Preservation of topoisomerase genetic sequences during in vivo and in vitro development of high-level resistance to ciprofloxacin in isogenic Stenotrophomonas maltophilia strains

Sylvia Valdezate1, Ana Vindel1, Juan Antonio Saéz-Nieto1, Fernando Baquero2 and Rafael Cantón2,*

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


* Correspondence address. Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Carretera de Colmenar Km 9.1, 28034-Madrid, Spain; Tel: +34-913368330; Fax: +34-913368809; E-mail: rcanton.hrc{at}salud.madrid.org

Received 24 January 2005; returned 17 March 2005; revised 28 April 2005; accepted 29 April 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: To ascertain the participation of topoisomerase mutations in the development of ciprofloxacin resistance in isogenic Stenotrophomonas maltophilia mutants.

Methods: gyrAB and parCE sequences in three paired in vivo isogenic ciprofloxacin-susceptible (MIC range 0.5–4 mg/L) and resistant (16–128 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


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In addition to co-trimoxazole, fluoroquinolones are used in severe Stenotrophomonas maltophilia infection therapy. However, this alternative should be used with caution as the appearance of resistant strains during fluoroquinolone treatment has been reported and the easy development of in vitro resistant mutants (10–5–10–7 frequency) has been documented.1 It has been previously shown that quinolone resistance in S. maltophilia has mainly been related to a low grade of permeability and over-expression of efflux-based mechanisms, but not to specific mutations located in the quinolone resistance determining region (QRDR) in the subunits that constitute the topoisomerases II (GyrA and GyrB) and IV (ParC and ParE).24

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Clinical isogenic strains

Three isogenic S. maltophilia clinical strain sets displaying different ciprofloxacin susceptibilities (MIC range, 0.5–128 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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Table 1. Epidemiological characteristics and susceptibility to the studied quinolones against paired in vivo isogenic S. maltophilia strains

 
Laboratory-derived isogenic ciprofloxacin resistant mutants

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 Mueller–Hinton 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 10–5 cfu/mL (bacterial density adjusted with saline solution) were subcultured onto Mueller–Hinton agar containing 2, 3, 4, 5 and 6 x MICs of ciprofloxacin displayed by each strain. After 24–48 h of incubation at 35°C, mutant (15–35 colonies per clinical isolate) stability was verified in Mueller–Hinton agar plates containing the same concentration as the mutants selected. Mutants were subcultured into ciprofloxacin-free Mueller–Hinton 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, 3–7 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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Table 2. Correlations of susceptibility to nalidixic acid and ciprofloxacin and their efflux pump inhibitor combinations with amino acid changes in topoisomerases II and IV in in vivo and in vitro paired isogenic S. maltophilia strains with respect to the corresponding S. maltophilia ATCC 13637 topoisomerases

 
Susceptibility testing and presence of active efflux mechanisms

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.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The present work was designed to complement a previous study in which no correlation between higher ciprofloxacin MIC values (4–64 mg/L) in S. maltophilia clinical strains and target-based mutations in topoisomerases was detected.4 In the present study, we selected three sets of paired isogenic clinical S. maltophilia strains from one non-cystic fibrosis patient and two cystic fibrosis patients according to quinolone susceptibility patterns (Table 1). In addition, stable in vitro ciprofloxacin-resistant strains displaying higher ciprofloxacin MIC values than those from the parent strains obtained from each wild-type strain were also studied. The in vitro activities of nalidixic acid and ciprofloxacin with and without different efflux pump inhibitors against S. maltophilia clinical strains and the corresponding laboratory mutants and QRDR sequences are indicated in Table 2.

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 4–20 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 8–32 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 3–16 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-119->Thr, 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 2–3 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 2–5-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 (resistance–nodulation–division) 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


    Acknowledgements
 
This research was partially supported by grants from Consejeria de Educación y Ciencia. Comunidad de Madrid (2114/98), Red Española de Investigación en Patología Infecciosa (REIPI) from the Ministerio de Sanidad y Consumo (FIS C03/014) and Microbial Sciences Foundation Madrid, Spain.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1. Lecso-Bonet M, Pierre J, Sarkis-Karam D et al. Susceptibility of Xanthomonas maltophilia to six quinolones and study of outer membrane proteins in resistant mutants selected in vitro. Antimicrob Agents Chemother 1992; 36: 669–71.[Abstract]

2. Alonso A, Martinez JL. Cloning and characterization of SmeDEF, a novel multidrug efflux pump from Stenotrophomonas maltophilia. Antimicrob Agents Chemother 2000; 44: 3079–86.[Abstract/Free Full Text]

3. Zhang L, Li XZ, Poole K. Multiple antibiotic resistance in Stenotrophomonas maltophilia: involvement of a multidrug efflux system. Antimicrob Agents Chemother 2000; 44: 287–93.[Abstract/Free Full Text]

4. Valdezate S, Vindel A, Echeita A et al.. Topoisomerase II and IV quinolone-resistance determining regions in Stenotrophomonas maltophilia clinical isolates with different levels of quinolone susceptibility. Antimicrob Agents Chemother 2002; 46: 665–71.[Abstract/Free Full Text]

5. Doss SA, Tillotson G, Barg S et al. In-vitro and in-vivo selection of Staphylococcus aureus mutants resistant to ciprofloxacin. J Antimicrob Chemother 1995; 35: 95–102.[Abstract]

6. Friedman SM, Lu T, Drlica K. Mutation in the DNA gyrase A Gene of Escherichia coli that expands the quinolone resistance-determining region. Antimicrob Agents Chemother 2001; 45: 2378–80.[Abstract/Free Full Text]

7. Ribera A, Ruíz J, Jiménez de Anta MT et al. Effect of an efflux pump inhibitor on the MIC of nalidixic acid for Acinetobacter baumannii and Stenotrophomonas maltophilia clinical isolates. J Antimicrob Chemother 2002; 49: 697–8.[Free Full Text]

8. Lomovskaya O, Warren MS, Lee A et al. Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrob Agents Chemother 2001; 45: 105–16.[Abstract/Free Full Text]

9. Sánchez P, Le U, Martinez JL. The efflux pump inhibitor Phe-Arg-beta-naphthylamide does not abolish the activity of the Stenotrophomonas maltophilia SmeDEF multidrug efflux pump. J Antimicrob Chemother 2003; 51: 1042–5.[Free Full Text]

10. Martínez JL, Baquero F. Mutation frequencies and antibiotic resistance. Antimicrob Agents Chemother 2002; 44: 1771–7.[CrossRef]





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