Vibrio cholerae SXT element, Laos.

To the Editor: The SXT element is a Vibrio cholerae-derived ICE (integrating and conjugative element), which has also been referred to as a conjugative transposon (1) or a constin (2). ICEs excise from the chromosomes of their hosts, transfer to a new host through conjugation, and then integrate into the chromosome again. SXT element was originally isolated in 1993 from a V. cholerae O39 clinical isolate (SX[T.sup.MO210]) (1). The [approximately equal to] 100-kbp SXT element confers resistance to sulfamethoxazole, trimethoprim, chloramphenicol, and streptomycin (1). Since 1994, V. cholerae isolates from Bangladesh, India, and Mozambique have also contained the SXT element (2-4). In SX[T.sup.MO10]), resistance genes are embedded near the 5' end, in a [approximately equal to] 17.2-kbp composite transposon-like element that interrupts the SXT-encoded rumAB operon. In contrast, in El Tor O1 V. cholerae strains isolated in India and Bangladesh, the resistance genes are located in SX[T.sup.ET], which is closely related but not identical to SX[T.sup.MO10] (2). Comparison of 2 related ICEs, SXT of V. cholerae and R391 of Providencia rettgeri (5), showed that the conserved backbone apparently contains 3 hot spots for insertions of additional DNA sequences: the first between sO43 and traL, the second between trA and sO54, and the third between sO73 and traF. R391 contains an intact rumAB operon and a transposon-associated kanamycin resistance gene located [approximately equal to] 3.5 kbp from the rumAB operon (6). Mobile genetic elements such as SXT have a crucial role in spreading antimicrobial drug resistance genes among microbial populations, and our understanding of these genetic elements would help to control the emergence of antimicrobial drug resistance.

We have been monitoring the drug sensitivity pattern in the Lao People's Democratic Republic (Laos) since 1993, and we have found that V. cholerae O1 strains isolated after 1997 were resistant to tetracycline, sulfamethoxazole, trimethoprim, chloramphenicol, and streptomycin (7). Analysis of the genetic determinants encoding antimicrobial drug resistance showed an SXT element (SX[T.sup.LAOS]), which is different from the previously reported SXTs (8). SX[T.sup.LAOS] contains 2 novel open reading frames (ORFs) in the third hot spot (between sO73 and traF). SX[T.sup.ET] contains a class 9 integron in hot spot sO73-traF that harbors dfrA1 as a gene cassette (2). In SX[T.sup.MO10], the gene encoding trimethoprim resistance (dfr18) is encoded in the [approximately equal to] 17.2-kbp composite transposon-like element that interrupts the SXT-encoded rumAB operon. SX[T.sup.LAOS] does not encode dfr18 or dfrA1, and the gene encoding trimethoprim resistance has not been identified. In this study, we analyzed hot spot sO43-traL and hot spot traA-sO54 to better characterize SX[T.sup.LAOS.]

Two sets of primers were designed to amplify the hot spot regions. Primer HS1-F, which anneals to sO43, was 5' GGC TAT TCC ACC GGT GGT G 3'; primer HS1-R, which anneals to traL, was 5' TGC CGA TCA CTA GCC CCA AC 3'; primer HS2-F, which anneals to traA, was 5' ATG GGT CTC TAC AAT ACG CC 3'; and primer HS2-R, which anneals to sO54, was 5' GGA GAC AGC GCA AGC GCC AG 3'. Polymerase chain reaction (PCR) amplifications on genomic DNA extracted from the V. cholerae O1 strain isolated in Laos (strain 00LA1) with primers HS1-F and HS1-R yielded an amplicon of [approximately equal to] 1100 bp, which is slightly different from the amplicon obtained with DNA extracted from V. cholerae O139, strain MO10 ([approximately equal to] 1,000 bp). PCR amplification using primers HS2-F and HS2-R gave amplicons of similar size ([approximately equal to] 2,200 bp) for both strains. The [approximately equal to] 1,000-bp and [approximately equal to] 2,200-bp PCR products from strain 00LA1 were cloned independently into the pCR 2.1 vector and tested to determine if recombinant plasmids confer trimethoprim resistance after transformation to Escherichia coli. No trimethoprim-resistant colonies were observed after transformation. The nucleotide sequences of the inserted fragments were analyzed. The region between sO43 and traL showed 97% identity to the corresponding region of P. rettgeri R391 (accession no. AY090559), which encodes 2 hypothetical proteins (ORF 37 and ORF 38). The region between traA and sO54 showed 97% identity to the corresponding region of SX[T.sup.MO10] (accession no. AY055428). Since the gene encoding trimethoprim resistance was not located in any of the hot spot regions proposed by Beaber et al. (5), we also analyzed the region between sO26 and sO27, which in R391 contains the kanamycin resistance gene. Primers sO26-F (5' GAG CAA TGG GCG AGA GTT CC) and sO27-R (5' TCA GCG ACA ACC GGA GAA TG) gave an amplicon of 409 bp for SX[T.sup.MO10], as expected, while no PCR product was obtained for SX[T.sup.LAOS]. This result suggested that the region between sO26 and sO27 in SX[T.sup.LAOS] is also different from SX[T.sup.MO10.]

V. cholerae O139 has not been isolated in Laos, and the SXT element was not likely transmitted from a V. cholerae O139 strain to a V. cholerae O1 strain. Since SX[T.sup.LAOS] has a hot spot that is identical to R391, we show evidence for a possible independent emerging of SX[T.sup.LAOS]. Further analysis is needed to understand the evolution and relationship between different ICEs and the emergence of new variants.

In a previous study (8), we confirmed experimentally that trimethoprim resistance was also transferred by conjugation, and we hypothesized that the responsible gene is located within SX[T.sup.LAOS]. However, the gene was not found in any of the proposed hot spot regions. The possibility that the trimethoprim resistance determinant is located on the chromosome outside the SXT element and cotransfers with the SXT in an Hfr-like manner cannot be ruled out (9). Therefore, additional hot spot regions may exist in SXT elements for insertion of DNA; otherwise the trimethoprim resistance gene is not encoded within SX[T.sup.LAOS].

The nucleotide sequence data reported in this study will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB 185252 for the hot spot sO43-traL and AB 186353 for the hot spot traA-s054.

References

(1.) Waldor MK, Tschape H, Mekalanos JJ. A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139. J Bacteriol. 1996;178: 4157-65.

(2.) Hochhut B, Lotfi Y, Mazel D, Faruque SM, Woodgate R, Waldor MK. Molecular analysis of antibiotic resistance gene clusters in Vibrio cholerae 139 and O1 SXT constins. Antimicrob Agents Chemother. 2001;45:2991-3000.

(3.) Amita, Chowdhury SR, Thungapathra M, Ramamurthy T, Nair GB, Ghosh A. Class I integrons and SXT elements in El Tor strains isolated before and after 1992 Vibrio cholerae O139 outbreak, Calcutta, India. Emerg Infect Dis. 2003;9:500-2.

(4.) Dalsgaard A, Forslund A, Sandvang D, Arntzen L, Keddy K. Vibrio cholerae O1 outbreak in Mozambique and South Africa in 1998 are multiple-drug resistant, contain the SXT element and the aadA2 gene located on class 1 integrons. J Antimicrob Chemother. 2001 ;48:827-38.

(5.) Beaber JW, Burrus V, Hochhut B, Waldor MK. Comparison of SXT and R391, two conjugative integrating elements: definition of a genetic backbone for the mobilization of resistance determinants. Cell Mol Life Sci. 2002;59:2065-70.

(6.) Boltner D, Mac Mahon C, Pembroke JT, Strike P, Osborn A. R391: a conjugative integrating mosaic comprised of phage, plasmid, and transposon elements. J Bacteriol. 2002; 184:5158-59.

(7.) Phantouamath B, Sithivong N, Sisavath L, Munnalath K, Khampheng C, Insisiengmay S, et al. Transition of drug susceptibilities of Vibrio cholerae O1 in Lao People's Democratic Republic. Southeast Asian J Trop Med Public Health. 2001;32:95-9.

(8.) Iwanaga M, Toma C, Miyazato T, Insisiengmay S, Nakasone N, Ehara M. Antibiotic resistance conferred by a class I integron and SXT constin in Vibrio cholerae strains isolated in Laos. Antimicrob Agents Chemother. 2004;48:2364-9.

(9.) Hochuut B, Marrero J, Waldor MK. Mobilization of plasmids and chromosomal DNA mediated by the SXT element, a constin found in Vibrio cholerae O139. J Bacteriol. 2000;182:2043-7.

Address for correspondence: Claudia Toma, Division of Bacterial Pathogenesis, Department of Microbiology, Graduate School of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-0215, Japan; fax: [phone]; email: [e-mail]

Claudia Toma, * Noboru Nakasone, * Tianyan Song, * and Masaaki Iwanaga *

* University of the Ryukyus, Okinawa, Japan