Impact of Enhanced Staphylococcus DNA Extraction on Microbial Community Measures in Cystic Fibrosis Sputum

Staphylococcus aureus is a common constituent of the bacterial community inhabiting the airways of persons with cystic fibrosis (CF). Culture-independent studies have shown that this species is often present in relatively high abundance and would therefore be expected to exert a pronounced effect on measures of CF airway bacterial community structure. We investigated the impact of DNA extraction method on pyrosequencing-based measures of Staphylococcus abundance and bacterial community structure in 17 sputum samples from five CF patients. Staphylococcus was detected in fewer samples when DNA was extracted using a standard bacterial lysis method compared to when DNA was extracted using a lysis buffer amended with lysostaphin and lysozyme. The standard lysis method resulted in significantly lower measures of Staphylococcus relative abundance and higher levels of community diversity, richness, and evenness compared to the lysostaphin-lysozyme modified method. Measures of community dynamics in serial sputum samples from the same individual were nevertheless highly concordant between the two DNA extraction methods. These results illustrate the impact of DNA preparation method on measures of Staphylococcus abundance and bacterial community structures in studies of the airways microbiota in CF

The cystic fibrosis microbiome in an ecological perspective and its impact in antibiotic therapy

The recent focus on the cystic fibrosis (CF) complex microbiome has led to the recognition that the microbes can interact between them and with the host immune system, affecting the disease progression and treatment routes. Although the main focus remains on the interactions between traditional pathogens, growing evidence supports the contribution and the role of emergent species. Understanding the mechanisms and the biological effects involved in polymicrobial interactions may be the key to improve effective therapies and also to define new strategies for disease control. This review focuses on the interactions between microbe-microbe and host-microbe, from an ecological point of view, discussing their impact on CF disease progression. There are increasing indications that these interactions impact the success of antimicrobial therapy. Consequently, a new approach where therapy is personalized to patients by taking into account their individual CF microbiome is suggested.Portuguese Foundation for Science and Technology (FCT), the strategic funding of UID/BIO/04469/2013-CEB and UID/EQU/00511/2013-LEPABE units. This study was also supported by FCT and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects “DNA mimics” PIC/IC/82815/2007, RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), “BioHealth—Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027 and NORTE-07-0124-FEDER-000025—RL2_ Environment and Health, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. The authors also acknowledge the grant of Susana P. Lopes (SFRH/BPD/95616/2013) and of the COST-Action TD1004: Theragnostics for imaging and therapy

Respiratory microbiota resistance and resilience to pulmonary exacerbation and subsequent antimicrobial intervention

© 2016 International Society for Microbial Ecology All rights reserved. Pulmonary symptoms in cystic fibrosis (CF) begin in early life with chronic lung infections and concomitant airway inflammation leading to progressive loss of lung function. Gradual pulmonary function decline is interspersed with periods of acute worsening of respiratory symptoms known as CF pulmonary exacerbations (CFPEs). Cumulatively, CFPEs are associated with more rapid disease progression. In this study multiple sputum samples were collected from adult CF patients over the course of CFPEs to better understand how changes in microbiota are associated with CFPE onset and management. Data were divided into five clinical periods: pre-CFPE baseline, CFPE, antibiotic treatment, recovery, and post-CFPE baseline. Samples were treated with propidium monoazide prior to DNA extraction, to remove the impact of bacterial cell death artefacts following antibiotic treatment, and then characterised by 16S rRNA gene-targeted high-throughput sequencing. Partitioning CF microbiota into core and rare groups revealed compositional resistance to CFPE and resilience to antibiotics interventions. Mixed effects modelling of core microbiota members revealed no significant negative impact on the relative abundance of Pseudomonas aeruginosa across the exacerbation cycle. Our findings have implications for current CFPE management strategies, supporting reassessment of existing antimicrobial treatment regimens, as antimicrobial resistance by pathogens and other members of the microbiota may be significant contributing factors

Lung Microbiota Changes Associated with Chronic Pseudomonas aeruginosa Lung Infection and the Impact of Intravenous Colistimethate Sodium

Exacerbations associated with chronic lung infection with Pseudomonas aeruginosa are a major contributor to morbidity, mortality and premature death in cystic fibrosis. Such exacerbations are treated with antibiotics, which generally lead to an improvement in lung function and reduced sputum P. aeruginosa density. This potentially suggests a role for the latter in the pathogenesis of exacerbations. However, other data suggesting that changes in P. aeruginosa sputum culture status may not reliably predict an improvement in clinical status, and data indicating no significant changes in either total bacterial counts or in P. aeruginosa numbers in sputum samples collected prior to pulmonary exacerbation sheds doubt on this assumption. We used our recently developed lung segmental model of chronic Pseudomonas infection in sheep to investigate the lung microbiota changes associated with chronic P. aeruginosa lung infection and the impact of systemic therapy with colistimethate sodium (CMS).We collected protected specimen brush (PSB) samples from sheep (n = 8) both prior to and 14 days after establishment of chronic local lung infection with P aeruginosa. Samples were taken from both directly infected lung segments (direct) and segments spatially remote to such sites (remote). Four sheep were treated with daily intravenous injections of CMS between days 7 and 14, and four were treated with a placebo. Necropsy examination at d14 confirmed the presence of chronic local lung infection and lung pathology in every direct lung segment. The predominant orders in lung microbiota communities before infection were Bacillales, Actinomycetales and Clostridiales. While lung microbiota samples were more likely to share similarities with other samples derived from the same lung, considerable within- and between-animal heterogeneity could be appreciated. Pseudomonadales joined the aforementioned list of predominant orders in lung microbiota communities after infection. Whilst treatment with CMS appeared to have little impact on microbial community composition after infection, or the change undergone by communities in reaching that state, when Gram negative organisms (excluding Pseudomonadales) were considered together as a group there was a significant decrease in their relative proportion that was only observed in the sheep treated with CMS. With only one exception the reduction was seen in both direct and remote lung segments. This reduction, coupled with generally increasing or stable levels of Pseudomonadales, meant that the proportion of the latter relative to total Gram negative bacteria increased in all bar one direct and one remote lung segment.The proportional increase in Pseudomonadales relative to other Gram negative bacteria in the lungs of sheep treated with systemic CMS highlights the potential for such therapies to inadvertently select or create a niche for bacteria seeding from a persistent source of chronic infection

Predominant pathogen competition and core microbiota divergence in chronic airway infection

© 2015 International Society for Microbial Ecology All rights reserved. Chronic bacterial lung infections associated with non-cystic fibrosis bronchiectasis represent a substantial and growing health-care burden. Where Pseudomonas aeruginosa is the numerically dominant species within these infections, prognosis is significantly worse. However, in many individuals, Haemophilus influenzae predominates, a scenario associated with less severe disease. The mechanisms that determine which pathogen is most abundant are not known. We hypothesised that the distribution of H. influenzae and P. aeruginosa would be consistent with strong interspecific competition effects. Further, we hypothesised that where P. aeruginosa is predominant, it is associated with a distinct 'accessory microbiota' that reflects a significant interaction between this pathogen and the wider bacterial community. To test these hypotheses, we analysed 16S rRNA gene pyrosequencing data generated previously from 60 adult bronchiectasis patients, whose airway microbiota was dominated by either P. aeruginosa or H. influenzae. The relative abundances of the two dominant species in their respective groups were not significantly different, and when present in the opposite pathogen group the two species were found to be in very low abundance, if at all. These findings are consistent with strong competition effects, moving towards competitive exclusion. Ordination analysis indicated that the distribution of the core microbiota associated with each pathogen, readjusted after removal of the dominant species, was significantly divergent (analysis of similarity (ANOSIM), R=0.07, P=0.019). Taken together, these findings suggest that both interspecific competition and also direct and/or indirect interactions between the predominant species and the wider bacterial community may contribute to the predominance of P. aeruginosa in a subset of bronchiectasis lung infections

The exclusion of dead bacterial cells is essential for accurate molecular analysis of clinical samples

AbstractThe DNA-based techniques used to detect bacteria in clinical samples are unable to discriminate between live bacteria, dead bacteria, and extracellular DNA. This failure to limit analysis to viable bacterial cells represents a significant problem, leading to false-positive results, as well as a failure to resolve the impact of antimicrobial therapy. The use of propidium monoazide treatment significantly reduces the contribution of dead cells and extracellular DNA to such culture-independent analyses. Here, the increased ability to resolve the impact of antibiotic therapy on Pseudomonas aeruginosa load in cystic fibrosis respiratory samples reveals statistically significant changes that would otherwise go undetected

Comparative Analysis of Bacterial Community Composition and Structure in Clinically Symptomatic and Asymptomatic Central Venous Catheters

International audienceTotally implanted venous access ports (TIVAPs) are commonly used catheters for the management of acute or chronic pathologies. Although these devices improve health care, repeated use of this type of device for venous access over long periods of time is also associated with risk of colonization and infection by pathogenic bacteria, often originating from skin. However, although the skin microbiota is composed of both pathogenic and nonpathogenic bacteria, the extent and the consequences of TIVAP colonization by nonpathogenic bacteria have rarely been studied. Here, we used culture-dependent and 16S rRNA gene-based culture-independent approaches to identify differences in bacterial colonization of TIVAPs obtained from two French hospitals. To explore the relationships between nonpathogenic organisms colonizing TIVAPs and the potential risk of infection, we analyzed the bacterial community parameters between TIVAPs suspected (symptomatic) or not (asymptomatic) of infection. Although we did not find a particular species assemblage or community marker to distinguish infection risk on an individual sample level, we identified differences in bacterial community composition, diversity, and structure between clinically symptomatic and asymptomatic TIVAPs that could be explored further. This study therefore provides a new view of bacterial communities and colonization patterns in intravascular TIVAPs and suggests that microbial ecology approaches could improve our understanding of device-associated infections and could be a prognostic tool to monitor the evolution of bacterial communities in implants and their potential susceptibility to infections. IMPORTANCE Totally implanted venous access ports (TIVAPs) are commonly used implants for the management of acute or chronic pathologies. Although their use improves the patient's health care and quality of life, they are associated with a risk of infection and subsequent clinical complications, often leading to implant removal. While all TIVAPs appear to be colonized, only a fraction become infected, and the relationship between nonpathogenic organisms colonizing TIVAPs and the potential risk of infection is unknown. We explored bacteria present on TIVAPs implanted in patients with or without signs of TIVAP infection and identified differences in phylum composition and community structure. Our data suggest that the microbial ecology of intravascular devices could be predictive of TIVAP infection status and that ultimately a microbial ecological signature could be identified as a tool to predict TIVAP infection susceptibility and improve clinical management