The relative contribution of efflux and target gene mutations to fluoroquinolone resistance in recent clinical isolates of Pseudomonas aeruginosa

  • S. A. Dunham
  • C. J. McPherson
  • A. A. MillerEmail author


The clinical utility of fluoroquinolones (FQs) for the treatment of Pseudomonas aeruginosa (PA) and other serious Gram-negative infections is currently decreasing due to the rapid emergence of resistance. Because previous studies have shown that efflux is a common mechanism contributing to FQ resistance in PA, one suggested approach to extend the longevity of this class of drugs is combination therapy with an efflux pump inhibitor (EPI). In order to determine the viability of this approach, it is necessary to understand the relative contribution of efflux- vs. target-mediated mechanisms of FQ resistance in the clinic. A set of 26 recent PA clinical isolates were characterized for antibiotic resistance profiles, efflux pump expression, topoisomerase mutations, and FQ susceptibility with and without an EPI. The contribution of OprM to the overall antibiotic resistance was assessed in a subset of these strains. Our results suggest that the co-administration of an EPI with FQs or other antibiotics currently in use would not be sufficient to combat the complexity of resistance mechanisms now present in many clinical isolates.


Clinical Isolate Levofloxacin Efflux Pump Target Mutation Clinafloxacin 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We wish to acknowledge Amy Tait-Kamradt for her help with the genomic sequencing and Andrew Tomaras and John Quinn for their critical reading of the manuscript. These studies were conducted at and funded by Pfizer Global Research and Development.


  1. 1.
    Bonomo RA, Szabo D (2006) Mechanisms of multidrug resistance in Acinetobacter species and Pseudomonas aeruginosa. Clin Infect Dis 43:S49–S56CrossRefPubMedGoogle Scholar
  2. 2.
    Hancock REW, Speert DP (2000) Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment. Drug Resist Updat 3:247–255CrossRefPubMedGoogle Scholar
  3. 3.
    Mesaros N, Nordmann P, Plésiat P et al (2007) Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium. Clin Microbiol Infect 13:560–578CrossRefPubMedGoogle Scholar
  4. 4.
    Rahal JJ (2006) Novel antibiotic combinations against infections with almost completely resistant Pseudomonas aeruginosa and Acinetobacter species. Clin Infect Dis 43:S95–S99CrossRefPubMedGoogle Scholar
  5. 5.
    Rice LB (2006) Challenges in identifying new antimicrobial agents effective for treating infections with Acinetobacter baumannii and Pseudomonas aeruginosa. Clin Infect Dis 43:S100–S105CrossRefPubMedGoogle Scholar
  6. 6.
    Rossolini GM, Mantengoli E (2005) Treatment and control of severe infections caused by multiresistant Pseudomonas aeruginosa. Clin Microbiol Infect 11(Suppl 4):17–32CrossRefPubMedGoogle Scholar
  7. 7.
    Bratu S, Quale J, Cebular S et al (2005) Multidrug-resistant Pseudomonas aeruginosa in Brooklyn, New York: molecular epidemiology and in vitro activity of polymyxin B. Eur J Clin Microbiol Infect Dis 24:196–201CrossRefPubMedGoogle Scholar
  8. 8.
    Henrichfreise B, Wiegand I, Pfister W et al (2007) Resistance mechanisms of multiresistant Pseudomonas aeruginosa strains from Germany and correlation with hypermutation. Antimicrob Agents Chemother 51:4062–4070CrossRefPubMedGoogle Scholar
  9. 9.
    Obritsch MD, Fish DN, MacLaren R et al (2005) Nosocomial infections due to multidrug-resistant Pseudomonas aeruginosa: epidemiology and treatment options. Pharmacotherapy 25:1353–1364CrossRefPubMedGoogle Scholar
  10. 10.
    Polk RE, Johnson CK, McClish D et al (2004) Predicting hospital rates of fluoroquinolone-resistant Pseudomonas aeruginosa from fluoroquinolone use in US hospitals and their surrounding communities. Clin Infect Dis 39:497–503CrossRefPubMedGoogle Scholar
  11. 11.
    Mouneimné H, Robert J, Jarlier V et al (1999) Type II topoisomerase mutations in ciprofloxacin-resistant strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother 43:62–66PubMedGoogle Scholar
  12. 12.
    Wolfson JS, Hooper DC (1989) Bacterial resistance to quinolones: mechanisms and clinical importance. Rev Infect Dis 11:S906–S968Google Scholar
  13. 13.
    Oh H, Stenhoff J, Jalal S et al (2003) Role of efflux pumps and mutations in genes for topoisomerases II and IV in fluoroquinolone-resistant Pseudomonas aeruginosa strains. Microb Drug Resist 9:323–328CrossRefPubMedGoogle Scholar
  14. 14.
    Aeschlimann JR (2003) The role of multidrug efflux pumps in the antibiotic resistance of Pseudomonas aeruginosa and other gram-negative bacteria. Insights from the Society of Infectious Diseases Pharmacists. Pharmacotherapy 23:916–924CrossRefPubMedGoogle Scholar
  15. 15.
    Kriengkauykiat J, Porter E, Lomovskaya O et al (2005) Use of an efflux pump inhibitor to determine the prevalence of efflux pump-mediated fluoroquinolone resistance and multidrug resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 49:565–570CrossRefPubMedGoogle Scholar
  16. 16.
    Köhler T, Michéa-Hamzehpour M, Plésiat P et al (1997) Differential selection of multidrug efflux systems by quinolones in Pseudomonas aeruginosa. Antimicrob Agents Chemother 41:2540–2543PubMedGoogle Scholar
  17. 17.
    Masuda N, Sakagawa E, Ohya S et al (2000) Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux pumps in Pseudomonas aeruginosa. Antimicrob Agents Chemother 44:3322–3327CrossRefPubMedGoogle Scholar
  18. 18.
    Poole K, Gotoh N, Tsujimoto H et al (1996) Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa. Mol Microbiol 21:713–724CrossRefPubMedGoogle Scholar
  19. 19.
    Poole K, Srikumar R (2001) Multidrug efflux in Pseudomonas aeruginosa: components, mechanisms and clinical significance. Curr Top Med Chem 1:59–71CrossRefPubMedGoogle Scholar
  20. 20.
    Dumas J-L, van Delden C, Perron K et al (2006) Analysis of antibiotic resistance gene expression in Pseudomonas aeruginosa by quantitative real-time-PCR. FEMS Microbiol Lett 254:217–225CrossRefPubMedGoogle Scholar
  21. 21.
    Fukuda H, Hosaka M, Iyobe S et al (1995) NfxC-type quinolone resistance in a clinical isolate of Pseudomonas aeruginosa. Antimicrob Agents Chemother 39:790–792PubMedGoogle Scholar
  22. 22.
    Higgins PG, Fluit AC, Milatovic D et al (2003) Mutations in GyrA, ParC, MexR and NfxB in clinical isolates of Pseudomonas aeruginosa. Int J Antimicrob Agents 21:409–413CrossRefPubMedGoogle Scholar
  23. 23.
    Jalal S, Wretlind B (1998) Mechanisms of quinolone resistance in clinical strains of Pseudomonas aeruginosa. Microb Drug Resist 4:257–261CrossRefPubMedGoogle Scholar
  24. 24.
    Sobel ML, Hocquet D, Cao L et al (2005) Mutations in PA3574 (nalD) lead to increased mexab-oprm expression and multidrug resistance in laboratory and clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother 49:1782–1786CrossRefPubMedGoogle Scholar
  25. 25.
    Hocquet D, Roussel-Delvallez M, Cavallo J-D et al (2007) MexAB-OprM- and MexXY-overproducing mutants are very prevalent among clinical strains of Pseudomonas aeruginosa with reduced susceptibility to ticarcillin. Antimicrob Agents Chemother 51:1582–1583CrossRefPubMedGoogle Scholar
  26. 26.
    Nakajima A, Sugimoto Y, Yoneyama H et al (2002) High-level fluoroquinolone resistance in Pseudomonas aeruginosa due to interplay of the MexAB-OprM efflux pump and the DNA gyrase mutation. Microbiol Immunol 46:391–395PubMedGoogle Scholar
  27. 27.
    Amaral L, Pagès J-M (2008) Control and regulation of permeability of MDR bacterial pathogens to antibiotics. Curr Drug Targets 9:718CrossRefPubMedGoogle Scholar
  28. 28.
    Barrett JF (2001) MC-207110 Daiichi Seiyaku/Microcide Pharmaceuticals. Curr Opin Investig Drugs 2:212–215PubMedGoogle Scholar
  29. 29.
    Kaatz GW (2002) Inhibition of bacterial efflux pumps: a new strategy to combat increasing antimicrobial agent resistance. Expert Opin Emerg Drugs 7:223–233CrossRefPubMedGoogle Scholar
  30. 30.
    Lomovskaya O, Warren MS, Lee A et al (2001) Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrob Agents Chemother 45:105–116CrossRefPubMedGoogle Scholar
  31. 31.
    Lomovskaya O, Bostian KA (2006) Practical applications and feasibility of efflux pump inhibitors in the clinic—a vision for applied use. Biochem Pharmacol 71:910–918CrossRefPubMedGoogle Scholar
  32. 32.
    Lomovskaya O, Zgurskaya HI, Totrov M et al (2007) Waltzing transporters and ‘the dance macabre’ between humans and bacteria. Nat Rev Drug Discov 6:56–65CrossRefPubMedGoogle Scholar
  33. 33.
    Griffith D, Lomovskaya O, Lee V et al (2000) Potentiation of levofloxacin by MC-02595, a broad-spectrum efflux pump inhibitor (EPI) in mouse models of infection due to Pseudomonas aeruginosa with combinations of different Mex pump expression and gyrA mutation. In: Interscience Conference on Antimicrobial Agents in Chemotherapy, Toronto, Ontario, Canada, September 2000Google Scholar
  34. 34.
    Lomovskaya O, Lee A, Hoshino K et al (1999) Use of a genetic approach to evaluate the consequences of inhibition of efflux pumps in Pseudomonas aeruginosa. Antimicrob Agents Chemother 43:1340–1346PubMedGoogle Scholar
  35. 35.
    Jung R, Fish DN, Obritsch MD et al (2004) Surveillance of multi-drug resistant Pseudomonas aeruginosa in an urban tertiary-care teaching hospital. J Hosp Infect 57:105–111CrossRefPubMedGoogle Scholar
  36. 36.
    Pournaras S, Maniati M, Spanakis N et al (2005) Spread of efflux pump-overexpressing, non-metallo-beta-lactamase-producing, meropenem-resistant but ceftazidime-susceptible Pseudomonas aeruginosa in a region with blaVIM endemicity. J Antimicrob Chemother 56:761–764CrossRefPubMedGoogle Scholar
  37. 37.
    Yoshida K, Nakayama K, Ohtsuka M et al (2007) MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate. Bioorg Med Chem 15:7087–7097CrossRefPubMedGoogle Scholar
  38. 38.
    Stover CK, Pham XQ, Erwin AL et al (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964CrossRefPubMedGoogle Scholar
  39. 39.
    Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Biotechnology 1:784–791CrossRefGoogle Scholar
  40. 40.
    Approved Standard M7-A2 (1990) In: Standards. NCfCL, ed. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. NCCLS, VillanovaGoogle Scholar
  41. 41.
    Mesaros N, Glupczynski Y, Avrain L et al (2007) A combined phenotypic and genotypic method for the detection of Mex efflux pumps in Pseudomonas aeruginosa. J Antimicrob Chemother 59:378–386CrossRefPubMedGoogle Scholar
  42. 42.
    Okamoto K, Gotoh N, Nishino T (2002) Extrusion of penem antibiotics by multicomponent efflux systems MexAB-OprM, MexCD-OprJ, and MexXY-OprM of Pseudomonas aeruginosa. Antimicrob Agents Chemother 46:2696–2699CrossRefPubMedGoogle Scholar
  43. 43.
    Okamoto K, Gotoh N, Nishino T (2002) Alterations of susceptibility of Pseudomonas aeruginosa by overproduction of multidrug efflux systems, MexAB-OprM, MexCD-OprJ, and MexXY/OprM to carbapenems: substrate specificities of the efflux systems. J Infect Chemother 8:371–373CrossRefPubMedGoogle Scholar
  44. 44.
    Masuda N, Gotoh N, Ohya S et al (1996) Quantitative correlation between susceptibility and OprJ production in NfxB mutants of Pseudomonas aeruginosa. Antimicrob Agents Chemother 40:909–913PubMedGoogle Scholar
  45. 45.
    Köhler T, Michéa-Hamzehpour M, Henze U et al (1997) Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Mol Microbiol 23:345–354CrossRefPubMedGoogle Scholar
  46. 46.
    Wolter DJ, Hanson ND, Lister PD (2005) AmpC and OprD are not involved in the mechanism of imipenem hypersusceptibility among Pseudomonas aeruginosa isolates overexpressing the mexCD-oprJ efflux pump. Antimicrob Agents Chemother 49:4763–4766CrossRefPubMedGoogle Scholar
  47. 47.
    Quale J, Bratu S, Gupta J et al (2006) Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother 50:1633–1641CrossRefPubMedGoogle Scholar
  48. 48.
    Wolter DJ, Hanson ND, Lister PD (2004) Insertional inactivation of oprD in clinical isolates of Pseudomonas aeruginosa leading to carbapenem resistance. FEMS Microbiol Lett 236:137–143CrossRefPubMedGoogle Scholar
  49. 49.
    Akasaka T, Tanaka M, Yamaguchi A et al (2001) Type II topoisomerase mutations in fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa isolated in 1998 and 1999: role of target enzyme in mechanism of fluoroquinolone resistance. Antimicrob Agents Chemother 45:2263–2268CrossRefPubMedGoogle Scholar
  50. 50.
    DeRyke CA, Kuti JL, Nicolau DP (2007) Reevaluation of current susceptibility breakpoints for Gram-negative rods based on pharmacodynamic assessment. Diagn Microbiol Infect Dis 58:337–344CrossRefPubMedGoogle Scholar
  51. 51.
    Frei CR, Wiederhold NP, Burgess DS (2008) Antimicrobial breakpoints for Gram-negative aerobic bacteria based on pharmacokinetic-pharmacodynamic models with Monte Carlo simulation. J Antimicrob Chemother 61:621–628CrossRefPubMedGoogle Scholar
  52. 52.
    Ball P (2003) Adverse drug reactions: implications for the development of fluoroquinolones. J Antimicrob Chemother 51:21–27CrossRefPubMedGoogle Scholar
  53. 53.
    Hocquet D, Nordmann P, El Garch F et al (2006) Involvement of the MexXY-OprM efflux system in emergence of cefepime resistance in clinical strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother 50:1347–1351CrossRefPubMedGoogle Scholar
  54. 54.
    Linares JF, López JA, Camafeita E et al (2005) Overexpression of the multidrug efflux pumps MexCD-OprJ and MexEF-OprN is associated with a reduction of type III secretion in Pseudomonas aeruginosa. J Bacteriol 187:1384–1391CrossRefPubMedGoogle Scholar
  55. 55.
    Wolter DJ, Smith-Moland E, Goering RV et al (2004) Multidrug resistance associated with mexXY expression in clinical isolates of Pseudomonas aeruginosa from a Texas hospital. Diagn Microbiol Infect Dis 50:43–50CrossRefPubMedGoogle Scholar
  56. 56.
    Mueller MR, Hayden MK, Fridkin SK et al (2008) Nosocomial acquisition of Pseudomonas aeruginosa resistant to both ciprofloxacin and imipenem: a risk factor and laboratory analysis. Eur J Clin Microbiol Infect Dis 27:565–570CrossRefPubMedGoogle Scholar
  57. 57.
    Adams-Haduch JM, Paterson DL, Sidjabat HE et al (2008) Genetic basis of multidrug resistance in Acinetobacter baumannii clinical isolates at a tertiary medical center in Pennsylvania. Antimicrob Agents Chemother 52:3837–3843CrossRefPubMedGoogle Scholar
  58. 58.
    Jones GL, Warren RE, Skidmore SJ et al (2008) Prevalence and distribution of plasmid-mediated quinolone resistance genes in clinical isolates of Escherichia coli lacking extended-spectrum beta-lactamases. J Antimicrob Chemother 62:1245–1251CrossRefPubMedGoogle Scholar
  59. 59.
    Shin JH, Jung HJ, Lee JY et al (2008) High rates of plasmid-mediated quinolone resistance QnrB variants among ciprofloxacin-resistant Escherichia coli and Klebsiella pneumoniae from urinary tract infections in Korea. Microb Drug Resist 14:221–226CrossRefPubMedGoogle Scholar
  60. 60.
    El Amin N, Giske CG, Jalal S et al (2005) Carbapenem resistance mechanisms in Pseudomonas aeruginosa: alterations of porin OprD and efflux proteins do not fully explain resistance patterns observed in clinical isolates. APMIS 113:187–196CrossRefPubMedGoogle Scholar
  61. 61.
    Michéa-Hamzehpour M, Lucain C, Pechere JC (1991) Resistance to pefloxacin in Pseudomonas aeruginosa. Antimicrob Agents Chemother 35:512–518PubMedGoogle Scholar
  62. 62.
    Huang H, Hancock RE (1993) Genetic definition of the substrate selectivity of outer membrane porin protein OprD of Pseudomonas aeruginosa. J Bacteriol 175:7793–7800PubMedGoogle Scholar
  63. 63.
    Mahamoud A, Chevalier J, Alibert-Franco S et al (2007) Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy. J Antimicrob Chemother 59:1223–1229CrossRefPubMedGoogle Scholar
  64. 64.
    Hirakata Y, Srikumar R, Poole K et al (2002) Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa. J Exp Med 196:109–118CrossRefPubMedGoogle Scholar
  65. 65.
    Piddock LJV (2006) Multidrug-resistance efflux pumps—not just for resistance. Nat Rev Micro 4:629–636CrossRefGoogle Scholar
  66. 66.
    Tegos GP, Masago K, Aziz F et al (2008) Inhibitors of bacterial multidrug efflux pumps potentiate antimicrobial photoinactivation. Antimicrob Agents Chemother 52:3202–3209CrossRefPubMedGoogle Scholar
  67. 67.
    Schweizer HP (1998) Intrinsic resistance to inhibitors of fatty acid biosynthesis in Pseudomonas aeruginosa is due to efflux: application of a novel technique for generation of unmarked chromosomal mutations for the study of efflux systems. Antimicrob Agents Chemother 42:394–398PubMedGoogle Scholar
  68. 68.
    Chuanchuen R, Beinlich K, Hoang TT et al (2001) Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible mutant strain to triclosan selects nfxB mutants overexpressing MexCD-OprJ. Antimicrob Agents Chemother 45:428–432CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • S. A. Dunham
    • 1
  • C. J. McPherson
    • 2
  • A. A. Miller
    • 2
    Email author
  1. 1.Pfizer Animal HealthKalamazooUSA
  2. 2.Antibacterials BiologyPfizer Global Research and DevelopmentGrotonUSA

Personalised recommendations