) it was shown that Pseudomonas aeruginosa undergoes intense genetic adaptation during chronic respiratory infection (CRI) in cystic fibrosis (CF) patients. We used the same collection of isolates to explore the role of hypermutation in this process, since one of the hallmarks of CRI is the high prevalence of DNA mismatch repair (MMR) system-deficient mutator strains. The presence of mutations in 34 genes (many of them positively linked to adaptation in CF patients) in the study collection of 90 P. aeruginosa isolates obtained longitudinally from 29 CF patients was not homogeneous; on the contrary, mutations were significantly concentrated in the mutator lineages, which represented 17% of the isolates (87% MMR deficient). While sequential nonmutator lineages acquired a median of only 0.25 mutation per year of infection, mutator lineages accumulated more than 3 mutations per year. On the whole-genome scale, data for the first fully sequenced late CF isolate, which was also shown to be an MMR-deficient mutator, also support these findings. Moreover, for the first time the predicted amplification of mutator populations due to hitchhiking with adaptive mutations in the course of natural human infections is clearly documented. Interestingly, increased accumulation of mutations in mutator lineages was not a consequence of overrepresentation of mutations in genes involved in antimicrobial resistance, the only adaptive trait linked so far to hypermutation in CF patients, demonstrating that hypermutation also plays a major role in P. aeruginosa genome evolution and adaptation during CRI.Chronic respiratory infection (CRI) with Pseudomonas aeruginosa is the main driver of morbidity and mortality in cystic fibrosis (CF) patients (4, 15). The establishment of P. aeruginosa CRI is mediated by a complex adaptive process that includes physiological changes produced by the activation of specific regulatory pathways, including the induction of the biofilm mode of growth or the differential expression of virulence genes (39), and genetic changes leading to selection of an important number of adaptive mutations required for long-term persistence (19,27,34).Although CF patients with CRI are generally infected by a single P. aeruginosa strain that in most cases persists throughout the patient's life (32), one of the hallmarks of such infections is the emergence and fixation over time of multiple phenotypic variants of the underlying clonal populations (24), a process known as adaptive radiation (30). Many of the selected phenotypes have been clearly linked to adaptation to the lung environment that favors lifelong persistence of CRI (27). Indeed, once the adaptation stage is reached and the CRI is fully established, eradication is generally no longer possible. At this point, with resignation, the therapeutic goals change from attempting to cure the infection to slowing the decline of lung function and improving the patient's quality of life.The intense genetic adaptation process that takes place during the establishment of CRI has r...

Hypermutable or mutator microorganisms are those that have an increased spontaneous mutation rate as a result of defects in DNA repair or error avoidance systems. Over the last two decades, several studies have provided strong evidence for a relevant role of mutators in the evolution of natural bacterial populations, particularly in the field of infectious diseases. Among them, chronic respiratory infection with Pseudomonas aeruginosa in cystic fibrosis (CF) patients was the first natural environment to reveal the high prevalence and important role of mutators. A remarkable positive selection of mutators during the course of the chronic infection has been reported, mainly as a result of the emergence of DNA mismatch repair system (mutS, mutL or mutU)-deficient mutants, although strains defective in the GO system (mutM, mutY and mutT) have also been observed. High frequencies of mutators have also been noted among other pathogens in the CF setting, particularly Staphylococcus aureus and Haemophilus influenzae. Enhanced antimicrobial resistance development is the most thoroughly studied consequence of mutators in CF and other chronic infections, although recent studies show that mutators may additionally have important effects on the evolution of virulence, genetic adaptation to the airways of CF patients, persistence of colonization, transmissibility, and perhaps lung function decline. Further prospective clinical studies are nevertheless still needed for an in-depth evaluation of the impact of mutators on disease progression and outcome.

Clinical isolates of Pseudomonas aeruginosa that hyperproduce a dark-brown pigment are quite often found in the lungs of chronically infected patients, suggesting that they may have an adaptive advantage in chronic infections. We have screened a library of random transposon insertions in P. aeruginosa. Transposon insertions resulting in the hyperproduction of a darkbrown pigment were found to be located in the hmgA gene, which putatively encodes the enzyme homogentisate-1,2-dioxygenase. Complementation studies indicate that hmgA disruption is responsible for the hyperproduction of pyomelanin in both laboratory and clinical isolates. A relationship between hmgA disruption and adaptation to chronic infection was explored and our results show that the inactivation of hmgA produces a slight reduction of killing ability in an acute murine model of lung infection. On the other hand, it also confers decreased clearance and increased persistence in chronic lung infections. Whether pyomelanin production is the cause of the increased adaptation to chronicity or just a side effect of hmgA inactivation is a question to be studied in future; however, this adaptation is consistent with the higher resistance to oxidative stress conferred in vitro by the pyomelanin pigment. Our results clearly demonstrate that hmgA inactivation leads to a better adaptation to chronic infection, and strongly suggest that this mechanism may be exploited in naturally occurring P. aeruginosa strains.

All extended-spectrum ␤-lactamase (ESBL)-producing Enterobacteriaceae isolates from patients admitted to and adult intensive care unit were prospectively documented from 2002 to 2005, when a large outbreak (51 patients affected) of multiresistant ESBL-producing Klebsiella pneumoniae infection was detected. The involvement of a single K. pneumoniae clone was demonstrated by pulsed-field gel electrophoresis. In addition to the ESBL-mediated resistance, the epidemic strain uniformly showed crossresistance to ciprofloxacin, gentamicin, tobramycin, trimethoprim-sulfamethoxazole, and tetracycline, whereas resistance to the ␤-lactam-␤-lactamase inhibitor combinations was variable. The ESBL involved was CTX-M-1, as demonstrated by isoelectric focusing, PCR amplification, and sequencing. CTX-M-1 as well as the aminoglycoside resistance determinants were encoded in a 50-kb plasmid that could be transferred to Escherichia coli only by transformation. In two of the infected patients, carbapenem resistance development (MICs of 8 to 12, 16, and >32 g/ml for imipenem, meropenem, and ertapenem, respectively) was documented, both in clinical samples and in intestinal colonization studies. The analysis of the outer membrane proteins of the carbapenem-susceptible and -resistant isolates revealed that the former expressed only one of the two major porins, OmpK36, whereas the latter did not express either of them. In one of the cases, the lack of expression of OmpK36 was demonstrated to be mediated by the interruption of the coding sequence by the insertion sequence IS26. This is the first report of a large outbreak of CTX-M-1-producing Enterobacteriaceae and, curiously, the first documented description in the literature of CTX-M-1 in K. pneumoniae, despite the fact that this enzyme has been found in multiple species. Furthermore, we document and characterize for the first time carbapenem resistance development in CTX-M-1-producing Enterobacteriaceae.

The high prevalence of hypermutable (mismatch repair-deficient) Pseudomonas aeruginosa strains in patients with cystic fibrosis (CF) is thought to be driven by their co-selection with adaptive mutations required for long-term persistence. Whether the increased mutation rate of naturally hypermutable strains is associated with a biological benefit or cost for the colonization of secondary environments is not known. Thirty-nine P. aeruginosa strains were collected from ten patients with CF during their course of chronic lung infections and screened for hypermutability. Seven hypermutable P. aeruginosa strains (18 %) isolated from six patients with CF (60 %) were identified and assigned to five different genotypes. Complementation and sequence analysis in the mutS, mutL and uvrD genes of these hypermutable P. aeruginosa strains revealed novel mutations. To understand the consequences of hypermutation for the fitness of the organisms, five pairs of clinical wild-type/hypermutable, clonally related P. aeruginosa strains and the laboratory strains PAO1/ PAO1DmutS were subjected to competition in vitro and in the agar-beads mouse model of chronic airway infection. When tested in competition assay in vitro, the wild-type outcompeted four clinical hypermutable strains and the PAO1DmutS strain. In vivo, all of the hypermutable strains were less efficient at establishing lung infection than their wild-type clones. These results suggest that P. aeruginosa hypermutation is associated with a biological cost, reducing the potential for colonization of new environments and therefore strain transmissibility.