1 Departamento de Bacteriología, Centro Nacional de Microbiología, Instituto Carlos III, Majadahonda, Madrid-28220, Spain; 2 Servicio de Microbiología, Hospital Ramón y Cajal, Crta. Colmenar, Km 9, Madrid-28034, Spain
Received 24 January 2005; returned 17 March 2005; revised 28 April 2005; accepted 29 April 2005
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Abstract |
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Methods: gyrAB and parCE sequences in three paired in vivo isogenic ciprofloxacin-susceptible (MIC range 0.54 mg/L) and resistant (16128 mg/L) S. maltophilia strains (PFGE-characterized) sequentially isolated from three patients, and their corresponding in vitro mutants (ciprofloxacin MIC range 2>128 mg/L), were studied. Efflux phenotype was also investigated.
Results: Despite different quinolone susceptibilities, each paired clinical strain displayed identical gyrAB and parCE sequences as well as their corresponding in vitro mutants. Up to 50% (18/36) of in vitro mutants displayed a positive efflux phenotype when nalidixic acid was combined with MC-207,110, while 6% (2/36) showed the phenotype when exposed to nalidixic acid and reserpine. Carbonyl cyanide m-chlorophenylhydrazone or arsenite failed to alter quinolone MICs.
Conclusions: The increase of ciprofloxacin MICs in in vivo and in vitro isogenic S. maltophilia mutant strains was not related to quinolone resistance determining region mutations. Highly effective efflux mechanisms might preserve topoisomerase targets from a ciprofloxacin challenge in S. maltophilia.
Keywords: QRDR , fluoroquinolone resistance , efflux pump inhibitors
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Introduction |
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In the present work, we have analysed the potential changes in the amino acid sequences of the QRDRs in paired isogenic clinical S. maltophilia strains expressing different levels of ciprofloxacin susceptibilities and also in laboratory-derived mutants selected after ciprofloxacin exposure. To the best of our knowledge the role of topoisomerase mutations in both in vivo and in vitro S. maltophilia isogenic mutants has not been studied previously. The potential role of an efflux-based resistance mechanism using a phenotypic approach with efflux pump inhibitors was also studied. This phenotypic assay included proton motive force (CCCP, MC207,110 and arsenite) and ion motive ATPase (reserpine) inhibitors.
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Materials and methods |
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Three isogenic S. maltophilia clinical strain sets displaying different ciprofloxacin susceptibilities (MIC range, 0.5128 mg/L) were collected from two cystic fibrosis patients and one surgical patient: A-Sm226/A-Smm227, B-Sm230/B-Sm204, C-Sm112/C-Sm125 (Table 1). Identification was performed with API-20NE (BioMerieux, La Balme, Les Grottes, France) and WIDER (Fco. Soria Melguizo, Madrid, Spain) systems. The isogenicity (identical profile) of each set was confirmed by PFGE (CHEF-DRII system, Bio-Rad, Hemel Hempstead, UK) under XbaI restriction (Roche Diagnostic, Barcelona, Spain).
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Both paired isogenic clinical strains from each set, including susceptible and resistant isolates, were inoculated from an overnight culture twice consecutively into 20 mL of MuellerHinton broth (Oxoid, Basingstoke, UK) supplemented with their corresponding 0.5 x MIC of ciprofloxacin. After 18 h of incubation at 35°C, 0.1 mL aliquots with 105 cfu/mL (bacterial density adjusted with saline solution) were subcultured onto MuellerHinton agar containing 2, 3, 4, 5 and 6 x MICs of ciprofloxacin displayed by each strain. After 2448 h of incubation at 35°C, mutant (1535 colonies per clinical isolate) stability was verified in MuellerHinton agar plates containing the same concentration as the mutants selected. Mutants were subcultured into ciprofloxacin-free MuellerHinton agar. After 10 serial passages onto this antibiotic-free media, the nalidixic acid and ciprofloxacin MICs were determined to ascertain resistance stability.5 For further studies, 37 mutants selected in vitro from each paired clinical strain set were chosen according to the different ciprofloxacin MIC values. S. maltophilia ATCC 13637 and laboratory-selected quinolone resistant mutants were also included. Final numbers of resistant mutants are indicated in Table 2.
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Susceptibility to different quinolones (Tables 1 and 2) was determined by the agar dilution method with a final inoculum of 105 cfu/spot.4 MICs were read after 24 h of incubation at 35°C. Antibiotics were provided by the manufacturer or purchased from Sigma Chemical Co. (St Louis, MO, USA).
The presence of an active efflux mechanism in in vivo (n = 6) and in vitro (n = 36) derived isogenic mutant strains was phenotypically determined by studying nalidixic acid and ciprofloxacin MIC values with and without the following efflux pump inhibitors: 0.5 mg/L of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 20 mg/L of reserpine, 20 mg/L of Phe-Arg-ß-naphthylamide (MC-207,110 compound) and 0.1 mM sodium arsenite. An efflux mechanism was inferred to be present when the quinolone MIC value in the presence of any efflux pump inhibitor was at least twofold dilutions lower than the corresponding MIC in the absence of these compounds. Efflux pumps inhibitors were purchased from Sigma. S. maltophilia ATCC 13637 and Pseudomonas aeruginosa ATCC 27853 were used as controls.
Amplification and QRDR region sequencing
The presence of amino acid changes in the gyrA, gyrB, parC and parE sequences, including the QRDR sequence, that could explain the increase in fluoroquinolone MIC values in clinical strains (n = 6) was determined as previously described.4 In the case of laboratory-derived mutants (n = 36), gyrA and parC sequences were determined.
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Results and discussion |
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In a 30-year-old cystic fibrosis patient (patient A), two S. maltophilia strains were collected with a 6 month interval. It is of note that the initial strain (A-Sm226) was more resistant to quinolones than the final strain (A-Sm227), with ranges of 420 proportional MIC decreases (initial MIC/final MIC) for the studied quinolones (Table 1), while susceptibilities for other antimicrobials were similar (data not shown). During this period, the patient was not exposed to any fluoroquinolone, but received a 2 week course of co-trimoxazole plus tobramycin.
In a 23-year-old cystic fibrosis patient (patient B), two S. maltophilia strains were recovered with a 38 month interval. The initial strain, B-Sm230, was more susceptible to quinolones than the final strain, B-Sm204, with ranges of 832 proportional MIC increases, the highest being for trovafloxacin (Table 1). During the isolation interval, this patient received three ciprofloxacin courses of 2 weeks each.
In an adult surgical patient (patient C), two S. maltophilia strains were recovered within a 1 month period. The initial strain, C-Sm112, was more susceptible to quinolones than the final strain, C-Sm125, with ranges of 316 proportional MIC increases for the studied quinolones, the highest being for grepafloxacin (Table 1). This patient had a ciprofloxacin course of 2 weeks prior to the first S. maltophilia isolation.
Paired S. maltophilia clinical strains displayed identical QRDR sequences of gyrA, gyrB, parC and parE, despite different quinolone susceptibilities (Table 2). No difference was found in the partial GyrA and ParC sequences, including QRDR, of the corresponding in vitro-derived mutants obtained after ciprofloxacin exposure to the susceptible and resistant clinical strains. Moreover, with respect to the corresponding proteins described for S. maltophilia ATCC 13637,4 only a GyrA substitution, Ala-119Thr, was detected in the paired B-Sm230 and B-Sm204 strains and in their corresponding derived mutants. However, the same apparent ParE tetramutation (Met-437
Leu, Ile-465
Val, Ser-477
Thr and Ile-485
Val) appeared in both sets of S. maltophilia strains recovered from the cystic fibrosis patients and in their corresponding derived mutants. As previously reported, this event could probably be linked to polymorphisms rather than resistance.4 In contrast, in the other paired strains (C-Sm112/C-Sm125) and in their in vitro mutants, no QRDR amino acid changes were detected. Although we expanded the sequenced QRDR, we cannot rule out the possibility of mutations outside this region, as previously suggested.6
Nowadays, the use of efflux pump inhibitors is driving the study of prevalence and contribution of efflux systems in intrinsic and acquired multiresistance in Gram-negative bacteria. It is noteworthy that in half of the in vitro-derived mutant strains a positive efflux phenotype was detected with the combination of nalidixic acid and MC-207,110, which conferred a 23 twofold decrease in MIC values. Meanwhile, with ciprofloxacin and MC-207,110 this phenotype was only observed in 11% (4/36) of the strains (Table 2), but with a 25-fold decrease. This effect was rarely observed with reserpine (2/36) and was absent with CCCP or arsenite. MC-207,110, a broad-spectrum compound active against RND (resistancenodulationdivision) efflux pumps such as Mex systems from P. aeruginosa (MexAB-OprM, MexCD-OprJ and MexEF-OprN), increases accumulation of efflux pump substrates inside the cell.7 This compound increases levofloxacin activity against P. aeruginosa laboratory strains and clinical isolates with overexpression of efflux pumps and multiple target-based mutations that confer resistance to quinolones. Also, the frequency of emergence of fluoroquinolone resistance is diminished.8 In our study, using a phenotypic approach, the involvement of a possible role of MC-207,110 acting as an efflux inhibitor was observed with nalidixic acid in 50% of in vitro-derived S. maltophilia mutants and in one of the three in vivo resistant mutants. An efflux-positive phenotype was also inferred with an MC-207,110 and ciprofloxacin combination in 11% of strains. Other authors using clinical S. maltophilia strains showed this effect with nalidixic acid and MC-207,110 in 18% of studied isolates but not with ciprofloxacin.7 It is worth noting that MC-207,110 does not abolish the activity of all S. maltophilia efflux pumps that cannot be phenotypically detected with the addition of ciprofloxacin.9 Interestingly and as previously reported, those strains displaying a positive efflux phenotype with MC-207,110 did not demonstrate this effect with reserpine (Table 2), denoting the possibility of the emergence of different resistant mutants overexpressing different efflux pumps affecting quinolone susceptibilities.7
In summary, we confirm that unlike other organisms, the increase in the quinolone MIC values in clinical and laboratory S. maltophilia isogenic mutant strains is not related to amino acid replacement in topoisomerases. These results confirmed our previous observations showing that clinical S. maltophilia strains displaying high quinolone resistance showed identical topoisomerase sequences to those of susceptible strains.4 It can be suggested that the high efficiency of several efflux pumps in S. maltophilia reduces the intracellular ciprofloxacin concentration to a level at which the ciprofloxacin targets are not under challenge, and therefore there may be no selective pressure for target genetic modification. In general, our results indicate that mutations decreasing drug entry could be protective for target mutations in some bacterial organisms.10
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Acknowledgements |
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