1 Enteric Microbiology Laboratory, Laboratory Sciences Division, ICDDR, B: Centre for Health and Population Research, GPO Box-128, Dhaka-1000, Bangladesh; 2 National Public Health Laboratory, Kathmandu, Nepal; 3 National Institute of Cholera and Enteric Diseases, Kolkata, India; 4 Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1231, Shinjuku-ku, Tokyo 162, Japan
Received 12 April 2004; returned 13 June 2004; revised 2 August 2004; accepted 10 August 2004
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Abstract |
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Methods: The antimicrobial susceptibilities were examined by NCCLS methods. Molecular epidemiological characterization was performed by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and mutation analysis of the quinolone resistance-determining region (QRDR) of gyrA by sequencing.
Results: Plasmid patterns of the current ciprofloxacin-resistant strains from India, Nepal and Bangladesh were very similar to those of the 1978, 1984 and 1994 epidemic isolates of S. dysenteriae 1, except for the presence of a new plasmid of 2.6 MDa, which was found in one recent ciprofloxacin-resistant strain isolated in Bangladesh. PFGE analysis showed that the ciprofloxacin-resistant strains isolated in Bangladesh, India and Nepal belonged to a PFGE type (type A), which was possibly related to that of the 1984 and 1994 clone of S. dysenteriae 1, but different from 1978 epidemic strains. The current ciprofloxacin-resistant strains belong to five subtypes (A3A7), all of which were found in India, but in Bangladesh and Nepal, only A3 existed. Mutation analysis of the QRDR of gyrA revealed that amino acid substitutions at positions 83 and 87 of ciprofloxacin-resistant strains isolated in Bangladesh were similar to those of the strains isolated in Nepal, but different (at position 87) from ciprofloxacin-resistant strains isolated in India.
Conclusions: PFGE and mutation analysis of gyrA showed differences between the current ciprofloxacin-resistant S. dysenteriae 1 strains isolated in south Asia and those associated with epidemics in 1978, 1984 and 1994.
Keywords: pulsed-field gel electrophoresis , gyrA , QRDRs , epidemics
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Introduction |
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In this study, in order to ascertain its genesis, we compared the antimicrobial resistance pattern, genetic fingerprint and QRDR of gyrA of the current ciprofloxacin-resistant S. dysenteriae 1 strains isolated in south Asia, and the strains of S. dysenteriae 1 associated with the epidemics of 1978, 1984 and 1994.
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Materials and methods |
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The origin and period of isolation of the strains in the present study are displayed in Table 1. Five strains of S. dysenteriae type 1 used in this study were isolated from patients attending the Dhaka and Matlab treatment centre operated by ICDDR, B; and three strains from Nepal were sent to us from the National Public Health Laboratory, Kathmandu, Nepal. Previous epidemic strains of S. dysenteriae 1 in Bangladesh and from India (Bombay and Kolkata) were collected from our stock; ciprofloxacin-resistant outbreak strains from Eastern India were gifted by the National Institute of Cholera and Enteric Diseases, Kolkata, India. Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were used as control strains for susceptibility studies.
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According to the guidelines of the National Committee for Clinical Laboratory Standards,8 antibiotic susceptibility testing was conducted by disc diffusion using commercially available antibiotic discs (Oxoid, Basingstoke, UK). The antibiotic discs used in this study were tetracycline (30 mg), ampicillin (10 mg), sulfamethoxazole/trimethoprim (25 mg), nalidixic acid (30 mg), ciprofloxacin (5 mg), norfloxacin (10 mg), ofloxacin (5 mg), mecillinam (25 mg), azithromycin (15 mg) and ceftriaxone (30 mg). The MIC of ciprofloxacin and nalidixic acid were determined by the Etest (AB Biodisk, Solna, Sweden).
Plasmid profile analysis
Plasmid DNA was prepared by the alkaline lysis method of Kado & Liu, with some modifications.9 The molecular weight of the unknown plasmid DNA was assessed by comparison with the mobilities of plasmids of known molecular weights.1 Plasmids present in the previously described strains E. coli PDK-9, R1, RP4, Sa and V5171 were used as molecular weight standards.
Pulsed-field gel electrophoresis
Intact agarose-embedded chromosomal DNA of S. dysenteriae 1 was prepared according to the procedures described earlier.9 It was digested with XbaI restriction enzyme (Gibco-BRL, Gaithersburg, MD, USA); the restriction fragments were then resolved using CHEF-DRII system apparatus (Bio-Rad Laboratories, Richmond, CA, USA) in 1% pulsed-field certified agarose in 0.5 x TBE (Tris/borate/EDTA) buffer with the following pulse times, 110 s for 10 h, 328 s for 10 h, 335 s for 5 h and 570 s for 15 h. A photograph was taken using a gel documentation system, and banding patterns were established using the criteria described previously.10 Commercially available DNA of bacteriophage ladder (size range, 48.51000 kb; Bio-Rad Laboratories) and Saccharomyces cerevisiae (size range, 2252200 kb; Bio-Rad Laboratories) were used as the size standards.
PCR amplification of gyrA
Chromosomal DNA was prepared and purified by procedures described earlier.9 Previously recommended primers were used to amplify the QRDRs of gyrA,11 to produce a 648 bp product. Thirty µL of each reaction mixture contained 3.0 µL 10x PCR buffer (Invitrogen), 2.0 µL dNTPs (Invitrogen), 10 pmol of each primer, (Integrated DNA Technologies, Inc. USA), 1 µL of chromosomal DNA and 1 U of Taq DNA polymerase enzyme (Invitrogen). PCR conditions were adopted from elsewhere.11
Nucleotide sequencing
The PCR amplicons were purified with the GFX PCR DNA and gel band purification kit (Amersham Pharmacia, USA). They were then sequenced using the dideoxynucleotide chain termination method with an ABI PRISM BigDye Terminator Cycle Sequencing Reaction kit (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) on an automated sequencer (ABI PRISM 310) at the ICDDR, B core sequencing facility.
DNA and protein sequence analysis
The chromatogram sequencing files were inspected using Chromas 2.23 (Technelysium, Queensland, Australia), and contiguous sequences were prepared using SeqMan II (DNASTAR, Madison, WI, USA). Nucleotide and protein sequence similarity searches were performed using the National Center for Biotechnology Information (NCBI, National Institutes of Health, Bethesda, MD, USA) BLAST (Basic Local Alignment Search Tool) server on GenBank database, release 138.0.12 Multiple sequence alignments were developed using CLUSTALX 1.81.13 Sequences were manually edited in the GeneDoc version 2.6.002 alignment editor.
Nucleotide sequences accession number
The nucleotide sequences of gyrA reported in this paper were submitted to GenBank using the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) Sequin, version 5.26 under accession numbers AY69248084 and AY69582932.
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Results and discussion |
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Analysis of plasmid DNA showed that 140, 6 and 2 MDa plasmids were commonly present in all the strains regardless of origin of isolation. Based on the number and molecular mass of the plasmid, strains were categorized into four different patterns (P1P4). Among five strains in Bangladesh, four were characterized as P1 (140, 6, 4, 2) and the remaining one was P4 (140, 52, 6, 4, 2.6, 2). In Nepal, two strains were characterized as P1 and the remaining one was P3 (140, 6, 2). Plasmid patterns P1 and P2 (140, 9020, 6, 4, 2) were found in the previous epidemic isolates from India and Bangladesh (Table 1). The plasmid patterns of previous epidemic isolates from India and Bangladesh, recent outbreak strains in India and recent ciprofloxacin-resistant strains in Bangladesh and Nepal were almost identical except for one recent ciprofloxacin-resistant strain in Bangladesh, which had a 2.6 MDa plasmid in addition to the core plasmids of S. dysenteriae 1 (Table 1).1
In order to scrutinize the chromosomal variation and clonal distribution of the strains from different origins and periods, we employed PFGE analysis. According to the interpretation criteria described by Tenover et al.,10 two different PFGE types designated as A and B were identified among the epidemic and sporadic strains isolated at distinct time intervals from different geographical locations (Figure 1). Type A was further subdivided into seven subtypes (subtypes A1A7); of these, A1 and A2 were found among the strains isolated in Bangladesh and India during the epidemic of 1984. Epidemic clone A1 reemerged in Bangladesh in the following epidemic of 1994. Recent ciprofloxacin-resistant strains were grouped into subtypes A3A7, all of which were present among the outbreak strains in India, whereas only subtype A3 was found in Bangladesh and Nepal (Table 1). On the other hand, Type B, subdivided into two subtypes (subtypes B1 and B2), was found only in Bombay during the epidemic of 1978. It appeared that ciprofloxacin-resistant S. dysenteriae 1 strains are clustered within a single PFGE type, and the existence of identical clones in Bangladesh and Nepaland to some extent in Indiaare more suggestive of an epidemiologically relevant link between cases.
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Ciprofloxacin-resistant strains of S. dysenteriae 1 are found in different countries within a limited duration of time, suggesting that the strain may spread to other parts of the world very rapidly, probably traversing a similar path as previous epidemic strains. Given this knowledge, public health officials and clinicians should start preparing to forestall the spread of the epidemic.
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Acknowledgements |
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Footnotes |
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References |
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