A Dose-Response Study of Antibiotic Resistance inPseudomonas aeruginosa Biofilms

ABSTRACT Bacterial biofilms show enormous levels of antibiotic resistance, but little is known about the underlying molecular mechanisms. Multidrug resistance pumps (MDRs) are responsible for the extrusion of chemically unrelated antimicrobials from the bacterial cell. Contribution of the MDR-mediated efflux to antibiotic resistance ofPseudomonas aeruginosa biofilms was examined by using strains overexpressing and lacking the MexAB-OprM pump. Resistance ofP. aeruginosa biofilms to ofloxacin was dependent on the expression of MexAB-OprM but only in the low concentration range. Unexpectedly, biofilm resistance to ciprofloxacin, another substrate of MexAB-OprM, did not depend on the presence of this pump. Dose-dependent killing indicated the presence of a small “superresistant” cell fraction. This fraction was primarily responsible for very high resistance of P. aeruginosa biofilms to quinolones. Bacterial cells recovered from a biofilm and tested under nongrowing conditions with tobramycin exhibited higher resistance levels than planktonic cells but lower levels than cells of an intact biofilm.

[1]  H. Ceri,et al.  The Calgary Biofilm Device: New Technology for Rapid Determination of Antibiotic Susceptibilities of Bacterial Biofilms , 1999, Journal of Clinical Microbiology.

[2]  S. Gander.  Bacterial biofilms: resistance to antimicrobial agents. , 1996, The Journal of antimicrobial chemotherapy.

[3]  J. Hearst,et al.  The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals , 1996, Molecular microbiology.

[4]  N. Masuda,et al.  Cross-resistance to meropenem, cephems, and quinolones in Pseudomonas aeruginosa , 1992, Antimicrobial Agents and Chemotherapy.

[5]  S. Taylor,et al.  A protein that activates expression of a multidrug efflux transporter upon binding the transporter substrates. , 1994, The Journal of biological chemistry.

[6]  A. Matin,et al.  EmrR is a negative regulator of the Escherichia coli multidrug resistance pump EmrAB , 1995, Journal of bacteriology.

[7]  Roberto Kolter,et al.  Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis , 1998, Molecular microbiology.

[8]  I. Paulsen,et al.  Proton-dependent multidrug efflux systems , 1996, Microbiological reviews.

[9]  D. Livermore,et al.  Mechanisms of resistance to beta-lactam antibiotics amongst Pseudomonas aeruginosa isolates collected in the UK in 1993. , 1995, Journal of medical microbiology.

[10]  K. Poole,et al.  β-Lactamase Inhibitors Are Substrates for the Multidrug Efflux Pumps of Pseudomonas aeruginosa , 1998, Antimicrobial Agents and Chemotherapy.

[11]  V. Deretic,et al.  Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. , 1996, Microbiological reviews.

[12]  H L Walmsley,et al.  The penetration of antibiotics into aggregates of mucoid and non-mucoid Pseudomonas aeruginosa. , 1989, Journal of general microbiology.

[13]  D. Musher,et al.  Vancomycin penetration into biofilm covering infected prostheses and effect on bacteria. , 1994, The Journal of infectious diseases.

[14]  J. Costerton,et al.  The involvement of cell-to-cell signals in the development of a bacterial biofilm. , 1998, Science.

[15]  T. Nishino,et al.  nfxC-type quinolone resistance in a clinical isolate of Pseudomonas aeruginosa , 1995, Antimicrobial agents and chemotherapy.

[16]  J. Struthers,et al.  The growth of Gardnerella vaginalis and Lactobacillus acidophilus in Sorbarod biofilms. , 1998, Journal of medical microbiology.

[17]  K. Poole.  Bacterial multidrug resistance--emphasis on efflux mechanisms and Pseudomonas aeruginosa. , 1994, The Journal of antimicrobial chemotherapy.

[18]  H. Nikaido,et al.  Involvement of an Active Efflux System in the Natural Resistance of Pseudomonas aeruginosa to Aminoglycosides , 1999, Antimicrobial Agents and Chemotherapy.

[19]  B. Iglewski,et al.  Active Efflux and Diffusion Are Involved in Transport of Pseudomonas aeruginosa Cell-to-Cell Signals , 1999, Journal of bacteriology.

[20]  S. Mitsuhashi,et al.  Mechanisms of high-level resistance to quinolones in urinary tract isolates of Pseudomonas aeruginosa , 1994, Antimicrobial Agents and Chemotherapy.

[21]  B. Iglewski,et al.  Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes , 1997, Journal of bacteriology.

[22]  H. Hashimoto,et al.  Occurrence of the nfxB type mutation in clinical isolates of Pseudomonas aeruginosa , 1992, Antimicrobial Agents and Chemotherapy.

[23]  S. Gander.  Bacterial biofllms: resistance to antimicrobial agents J Anlimicrob Chemother 1996; 37: 1047–1050 , 1996 .

[24]  Richard A. Moore,et al.  Efflux-Mediated Aminoglycoside and Macrolide Resistance in Burkholderia pseudomallei , 1999, Antimicrobial Agents and Chemotherapy.

[25]  A. Brooun,et al.  Purification and Ligand Binding of EmrR, a Regulator of a Multidrug Transporter , 1999, Journal of bacteriology.

[26]  K. Poole,et al.  Overexpression of the mexC–mexD–oprJ efflux operon in nfxB‐type multidrug‐resistant strains of Pseudomonas aeruginosa , 1996, Molecular microbiology.

[27]  J. Costerton,et al.  Bacterial biofilms: a common cause of persistent infections. , 1999, Science.

[28]  K. Lewis,et al.  Multidrug resistance pumps in bacteria: variations on a theme. , 1994, Trends in biochemical sciences.

[29]  H. Nikaido.  Multidrug efflux pumps of gram-negative bacteria , 1996, Journal of bacteriology.

[30]  R. Kolter,et al.  Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development , 1998, Molecular microbiology.

[31]  J. Costerton,et al.  The biofilm glycocalyx as a resistance factor. , 1990, The Journal of antimicrobial chemotherapy.

[32]  J. Costerton,et al.  Induction of beta-lactamase production in Pseudomonas aeruginosa biofilm , 1991, Antimicrobial Agents and Chemotherapy.

[33]  H. Nikaido,et al.  Role of mexA-mexB-oprM in antibiotic efflux in Pseudomonas aeruginosa , 1995, Antimicrobial agents and chemotherapy.

[34]  J. Lacroix,et al.  Effects of metronidazole on Porphyromonas gingivalis biofilms. , 1997, Journal of periodontal research.

[35]  K. Poole,et al.  Influence of the MexAB-OprM Multidrug Efflux System on Quorum Sensing in Pseudomonas aeruginosa , 1998, Journal of bacteriology.

[36]  R. Kolter,et al.  Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili , 1998, Molecular microbiology.

[37]  S. J. Knott,et al.  Effect of antibiotics on non-growing planktonic cells and biofilms of Escherichia coli. , 1994, The Journal of antimicrobial chemotherapy.

[38]  P. Stewart,et al.  Theoretical aspects of antibiotic diffusion into microbial biofilms , 1996, Antimicrobial agents and chemotherapy.

[39]  I. Paulsen,et al.  QacR Is a Repressor Protein That Regulates Expression of theStaphylococcus aureus Multidrug Efflux Pump QacA* , 1998, The Journal of Biological Chemistry.

[40]  H. Nikaido,et al.  AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants , 1996, Journal of bacteriology.

[41]  N. Gotoh,et al.  Characterization of MexE–MexF–OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa , 1997, Molecular microbiology.

[42]  C. Potera.  Forging a Link Between Biofilms and Disease , 1999, Science.