Fluoroquinolone susceptibilities of efflux-mediated multidrug-resistant Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia

Li Zhang, Xian-Zhi Li and Keith Poole,*

Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The antibacterial activities of seven fluoroquinolones (ciprofloxacin, BAYy3118, clinafloxacin, gemifloxacin, moxifloxacin, sparfloxacin and trovafloxacin) against isogenic efflux-mediated multidrug-resistant strains of Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia, were compared. The results indicate that these fluoroquinolones are all substrates for the multidrug efflux systems of these organisms. Clinafloxacin was found generally to be the most active agent against multidrug-resistant strains.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Antibiotic extrusion by efflux pumps is a major determinant of antimicrobial resistance in bacteria. In Gramnegative organisms, multidrug efflux pumps with broad specificities function synergically with the outer membrane barrier to provide intrinsic and/or acquired multidrug resistance (MDR).1,2 This is particularly true for organisms such as Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia, which are highly multidrug resistant (reviewed in Poole2). Previously classified as members of the Pseudomonas genus, all three species are opportunistic human pathogens characterized by innate and mutationally acquired resistance to a variety of antimicrobial agents including quinolones, ß-lactams, aminoglycosides, macrolides, tetracyclines, chloramphenicol, detergents, dyes and/or organic solvents.1,2 Four multidrug efflux systems have been identified in P. aeruginosa, and two of these, MexAB-OprM and MexXY-OprM, contribute significantly to the intrinsic drug resistance of this organism.2 These two efflux systems and two additional ones, MexCD-OprJ and MexEF-OprN, mediate acquired MDR in P. aeruginosa as a result of mutational hyperexpression of the corresponding efflux genes.2 Homologues of these tripartite efflux systems have been described in S. maltophilia35 and B. cepacia,6 where MDR is also seen.

In vitro- and in vivo-selected fluoroquinolone-resistant strains of these organisms often display an MDR phenotype, consistent with MDR efflux systems playing a role in fluoroquinolone resistance.2 Indeed, it is now known that the multidrug efflux systems of P. aeruginosa are one of the primary determinants of resistance to quinolones.2

The fluoroquinolone compounds are an important group of antimicrobial agents that have been developed extensively over the past decade.7 While older fluoroquinolones are generally effective against aerobic Gram-negative bacteria, newer fluoroquinolones have a broader spectrum of activity against Gram-negative and Grampositive bacteria and/or mycobacteria.7 In this report, we assess the fluoroquinolone susceptibilities of efflux-mediated multidrug-resistant strains of P. aeruginosa, S. maltophilia and B. cepacia. The results indicate that the newer fluoroquinolone agents are substrates for the MDR efflux pumps of these organisms.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial strains and growth conditions

Bacterial strains used in this study are described in the TableGo. P. aeruginosa strains PAO1, ML5087 and PAO2375, S. maltophilia strains ATCC 13637 and ULA-511, and B. cepacia strains K127 and K131 were used as wild-type and reference strains.3,8,9 Most multidrug-resistant or efflux pump-deficient mutants derived from these strains were selected or constructed as reported previously,3,8,9 and their susceptibilities to ß-lactams, ciprofloxacin, aminoglycosides, macrolides, tetracycline and/or chloramphenicol have been reported.3,8,9 The PAO1 nfxB mutant, strain K1536, was selected on Luria–Bertani (LB) agar supplemented with ciprofloxacin (0.4 mg/L) (L. Adewoye, unpublished data), and hyperexpression of MexCD-OprJ in this mutant was confirmed by western immunoblotting with antibodies specific to the outer membrane efflux component, OprJ (X.-Z. Li, unpublished data). PAO2375 and its nfxC mutant (strain K1240) were kindly provided by N. Gotoh of Kyoto Pharmaceutical University, Japan.10 Multidrug-resistant strains K1433 and K1443 of S. maltophilia were selected, respectively, on tetracycline- or ciprofloxacin-containing LB agar, as described previously.3 These mutants were shown to have reduced susceptibilities to fluoroquinolones, tetracyclines and macrolides.3 Strain K1433, but not strain K1443, was shown also to be more resistant to aminoglycoside antibiotics.3 Multidrug-resistant mutants (K1578, K1580 and K1584) of B. cepacia were selected on LB agar containing ciprofloxacin (8 mg/L). These mutants are resistant to ciprofloxacin, chloramphenicol and trimethoprim (four- to 16-fold increases in MICs) (R. Srikumar and P. Segal, unpublished data). Strain K1584, but not strain K1580, was shown also to be more resistant to ß-lactams such as piperacillin, ceftazidime and cefepime (greater than eight- to 16-fold increases in MICs) than the parent strain K131 (R. Srikumar and P. Segal, unpublished data). Bacteria were cultured in LB broth [1% (w/v) Difco tryptone, 0.5% (w/v) Difco yeast extract, 0.5% (w/v) NaCl] at 37°C, except S. maltophilia ATCC 13637 and its derivatives (strains K1433 and K1443), which were grown at their optimal growth temperature, 30°C.


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Table. Efflux-mediated fluoroquinolone resistance in P. aeruginosa, S. maltophilia and B. cepacia
 
Fluoroquinolone agents

Fluoroquinolones were purchased or kindly provided by manufacturers as follows: ciprofloxacin from Sigma– Aldrich Canada (Oakville, Ontario, Canada); moxifloxacin and BAYy3118 (a monofluorinated quinolone) from Bayer AG (Leverkusen, Germany); clinafloxacin from Parke-Davis Pharmaceutical Research (Ann Arbor, MI, USA); trovafloxacin from Pfizer Inc. (Groton, CT, USA); and gemifloxacin (SB-265805) from SmithKline Beecham (Frythe, UK).

Antimicrobial susceptibility testing

Antibiotic susceptibility testing was carried out using the two-fold serial broth dilution method with an inoculum of 5 x 105 cells/mL. Data are reported as MIC, which reflects the lowest concentration of antibiotic that inhibited the development of visible growth after 18 h incubation at 37°C or 30°C, as appropriate.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The fluoroquinolone susceptibilities of isogenic efflux pump-deficient and in vitro-selected multidrug-resistant mutants of P. aeruginosa, S. maltophilia and B. cepacia were determined (TableGo). In the case of P. aeruginosa, fluoroquinolone resistance to all fluoroquinolones tested correlated well with expression of the MexAB-OprM, MexCD-OprJ and MexEF-OprN multidrug efflux systems (TableGo). MexAB-OprM-deficient strains (i.e. K1119 and K1115) showed hypersusceptibility to the fluoroquinolones, while efflux pump-overproducing nalB (OCR1, K1112), nfxB (K1111, K1131, K1536) and nfxC mutants (K1117, K1240) displayed reduced susceptibilities to fluoroquinolones when compared with their parental strains. Overexpression of any given MDR efflux system in P. aeruginosa generally resulted in a four- to eight-fold increase in MICs (TableGo), consistent with all seven fluoroquinolones being substrates for the multidrug efflux systems of this organism. Of the seven fluoroquinolones, ciprofloxacin and clinafloxacin were the most active against P. aeruginosa strains (MICs are 0.1 mg/L for the wildtype strains), although clinafloxacin showed superior activity against nalB, nfxB and nfxC multidrug-resistant mutants hyperexpressing MexAB-OprM, MexCD-OprJ and MexEF-OprN, respectively. It is possible that clinafloxacin is an inferior substrate for these multidrug efflux pumps. The other fluoroquinolones tested were less active against P. aeruginosa, with a rank order of ciprofloxacin > BAYy3118 > trovafloxacin and gemifloxacin > sparfloxacin > moxifloxacin. It should be noted that certain of the newer generation fluoroquinolones (e.g. moxifloxacin and gemifloxacin) are used primarily against Grampositive bacteria. Their relatively poor activity against P. aeruginosa may reflect a limited ability to penetrate the outer membrane barrier,1,2 although the possibility that they are particularly good substrates for the P. aeruginosa multidrug efflux systems cannot be ruled out.

S. maltophilia is resistant to most currently available antimicrobial agents.2,3 Using an individual antibiotic (norfloxacin, ciprofloxacin, tetracycline or chloramphenicol) as the selective agent, we isolated a number of multidrug-resistant mutants that are resistant to fluoroquinolones (norfloxacin and ciprofloxacin), tetracycline and chloramphenicol, but that show different susceptibilities to aminoglycoside antibiotics.3 These multidrug-resistant phenotypes were attributed to multidrug efflux systems in these mutants.3 As shown in the TableGo, these multidrug-resistant mutants, derivatives of strains ATCC 13637 and ULA-511, showed elevated resistance to all fluoroquinolones tested. Clinical isolates of S. maltophilia described previously were also shown to be resistant to all the fluoroquinolones tested (TableGo).3 Intriguingly, ciprofloxacin, which is very active against P. aeruginosa (see above, TableGo), was the worst of all the fluoroquinolones tested against both wild-type and multidrug-resistant S. maltophilia (TableGo). In contrast, BAYy3118 and clinafloxacin showed relatively strong anti-S. maltophilia activity (TableGo).

B. cepacia also is highly resistant to many antimicrobial agents.2,6 Multidrug-resistant strains of B. cepacia selected on ciprofloxacin (strains K1578, K1580 and K1584) have reduced susceptibility to all of the fluoroquinolones tested (eight- to 16-fold increases in MICs) (TableGo). Clinafloxacin and BAYy3118 showed the best activities against wild-type and multidrug-resistant B. cepacia (TableGo). Although the MDR basis of B. cepacia mutants described here have yet to be attributed unequivocally to a multidrug efflux system(s), this is likely to be the case.

The multidrug-resistant phenotypes of the P. aeruginosa and S. maltophilia strains described here encompass all the fluoroquinolones tested, indicating that newer fluoroquinolones, like the older ones, are substrates for multidrug efflux systems. Overall, clinafloxacin was found to be the most effective fluoroquinolone against the multidrug-resistant mutants of all the strains tested, irrespective of bacterial species, indicating perhaps that it is a poor efflux pump substrate.

The results presented in this study demonstrate a strong correlation between fluoroquinolone resistance and expression of multidrug efflux systems in P. aeruginosa and S. maltophilia (and probably B. cepacia). Therefore, the development of fluoroquinolones that are not substrates for efflux systems and of fluoroquinolone–efflux pump inhibitor combinations will be important, if the use of fluoroquinolones to treat infections caused by these organisms is to continue. Fortunately, the first examples of broad-spectrum efflux pump inhibitors of the Mex efflux systems of P. aeruginosa have been reported and have been shown to potentiate the antibacterial activity of the fluoroquinolone levofloxacin.11 These pump inhibitors effectively reversed acquired fluoroquinolone resistance attributable to efflux pump as well as target site mutations, and markedly decreased the frequency with which high-level fluoroquinolone-resistant strains were selected in vitro.11


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The authors thank P. Heisig for the gift of moxifloxacin and BAYy3118, N. Gotoh for providing PAO2375 and its nfxC mutant, L. Adewoye for the isolation of K1536, R. Srikumar and P. Segal for the isolation of the B. cepacia multidrug-resistant mutants. This research was supported by operating grants from the Canadian Cystic Fibrosis Foundation (CCFF) and the Canadian Bacterial Disease Network (one of the Networks of Centers of Excellence). K.P. is a CCFF Scholar.


    Notes
 
* Corresponding author. Tel: +1-613-533-6677; Fax: +1-613-533-6796; E-mail: poolek{at}post.queensu.ca Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Nikaido, H. (1998). Antibiotic resistance caused by gramnegative multidrug efflux pumps. Clinical Infectious Diseases, Suppl. 1, S32–41.

2 . Poole, K. (2000). Efflux-mediated resistance to fluoroquinolones in gram-negative bacteria. Antimicrobial Agents and Chemotherapy 44, 2233–41.[Free Full Text]

3 . Zhang, L., Li, X.-Z. & Poole, K. (2000). Multiple antibiotic resistance in Stenotrophomonas maltophilia: involvement of a multidrug efflux system. Antimicrobial Agents and Chemotherapy 44, 287–93.[Abstract/Free Full Text]

4 . Alonso, A. & Martinez, J. L. (2000). Cloning and characterization of SmeDEF, a novel multidrug efflux pump from Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 44, 3079–86.[Abstract/Free Full Text]

5 . Li, X.-Z., Zhang, L. & Poole, K. (2000). Identification and characterization of a novel multidrug efflux system, SmeABC, regulated by a two-component regulatory system, SmeSR, in Stenotrophomonas maltophilia. In Abstracts of the 100th General Meeting of the American Society for Microbiology, Los Angeles, CA, USA, 2000, Abstract A33, p. 8.

6 . Burns, J. L., Wadsworth, C. D., Barry, J. J. & Goodall, C.P. (1996). Nucleotide sequence analysis of a gene from Burkholderia (Pseudomonas) cepacia encoding an outer membrane lipoprotein involved in multiple antibiotic resistance. Antimicrobial Agents and Chemotherapy 40, 304–13.

7 . Hooper, D. C. (2000). Mechanisms of action and resistance of older and newer fluoroquinolones. Clinical Infectous Diseases, Suppl. 2, S24–8.

8 . Li, X.-Z., Zhang, L., Srikumar, R. & Poole, K. (1998). ß-lactamase inhibitors are substrates for the multidrug efflux pumps of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 42, 399–403.[Abstract/Free Full Text]

9 . Srikumar, R., Li, X.-Z. & Poole, K. (1997). Inner membrane efflux components are responsible for ß-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa. Journal of Bacteriology 179, 7875–81.[Abstract]

10 . Li, X.-Z., Barré, N. & Poole, K. (2000). Influence of the MexA-MexB-OprM multidrug efflux system on expression of the MexC-MexD-OprJ and MexE-MexF-OprN multidrug efflux systems in Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 46, 885–93[Abstract/Free Full Text]

11 . Lomovskaya, O., Warren, M. S., Lee, A., Galazzo, J., Fronko, R., Lee, M. 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, 105–16.[Abstract/Free Full Text]

Received 2 January 2001; returned 28 March 2001; revised 5 April 2001; accepted 13 July 2001