Clonal Dissemination, Emergence of Mutator Lineages and Antibiotic Resistance Evolution in Pseudomonas aeruginosa Cystic Fibrosis Chronic Lung Infection

Abstract

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.

Antimicrobial Susceptibility of Pseudomonas aeruginosa Isolated from Cystic Fibrosis Patients in Northern Europe

“…For strains collected in the UK, higher resistance is clearly related to the high prevalence of the LES clone, which has been described as exhibiting a large proportion of MDR isolates (5). Of interest, we observed different resistance profiles within this clone, which is consistent with the previously described phenotypic variability among LES isolates (6). ST958, represented in the three countries, is also found among the MDR clonal complexes (7).…”

“…In contrast, other studies following the evolution of antibiotic susceptibility in successive isogenic isolates from a single patient suggest that resistance can occur either sporadically (50) or without correlation with the time of isolation (51). In these cases, the presence of mutator variants seems to predetermine the risk of developing resistance over time (6).…”

“…A further reason for the spread of antibiotic resistance in CF patients is the dissemination of MDR clones. The Liverpool epidemic strain (LES), first described in 1996 (4), has proven particularly successful at acquiring resistance mechanisms over the years (5,6) and at spreading from the United Kingdom (UK) to other countries, such as Canada, Spain, and Australia (7).…”

Sequential Treatment of Biofilms with Aztreonam and Tobramycin Is a Novel Strategy for Combating Pseudomonas aeruginosa Chronic Respiratory Infections

“…A total of 29 representative P. aeruginosa isolates from a well-characterized collection of cystic fibrosis (CF) strains from Hospital Son Espases (HSE), Palma de Mallorca, Spain (18) were screened for biofilm formation on microtiter plates using the crystal violet (CV) assay (data not shown) described by Christensen et al (23). Two hypermutable isolates showing robust biofilm formation and belonging to the LES-1 epidemic clone ST146 (146-HSE) and clone ST1089 (1089-HSE) (a hypermutable derivate from the epidemic clone ST274) (18) were selected as relevant representative CF strains for further studies.…”

“…P. aeruginosa phenotypes that are highly adapted and prevalent in CRI, such as mucoid and hypermutable strains, were used. Furthermore, we included two hypermutable clinical isolates deriving from two relevant CF epidemic clones, ST274 (18) and the multidrug-resistant (MDR) Liverpool epidemic strain (LES-1), which are likely the clones of most concern in CF worldwide (19).…”