-O139 O1 strains isolated in India. In contrast, most of the post-O139 O1 strains contained the SXT constin and were devoid of the class I integron. Although V. cholerae O139 has not been isolated in the Lao PDR, it seems that strains isolated in the Lao PDR after 1997 are similar to Indian post-O139 O1 strains, as they showed resistance to trimethoprim-sulfamethoxazole, chloramphenicol, and streptomycin, the resistance genes of which are encoded in an SXT constin. However, strains from Laos are tetracycline resistant, in constrast to Indian strains, which are susceptible to this antibiotic (2). Tetracycline-resistant V. cholerae O1 strains isolated in Mozambique and South Africa in 1998 were also reported by Dalsgaard et al. (10). Tetracycline resistance is encoded by tetA in African strains, as well as Laotian strains. In Laotian strains, as well as V. cholerae strain V21 isolated in Vietnam, tetA is located within the SXT element.
PCR analysis of the SXT constin showed that SXTLAOS is different from the SXTMO10 and SXTET reported previously (Fig. 3). Comparison of two conjugative integrating elements, SXT of V. cholerae and R391 of Providencia rettgeri (4), revealed that the conserved backbone apparently contains three hot spots for insertion of additional DNA sequences, the first between sO43 and traL, the second between traA and sO54, and the third between sO73 and traF. SXTET contains a class 9 integron in hot spot sO73-traF that harbors dfrA1 as a gene cassette (16). We first hypothesized that this integron structure has a different gene cassette in SXTLAOS; however, PCR of integrase IntI9 (16) failed to produce a reaction (data not shown). The nucleotide sequence of this hot-spot region of SXTLAOS revealed the presence of two ORFs possibly involved in conjugal transfer (5). A trimethoprim resistance determinant was not found in this region, and we therefore could not identify the gene responsible for trimethoprim resistance. Trimethoprim resistance was also transferred by conjugation, and we hypothesized that the responsible gene is located within SXTLAOS, as in previously reported SXTs. However, the possibility that the trimethoprim resistance determinant is located on the chromosome outside the SXT constin and cotransfers with the SXT constin in an Hfr-like manner cannot be ruled out (15). A rapidly changing antibiotic resistance pattern was also observed among V. cholerae O139 strains (12, 19; R. Mitra, A. Basu, D. Dutta, G. B. Nair, and Y. Takeda, Letter, Lancet 348:1181, 1996). V. cholerae O139 strains are becoming increasingly resistant to nalidixic acid but are susceptible to trimethoprim-sulfamethoxazole and streptomycin. Our results suggested that variation within sequences inserted in the hot spots and the antibiotic resistance gene cluster might occur in V. cholerae strains, allowing rapid adaptation to changing environments.
SXT constins are present in virtually all recent clinical V. cholerae isolates from Asia, and SXT variants are arising in a manner similar to the Salmonella genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona (6). The presence of IntSXT-positive, antimicrobial-susceptible strains should also be carefully monitored, since “empty SXT constins” (group D) are capable of inserting not only antibiotic resistance genes but also other virulence factors which could be easily transferred to other strains by conjugation.
ACKNOWLEDGMENTS
We thank Shinji Yamasaki (Osaka Prefecture University, Osaka, Japan) and Irene Martin (National Microbiology Laboratory, Winnipeg, Canada) for their help during the course of this study.
FOOTNOTES
- Received 8 August 2003.
- Returned for modification 2 January 2004.
- Accepted 28 March 2004.
- American Society for Microbiology
