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Antimicrobial Agents and Chemotherapy, November 2008, p. 3829-3836, Vol. 52, No. 11
0066-4804/08/$08.00+0     doi:10.1128/AAC.00375-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vibrio cholerae O139 Multiple-Drug Resistance Mediated by Yersinia pestis pIP1202-Like Conjugative Plasmids

Jing-Cao Pan,^1* Rong Ye,¹ Hao-Qiu Wang,¹ Hai-Qing Xiang,¹ Wei Zhang,¹ Xin-Fen Yu,¹ Dong-Mei Meng,¹ and Zhe-Sheng He²

Microbiology Laboratory, Hangzhou Center for Disease Control and Prevention,¹ Department of Bacteriology, Institute of Bioengineering, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang, People's Republic of China²

Received 19 March 2008/ Returned for modification 21 May 2008/ Accepted 25 July 2008

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A conjugative plasmid, pMRV150, which mediated multiple-drug resistance (MDR) to at least six antibiotics, including ampicillin, streptomycin, gentamicin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole, was identified in a Vibrio cholerae O139 isolate from Hangzhou, eastern China, in 2004. According to partial pMRV150 DNA sequences covering 15 backbone regions, the plasmid is most similar to pIP1202, an IncA/C plasmid in an MDR Yersinia pestis isolate from a Madagascar bubonic plague patient, at an identity of 99.99% (22,180/22,183 nucleotides). pMRV150-like plasmids were found in only 7.69% (1/13) of the O139 isolates tested during the early period of the O139 epidemic in Hangzhou (1994, 1996, and 1997); then the frequency increased gradually from 60.00% (3/5) during 1998 and 1999 to 92.16% (47/51) during 2000 to 2006. Most (42/51) of the O139 isolates bearing pMRV150-like plasmids were resistant to five to six antibiotics, whereas the plasmid-negative isolates were resistant only to one to three antibiotics. In 12 plasmid-bearing O139 isolates tested, the pMRV150-like plasmids ranged from approximately 140 kb to 170 kb and remained at approximately 1 or 2 copies per cell. High (4.50 x 10^–2 and 3.08 x 10^–2) and low (0.88 x 10^–8 to 3.29 x 10^–5) plasmid transfer frequencies, as well as no plasmid transfer (under the detection limit), from these O139 isolates to the Escherichia coli recipient were observed. The emergence of pMRV150-like or pIP1202-like plasmids in many bacterial pathogens and nonpathogens occupying diverse niches with global geographical distribution indicates an increasing risk to public health worldwide. Careful tracking of these plasmids in the microbial ecosystem is warranted.

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Cholera is caused by toxigenic strains of Vibrio cholerae serogroups O1 and O139. Cholera toxin, encoded by ctxAB in the genome of the lysogenic bacteriophage CTX on the V. cholerae chromosome, is responsible for secretory diarrhea. V. cholerae O139, which emerged in late 1992 in southern India and Bangladesh, is the only non-O1 serogroup that causes epidemic and pandemic cholera. The initial V. cholerae O139 isolates, recovered during 1992 to 1993, were sensitive to tetracycline and resistant to trimethoprim-sulfamethoxazole (SXT) and streptomycin, because they harbored an integrating conjugative element, termed the SXT element, on the chromosome (3, 13, 36). Subsequently, resistance to other drugs commonly used for prophylaxis and clinical therapy, such as tetracycline, ampicillin, chloramphenicol, gentamicin, furazolidone, ciprofloxacin, and others, has been reported at different rates among O139 strains, and multiple-drug-resistant (MDR) O139 strains have been isolated frequently (3, 12, 17, 26, 38).

It was believed that the bacterial mobile genetic elements harboring resistance genes, including conjugative plasmids, integrating conjugative elements, and integrons, were largely responsible for the shift of drug resistance patterns among both the O1 and O139 serogroups (9, 11, 13, 22, 24, 36). Recently, the molecular characterization of SXT elements and integrons in Vibrio has been reported (1, 2, 4-6, 10, 22, 24). Although the contribution of plasmids to resistance was recognized earlier than that of SXT elements or integrons, little genetic information on conjugative plasmids in V. cholerae has been reported. Molecular characterization of conjugative plasmids among V. cholerae and other bacteria and comparative analysis of these plasmids would provide deep insight into their role in the emergence of MDR strains. Additionally, this information would be useful as the basis for tracking the dissemination of these plasmids within the microbial ecosystem, allowing better evaluation of their impact on public health.

The first strain of V. cholerae O139 in Hangzhou, a coastal city in eastern China, was isolated from a patient with diarrhea in 1994, and O139 cholera cases have been reported continuously since 1996 (31). In this paper, we describe an approximately 150-kb conjugative plasmid (pMRV150) as a major contributor to MDR in the O139 strains prevailing in Hangzhou, China. This plasmid shares a common backbone with pIP1202, an IncA/C conjugative plasmid from a Madagascar MDR isolate of Yersinia pestis bv. Orientalis, and pIP1202-like plasmids found in Salmonella enterica serovar Newport and Yersinia ruckeri strains in the United States and in Photobacterium damselae subsp. piscicida strains in the United States and Japan (16, 25, 37). Because MDR-conferring pMRV150-like or pIP1202-like plasmids are emerging in an increasing number of human and zoonotic bacterial pathogens occupying distinct ecologic niches and originating in widely dispersed geographic sites, they pose a severe threat to global public health.

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V. cholerae O139 strains. The first O139 strain in Hangzhou emerged in 1994, 1 year after the first O139 strains in China were found in the Xinjiang Autonomous Region (33). A total of 179 ctxA-positive O139 strains (97 from patients, 74 from close contacts, and 8 from environmental specimens related to cholera cases) were isolated in Hangzhou during 1994 and 1996 to 2006 (Table 1). Due to the dramatic decrease in the number of serogroup O1 isolates, O139 has become the dominant V. cholerae serogroup in Hangzhou since 2000 (31). In 2004, O139 caused an outbreak from which 65 O139 strains were isolated. In the present study, 69 O139 isolates, including 3 of the 65 isolates from the 2004 outbreak and 66 of 114 isolates from sporadic cases and small outbreaks, were chosen for investigation of the relationship between resistance and mobile genetic elements. Generally, the collection of O139 isolates from sporadic cases and small outbreaks covered the early, middle, and late periods in each epidemic year and all years between 1994 and 2006 in which O139 cholera cases were reported in Hangzhou. Only one or two isolates from each small outbreak were chosen.

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TABLE 1. Distribution of pMRV150-like plasmids, class 1 integrons, and SXT elements among O139 isolates

Susceptibility testing. The susceptibilities of the O139 isolates to ampicillin, amikacin, streptomycin, gentamicin, tetracycline, chloramphenicol, SXT, and ciprofloxacin were tested by the disk diffusion method on Mueller-Hinton agar according to the guidelines of the NCCLS (now CLSI) (30). The interpretive criteria for susceptibilities to ampicillin, tetracycline, chloramphenicol, and SXT were based on the standards for V. cholerae established by the CLSI, and susceptibilities to amikacin, streptomycin, gentamicin, and ciprofloxacin were interpreted using the CLSI criteria for the Enterobacteriaceae (8). Escherichia coli ATCC 25922 was used as a quality control strain in these experiments. The susceptibility data for the V. cholerae isolates tested were recorded only when the zone diameter for E. coli ATCC 25922, tested in parallel, was within the acceptable range given in the CLSI guidelines.

Conjugation experiments. Conjugation experiments were performed by mixing the donor (O139 isolates resistant to tetracycline or gentamicin and susceptible to rifampin) and recipient (E. coli strain C600, resistant to rifampin and susceptible to tetracycline and gentamicin) strains at a ratio of 1:1, streaking 100 µl of the mixture onto a Millipore 0.45-µm-pore-size filter on an LB agar plate, and incubating the filter for 4 h at 37°C. After mating, bacteria were collected, washed, diluted, and plated on a medium containing appropriate drug concentrations (either 16 µg/ml tetracycline and 16 µg/ml rifampin or 8 µg/ml gentamicin and 16 µg/ml rifampin). The frequency of transfer was expressed as the number of transconjugants per donor cell in the mating mixture at the time of plating. To check for cotransfer of resistance, the resistance patterns of the transconjugants and the recipient strain were also determined by the disk diffusion method using the interpretive criteria for the Enterobacteriaceae (8).

Plasmid analysis. Plasmids of O139 isolates and transconjugants were extracted as described previously (38). The large conjugative plasmid R388 in E. coli DH5 (from D. Mazel) was used as a control for the extraction process.

Identification of conjugative elements in O139 isolates. To identify a hypothetical conjugative element in the transconjugants, random amplified polymorphic DNA (RAPD) analysis was performed with a transconjugant from O139 isolate 0468, with the most common resistance pattern, and the recipient strain (E. coli strain C600). The RAPD bands present in the transconjugant and absent in the recipient were purified, cloned into the pGEM-T Easy vector (Promega), and sequenced. Forty 10-mer RAPD primers (named S1 to S40) were purchased from Shanghai Sangon Co., Ltd. A primer was used in each RAPD experiment. The RAPD program was as follows: an initial cycle at 94°C for 4 min; then 35 cycles of 94°C for 30 s, 36°C for 30 s, and 72°C for 3 min; and a final step at 72°C for 5 min. The fingerprints of amplification products were viewed in ethidium bromide-stained agarose gels under UV light after electrophoresis.

When the sequences of two fragments of the hypothetical conjugative element were obtained, two pairs of specific PCR primers, RS22F-RS22R and RS27F-RS27R (Table 2), were designed for a duplex PCR, which was performed on O139 donors, their transconjugants, and the E. coli recipient to check for the presence of the two specific fragments.

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TABLE 2. PCR primers designed in this study

To characterize the hypothetical conjugative element, pulsed-field gel electrophoresis (PFGE) was applied to XbaI- or NotI-digested genomic DNA from O139 donors, their transconjugants, and the recipient, according to the 1-day standardized protocols for subtyping of E. coli O157:H7 and V. cholerae developed by PulseNet, Centers for Disease Control and Prevention, Atlanta, GA (http://www.cdc.gov/pulsenet/protocols.htm). Southern blot analysis was used to determine whether each of the conjugally transferred bands viewed in the PFGE patterns was specific to the sequence of one of the RAPD bands. XbaI-restricted genomic DNAs were transferred and fixed to a positively charged nylon membrane (Millipore), hybridized with a digoxigenin-labeled RS27F-RS27R fragment probe, and detected according to the manufacturer's manual for the DIG High Prime DNA labeling and detection kit (Roche).

Multisite PCRs with 13 primer sets (repAF-repAR and R1F-R1R to R12F-R12R) were used to scan for pIP1202 backbone regions in O139 donors and transconjugants as described previously (37). The full-length repA gene, encoding the protein necessary for plasmid replication and control, with its two flanking segments was amplified by the RepAF868-RepAR2619 primer set (Table 2); these primers are specific to two open reading frames (ORFs) upstream and downstream of repA on the pIP1202 backbone (GenBank accession number CP000603). These PCR products from O139 isolate 0468 were directly sequenced.

Plasmids, SXT elements, and class 1 integrons in V. cholerae isolates. A total of 69 ctxA-positive O139 isolates (collected in 1994 and 1996 to 2006) and 18 ctxA-positive O1 isolates (1 of serotype Inaba and 17 of serotype Ogawa; collected in 1997 to 2001 in Hangzhou) were screened by PCR for the presence of pIP1202-like plasmids, SXT elements, and class 1 integrons. Three primer sets, RS22F-RS22R, RS27F-RS27R, and repAF-repAR (37), were used to amplify markers of pIP1202. PCR with primers INT1 and INT2 for the SXT element integrase gene int_SXT was performed as described previously (22). Class 1 integrons were analyzed by PCR using three sets of primers as described previously: intI1L and intI1R, in-F and in-B, and qacE1-F and sul1-B, covering the 5' conserved segment, the cassette region, and the 3' conserved segment of the class 1 integron, respectively (32). The PCR products obtained by using primers in-F and in-B were sequenced to determine the gene cassette arrays of the class 1 integrons.

Nucleotide sequence analysis and accession number. Nucleotide sequence analysis was carried out using the basic local alignment search tool (http://www.ncbi.nlm.nih.gov/BLAST/). The sequences of 2 RAPD fragments, 12 PCR products (R1 to R12) obtained with primer sets R1F-R1R to R12F-R12R, and the full-length repA gene of the pIP1202-like plasmid pMRV150 from O139 isolate 0468 were deposited in GenBank under accession numbers EU116442, EU116443, and EU315225, respectively. Representative sequences of class 1 integron cassettes found in O139 isolates were deposited in GenBank under accession numbers DQ79597, DQ789598, EU1161140, and EU116441.

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Identification of the pIP1202-like plasmid pMRV150 in O139 isolate 0468. O139 isolate 0468, one of 65 isolates recovered from an outbreak in 2004 in Hangzhou, showed resistance to ampicillin, streptomycin, gentamicin, tetracycline, chloramphenicol, and SXT, which was the most frequent resistance pattern found in this study (Table 3). Cotransfer of these resistances from isolate 0468 to an E. coli recipient was observed with a transfer frequency of 5.12 x 10^–7 (Table 4). O139 isolate 0468 harbored the SXT element (positive for the integrase gene int_SXT). However, 10 transconjugants selected randomly were all negative for int_SXT, suggesting that another conjugative element might be a major contributor to mediating MDR in O139 isolate 0468 and conjugal resistance transmission between bacteria, rather than the SXT element.

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TABLE 3. Resistance patterns of O139 isolates with or without pMRV150-like plasmids

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TABLE 4. Conjugation experiments with pMRV150-like plasmid-bearing O139 isolates and characterization of pMRV150-like plasmids

No plasmid was identified in O139 isolate 0468 or its transconjugant by a method commonly used for Vibrio plasmid extraction (38) or by use of several commercial extraction kits based on alkaline lysis. We attempted to obtain some genetic information on the probable conjugative element by comparing the RAPD fingerprint of the 0468 transconjugant with that of the control recipient strain. Two bands of 916 bp and 1,185 bp, amplified by RAPD primers S22 (5'-TGCCGAGCTG-3') and S27 (5'-GAAACGGGTG-3'), respectively, were found to be unique to the transconjugant. Sequence analysis revealed that the two bands (termed the S22 and S27 fragments, respectively [GenBank accession numbers EU116442 and EU116443]) were highly similar to the corresponding regions of pIP1202 in Y. pestis bv. Orientalis strain IP275 (37), pP91278 in Photobacterium damselae subsp. piscicida strain USA91278 (25), pP99-018 in P. damselae subsp. piscicida strain PT99-018 (25), pYR1 in Y. ruckeri strain YP71 (37), and pSN254 in Salmonella serovar Newport strain SL254 (37) (Table 5). Thus, we hypothesized that there might be a pIP1202-like conjugative element in O139 isolate 0468.

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TABLE 5. Nucleotide identities of pMRV150 backbone regions with their counterparts in other characterized plasmids

The presence of the S22 and S27 fragments in both O139 isolate 0468 and its transconjugant was confirmed by duplex PCR with primer sets RS22F-RS22R and RS27F-RS27R, specific to the S22 and S27 fragments, respectively (Table 4). Moreover, O139 isolate 0468 and its transconjugant were also positive in all specific PCRs covering 13 backbone regions of pIP1202 (Table 4). The available sequences of these 15 PCR fragments from O139 isolate 0468, including the S22 fragment (GenBank accession number EU116442), the S27 fragment (EU116443), repA with its two flanking segments, and 12 PCR products (R1 to R12) obtained with primer sets R1F-R1R to R12F-R12R (EU315225), had a total length of 22,183 bp and were highly similar to their counterparts in pIP1202, pP99-018, pP91278, pSN254, and pYR1 (Table 5). The first 864 nucleotides and 288 amino acids of the putative repA gene and its product from O139 isolate 0468 were 93.87% (811/864) and 98.26% (283/288) identical, respectively, to those of the repA gene and protein of pRA1, an IncA/C plasmid, in E. coli (29), suggesting that the pIP1202-like conjugative element in O139 isolate 0468 belongs to the IncA/C plasmid incompatibility group. This evidence further supported our hypothesis. The pIP1202-like conjugative plasmid in O139 isolate 0468 was named pMRV150.

Resistance patterns of O139 isolates with or without pMRV150-like plasmids. Among 69 O139 isolates recovered during 1994 and 1996 to 2006, 51 isolates recovered during 1997 to 2006 were positive for all three markers of pMRV150 obtained with primer sets RS22F-RS22R, RS27F-RS27R, and repAF-repAR and thus were considered to bear pMRV150-like plasmids (Table 3). These plasmid-bearing isolates were resistant to two to six drugs, and most (42/51) were resistant to five to six drugs (Table 3). In contrast, 18 plasmid-negative isolates recovered during 1994, 1996 to 2000, and 2004 were resistant only to one to three drugs (Table 3). This indicated a strong relationship between the presence of pMRV150-like plasmids and the MDR phenotype in O139 isolates.

Characterization of pMRV150-like plasmids in O139 isolates. In conjugation experiments with 12 O139 isolates harboring pMRV150-like plasmids and resistant to three to six antibiotics, high plasmid transfer frequencies of 4.50 x 10^–2 and 3.08 x 10^–2 for 2 isolates and relatively lower frequencies, ranging from 0.88 x 10^–8 to 3.29 x 10^–5, for 8 isolates were observed, while no transconjugants could be obtained from 2 isolates (detection limits were 0.75 x 10^–8 and 0.47 x 10^–8, respectively) (Table 4). Resistance to ampicillin, streptomycin, gentamicin, tetracycline, chloramphenicol, and SXT in the 10 transconjugant-producing isolates could be cotransferred from donors to E. coli recipients along with pMRV150-like plasmids, except for resistance to streptomycin, SXT, and gentamicin in 2 isolates (Table 4). These pMRV150-like plasmids in the 12 isolates and their transconjugants were positive for all or almost all of 15 pIP1202 backbone regions tested by PCR screening (Table 4). Cotransfer of the class 1 integron along with pMRV150-like plasmids was also found by the conjugation experiments for all 9 class 1 integron-bearing and transconjugant-producing isolates, while cotransfer of the SXT element was not observed for any of the 10 SXT element-bearing and transconjugant-producing isolates (Table 4).

Compared with the PFGE pattern of the recipient, transferred DNA bands were identified at approximately 104 kb (isolate 9868), 140 kb (isolate 001), and 150 kb (the other 8 isolates) in the XbaI-digested PFGE patterns of the 10 transconjugant-producing O139 isolates and their transconjugants. In NotI-digested PFGE patterns, we observed bands of approximately 170 kb (isolate 9868), 130 kb (isolate 001), and 125 kb to 138 kb (the other eight isolates) (Fig. 1). The digoxigenin-labeled probe specific to the S27 fragment hybridized to the transferred DNA bands at these locations in Southern blot analysis, confirming that the bands contained pMRV150-like plasmids (Fig. 1). Therefore, the sizes of pMRV150-like plasmids in the O139 isolates were estimated as ranging from approximately 140 kb to 170 kb, with most being 150 kb. These transferred bands did not cause any perceptible change of the PFGE background pattern of the recipient in the transconjugants (Fig. 1); hence, these conjugative elements did not appear to be integrated into the chromosome of the recipient. Judging from the fact that the densities of these transfer bands were close to those of most background genomic bands of donors and transconjugants, the copy number of the pMRV150-like plasmids was very low, approximately 1 or 2 copies per cell.

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FIG. 1. Conjugative transfer of pMRV150-like elements as shown by PFGE and Southern blot analysis. (A) XbaI-digested PFGE patterns of V. cholerae O139 donors and transconjugants and the E. coli recipient. (B) Southern blot analysis with a probe specific to the S27 fragment on positively charged nylon membranes containing the XbaI-digested genomic DNAs transferred from PFGE gels. (C) NotI-digested PFGE patterns of V. cholerae O139 donors and transconjugants and the E. coli recipient. Strain names are given above the gels. Tc, transconjugant. Open arrowheads indicate transfer bands.

Distribution of pMRV150-like plasmids, SXT elements, and class 1 integrons in V. cholerae isolates. The SXT element emerged along with the first O139 isolate found in Hangzhou during 1994 and was present among almost all (68/69) tested O139 isolates collected after that time (Table 1). Unlike the SXT element, the pMRV150-like plasmid was first identified in 1 O139 isolate during 1997 and was found in only 1 (7.69%) of the 13 O139 isolates tested during the early period of the O139 epidemic in Hangzhou (1994, 1996, and 1997). The plasmid-bearing O139 isolates, however, gradually came to dominate, from 3 (60.00%) of 5 isolates during 1998 and 1999 to 47 (92.16%) of 51 isolates during 2000 to 2006 (Table 1). The emergence of the pMRV150-like plasmids correlated better than the presence of SXT elements with the appearance of MDR in the O139 isolates, suggesting a key role for the pMRV150-like plasmids in the MDR phenomenon of O139 isolates in Hangzhou.

Consistent with the finding of cotransfer of the class 1 integron along with pMRV150-like plasmids in conjugation experiments, all 46 class 1 integrons detected in this study were found only in the plasmid-bearing O139 isolates, although in a few (n = 5) plasmid-bearing O139 isolates (Table 1), the class 1 integron was missing. A total of four gene cassette arrays were identified. Most resistance genes in these cassettes were members of the aadA or dfrA family, encoding aminoglycoside adenylyltransferase or dihydrofolate reductase and conferring resistance to streptomycin/spectinomycin or trimethoprim, respectively. In the cassette array of dfrA12 and aadA2, detected in an isolate obtained in 1997, a retroelement, a group II intron, was found to insert into the attC site of the aadA2 cassette.

The SXT element also predominated in 15 (83.33%) of 18 ctxA-positive O1 isolates in Hangzhou during 1997 to 2001; however, both pMRV150-like plasmids and the class 1 integron were absent in these O1 isolates.

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Cholera and plague, caused by two gram-negative bacteria, V. cholerae and Y. pestis, respectively, are notorious as two of the most devastating pandemic diseases in human history. Effective antibiotic treatment can reduce the volume and duration of diarrhea and the period of pathogen excretion in cholera cases (34) and is essential to the dramatic drop in mortality in human plague cases (23). The emergence of MDR strains of V. cholerae and Y. pestis, however, is becoming a global public health concern. Our present study demonstrated that V. cholerae O139 isolates were resistant to one to three drugs during their early epidemic period in Hangzhou and have acquired resistance to as many as five to six drugs commonly used in cholera therapy and prophylaxis owing to the appearance of pMRV150-like conjugative plasmids. Moreover, the plasmid-bearing O139 isolates seemed to be selected for during the epidemic and have become dominant in Hangzhou since 2000. These plasmids had a backbone almost identical with that of pIP1202, an IncA/C conjugative plasmid originally found in a multiple-antibiotic-resistant Y. pestis isolate (strain IP275) from a bubonic plague patient in Madagascar, suggesting that they shared a recent common ancestor.

The pIP1202-like plasmids have also been found recently among many gram-negative bacteria occupying diverse niches and with global geographical distribution. These bacteria include isolates of multiple S. enterica serotypes, E. coli, and Klebsiella spp. from retail meats and E. coli isolates from food animals in several states of the United States, the fish pathogen Y. ruckeri in freshwater ecosystems (37), and strains of the marine fish pathogen P. damselae subsp. piscicida in the United States and Japan (25). The extended transmission of pIP1202-like plasmids in the microbial ecosystem might be caused by the increasing selective pressure of mass antibiotic usage in medicine and agriculture. The presence of these plasmids should be carefully monitored in order to comprehensively evaluate their impact on global public health and to prepare for the emerging risk posed by these elements.

O139 isolates with MDR patterns similar to those of the plasmid-bearing isolates from Hangzhou have been found in several coastal regions in southern and eastern China, including Fujian, Zhejiang, Shanghai, and Guangxi, since the late 1990s and early 2000s (7, 28, 35, 39). However, the role of pMRV150-like plasmids in MDR in these isolates remains to be confirmed. IncA/C conjugative plasmids mediating MDR to tetracycline, ampicillin, chloramphenicol, kanamycin, gentamicin, streptomycin, sulfamethoxazole, trimethoprim, and O/129 were found in six O139 strains from India as early as 1992 to 1993 (38). IncA/C plasmids were also detected in MDR O1 strains from Southeast Asia and the southern Soviet Union even before the mid-1970s (20, 21) and in O1 strains from Asia and Africa during the 1980s and 1990s (14, 15, 18, 19, 27). Recently, class 1 integron-bearing conjugative plasmids were found in O1 strains from Europe and Africa (6, 9). Unfortunately, these plasmids in O1 and O139 strains were not characterized sufficiently to identify the possible relationships among them or between them and plasmids in other bacteria.

In summary, we report that pMRV150-like plasmids, effective disseminators of multiple antibiotic resistance, play a major role in the appearance of resistance in V. cholerae O139 isolates. The emergence of these plasmids in many bacterial pathogens and nonpathogens that occupy diverse niches and have a widespread geographical distribution, and especially in two devastating human pathogens, suggests an increasing risk to public health worldwide. Careful tracking of these plasmids within microbial populations is needed.

 
This work was supported by Training Project 131 for Scientists, government of Hangzhou, China, and by the Zhejiang Provincial Natural Science Foundation of China under grant Y207076.

We thank David Dyer for careful review and revision of the manuscript.

 
* Corresponding author. Mailing address: Microbiology Laboratory, Hangzhou Center for Disease Control and Prevention, Hangzhou, Zhejiang, 310006, People's Republic of China. Phone: 86-571-85177696. Fax: 86-571-85165007. E-mail: jingcaopan{at}sina.com

Published ahead of print on 18 August 2008.

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Antimicrobial Agents and Chemotherapy, November 2008, p. 3829-3836, Vol. 52, No. 11
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