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.

While much attention has been focused on acquired antibiotic resistance genes, chromosomal mutations may be most important in chronic infections where isolated, persistently infecting lineages experience repeated antibiotic exposure. Here, we used experimental evolution and whole-genome sequencing to investigate chromosomally encoded mutations causing aztreonam resistance in Pseudomonas aeruginosa and characterized the secondary consequences of resistance development. We identified 19 recurrently mutated genes associated with aztreonam resistance. The most frequently observed mutations affected negative transcriptional regulators of the mexAB-oprM efflux system and the target of aztreonam, ftsI. While individual mutations conferred modest resistance gains, high-level resistance (1,024 µg/ml) was achieved through the accumulation of multiple variants. Despite being largely stable when strains were passaged in the absence of antibiotics, aztreonam resistance was associated with decreased in vitro growth rates, indicating an associated fitness cost. In some instances, evolved aztreonam-resistant strains exhibited increased resistance to structurally unrelated antipseudomonal antibiotics. Surprisingly, strains carrying evolved mutations which affected negative regulators of mexAB-oprM (mexR and nalD) demonstrated enhanced virulence in a murine pneumonia infection model. Mutations in these genes, and other genes that we associated with aztreonam resistance, were common in P. aeruginosa isolates from chronically infected patients with cystic fibrosis. These findings illuminate mechanisms of P. aeruginosa aztreonam resistance and raise the possibility that antibiotic treatment could inadvertently select for hypervirulence phenotypes.

SUMMARY Culture and sequencing have produced divergent hypotheses about cystic fibrosis (CF) lung infections. Culturing suggests that CF lungs are uninfected before colonization by a limited group of CF pathogens. Sequencing suggests diverse communities of mostly oral bacteria inhabit lungs early-on, and diversity decreases as disease progresses. We studied the lung microbiota of CF children using bronchoscopy and sequencing, with measures to reduce contamination. We found no evidence for oral bacterial communities in lung lavages that lacked CF pathogens. Lavage microbial diversity varied widely, but decreases in diversity appeared to be driven by increased CF pathogen abundance, which reduced the signal from contaminants. Streptococcus, Prevotella, and Veillonella DNA was detected in some lavages containing CF pathogens, but DNA from these organisms was vastly exceeded by CF pathogen DNA and was not associated with inflammation. These findings support the hypothesis that established CF pathogens are primarily responsible for CF lung infections.

Background/aims:Non-alcoholic fatty liver disease is one of the most common chronic liver diseases. Some risk factors are known to influence the development of non-alcoholic fatty liver disease, but the effect of tobacco smoking on the progression of non-alcoholic fatty liver disease is controversial. The main goal of this systematic review and meta-analysis is to investigate the association between smoking and non-alcoholic fatty liver disease.Method:Electronic databases (PubMed, Scopus, and ISI Web of Science) were searched to find published articles on non-alcoholic fatty liver disease and smoking until December 2016. All relevant studies were screened by inclusion and exclusion criteria and compatible studies were chosen. The Newcastle–Ottawa Scale was used to assess the methodological quality of eligible articles. Subsequently, information was gathered based on the following: author, publication year, keywords, country, inclusion and exclusion criteria, main results, study design, conclusion, and confounder variables (age, body mass index, gender, ethnicity, and diabetes). Finally, analyses were performed using Comprehensive Meta-Analysis Software.Results:Data were extracted from 20 observational studies (9 cross-sectional, 6 case-control, 4 cohort studies, and 1 retrospective cohort study). A significant association was observed between smoking and non-alcoholic fatty liver disease with a pooled odds ratio of 1.110 (95% confidence interval, 1.028–1.199), p-value = 0.008. The statistical heterogeneity was medium with an I2 of 40.012%, p-heterogeneity = 0.074. Also there was a significant relation between non-alcoholic fatty liver disease and passive smoking with a pooled odds ratio of 1.380 (95% confidence interval, 1.199–1.588; p-value = 0.001; I2 = 59.41; p-heterogeneity = 0.117).Conclusion:Our meta-analysis demonstrated that smoking is significantly associated with non-alcoholic fatty liver disease. Further prospective studies exploring the underlying mechanisms of this association should be pursued. Also passive smoking increases the risk of non-alcoholic fatty liver disease about 1.38-fold. The effects of smoking cigarettes on active smokers (current smoker, former smoker, and total smoker) are less than passive smokers. Further studies are needed to compare the of effects of passive and active smoking on non-alcoholic fatty liver disease.