RESUMO
The 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 ß-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.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genes Bacterianos , Loci Gênicos , Genoma Bacteriano , Mutação , Pseudomonas aeruginosa/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Clonais , Fibrose Cística/tratamento farmacológico , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/microbiologia , Sistema Respiratório/patologia , Alinhamento de SequênciaRESUMO
BACKGROUND: Early antimicrobial chemotherapy can prevent or at least delay chronic cystic fibrosis (CF) airways infections with Pseudomonas aeruginosa. METHODS: During a 10-year study period P. aeruginosa was detected for the first time in 54 CF patients regularly seen at the CF centre Hannover. Amplicon sequencing of 34 loci of the P. aeruginosa core genome was performed in baseline and post-treatment isolates of the 15 CF patients who had remained P. aeruginosa - positive after the first round of antipseudomonal chemotherapy. RESULTS: Deep sequencing uncovered coexisting alternative nucleotides at in total 33 of 55,284 examined genome positions including six non-synonymous polymorphisms in the lasR gene, a key regulator of quorum sensing. After early treatment 42 of 50 novel nucleotide substitutions had emerged in exopolysaccharide biosynthesis, efflux pump and porin genes. CONCLUSIONS: Early treatment selects pathoadaptive mutations in P. aeruginosa that are typical for chronic infections of CF lungs.