Patrizia Morelli

Changes in cystic fibrosis airway microbial community associated with a severe decline in lung function

Cystic fibrosis (CF) is a genetic disease resulting in chronic polymicrobial infections of the airways and progressive decline in lung function. To gain insight into the underlying causes of severe lung diseases, we aimed at comparing the airway microbiota detected in sputum of CF patients with stable lung function (S) versus those with a substantial decline in lung function (SD). Microbiota composition was investigated by using culture-based and culture-independent methods, and by performing multivariate and statistical analyses. Culture-based methods identified some microbial species associated with a worse lung function, i.e. Pseudomonas aeruginosa, Rothia mucilaginosa, Streptococcus pneumoniae and Candida albicans, but only the presence of S. pneumoniae and R. mucilaginosa was found to be associated with increased severe decline in forced expiratory volume in 1 second (FEV1). Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis revealed a higher bacterial diversity than that detected by culture-based methods. Molecular signatures with a statistically significant odds ratio for SD status were detected, and classified as Pseudomonas, Burkholderia and Shewanella, while for other Terminal Restriction Fragments (T-RFs) no species assignation was achieved. The analysis of T-RFLP data using ecological biodiversity indices showed reduced Evenness in SD patients compared to S ones, suggesting an impaired ecology of the bacterial community in SD patients. Statistically significant differences of the ecological biodiversity indices among the three sub-groups of FEV1 (normal/mild vs moderate vs severe) were also found, suggesting that the patients with moderate lung disease experienced changes in the airway assembly of taxa. Overall, changes in CF airway microbial community associated with a severe lung function decline were detected, allowing us to define some discriminatory species as well as some discriminatory T-RFs that represent good candidates for the development of predictive biomarkers of substantial decline in lung function.

Pyrosequencing Unveils Cystic Fibrosis Lung Microbiome Differences Associated with a Severe Lung Function Decline

Chronic airway infection is a hallmark feature of cystic fibrosis (CF) disease. In the present study, sputum samples from CF patients were collected and characterized by 16S rRNA gene-targeted approach, to assess how lung microbiota composition changes following a severe decline in lung function. In particular, we compared the airway microbiota of two groups of patients with CF, i.e. patients with a substantial decline in their lung function (SD) and patients with a stable lung function (S). The two groups showed a different bacterial composition, with SD patients reporting a more heterogeneous community than the S ones. Pseudomonas was the dominant genus in both S and SD patients followed by Staphylococcus and Prevotella. Other than the classical CF pathogens and the most commonly identified non-classical genera in CF, we found the presence of the unusual anaerobic genus Sneathia. Moreover, the oligotyping analysis revealed the presence of other minor genera described in CF, highlighting the polymicrobial nature of CF infection. Finally, the analysis of correlation and anti-correlation networks showed the presence of antagonism and ecological independence between members of Pseudomonas genus and the rest of CF airways microbiota, with S patients showing a more interconnected community in S patients than in SD ones. This population structure suggests a higher resilience of S microbiota with respect to SD, which in turn may hinder the potential adverse impact of aggressive pathogens (e.g. Pseudomonas). In conclusion, our findings shed a new light on CF airway microbiota ecology, improving current knowledge about its composition and polymicrobial interactions in patients with CF.

Upregulation of TMEM16A protein in bronchial epithelial cells by bacterial pyocyanin

Induction of mucus hypersecretion in the airway epithelium by Th2 cytokines is associated with the expression of TMEM16A, a Ca2+-activated Cl- channel. We asked whether exposure of airway epithelial cells to bacterial components, a condition that mimics the highly infected environment occurring in cystic fibrosis (CF), also results in a similar response. In cultured human bronchial epithelial cells, treatment with pyocyanin or with a P. aeruginosa culture supernatant caused a significant increase in TMEM16A function. The Ca2+-dependent Cl- secretion, triggered by stimulation with UTP, was particularly enhanced by pyocyanin in cells from CF patients. Increased expression of TMEM16A protein and of MUC5AC mucin by bacterial components was demonstrated by immunofluorescence in CF and non-CF cells. We also investigated TMEM16A expression in human bronchi by immunocytochemistry. We found increased TMEM16A staining in the airways of CF patients. The strongest signal was observed in CF submucosal glands. Our results suggest that TMEM16A expression/function is upregulated in CF lung disease, possibly as a response towards the presence of bacteria in the airways.

A Different Microbiome Gene Repertoire in the Airways of Cystic Fibrosis Patients with Severe Lung Disease

In recent years, next-generation sequencing (NGS) was employed to decipher the structure and composition of the microbiota of the airways in cystic fibrosis (CF) patients. However, little is still known about the overall gene functions harbored by the resident microbial populations and which specific genes are associated with various stages of CF lung disease. In the present study, we aimed to identify the microbial gene repertoire of CF microbiota in twelve patients with severe and normal/mild lung disease by performing sputum shotgun metagenome sequencing. The abundance of metabolic pathways encoded by microbes inhabiting CF airways was reconstructed from the metagenome. We identified a set of metabolic pathways differently distributed in patients with different pulmonary function; namely, pathways related to bacterial chemotaxis and flagellar assembly, as well as genes encoding efflux-mediated antibiotic resistance mechanisms and virulence-related genes. The results indicated that the microbiome of CF patients with low pulmonary function is enriched in virulence-related genes and in genes encoding efflux-mediated antibiotic resistance mechanisms. Overall, the microbiome of severely affected adults with CF seems to encode different mechanisms for the facilitation of microbial colonization and persistence in the lung, consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in patients with severe lung disease.

First Report of Chronic Pulmonary Infection by KPC-3-Producing and Colistin-Resistant Klebsiella pneumoniae Sequence Type 258 (ST258) in an Adult Patient with Cystic Fibrosis

ABSTRACT The spread of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae continues to increase, and the possible development of KPC-producing K. pneumoniae infections in cystic fibrosis (CF) patients is a matter of concern. Here, we describe the establishment of a chronic lung infection due to a colistin-resistant KPC-producing K. pneumoniae isolate in an Italian CF patient.

Increased expression of ATP12A proton pump in cystic fibrosis airways

Proton secretion mediated by ATP12A protein on the surface of the airway epithelium may contribute to cystic fibrosis (CF) lung disease by favoring bacterial infection and airway obstruction. We studied ATP12A in fresh bronchial samples and in cultured epithelial cells. In vivo, ATP12A expression was found almost exclusively at the apical side of nonciliated cells of airway epithelium and in submucosal glands, with much higher expression in CF samples. This could be due to bacterial infection and inflammation, since treating cultured cells with bacterial supernatants or with IL-4 (a cytokine that induces goblet cell hyperplasia) increased the expression of ATP12A in nonciliated cells. This observation was associated with upregulation and translocation of ATP1B1 protein from the basal to apical epithelial side, where it colocalizes with ATP12A. ATP12A function was evaluated by measuring the pH of the apical fluid in cultured epithelia. Under resting conditions, CF epithelia showed more acidic values. This abnormality was minimized by inhibiting ATP12A with ouabain. Following treatment with IL-4, ATP12A function was markedly increased, as indicated by strong acidification occurring under bicarbonate-free conditions. Our study reveals potentially novel aspects of ATP12A and remarks its importance as a possible therapeutic target in CF and other respiratory diseases.

99 Investigating the airway microbiome in cystic fibrosis patients with normal and severe pulmonary function decline: an opportunity for a personalized microbiome-based therapy

97 Nocardia and cystic fibrosis: the impact of Gram staining O. Tuncer, S. Olmez, B. Sancak, G.D. Tugcu, N. Emiralioglu, B. Otlu, B. Er, E. Yalcin, D. Dogru, U. Ozcelik, N. Kiper, B. Şener. Hacettepe University Medical Faculty, Clinical Microbiology, Ankara, Turkey; Hacettepe University Ihsan Dogramaci Children’s Hospital, Pediatric Pulmonology, Ankara, Turkey; Inonu University Medical Faculty, Clinical Microbiology, Malatya, Turkey; Hacettepe University Medical Faculty, Pulmonology, Ankara, Turkey

99 Active surveillance and containment of carbapenemase producing Enterobacteriaceae (CPE) in cystic fibrosis (CF) patients attending an Italian care centre

Objectives Carbapenemases, that hydrolyse most b-lactams, have emerged and spread worldwide. Currently are not many data of CPE in CF. The accesses to different hospitals, such as transplant centres, make these patients at high risk for transmission of CPE. The study aims to quantify and describe the CPE isolated from CF patients in a year about active surveillance. Methods During 2014, 147 rectal swabs were analyzed. The swabs were inoculated on McConkey agar plate with meropenem disk. The susceptibility testing was performed with automatic method (Phoenix). Carbapenemase were tested by disk diffusion synergy test. For molecular characterization was used GeneXpert. The fingerprinting of isolates were performed by Box-PCR. Results In ten samples we not evaluated the CPE because there was no bacterial growth. Three patients were colonized by CPE identified as E. cloacae. Clonal identity among strains was founded. The strains show a reduced susceptibility to carbapenems and the resistance to other b-lactams and aminoglycosides. The strains were susceptibility to fluoroquinolones, colistin and tigecycline. The molecular assay shows the presence of bla VIM gene. Conclusion The screening using culture methods has unsuitable for some CF patients while molecular method has more effective. Moreover the genotyping suggests a spread among patients. The CPE represents a danger for CF patients: this resistance may be transmitted to the multi-resistant pathogens that support CF pulmonary infection. Then an active surveillance, with an appropriate method of screening for CF patients, is needed to decrease and prevent the transmission of CPE.

125 Investigation of cystic fibrosis airway microbiome in patients showing a severe decline in lung function and not responding to conventional antimicrobial therapy

123 A shotgun metaproteomics approach to study the faecal microbiome of patients with cystic fibrosis reveals a reduction of butyrate-producing bacteria G. Debyser1, B. Mesuere2, L. Clement2, G. Duytschaever1, P. Van Hecke1, P. Dawyndt2, K. De Boeck3, P. Vandamme1, B. Devreese1. 1Ghent University, Department of Biochemistry and Microbiology, Ghent, Belgium; 2Ghent University, Department of Applied Mathematics and Computer Science, Ghent, Belgium; 3University Hospital of Leuven, Department of Pediatrics, Leuven, Belgium