J Med Microbiol 56 (2007), 346-351; DOI: 10.1099/jmm.0.46655-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Multiplex PCR for detection of antibiotic resistance genes and the SXT element: application in the characterization of Vibrio cholerae
Dhanya Ramachandran1,
R. Bhanumathi1,
and
Durg V. Singh2
1 Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram-695 014, India
2 Institute of Life Sciences, Nalco Square, Bhubaneswar-751 023, India
Correspondence
Durg V. Singh
durg_singh{at}yahoo.co.in
Received 3 April 2006
Accepted 26 October 2006
This study describes a multiplex PCR assay for the detection of antibiotic resistance genes and the SXT element in Vibrio cholerae. Conditions were optimized to amplify fragments of sulII (encoding sulfamethoxazole resistance), dfrA1 (O1-specific trimethoprim resistance), dfr18 (O139-specific trimethoprim resistance), strB (streptomycin B resistance) and the SXT element simultaneously in one PCR. This multiplex PCR was evaluated on 142 V. cholerae isolates and the results correlated with the phenotypic antibiotic data obtained using a disc diffusion assay and a colony blot assay. Thus this one-step PCR can be used as a simple, rapid and accurate method for identification of antibiotic resistance profiles and could be used for the surveillance of the spread of antibiotic resistance determinants in epidemiological and environmental studies.
Abbreviations: Cm, chloramphenicol; Fr, furazolidone; Str, streptomycin; Sul, sulfamethoxazole; Tmp, trimethoprim.
Present address: Patent Office, Chennai Branch, IPR Building, GST Road, Chennai-600 032, India. 
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INTRODUCTION
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Vibrio cholerae is the causative agent of cholera, a severe and sometimes lethal disease. Multiple drug resistance in V. cholerae has been reported frequently, usually after acquisition by strains of a conjugative plasmid (Falbo et al., 1999; Glass et al., 1983; Tabtieng et al., 1989). Other genetic elements, such as the class 1 integron and SXT element, have also been reported to carry genetic determinants for antimicrobial resistance (Dalsgaard et al., 2000, 2001; Hochhut et al., 2000, 2001; Iwanaga et al., 2004; Thungapathra et al., 2002). Historically, only the O1 serogroup of V. cholerae has been the causative agent of epidemic cholera. However, in late 1992, in India and Bangladesh, a novel serogroup designated V. cholerae O139 emerged, replacing V. cholerae O1, and gave rise to major cholera outbreaks (Ramamurthy et al., 1993). These strains were characteristically resistant to the antibiotics furazolidone (Fr), sulfamethoxazole (Sul), trimethoprim (Tmp), chloramphenicol (Cm) and low concentrations of streptomycin (Str). Genes encoding resistance to these antibiotics were found to be located on a novel transmissible genetic element, designated SXT (Waldor et al., 1996).
After the extensive cholera outbreaks caused by V. cholerae O139, V. cholerae O1 El Tor strains re-emerged in 1994 as the predominant cause of cholera on the Indian continent. In contrast to El Tor strains isolated before the O139 outbreak, the re-emerged El Tor strains, like the initial O139 isolates, were resistant to Fr, Sul, Tmp, Cm and Str (Yamamoto et al., 1994). The corresponding genes were found to be located in an integrating conjugative element that was closely related but not identical to the O139 SXT element (Hochhut & Waldor, 1999; Waldor et al., 1996). Variations were noted in recent V. cholerae O139 isolates from India, i.e. they were generally no longer resistant to Sul and Tmp, and showed varying resistance to Str (Mitra et al., 1996; Sinha et al., 2002; Yam et al., 1994).
As the SXT genetic element plays a role in the acquisition of antibiotic resistance, it is important to look for the presence of the sulII (encoding Sul resistance), dfrA1 (O1-specific Tmp resistance), dfr18 (O139-specific Tmp resistance) and strB (Str B resistance) genes and the SXT element (Bhanumathi et al., 2003; Falbo et al., 1999; Hochhut et al., 2001) in V. cholerae strains. To trace the changes in antibiotic resistance, we developed a multiplex PCR for the detection of antibiotic resistance genes and the SXT element and used it to characterize V. cholerae strains.
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METHODS
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Bacterial strains.
A total of 142 strains of V. cholerae were included in this study. Of these, 72 were serogroup O1, 62 were serogroup O139 and eight were non-O1, non-O139 serogroup obtained from laboratory stocks identified previously using standard bacteriological methods (World Health Organization, 1987). All isolates were examined for their oxidase reaction, and the identities of the V. cholerae O1 strains were confirmed by serogrouping using growth from triple-sugar iron agar slants with polyclonal O1 and monospecific Inaba and Ogawa antisera. V. cholerae strains that did not agglutinate with O1 antiserum were checked with O139 monoclonal antibody supplied by the WHO Regional Office of South East Asia, New Delhi, India. V. cholerae strains that did not agglutinate with either O1 or O139 antisera were assumed to belong to non-O1, non-O139 serogroups. V. cholerae O1 strains were further biotyped using multiplex PCR by exploiting sequence differences between classical and El Tor biotype tcpA genes to distinguish the two biotypes (Keasler & Hall, 1993). The cities in India from which the V. cholerae strains were obtained are shown in Fig. 1
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Fig. 1. Map showing the different cities in India from where the Vibrio cholerae strains were obtained. Not to scale.
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V. cholerae O139 strains ATCC 51394, CO594 and VO143 were used as the PCR positive controls for sulII, dfr18, strB and SXT; and O1 biotype El Tor strains KO194 for sulII, dfrA1, strB and SXT and TO267 for dfrA1 and SXT, respectively. V. cholerae O1 biotype El Tor strain VO101 was used as a negative control. All strains were maintained in tryptic soy broth (Difco) supplemented with 30 % glycerol and stored at –70 °C. Before use, the identification of each culture was confirmed by selected biochemical tests and serology (World Health Organization, 1987).
Multiplex PCR.
V. cholerae strains grown overnight at 37 °C in Luria–Bertani broth (USB, Amersham Life Technology) were boiled and stored at –20 °C until use. The bacterial cell lysate served as the source of template DNA. Primer sequences for the antibiotic resistance genes are shown in Table 1.
PCR amplification of target DNA was carried out in a thermal cycler (Bio-Rad) in a 200 µl PCR tube containing a reaction mixture volume of 30 µl. Each reaction contained 2.5 µl 10x amplification buffer [500 mM KCl, 100 mM Tris/HCl (pH 9.0), 1.0 % Triton X-100; Promega], 2.0 µl 25 mM MgCl2, 2.5 µl each 2.5 mM dATP, dCTP, dGTP and dTTP (Amersham Pharmacia Biotech), 0.9 µl 3 % DMSO (Sigma), 1.2 µl forward and reverse primers for SXT (20 ng µl–1), 0.25 µl Taq DNA polymerase (5 U µl–1; Promega), Milli-Q water to a final volume of 27.5 µl, and 2.5 µl cell lysate (template DNA). PCR cycling conditions were as follows: initial denaturation at 94 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 60.5 °C for 1 min and 72 °C for 1 min, with a final extension at 72 °C for 10 min.
Multiplex PCR was carried out by the simultaneous addition of primer pairs for sulII, dfr18, dfrA1 and strB in the same reaction mixture. In initial experiments, sulII, dfr18, dfrA1 and strB primer concentrations varied between 6 and 25 µM, keeping the SXT primer concentration fixed at either 12 or 15 µM in the reaction mixture of 30 µl. Optimum results were obtained with primer concentrations of 6 µM for sulII, 9 µM for dfr18, 10 µM for strB and 25 µM for dfrA1 and 12 µM for SXT. PCR conditions for amplification were maintained as described above. Amplified products were separated by agarose (1.5 %) gel electrophoresis in 0.5x TBE, stained in ethidium bromide and visualized with a Fluoro-S-MultiImager (Bio-Rad). PCR-negative and -positive strains were verified by Southern blot hybridization.
Probes and hybridization.
Colony blots were prepared using nylon membranes (Hybond-N+; Amersham International) and processed using a standard method (Sambrook & Russell, 2001). Briefly, colony blots were denatured in denaturing solution (0.5 M NaOH, 1.5 M NaCl), neutralized in neutralizing solution (0.5 M Tris/HCl, pH 8.0; 1.5 M NaCl) and the DNA was fixed on to nylon membranes by exposure to UV light for 2 min, in accordance with the manufacturer's instructions.
PCR-amplified products obtained from V. cholerae O139 strain ATCC 51394 for the sulII, dfr18 and strB genes and SXT and from V. cholerae O1 strain KO194 for the dfrA1 gene were used as probes and were randomly labelled (Feinberg & Vogelstein, 1984) with [
-32P]dCTP (3000 Ci mmol–1; BARC) and hybridized at 65 °C in phosphate buffer containing 500 mM Na2HPO4 (pH 7.2), 7 % SDS, 1 mM EDTA and 1 % BSA. Hybridized blots were washed once in 2x SSC for 5 min at room temperature, twice in 2x SSC/0.1 % SDS for 15 min at 65 °C and once in 0.1x SSC/0.1 % SDS for 15 min at 65 °C. Autoradiographs were developed from the hybridized filters with the Bio-Rad PhosphorImager screen and visualized in a PhosphorImager (Bio-Rad).
Antibiotic susceptibility testing.
V. cholerae strains were tested for antibiotic susceptibility using the method of Bauer et al. (1966) using the following antibiotics (Hi-Media Laboratories): ampicillin (10 µg), Cm (30 µg), ciprofloxacin (5 µg), Fr (100 µg), gentamicin (10 µg), neomycin (30 µg), norfloxacin (10 µg), nalidixic acid (30 µg), polymyxin B (50 U), Str (30 µg), Tmp–Sul (25 µg), Tmp (5 µg), tetracycline (30 µg) and the vibriostatic agent O/129 (10 and 150 µg). A 3 h culture of V. cholerae in Mueller–Hinton broth (Difco) was spread plated on well-dried Mueller–Hinton agar (Difco). Plates were incubated for 24 h at 37 °C. Characterization of strains as susceptible or resistant was based on the size of inhibition zone around each disc, according to the manufacturer's instructions, which matched the interpretive criteria recommended by the World Health Organization (1993).
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RESULTS
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V. cholerae strains belonging to serogroups O1, O139 and non-O1, non-O139 were examined for the presence of antibiotic resistance genes and the SXT element. Two strains of V. cholerae O1, one each isolated from Varanasi in 1976 and 1992, gave negative PCR results for the sulII, dfr18, dfrA1 and strB genes and the SXT element (Fig. 2a, b, and Table 2). These strains, when tested for antibiotic susceptibility, were sensitive to all of the antibiotics tested, whereas 51 strains of V. cholerae O1, of which six were isolated from Kottayam in 2000, 26 from Trivandrum in 2000 and 2003, and 19 from Varanasi in 1994, 1995, 1996, 1998 and 1999, gave positive results by PCR and amplified a 626 bp fragment of sulII, a 278 bp fragment of dfrA1, a 515 bp fragment of strB and a 1035 bp fragment of the SXT element (Fig. 2a, b). All of these strains showed resistance to ampicillin, Fr, nalidixic acid, Str, Tmp-Sul and Tmp (Table 2). Furthermore, 19 strains, of which seven were isolated from Alleppey in 2000 and 12 from Trivandrum in 2002, when tested by multiplex PCR, gave positive results for the dfrA1 gene and the SXT element. They were resistant to ampicillin, Fr and Tmp, but not to Tmp–Sul (Table 2). All of them were resistant to the vibriostatic agent O/129.
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Fig. 2. Agarose gels of multiplex PCR products stained with ethidium bromide. (a) Lanes: M, 100 bp DNA ladder; 1–3, V. cholerae O139 strains ATCC 51394 (isolated in Madras, 1992), CO594 (Kolkata, 1992) and VO143 (Varanasi, 1992) (SXT+ sulII+ dfr18+ strB+); 4, V. cholerae O1 strain VO101 (Varanasi, 1975) (SXT– sulII– dfrA1– strB–); 5, V. cholerae O1 El Tor strain KO194 (Kottayam, 2000) (SXT+ sulII+ dfrA1+ strB+); 6, V. cholerae O1 El Tor strain TO267 (Trivandrum, 2003) (SXT+ suIIl– dfrA1+ strB–). (b) Multiplex PCR products of representative V. cholerae O1, O139 and non-O1, non-O139 strains. Lanes: M, 100 bp DNA ladder; 1–4, V. cholerae O139 strains CO129 and CO130 (Kolkata, 1992), and VO135 and VO144 (Varanasi, 1992) (SXT+ sulII+ dfr18+ strB+); 5 and 6, V. cholerae O139 strains TO255 (Trivandrum, 2002) and WO167 (Wardha, 1998) (SXT+ sulII– dfr18– strB–); 7, V. cholerae non-O1, non-O139 strain VO222 (Varanasi, 1987) (SXT+ sulII– dfr18– strB–); 8, 10 and 12, V. cholerae O1 El Tor strains KO52 (Kottayam, 2000), TO69 (Trivandrum, 2000) and TO289 (Trivandrum, 2003) (SXT+ sulII+ dfrA1+ strB+); 9 and 11, V. cholerae O1 El Tor strains AO60 (Alleppey, 2000) and TO274 (Trivandrum, 2002) (SXT+ sulII– dfrA1+ strB–); 13, V. cholerae O1 El Tor strain VO102 (Varanasi, 1976) (SXT– sulII– dfrA1– strB–).
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Table 2. Results of analysis employing multiplex PCR to study Vibrio cholerae O1, O139 and non-O1, non-O139 strains isolated from different geographical locations in India
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Twenty-one V. cholerae O139 strains, of which one was isolated from Alleppey in 2000, two from Kolkata in 1992, and 18 from Varanasi in 1992, 1993 and 1994, gave positive results by PCR and amplified a 626 bp fragment of sulII, a 389 bp fragment of dfr18, a 515 bp fragment of strB and a 1035 bp fragment of the SXT element (Fig. 2a, b
). These strains were resistant to ampicillin, Fr, Str, Tmp-Sul and Tmp and to vibriostatic agent O/129 (Table 2). However, 41 strains of V. cholerae O139, comprising 14 from Alleppey, 11 from Trivandrum and 16 from Wardha, were SXT positive when tested by PCR. They were resistant to ampicillin, Fr and nalidixic acid and sensitive to all of the other antibiotics tested, including the vibriostatic agent O/129 (Table 2).
One of eight V. cholerae non-O1, non-O139 strains was SXT positive. The other strains were negative for the sulII, dfr18, dfrA1 and strB genes and for the SXT element (Fig. 2b). All eight strains were resistant to ampicillin (Table 2).
All V. cholerae O1, O139 and non-O1, non-O139 strains negative for the sulII, strB and/or dfr18/dfrA1 genes and for the SXT element by PCR were also negative in the colony blot assay, whereas representative V. cholerae O1, O139 and non-O1, non-O139 strains positive for the sulII, strB and/or dfr18/dfrA1 genes and for the SXT element by PCR were also positive in the colony blot assay (data not shown).
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DISCUSSION
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Antibiotic therapy shortens the duration of diarrhoea caused by cholera (Lindenbaum et al., 1967), but the use of antibiotics has contributed to resistance in V. cholerae. The SXT element is a 62 kb, conjugative, self-transmissible, integrating element encoding resistance to Sul, Tmp, Cm and Str (Waldor et al., 1996). Furthermore, SXT encodes an integrase at its 5' end required for SXT transfer. The SXTMO10 element was first detected in the newly emerged O139 serogroup of V. cholerae in 1992. Since 1994, V. cholerae O1 strains isolated from India, Bangladesh, Mozambique and Laos have been found to contain the SXT element (Amita et al., 2003; Dalsgaard et al., 2001; Hochhut et al., 2001; Iwanaga et al., 2004). In El Tor V. cholerae strains, the resistance genes are located in SXTET.
The results obtained in this study showed that V. cholerae O1 El Tor strains isolated from Alleppey, Kottayam and Trivandrum were resistant to Tmp and vibriostatic agent O/129 and positive by multiplex PCR for the dfrA1 gene, indicating that genes encoding resistance to Tmp and O/129 are linked, a finding further supported by the fact that O139 V. cholerae strains isolated up to 1994 were resistant to Tmp and vibriostatic agent and possessed the dfr18 gene, whereas strains isolated after 1996 from different regions of India were susceptible to Tmp and vibriostatic agent and lacked dfr18. However, further study is needed to confirm the presence of Tmp and vibriostatic agent O/129 genes in the SXT element.
In conclusion, there is a difference in the antibiotic resistance gene clusters in the SXT element in V. cholerae O1 and O139, corroborating the results of earlier workers (Hochhut et al., 2001) who reported differences in antibiotic resistance and suggested that these genes were not intrinsic features of this family of integrase, but rather appeared to have been inserted into these elements, becoming transmissible bacterial populations. At least one non-O1, non-O139 V. cholerae strain possessed the SXT element, suggesting a role in the acquisition of antibiotic resistance genes encoding Sul, Tmp and/or Str resistance. Thus one-step multiplex PCR can be used effectively to detect antibiotic resistance genes and could be used in surveillance of the spread of antibiotic resistance in epidemiological and environmental studies.
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ACKNOWLEDGEMENTS
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This research was supported by grant SR/SO/HS-51/2002 to D. V. S. from the Department of Science and Technology, New Delhi, India, and funds from the Department of Biotechnology, Government of India to Institute of Life Sciences, Bhubaneswar, India. D. R. and R. B. were supported by Senior Research Fellowships awarded by the Council of Scientific and Industrial Research, New Delhi, India. The authors are thankful to B. N. Shukla (Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India), P. Narang (Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Wardha, India) and G. B. Nair (National Institute of Cholera and Enteric Diseases, Kolkata, India) for providing the V. cholerae O1 and O139 strains. The authors are grateful to R. R. Colwell of the Maryland Institute for Advanced Computer Studies, University of Maryland at College Park, MD, USA, for critical reading of this manuscript.
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INTRODUCTION
METHODS
