Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients

Abstract

bThe chronic airway infections with Pseudomonas aeruginosa in people with cystic fibrosis (CF) are treated with aerosolized antibiotics, oral fluoroquinolones, and/or intravenous combination therapy with aminoglycosides and ␤-lactam antibiotics. An international strain collection of 361 P. aeruginosa isolates from 258 CF patients seen at 30 CF clinics was examined for mutations in 17 antimicrobial susceptibility and resistance loci that had been identified as hot spots of mutation by genome sequencing of serial isolates from a single CF clinic. Combinatorial amplicon sequencing of pooled PCR products identified 1,112 sequence variants that were not present in the genomes of representative strains of the 20 most common clones of the global P. aeruginosa population. A high frequency of singular coding variants was seen in spuE, mexA, gyrA, rpoB, fusA1, mexZ, mexY, oprD, ampD, parR, parS, and envZ (amgS), reflecting the pressure upon P. aeruginosa in lungs of CF patients to generate novel protein variants. The proportion of nonneutral amino acid exchanges was high. Of the 17 loci, mexA, mexZ, and pagL were most frequently affected by independent stop mutations. Private and de novo mutations seem to play a pivotal role in the response of P. aeruginosa populations to the antimicrobial load and the individual CF host. C ystic fibrosis (CF) is a severe autosomal recessive trait that is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) (1). CFTR dysfunction affects many organs, but lung disease is responsible for the vast majority of morbidity and mortality in people with CF. The basic defect in CF predisposes to viral and bacterial airway infections (2). The most prevalent airway pathogen in adolescents and adults with CF is Pseudomonas aeruginosa (3, 4). The chronic airway colonization with P. aeruginosa has been associated with a rapid decline in lung function, heightened lung inflammation, and worse prognosis (1).Antipseudomonal chemotherapy in CF comprises intravenous combination therapy with ␤-lactam antibiotics and aminoglycosides, oral fluoroquinolones, immunomodulatory treatment with azithromycin and aerosolized antibiotics such as tobramycin, aztreonam, or colistin (4-8). The high antimicrobial pressure exerted on the microorganism, which can persist and diversify in the patient's airways for decades, drives the emergence of drug resistance phenotypes (9, 10). Typical targets are elements of replication, transcription, and translation, the cell wall, and transport and its regulation (9-11).During ongoing studies on the long-term microevolution of P. aeruginosa in lungs of 12 CF patients, we have identified 17 loci to be repetitively hit by mutations. These loci are known from the literature to be associated with the emergence of drug resistance in P. aeruginosa (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Hence, we became interested to know if and to what extent the global P. aeruginosa population in lungs of patients (CF lungs) carries mutations in these ge...

Genomic Analysis Identifies Novel Pseudomonas aeruginosa Resistance Genes under Selection during Inhaled Aztreonam Therapy In Vivo

“…Although loss of MexA resulted in aztreonam hypersensitivity (Table 3), observed mutations were not recurrent or spatially restricted to specific regions of the protein, making them less likely to fit the profile of gain-of-function changes. Though these results appear paradoxical, they are consistent with prior analyses of CF clinical isolates which have shown frequent missense and loss-of-function mutations in MexA (22,50). Given the correlation of MexAB-OprM overproduction with increased virulence (12), it is possible that inactivating mexA mutations reflect selection for attenuation during aztreonam therapy (51) rather than increased resistance to aztreonam itself.…”

Mutations in Gene fusA1 as a Novel Mechanism of Aminoglycoside Resistance in Clinical Strains of Pseudomonas aeruginosa

“…Thus, it was suggested that these genetic alterations could contribute to the development of a high resistance to aminoglycosides in the CF context (20,(34)(35)(36). For instance, 52 of 361 nonrelated CF isolates appeared to harbor nonsynonymous mutations in fusA1 (34). In another study, the gene was found to be mutated in 13 isolates belonging to international clone CC274, within a collection of 29 CF strains (20).…”

“…A number of studies on the genomic evolution of P. aeruginosa strains in CF patients have reported that the fusA1 gene is one of the most repeatedly hit by mutations. Thus, it was suggested that these genetic alterations could contribute to the development of a high resistance to aminoglycosides in the CF context (20,(34)(35)(36). For instance, 52 of 361 nonrelated CF isolates appeared to harbor nonsynonymous mutations in fusA1 (34).…”

“…Our analysis of the single nucleotide polymorphisms (SNPs) found in various CF and non-CF strains revealed that three of the amino acid substitutions predicted to occur in EF-G1A were identical to ones substitutions in the present study (Val93Ala, Thr456Ala, and Thr671Ala), thus reinforcing the notion that such variations represent a common mechanism of aminoglycoside resistance in P. aeruginosa. To assess the phenotypic impact of other reported fusA1 mutations (34,35,37), site-directed mutagenesis experiments were carried out to generate fusA1 alleles encoding EF-G1A variants with Leu40Gln (domain G), Gly118Ser (domain G), and Asn592Ile (domain IV) changes, respectively (Fig. 2).…”

Emergence of Antimicrobial Resistance among Pseudomonas aeruginosa: Implications for Therapy

“…PA (particularly mucoid strains) is the most common respiratory pathogen in CF 145 and has been associated with increased frequency of exacerbations, disease progression, and mortality in both CF 105,146,147 and non-CF bronchiec-tases. 39,40,[148][149][150][151][152] Antimicrobial resistance rates are particularly high in patients with CF 38,153,154 and MDR-PA strains may limit therapeutic options. 155 Several distinct epidemic clones of MDR-PA have disseminated globally among CF patients and centers.…”