Summary Bacterial lineages that chronically infect cystic fibrosis (CF) patients genetically diversify during infection. However, the mechanisms driving diversification are unknown. By dissecting 10 CF lung pairs and studying ~12,000 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 type 3 secretion systems. 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.

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.

The essential functions of a bacterial pathogen reflect the most basic processes required for its viability and growth, and represent potential therapeutic targets. Most screens for essential genes have assayed a single condition-growth in a rich undefined medium-and thus have not distinguished genes that are generally essential from those that are specific to this particular condition. To help define these classes for Pseudomonas aeruginosa, we identified genes required for growth on six different media, including a medium made from cystic fibrosis patient sputum. The analysis used the Tn-seq circle method to achieve high genome coverage and analyzed more than 1,000,000 unique insertion positions (an average of one insertion every 6.0 bp). We identified 352 general and 199 condition-specific essential genes. A subset of assignments was verified in individual strains with regulated expression alleles. The profile of essential genes revealed that, compared with Escherichia coli, P. aeruginosa is highly vulnerable to mutations disrupting central carbon-energy metabolism and reactive oxygen defenses. These vulnerabilities may arise from the stripped-down architecture of the organism's carbohydrate utilization pathways and its reliance on respiration for energy generation. The essential function profile thus provides fundamental insights into P. aeruginosa physiology as well as identifying candidate targets for new antibacterial agents.Tn-seq | ESKAPE | cystic fibrosis | antibiotic target | sputum

Our findings suggest that conditions associated with several CF pathogenesis hypotheses could cause the noninvasive and resistant infection phenotype, independently of the bacterial functions needed for biofilm formation.

Summary Biofilm growth increases the fitness of bacteria in harsh conditions. However, bacteria from clinical and environmental biofilms can exhibit impaired growth in culture, even when the species involved are readily cultureable, and permissive conditions are used. Here we show that culture-impaired variants of Pseudomonas aeruginosa rapidly and abundantly evolve in laboratory biofilms. The culture-impaired phenotype is caused by mutations that alter the outer-membrane lipopolysaccharide structure. Within biofilms, the lipopolysaccharide mutations markedly increase bacterial fitness. However, outside the protected biofilm environment, the mutations sensitize the variants to killing by a self-produced antimicrobial agent. Thus, a biofilm-mediated adaptation produces a stark fitness trade off that compromises bacterial survival in culture. Trade offs like this could limit the ability of bacteria to transition between biofilm growth and the free-living state, and produce bacterial populations that escape detection by culture-based sampling.