a Laboratoire de Microbiologie, Hôpital Robert Debré, 48 Boulevard Serurier, F-75395 Paris cedex 19; b Laboratoire de Bactériologie, Hôpital Jean Minjoz, F-25030 Besançon, France
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Abstract |
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Introduction |
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Materials and methods |
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Two clinical isolates of P. aeruginosa were recovered from swabs of a surgical site infection in an 18-year-old patient admitted for post-rhabdomyosarcoma mandibuloplasty, before (P1) and after (P2) a 4 day course of ciprofloxacin (20 mg/kg/day) monotherapy. The bacteria were identified by conventional methods, and their O type lipopolysaccharide was determined with specific antisera according to the International Antigenic Typing System (Diagnostics Pasteur, Marne la Coquette, France).
Antibiotic susceptibility testing
Susceptibility of the isolates to ticarcillin, cefsulodin, ceftazidime, aztreonam, imipenem, ciprofloxacin, tobramycin and amikacin was evaluated using the disc diffusion method (Diagnostics Pasteur). MICs of ticarcillin and ciprofloxacin were further determined according to the NCCLS agar dilution procedure.4 NCCLS breakpoints are as follows: ticarcillin, susceptible 16 mg/L and resistant
64 mg/L; ciprofloxacin, susceptible
1 mg/L and resistant
4 mg/L.
Western blotting (immunoblotting)
Ten micrograms of purified outer membrane were separated by SDSPAGE and transferred electrophoretically on to nitrocellulose filters. These filters were subsequently blocked with 3% (w/v) gelatin and hybridized for 1 h with an OprM-specific polyclonal rabbit antiserum diluted 1:5000 in phosphate-buffered saline.5 Development of membranes was carried out with alkaline phosphatase conjugated to an anti-rabbit secondary antibody by using the AP colour reagent kit from Bio-Rad (Bio-Rad S.A., Ivry sur Seine, France).
Randomly amplified polymorphic DNA analysis
The two P. aeruginosa isolates P1 and P2 were genotypically compared by random amplified polymorphism DNA (RAPD) analysis, as described previously6 using the homemade PCR primer 5'-GCCCCCAGGGGCACAGT-3'. Reactions were each conducted in a 50 µL mixture consisting of 100 mM TrisHCl buffer (pH 8.3), 50 mM KCl, 4 mM MgCl2, 0.4 mM deoxynucleoside triphosphate, 3 µM primer, 50 ng of DNA and 2.5 U of Taq DNA polymerase (ATGC Biotechnologie, Noisy-le-Grand, France). Amplification was carried out with a Perkin-Elmer GeneAmp 9600 thermal cycler (Applied Biosystems, Warrington, UK) programmed for 5 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 36°C and 1 min at 72°C; and a final extension step of 3 min at 72°C. Amplification products were resolved by electrophoresis in a 2% agarose gel and revealed under UV after staining with ethidium bromide. Isolates that differed by two or more prominent bands are considered sufficiently divergent to warrant separate strain designations; identical profiles are considered as the same isolate.6
PCR amplification and DNA sequencing
A first set of primers GYRB.1 (5'-GCGCGTGAGATGACCCGCCGT-3') and GYRB.2 (5'-CTGGCGGTAGAAGAAGGTCAG-3') previously designed by Mouneimné et al.2 was used to amplify a 390 bp fragment of P. aeruginosa gyrB encompassing the sequence coding for the quinolone resistance-determining region (QRDR) of the GyrB protein under the following conditions: denaturation for 4 min at 94°C, 30 cycles of 5 s at 94°C and 10 s at 65°C, and a final extension step of 5 min at 72°C. Two other pairs of primers PSE1 (5'-GACGGCCTGAAGCCGGTGCAC-3') and PYO1 (5'-GCCCACGGCGATACCGC TGGA-3'), PARC1 (5'-CTGGATGCCGATTCCAAG-AC-3') and PARC2 (5'-GAAGGACTTGGGATCGT CCGG-3') were also utilized under the same conditions for the amplification of a 417 bp fragment of gyrA corresponding to the QRDR of GyrA and a 186 bp fragment of parC corresponding to the QRDR of ParC, respectively.2 Once purified, the amplicons were sequenced by the ESGS Society (Evry, France).
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Results |
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Both isolates exhibited the same serotype (O6) and were found to be genetically related by RAPD genotyping thus confirming the emergence of a multidrug-resistant mutant in our patient (Figure 1).
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Discussion |
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A recent cohort study of 271 patients with P. aeruginosa infections showed that fluoroquinolone resistance tends to develop quite frequently (8%) during ciprofloxacin treatment.7 In the case of our patient, it could be tempting to draw the conclusion that fluoroquinolone monotherapy is more prone than combination therapy to select multidrug-resistant mutants in vivo and thus is not a valid option in P. aeruginosa infections. However, emergence of MexAB-OprM mutants has been observed in patients treated with ß-lactams and fluoroquinolones, ß-lactams and aminoglycosides, or fluoroquinolones and aminoglycosides.5 Similarly, in another study, combination therapy with aminoglycosides did not appear to prevent the emergence of resistance to ciprofloxacin.7
Overexpression of the multidrug efflux system MexAB-OprM3,5,8,9 as well as occurrence of mutations in gyrB2 are known to confer moderate resistance to fluoroquinolones (e.g. MIC of ciprofloxacin <4 mg/L). Importantly, our observation demonstrates that acquisition of both mechanisms may provide clinical strains with a high degree of fluoroquinolone resistance and lead to clinical failure. Other examples of cumulative effects between efflux pumps (MexAB-OprM or MexCD-OprJ) and target alterations (gyrA, parC) have been reported recently.3,10 Thus, it becomes more and more evident that high-level resistance to fluoroquinolones in P. aeruginosa not only results from sequential acquisition of mutations in the target genes gyrA, gyrB and parC, but may also be associated with overexpression of efflux pumps.
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Notes |
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References |
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2
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Mouneimne, H., Robert, J., Jarlier, V. & Cambau, E. (1999). Type II topoisomerase mutation in ciprofloxacin-resistant strains of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 43, 626.
3
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Lomovskaya, O., Lee, A., Hoshino, K., Ishida, H., Mistry, A., Warren, M. S. et al. (1999). Use of a genetic approach to evaluate the consequences of inhibition of efflux pumps in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 43, 13406.
4 . National Commitee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A4. NCCLS, Villanova, PA.
5
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Ziha-Zarifi, I., Llanes, C., Köhler, T., Péchere, J. C. & Plésiat, P. (1999). In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM. Antimicrobial Agents and Chemotherapy 43, 28791
6 . Bingen, E., Bonacorsi, S., Rohrlich, P., Duval, M., Lhopital, S., Brahimi, N. et al. (1996). Molecular epidemiology provides evidence of genotypic heterogeneity of multidrug-resistant Pseudomonas aeruginosa serotype O: 12 outbreak isolates from a pediatric hospital. Journal of Clinical Microbiology 34, 32269.[Abstract]
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Carmeli, Y., Troillet, N., Eliopoulos, G. M. & Samore, M. H. (1999). Emergence of antibiotic-resistant Pseudomonas aeruginosa: comparison of risks associated with different antipseudomonal agents. Antimicrobial Agents and Chemotherapy 43, 137982.
8 . Li, X. Z., Nikaido, H. & Poole, K. (1995). Role of MexA-MexB-OprM in antibiotic efflux in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 194853.[Abstract]
9 . Masuda, N. & Ohya, S. (1992). Cross-resistance to meropenem, cephems, and quinolones in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 36, 184751.[Abstract]
10 . Yoshida, T., Muratani, T., Iyobe, S. & Mitsuhashi, S. (1994). Mechanisms of high level resistance to quinolones in urinary tract isolates of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 38, 14669.[Abstract]
Received 12 March 2001; returned 18 May 2001; revised 12 July 2001; accepted 26 July 2001