RESUMO
Recent work using culture-independent methods suggests that the lungs of cystic fibrosis (CF) patients harbor a vast array of bacteria not conventionally implicated in CF lung disease. However, sampling lung secretions in living subjects requires that expectorated specimens or collection devices pass through the oropharynx. Thus, contamination could confound results. Here, we compared culture-independent analyses of throat and sputum specimens to samples directly obtained from the lungs at the time of transplantation. We found that CF lungs with advanced disease contained relatively homogenous populations of typical CF pathogens. In contrast, upper-airway specimens from the same subjects contained higher levels of microbial diversity and organisms not typically considered CF pathogens. Furthermore, sputum exhibited day-to-day variation in the abundance of nontypical organisms, even in the absence of clinical changes. These findings suggest that oropharyngeal contamination could limit the accuracy of DNA-based measurements on upper-airway specimens. This work highlights the importance of sampling procedures for microbiome studies and suggests that methods that account for contamination are needed when DNA-based methods are used on clinical specimens.
Assuntos
Fibrose Cística/genética , Pulmão/microbiologia , Metagenoma/fisiologia , Escarro/microbiologia , Traqueia/microbiologia , Antibacterianos/farmacologia , Bactérias/genética , Humanos , Pulmão/metabolismo , Pneumologia/métodos , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNA , Especificidade da Espécie , Manejo de EspécimesRESUMO
Bacterial lineages that chronically infect cystic fibrosis (CF) patients genetically diversify during infection. However, the mechanisms driving diversification are unknown. By dissecting ten CF lung pairs and studying â¼12,000 regional isolates, we were able to investigate whether clonally related Pseudomonas aeruginosa inhabiting different lung regions evolve independently and differ functionally. Phylogenetic analysis of genome sequences showed that regional isolation of P. aeruginosa drives divergent evolution. We investigated the consequences of regional evolution by studying isolates from mildly and severely diseased lung regions and found evolved differences in bacterial nutritional requirements, host defense and antibiotic resistance, and virulence due to hyperactivity of the type 3 secretion system. These findings suggest that bacterial intermixing is limited in CF lungs and that regional selective pressures may markedly differ. The findings also may explain how specialized bacterial variants arise during infection and raise the possibility that pathogen diversification occurs in other chronic infections characterized by spatially heterogeneous conditions.