is the most relevant pathogen producing chronic lung infections in patients with chronic underlying diseases such as CF and bronchiectasis and is beginning to be recognized as a relevant pathogen in COPD patients with advanced airflow obstruction. While long-term treatment with antipseudomonal agents is crucial to avoid a fast decline in the respiratory function of the infected patients, mutants resistant to multiple antimicrobials almost invariably evolve and frequently lead to treatment failure. Unlike many other bacteria, P. aeruginosa
can, by changes in single genes, generate mutants that are resistant to clinical concentrations of all antimicrobial agents used for therapy (20
In vitro and theoretical approaches suggest that the acquisition of a stable mutator phenotype may confer a selective advantage for bacteria, particularly in stressful and fluctuating environments (41
). In vivo experiments have also shown a potential benefit of hypermutation for bacterial adaptation to new environments, although once adapted, this advantage tends to disappear, and the transmissibility of the mutator strains is considerably reduced (8
). In natural bacterial populations, the presence of hypermutable strains has been linked to resistance to host immunological defenses, such as mutation-dependent phase variation in Neisseria meningitidis
) or resistance to antibiotics in P. aeruginosa
isolates from chronically infected CF patients (29
). Up to 37% of the CF patients were colonized by hypermutable strains in the mentioned study (29
), whereas not a single hypermutable strain was found in 75 acutely infected patients. Recently, results from another study have suggested that chronicity itself maybe a key factor for the selection of hypermutable P. aeruginosa
strains: despite the fact that mutational-antibiotic resistance development was a frequent outcome in a series of 103 acutely infected ICU patients, the prevalence of hypermutable strains was found to be lower than 1% (10
). Certainly, the extremely high prevalence of hypermutable strains found in the present work suggests that hypermutation plays a role in the bacterial adaptation to the lung environment required for long-term persistence because hypermutation increases the rates of generation of any mutant phenotype (not only antibiotic resistance), including those involved in the development of resistance to the host immune system. In this sense, it has recently been found in vitro that mismatch repair disruption in P. aeruginosa
determines the emergence at a high frequency of multiple morphological variants resembling what naturally occurs in chronic infections (39
). Furthermore, the link between hypermutation and chronic infections seems not to be restricted to just P. aeruginosa
, since recent works have found a significantly higher prevalence (14%) of hypermutable Staphylococcus aureus
and Haemophilus influenzae
strains in CF patients than in patients with other infections (1%) (32
Whether the impressive high frequency of hypermutable P. aeruginosa strains that we have found in chronic infections is a consequence of (i) coselection of the hypermutable variants with the adaptive mutations required for long-term bacterial persistence, (ii) their coselection with antibiotic resistance mutations required for survival during the frequent antibiotic treatments received by these patients, or, more likely, (iii) both selecting forces acting synergistically remains to be elucidated. Nevertheless, results from this work strongly suggest that hypermutation is the main driver for the development of antibiotic resistance in chronic P. aeruginosa infections, since most of the strains resistant to any of the antibiotics and all of the strains resistant to multiple antibiotics were hypermutable.
In the last 2 years, the role of hypermutation in antibiotic resistance development has begun to be acknowledged as a potentially concerning problem (1
). A recent in vitro work has shown that mutS
deficiency in P. aeruginosa
determines the immediate development of resistance to every single antipseudomonal agent, due to the ascent to dominance of resistant mutants in a few hours during drug exposure, and therefore monotherapy should be avoided in the treatment of infections by hypermutable strains (31
). These in vitro results, together with those obtained in the present study, certainly demonstrate that hypermutation is a major negative factor for the treatment of chronic infections not only because hypermutable strains are more frequently resistant to the antibiotics, limiting our therapeutic arsenal, but also because development of resistance to additional antibiotics is expected to be extremely fast during therapy.
Early treatment of P. aeruginosa
lung colonization in patients with underlying chronic respiratory diseases before the typical markers of chronic P. aeruginosa
infection, among them hypermutation, are selected should be the main therapeutic strategy (35
). Once the chronic infection is established and the hypermutable strains selected, eradication is expected to be almost impossible, and avoidance of treatment failure due to multiple-antimicrobial resistance development should be the main objective. Maintenance inhaled-tobramycin treatment has been found to have a positive effect on the pulmonary function of CF patients, whereas resistance selection has been found to be relatively low due to the higher (up to 100-fold) antibiotic concentrations reached in the colonization site than by the systemic route (34
). The high antibiotic concentrations may prevent resistance development even in the hypermutable strains, since the accumulation of multiple resistance mutations is required for reaching such high-level resistance.
In summary, in this work we find that hypermutable P. aeruginosa strains are extremely frequent in chronic lung infections, in contrast to what is observed in acute processes. Furthermore, hypermutation is found to be the main driver for the development of multiple-antimicrobial resistance in patients with chronic P. aeruginosa infections. In order to avoid this negative outcome, treatment strategies should be directed to the avoidance of the selection of hypermutable variants.