Biofilms are organized bacterial communities embedded in an extracellular polymeric matrix attached to living or abiotic surfaces. The development of biofilms is currently recognized as one of the most relevant drivers of persistent infections. Among them, chronic respiratory infection by Pseudomonas aeruginosa in cystic fibrosis patients is probably the most intensively studied. The lack of correlation between conventional susceptibility test results and therapeutic success in chronic infections is probably a consequence of the use of planktonically growing instead of biofilm-growing bacteria. Therefore, several in vitro models to evaluate antimicrobial activity on biofilms have been implemented over the last decade. Microtitre plate-based assays, the Calgary device, substratum suspending reactors and the flow cell system are some of the most used in vitro biofilm models for susceptibility studies. Likewise, new pharmacodynamic parameters, including minimal biofilm inhibitory concentration, minimal biofilm-eradication concentration, biofilm bactericidal concentration, and biofilm-prevention concentration, have been defined in recent years to quantify antibiotic activity in biofilms. Using these parameters, several studies have shown very significant quantitative and qualitative differences for the effects of most antibiotics when acting on planktonic or biofilm bacteria. Nevertheless, standardization of the procedures, parameters and breakpoints, by official agencies, is needed before they are implemented in clinical microbiology laboratories for routine susceptibility testing. Research efforts should also be directed to obtaining a deeper understanding of biofilm resistance mechanisms, the evaluation of optimal pharmacokinetic/pharmacodynamic models for biofilm growth, and correlation with clinical outcome.

Pseudomonas aeruginosa is the most relevant pathogen producing chronic lung infections in patients with chronic underlying diseases such as cystic fibrosis (CF), bronchiectasis, and chronic obstructive pulmonary disease (COPD). Hypermutable (or mutator) P. aeruginosa strains, characterized by increased (up to 1,000-fold) spontaneous mutation rates due to alterations of the DNA mismatch repair (MMR) system have been found at high frequencies in the lungs of CF patients, but their role in other chronic processes is still unknown. Sixty-two P. aeruginosa isolates from 30 patients with underlying non-CF chronic respiratory diseases (22 with bronchiectasis and 8 with COPD) and documented chronic infection were studied. Antibiotic susceptibility profiles and mutation frequencies were determined, and complementation assays using the cloned wild-type mutS gene and molecular epidemiology studies (pulsed-field electrophoresis, [PFGE]) were performed with these strains. Thirty-three (53%) of the isolates were hypermutable, and 17 (57%) of the 30 patients were colonized by hypermutable strains. Strains from 11 of the 17 patients were found to be defective in the MMR mutS gene by complementation assays. Interpatient transmission of strains was ruled out by PFGE. Multipleantimicrobial resistance was documented in 42% of the hypermutable strains in contrast to 0% resistance in the nonhypermutable strains (P < 0.0001). Hypermutable P. aeruginosa strains are extremely prevalent in chronic infections in contrast to what has been described in acute processes, suggesting a role of hypermutation in bacterial adaptation for long-term persistence. Furthermore, hypermutation is found to be a key factor for the development of multiple-antimicrobial resistance, and therefore these findings are expected to have important consequences for the treatment of chronic infections.

Bacteria with greatly elevated mutation rates (mutators) are frequently found in natural and laboratory populations, and are often associated with clinical infections. Although mutators may increase adaptability to novel environmental conditions, they are also prone to the accumulation of deleterious mutations. The long-term maintenance of high bacterial mutation rates is therefore likely to be driven by rapidly changing selection pressures, in addition to the possible slow transition rate by point mutation from mutators to non-mutators. One of the most likely causes of rapidly changing selection pressures is antagonistic coevolution with parasites. Here we show whether coevolution with viral parasites could drive the evolution of bacterial mutation rates in laboratory populations of the bacterium Pseudomonas fluorescens. After fewer than 200 bacterial generations, 25% of the populations coevolving with phages had evolved 10- to 100-fold increases in mutation rates owing to mutations in mismatch-repair genes; no populations evolving in the absence of phages showed any significant change in mutation rate. Furthermore, mutator populations had a higher probability of driving their phage populations extinct, strongly suggesting that mutators have an advantage against phages in the coevolutionary arms race. Given their ubiquity, bacteriophages may play an important role in the evolution of bacterial mutation rates.

Bacterial biofilms are associated with a wide range of infections, from those related to exogenous devices, such as catheters or prosthetic joints, to chronic tissue infections such as those occurring in the lungs of cystic fibrosis patients. Biofilms are recalcitrant to antibiotic treatment due to multiple tolerance mechanisms (phenotypic resistance). This causes persistence of biofilm infections in spite of antibiotic exposure which predisposes to antibiotic resistance development (genetic resistance). Understanding the interplay between phenotypic and genetic resistance mechanisms acting on biofilms, as well as appreciating the diversity of environmental conditions of biofilm infections which influence the effect of antibiotics are required in order to optimize the antibiotic treatment of biofilm infections. Here, we review the current knowledge on phenotypic and genetic resistance in biofilms and describe the potential strategies for the antibiotic treatment of biofilm infections. Of note is the optimization of PK/PD parameters in biofilms, high-dose topical treatments, combined and sequential/alternate therapies or the use antibiotic adjuvants.

These results provide the first evidence supporting the hypothesis that P. aeruginosa causes chronic infections in COPD, with patterns of infection and evolution that resemble those observed in cystic fibrosis. Experience gained from treating cystic fibrosis might be useful for implementing new procedures for the prevention, diagnosis, and treatment of infection due to P. aeruginosa in COPD.

The prevalence and impact of the overexpression of AmpC and efflux pumps were evaluated with a collection of 190 Pseudomonas aeruginosa isolates recovered from bloodstream infections in a 2008 multicenter study (10 hospitals) in Spain. The MICs of a panel of 13 antipseudomonal agents were determined by microdilution, and the expressions of ampC, mexB, mexY, mexD, and mexF were determined by real-time reverse transcription (RT)-PCR. Up to 39% of the isolates overexpressed at least one of the mechanisms. ampC overexpression (24.2%) was the most prevalent mechanism, followed by mexY (13.2%), mexB (12.6%), mexF (4.2%), and mexD (2.2%). The overexpression of mexB plus mexY, documented for 5.3% of the isolates, was the only combination showing a significantly (P ‫؍‬ 0.02) higher prevalence than expected from the frequencies of the individual mechanisms (1.6%). Additionally, all imipenem-resistant isolates studied (25 representative isolates) showed inactivating mutations in oprD. Most of the isolates nonsusceptible to piperacillin-tazobactam (96%) and ceftazidime (84%) overexpressed ampC, while mexB (25%) and mexY (29%) overexpressions gained relevance among cefepime-nonsusceptible isolates. Nevertheless, the prevalence of mexY overexpression was highest among tobramycin-nonsusceptible isolates (37%), and that of mexB was highest among meropenem-nonsusceptible isolates (33%). Regarding ciprofloxacin-resistant isolates, besides the expected increased prevalence of efflux pump overexpression, a highly significant link to ampC overexpression was documented for the first time: up to 52% of ciprofloxacin-nonsusceptible isolates overexpressed ampC, sharply contrasting with the 24% documented for the complete collection (P < 0.001). In summary, mutation-driven resistance was frequent in P. aeruginosa isolates from bloodstream infections, whereas metallo-␤-lactamases, detected in 2 isolates (1%) producing VIM-2, although with increasing prevalences, were still uncommon.The increasing prevalence of nosocomial infections produced by multidrug-resistant (MDR) Pseudomonas aeruginosa strains severely compromises the selection of appropriate treatments and is therefore associated with significant morbidity and mortality (17,21,25). The growing threat of antimicrobial resistance in P. aeruginosa results from the extraordinary capacity of this microorganism for developing resistance to almost any available antibiotic by the selection of mutations in chromosomal genes and from the increasing prevalence of transferrable resistance determinants, particularly those encoding class B carbapenemases (or metallo-␤-lactamases [MBLs]) or extended-spectrum ␤-lactamases (ESBLs), frequently cotransferred with genes encoding aminoglycoside-modifying enzymes (18,19). Among the mutation-mediated resistance mechanisms, particularly noteworthy are those leading to the repression or inactivation of the carbapenem porin OprD, the hyperproduction of the chromosomal cephalosporinase AmpC, or the upregulation of one of the several efflux pumps encoded in t...

The molecular mechanisms of ␤-lactam resistance mediated by AmpC hyperproduction in natural strains of Pseudomonas aeruginosa were investigated in a collection of 10 isogenic, ceftazidime-susceptible and -resistant pairs of isolates, each sequentially recovered from a different intensive care unit patient treated with ␤-lactams. All 10 ceftazidime-resistant mutants hyperproduced AmpC (␤-lactamase activities were 12-to 657-fold higher than those of the parent strains), but none of them harbored mutations in ampR or the ampC-ampR intergenic region. On the other hand, six of them harbored inactivating mutations in ampD: four contained frameshift mutations, one had a C3T mutation, creating a premature stop codon, and finally, one had a large deletion, including the complete ampDE region. Complementation studies revealed that only three of the six ampD mutants could be fully transcomplemented with either ampD-or ampDE-harboring plasmids, whereas one of them could be transcomplemented only with ampDE and two of them (including the mutant with the deletion of the ampDE region and one with an ampD frameshift mutation leading to an ampDE-fused open reading frame) could not be fully transcomplemented with any of the plasmids. Finally, one of the four mutants with no mutations in ampD could be transcomplemented, but only with ampDE. Although the inactivation of AmpD is found to be the most frequent mechanism of AmpC hyperproduction in clinical strains, our findings suggest that for certain types of mutations, AmpE plays an indirect role in resistance and that there are other unknown genes involved in AmpC hyperproduction, with at least one of them apparently located close to the ampDE operon.

Hypermutable Pseudomonas aeruginosa strains are found with high frequency in the lungs of patients with chronic infections and are associated with high antibiotic resistance rates. The in vivo consequences of hypermutation for treatment in a mouse model of lung infection using strain PAO1 and its hypermutable derivative PAO⌬mutS are investigated. Groups of 30 mice were treated for 3 days with humanized regimens of ciprofloxacin (CIP), tobramycin (TOB), CIP plus TOB, or placebo, and mortality, total lung bacterial load, and 4؋-and 16؋-MIC mutants were recorded. The rates of mutation and the initial in vivo frequencies of mutants (at the onset of treatment) were also estimated and the in vitro-and in vivo-selected mutants characterized. Since both strains had identical MICs, the same pharmacokinetic/pharmacodynamic (PK/PD) parameters were obtained: area under the 24-h concentration-time curve (fAUC)/MIC ‫؍‬ 385 for CIP and maximum concentration of drug in serum (fC max )/MIC ‫؍‬ 19 for TOB. Despite adequate PK/PD parameters, persistence of high bacterial numbers and amplification (50,000-fold) of resistant mutants (MexCD-OprJ hyperexpression) were documented with CIP treatment for PAO⌬mutS, in contrast to complete resistance suppression for PAO1 (P < 0.01), showing that conventional PK/PD parameters may not be applicable to infections by hypermutable strains. On the other hand, the efficacy of TOB monotherapy in terms of mortality reduction and bacterial load was very low regardless of the strain but not due to resistance development, since mutants were not selected for PAO1 and were only modestly amplified for PAO⌬mutS. Finally, the CIP-plus-TOB combination was synergistic, further reducing mortality and bacterial load and completely preventing resistance even for PAO⌬mutS (P < 0.01 compared to monotherapy), showing that it is possible to suppress resistance selection in infections by hypermutable P. aeruginosa using appropriate combined regimens.