Hypermutation Is a Key Factor in Development of Multiple-Antimicrobial Resistance in Pseudomonas aeruginosa Strains Causing Chronic Lung Infections

Pseudomonas aeruginosa is the most relevant pathogen producing chronic lung infections in patients with chronic underlying diseases such as cystic fibrosis (CF), bronchiectasis, and chronic obstructive pulmonary disease (COPD). Hypermutable (or mutator) P. aeruginosa strains, characterized by increased (up to 1,000-fold) spontaneous mutation rates due to alterations of the DNA mismatch repair (MMR) system have been found at high frequencies in the lungs of CF patients, but their role in other chronic processes is still unknown. Sixty-two P. aeruginosa isolates from 30 patients with underlying non-CF chronic respiratory diseases (22 with bronchiectasis and 8 with COPD) and documented chronic infection were studied. Antibiotic susceptibility profiles and mutation frequencies were determined, and complementation assays using the cloned wild-type mutS gene and molecular epidemiology studies (pulsed-field electrophoresis, [PFGE]) were performed with these strains. Thirty-three (53%) of the isolates were hypermutable, and 17 (57%) of the 30 patients were colonized by hypermutable strains. Strains from 11 of the 17 patients were found to be defective in the MMR mutS gene by complementation assays. Interpatient transmission of strains was ruled out by PFGE. Multiple-antimicrobial resistance was documented in 42% of the hypermutable strains in contrast to 0% resistance in the nonhypermutable strains (P < 0.0001). Hypermutable P. aeruginosa strains are extremely prevalent in chronic infections in contrast to what has been described in acute processes, suggesting a role of hypermutation in bacterial adaptation for long-term persistence. Furthermore, hypermutation is found to be a key factor for the development of multiple-antimicrobial resistance, and therefore these findings are expected to have important consequences for the treatment of chronic infections

Clonal dissemination, emergence of mutator lineages and antibiotic resistance evolution in Pseudomonas aeruginosa cystic fibrosis chronic lung infection.

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 results and resistant mechanisms detected in first and last studied isolates from each patient and clone.

a<p>PAO1 and PA14 were used as reference wild-types sequences (<a href="http://www.pseudomonas.com" target="_blank">www.pseudomonas.com</a>).</p>b<p>1.2 Kb IS located in <i>mexX-mexZ</i> intergenic region (nt -72 respect <i>mexZ</i> coding sequence). Encodes a putative transposase identical to that previously reported in <i>Pseudomonas pseudoalcaligenes</i> CECT 5344 (ref ZP_10763279.1).</p

Schematic representation of the 10 sequential isolates from each of the 10 CF patients in the time frame of the study period.

<p>Each different clone is represented by a different colour. Resistance profiles and presence mutator phenotypes are indicated for each isolate. Results of MLST analysis are also provided for specific isolates.</p

Allelic profiles and relevant features of the different ST detected.

a<p>Nonpreviously described alleles are shown in bold.</p

Antimicrobial susceptibility of the studied <i>P. aeruginosa</i> isolates.

<p>Antimicrobial susceptibility of the studied <i>P. aeruginosa</i> isolates.</p

Bronchopulmonary infection-colonization patterns in Spanish cystic fibrosis patients: Results from a national multicenter study

Background: Clinical and demographical knowledge on Spanish cystic fibrosis (CF) patients is incomplete as no national registry exists. CF-microbiology has not been studied at national level. The results of the first Spanish multicenter study on CF microbiology are presented.Methods: 24 CF-Units for adult (n = 12) and pediatric (n = 12) patients from 17 hospitals provided sputa and clinical data from 15 consecutive patients. Cultures and susceptibility testing were performed. Colonization impact on pulmonary function was assessed.Results: 341 patients [mean (SD) age 21 (11) years, 180 >= 18 years, mean (SD) FEV1 = 68 (25)%] were included. Pseudomonas aeruginosa was reported as chronic, intermittent or absent in 46%, 22% and 32% of patients, respectively. The annual prevalence was 62%. Positive P. aeruginosa and methicillin-resistant Staphylococcus aureus cultures were significantly associated with lower FEV1 (p = 18 years, mean (SD) FEV1 = 68 (25)%] were included. Pseudomonas aeruginosa was reported as chronic, intermittent or absent in 46%, 22% and 32% of patients, respectively. The annual prevalence was 62%. Positive P. aeruginosa and methicillin-resistant Staphylococcus aureus cultures were significantly associated with lower FEV1 (