Departments of 1 Microbiology and 3 Urology, Kaohsiung Medical University, Kaohsiung; 2 Department of Food Science and Technology, Tajen Institute of Technology, Pingtung, Taiwan
Received 14 August 2003; returned 6 October 2003; revised 30 November 2003; accepted 1 December 2003
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Abstract |
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Materials and methods: Ninety-three S. maltophilia clinical isolates were studied. PCR and direct sequencing were used to detect the presence of integrons. Real-time PCR was performed to assess and quantify the expression of the Sme efflux pumps of S. maltophilia.
Results: Class 1 integrons were detected in 22% of clinical isolates and carried cassettes conferring resistance mainly to aminoglycosides and trimethoprim. The small multidrug resistance gene, smr, was found on class 1 integrons in six isolates. Thirty-one percent of the isolates overexpressed the smeDEF gene, as compared with a control strain, and 59% overexpressed the smeABC gene. Extrusion of ciprofloxacin and meropenem was specific to the SmeABC and SmeDEF pumps, respectively.
Conclusion: SmeABC and SmeDEF efflux pumps play important roles in resistance of S. maltophilia to ciprofloxacin and meropenem.
Keywords: real-time PCR, class 1 integrons, plasmids
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Introduction |
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In many other Gram-negative bacteria, integrons carrying antibiotic resistance genes have been identified on mobile elements, such as transposons and plasmids, and these facilitate transfer of antibiotic resistance genes between different species.6
In the present study, the distribution of class 1 and 2 integrons was examined in a collection of clinical isolates of S. maltophilia. The levels of expression of the SmeABC and SmeDEF pumps were also analysed and were correlated with the antibiotic susceptibilities of the isolates.
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Material and methods |
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Antimicrobial susceptibilities were determined by the agar dilution methods described by the NCCLS.7
The template DNA for PCR was prepared as described by Bass et al.8 Integrons were detected with integrase-specific primers for intI 1 (F: 5'-CCTCCCGCACGATGATC-3', R: 5'-TCCACGCATCGTCAG- GC-3') or intI 2 (F: 5'-ATGTCTAACAGTCCATTTTTAAATT-3', R: 5'-GTAGCAAACGAGTGACGAAATG-3'). Gene cassettes within integrons were amplified with primers specific for the integron 5' conserved segment (5'-GGCATCCAAGCAGCAAG-3') and 3' conserved segment (5'-AAGCAGACTTGACCTGA-3'). The amplicons were then sequenced directly and sequence comparisons were made with the BLAST search program.
Plasmid DNA was extracted by the method of Kado & Liu.9 Hybridization was performed with a digoxigenin-labelled probe specific for the integrase gene intI 1.
In real-time PCR, the sequences of the primers for the target genes were as follows: smeB (F: 5'-GGGCCGGAAAGCTACGA-3', R: 5'-AGC- GAAATGGTCACGAATGG-3'); and smeF (F:5'-CCAACGCGGAT- CGTGATATC-3', R: 5'-TGCTCATCCAGGCTGACATTC-3'). The primer sequences for the endogenous control gene were: rDNA (F: 5'-GAC- CTTGCGCGATTGAATG-3', R: 5'- CGGATCGTCGCCTTGGT-3').
Total RNA was isolated using the High Pure RNA Isolation Kit (Roche, Mannheim, Germany) and reverse transcribed using oligo (dT)15 and avian myeloblastosis virus reverse transcriptase (Promega, Madison, USA) in a 20 µL reaction. Then, real-time PCR reactions were performed on an ABI Prism 7900 thermal cycler (Applied Biosystems, Foster, CA, USA). Amplification mixtures (50 µL) for smeB, smeF and rDNA quantification contained template cDNA, 2¥SYBR Green I Master Mix (Applied Biosystems) and primers. PCR was accomplished after a 10 min activation and denaturation step at 95°C, followed by 40 cycles of 15 s at 95°C, and 1 min at 59°C (for smeB) or 58°C (for smeF and rDNA) for annealing and extension. The parameter Ct was defined as the threshold cycle number at which the fluorescence generated by the binding of SYBR Green I dye to double-stranded DNA began to increase exponentially.
Clinical S. maltophilia isolate 14109 was chosen to construct the standard curve of smeABC, smeDEF and rDNA. S. maltophilia ATCC 13637 was used as calibrator to normalize the relative expression level of smeB and smeF genes in clinical isolates. Final results, expressed as n-fold differences in expression of smeABC or smeDEF genes, were determined as follows:
Values of n < 1 were considered to indicate overexpression of the Sme efflux system.
Statistical analysis
t-Tests (two-tailed) and ANOVA tests were used to determine the correlation between overexpression of the Sme efflux pumps and drug resistance in clinical isolates of S. maltophilia.
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Results |
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Twenty (22%) isolates carried detectable class 1 integrons; class 2 integrons were not found. Four different resistance cassette arrays were identified: aacA4 only (resistance to aminoglycosides, including amikacin) in five isolates; dfrIIa only (trimethoprim) in seven isolates; and the small multidrug resistance gene, smr, alone in four isolates and with aacA4 in two others. Two isolates contained empty integrons that lacked resistance cassettes. The six smr-positive isolates showed an four-fold increase in MICs of ciprofloxacin in comparison with isolates that did not carry this gene. Ten (11%) isolates possessed plasmid DNA, but Southern hybridization revealed that only one isolate carried a plasmid-mediated integron; this harboured an aacA4 gene cassette.
Real-time PCR methods were performed to assess and quantify expression of the Sme efflux systems among S. maltophilia. Our data indicated that smeF was overexpressed by 29 (31%) of the clinical isolates analysed. MICs of meropenem were significantly higher for isolates in which smeF was detectable by real-time PCR than for isolates in which it was not detectable (Table 1). The SmeABC efflux system was not expressed in wild-type S. maltophilia ATCC 13637. Fifty-five (59%) clinical isolates overexpressed smeABC and showed significantly increased resistance to ciprofloxacin (Table 1). In Table 2, we used an ANOVA method to compare the resistance of isolates in three categories; those containing both smeB and smeF, those containing only one of these genes and those with neither gene. The test would regard as significant only those values where P < 0.01. However, none of the results reached this level of significance.
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Discussion |
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Previous reports have detected class 1 integrons in many (40%60%) Gram-negative clinical isolates, with plasmids or transposons contributing to their dissemination.6 However, in this study, only 22% (20/93) of S. maltophilia clinical isolates had class 1 integrons, and only one of these was located on a plasmid. The results are consistent with our previous report analysing plasmid content of P. aeruginosa, where 15.2% of isolates carried plasmids, but only 13.3% of plasmid-carrying strains contained R plasmids.10 These results suggest that integrons and plasmids might not play important roles in the multidrug resistance of S. maltophilia. Interestingly, an smr gene was detected on integrons in six isolates. Proteins of the SMR family have been characterized in Escherichia coli (EmrEEco) and P. aeruginosa (EmrEPae); they contribute to resistance to multiple toxic compounds and antimicrobial agents, especially aminoglycosides.11 The role and contribution of smr to the drug resistance of S. maltophilia merits further study.
Using antibiotics as the selective agent, Zhang et al.5 reported the isolation of a number of multidrug-resistant strains of S. maltophilia resistant to fluoroquinolones, tetracycline and chloramphenicol. The multidrug-resistant phenotypes of the mutants were attributed to multidrug efflux systems. Two efflux systems, SmeABC and SmeDEF, have been identified in S. maltophilia.4,5 Alonso & Martinez2 analysed the expression of the SmeF protein by western blotting with an anti-SmeF antibody and showed that 47% of the strains examined overproduced this protein. Furthermore, the MICs of tetracycline, chloramphenicol, erythromycin and the quinolones were higher for strains in which expression of SmeF was detectable by western blotting. Similarly, Li & Poole4 used deletion experiments to indicate that SmeABC also contributes to antimicrobial resistance. In addition, overexpression of smeABC genes in a constructed multidrug resistant strain induced resistance to several antimicrobials, including aminoglycosides, ß-lactams and fluoroquinolones.4 In the present study, real-time PCR was used to quantify and compare the expression of SmeABC and SmeDEF efflux pumps in clinical isolates. We showed that the SmeABC and SmeDEF efflux pumps play a role in resistance of S. maltophilia to ciprofloxacin and meropenem, respectively.
In summary, the driving force behind the accelerated evolution of multidrug resistance in clinical isolates of S. maltophilia remains unclear, but could in part be the use of antibiotics in clinical settings and the presence of efflux pump systems. Increasingly, combinations of antimicrobial agents may be needed for the therapy of resistant S. maltophilia infections. In addition, effective antibiotic therapy of S. maltophilia infections may require the development of derivatives that are poor substrates for the efflux pumps and of pump inhibitors to restore susceptibility to available antimicrobial agents.12
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Acknowledgements |
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Footnotes |
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References |
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2
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Alonso, A. & Martinez, J. L. (2001). Expression of multidrug efflux pump SmeDEF by clinical isolates of Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 45, 187981.
3 . Nikaido, H. (1994). Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264, 3828.[ISI][Medline]
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Li, X. Z. & Poole, K. (2002). SmeC, an outer membrane multidrug efflux protein of Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 46, 33343.
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Li, X. Z., Poole, K. & Nikaido, H. (2003). Contributions of MexAB- OprM and an EmrE homolog to intrinsic resistance of Pseudomonas aeruginosa to aminoglycosides and dyes. Antimicrobial Agents and Chemotherapy 47, 2733.
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Lomovskaya, O., Warren, M. S., Lee, A. et al. (2001). Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrobial Agents and Chemotherapy 45, 10516.