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.

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.

Chronic respiratory infection by Pseudomonas aeruginosa is a major cause of mortality in cystic fibrosis (CF). We investigated the interplay between three key microbiological aspects of these infections: the occurrence of transmissible and persistent strains, the emergence of variants with enhanced mutation rates (mutators) and the evolution of antibiotic resistance. For this purpose, 10 sequential isolates, covering up to an 8-year period, from each of 10 CF patients were studied. As anticipated, resistance significantly accumulated overtime, and occurred more frequently among mutator variants detected in 6 of the patients. Nevertheless, highest resistance was documented for the nonmutator CF epidemic strain LES-1 (ST-146) detected for the first time in Spain. A correlation between resistance profiles and resistance mechanisms evaluated [efflux pump (mexB, mexD, mexF, and mexY) and ampC overexpression and OprD production] was not always obvious and hypersusceptibility to certain antibiotics (such as aztreonam or meropenem) was frequently observed. The analysis of whole genome macrorestriction fragments through Pulsed-Field Gel Electrophoresis (PFGE) revealed that a single genotype (clone FQSE-A) produced persistent infections in 4 of the patients. Multilocus Sequence typing (MLST) identified clone FQSE-A as the CF epidemic clone ST-274, but striking discrepancies between PFGE and MLST profiles were evidenced. While PFGE macrorestriction patterns remained stable, a new sequence type (ST-1089) was detected in two of the patients, differing from ST-274 by only two point mutations in two of the genes, each leading to a nonpreviously described allele. Moreover, detailed genetic analyses revealed that the new ST-1089 is a mutS deficient mutator lineage that evolved from the epidemic strain ST-274, acquired specific resistance mechanisms, and underwent further interpatient spread. Thus, presented results provide the first evidence of interpatient dissemination of mutator lineages and denote their potential for unexpected short-term sequence type evolution, illustrating the complexity of P. aeruginosa population biology in CF.

Chronic respiratory infection is the main cause of morbidity and mortality in cystic fibrosis (CF) patients. One of the hallmarks of these infections, led by the opportunistic pathogen Pseudomonas aeruginosa, is their long-term (lifelong) persistence despite intensive antimicrobial therapy. Antimicrobial resistance in CF is indeed a multifactorial problem, which includes physiological changes, represented by the transition from the planktonic to the biofilm mode of growth and the acquisition of multiple (antibiotic resistance) adaptive mutations catalyzed by frequent mutator phenotypes. Emerging multidrug-resistant CF pathogens, transmissible epidemic strains and transferable genetic elements (such as those encoding class B carbapenemases) also significantly contribute to this concerning scenario. Strategies directed to combat biofilm growth, prevent the emergence of mutational resistance, promote the development of novel antimicrobial agents against multidrug-resistant strains and implement strict infection control measures are thus needed.

Traditional therapeutic strategies to control chronic colonization in cystic fibrosis (CF) patients are based on the use of a single nebulized antibiotic. In this study, we evaluated the therapeutic efficacy and dynamics of antibiotic resistance in Pseudomonas aeruginosa biofilms under sequential therapy with inhaled aztreonam (ATM) and tobramycin (TOB). Laboratory strains PAO1, PAOMS (hypermutable), PAOMA (mucoid), and PAOMSA (mucoid and hypermutable) and two hypermutable CF strains, 146-HSE (Liverpool epidemic strain [LES-1]) and 1089-HSE (ST1089), were used. Biofilms were developed using the flow cell system. Mature biofilms were challenged with peak and 1/10-peak concentrations of ATM (700 mg/liter and 70 mg/liter), TOB (1,000 mg/liter and 100 mg/liter), and their alternations (ATM/TOB/ATM and TOB/ATM/TOB) for 2 (t ‫؍‬ 2), 4 (t ‫؍‬ 4), and 6 days (t ‫؍‬ 6). The numbers of viable cells (CFU) and resistant mutants were determined. Biofilm structural dynamics were monitored by confocal laser scanning microscopy and processed with COMSTAT and IMARIS software programs. TOB monotherapy produced an intense decrease in CFU that was not always correlated with a reduction in biomass and/or a bactericidal effect on biofilms, particularly for the CF strains. The ATM monotherapy bactericidal effect was lower, but effects on biofilm biomass and/or structure, including intense filamentation, were documented. The alternation of TOB and ATM led to an enhancement of the antibiofilm activity against laboratory and CF strains compared to that with the individual regimens, potentiating the bactericidal effect and/or the reduction in biomass, particularly at peak concentrations. Resistant mutants were not documented in any of the regimens at the peak concentrations and only anecdotally at the 1/10-peak concentrations. These results support the clinical evaluation of sequential regimens with inhaled antibiotics in CF, as opposed to the current maintenance treatments with just one antibiotic in monotherapy.