The lungs of cystic fibrosis (CF) patients are chronically infected for years by one or a few lineages of Pseudomonas aeruginosa. These bacterial populations adapt to the highly compartmentalized and anatomically deteriorating lung environment of CF patients, as well as to the challenges of the immune defenses and antibiotic therapy. These selective conditions are precisely those that recent theoretical studies predict for the evolution of mechanisms that augment the rate of variation. Determination of spontaneous mutation rates in 128 P. aeruginosa isolates from 30 CF patients revealed that 36% of the patients were colonized by a hypermutable (mutator) strain that persisted for years in most patients. Mutator strains were not found in 75 non-CF patients acutely infected with P. aeruginosa . This investigation also reveals a link between high mutation rates in vivo and the evolution of antibiotic resistance.

Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies.

It has long been recognized that the modification of penicillin-binding proteins (PBPs) to reduce their affinity for β-lactams is an important mechanism (target modification) by which Gram-positive cocci acquire antibiotic resistance. Among Gram-negative rods (GNR), however, this mechanism has been considered unusual, and restricted to clinically irrelevant laboratory mutants for most species. Using as a model Pseudomonas aeruginosa, high up on the list of pathogens causing life-threatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in β-lactam resistance in GNR, but through a totally distinct mechanism. Through a detailed genetic investigation, including whole-genome analysis approaches, we demonstrate that high-level (clinical) β-lactam resistance in vitro, in vivo, and in the clinical setting is driven by the inactivation of the dacB-encoded nonessential PBP4, which behaves as a trap target for β-lactams. The inactivation of this PBP is shown to determine a highly efficient and complex β-lactam resistance response, triggering overproduction of the chromosomal β-lactamase AmpC and the specific activation of the CreBC (BlrAB) two-component regulator, which in turn plays a major role in resistance. These findings are a major step forward in our understanding of β-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases. Decades after their discovery, β-lactams remain key components of our antimicrobial armamentarium for the treatment of infectious diseases. Nevertheless, resistance to these antibiotics is increasing alarmingly. There are two major bacterial strategies to develop resistance to β-lactam antibiotics: the production of enzymes that inactivate them (β-lactamases), or the modification of their targets in the cell wall (the essential penicillin-binding proteins, PBPs). Using the pathogen Pseudomonas aeruginosa as a model microorganism, we show that high-level (clinical) β-lactam resistance in vitro and in vivo frequently occurs through a previously unrecognized, totally distinct resistance pathway, driven by the mutational inactivation of a nonessential PBP (PBP4) that behaves as a trap target for β-lactams. We show that mutation of this PBP determines a highly efficient and complex β-lactam resistance response, triggering overproduction of the chromosomal β-lactamase AmpC and the specific activation of a two-component regulator, which in turn plays a key role in resistance. These findings are a major step forward in our understanding of β-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases.

Clinical antimicrobial susceptibility breakpoints are used to predict the clinical outcome of antimicrobial treatment. In contrast, microbiologic breakpoints are used to identify isolates that may be categorized as susceptible when applying clinical breakpoints but harbor resistance mechanisms that result in their reduced susceptibility to the agent being tested. Currently, the National Committee for Clinical Laboratory Standards (NCCLS) guidelines utilize clinical breakpoints to characterize the activity of the fluoroquinolones against Streptococcus pneumoniae. To determine whether levofloxacin breakpoints can identify isolates that harbor recognized resistance mechanisms, we examined 115 S. pneumoniae isolates with a levofloxacin MIC of >2 μg/mL for first-step parC mutations. A total of 48 (59%) of 82 isolates with a levofloxacin MIC of 2 μg/mL, a level considered susceptible by NCCLS criteria, had a first-step mutation in parC. Whether surveillance programs that use levofloxacin data can effectively detect emerging resistance and whether fluoroquinolones can effectively treat infections caused by such isolates should be evaluated.

The evolution and spread of antibiotic resistance depends on the antibiotic pressure exerted in the microbial environment. Selective effects occur in