Development of Novel Membrane Disrupting Lipoguanidine Compounds Sensitizing Gram-Negative Bacteria to Antibiotics.

A new class of amphiphilic molecules, the lipoguanidines, designed as hybrids of guanidine and fatty acid compounds, has been synthesized and developed. The new molecules present both a guanidine polar head and a lipophilic tail that allow them to disrupt bacterial membranes and to sensitize Gram-negative bacteria to the action of the narrow-spectrum antibiotics rifampicin and novobiocin. The lipoguanidine 5g sensitizes Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli to rifampicin, thereby reducing the antibiotic minimum inhibitory concentrations (MIC) up to 256-fold. Similarly, 5g is able to potentiate novobiocin up to 64-fold, thereby showing a broad spectrum of antibiotic potentiating activity. Toxicity and mechanism studies revealed the potential of 5g to work synergistically with rifampicin through the disruption of bacterial membranes without affecting eukaryotic cells.

Design and manufacture of a lyophilised faecal microbiota capsule formulation to GMP standards.

Faecal microbiota transplant (FMT) is an established and effective treatment for recurrent Clostridioides difficile infection (CDI) and has many other potential clinical applications. However, preparation and quality of FMT is poorly standardised and clinical studies are hampered by a lack of well-defined FMT formulations that meet regulatory standards for medicines. As an alternative to FMT suspensions for administration by nasojejunal tube or colonoscopy, which is invasive and disliked by many patients, this study aimed to develop a well-controlled, standardised method for manufacture of lyophilised FMT capsules and to provide stability data allowing storage for extended time periods. Faecal donations were collected from healthy, pre-screened individuals, homogenised, filtered and centrifuged to remove dietary matter. The suspension was centrifuged to pellet bacteria, which were resuspended with trehalose and lyophilised to produce a powder which was filled into 5 enteric-coated capsules (size 0). Live-dead bacterial cell quantitative PCR assay showed <10 fold viable bacterial load reduction through the manufacturing process. No significant loss of viable bacterial load was observed after storage at -80 °C for 36 weeks (p = 0.24, n = 5). Initial clinical experience demonstrated that the capsules produced clinical cure in patients with CDI with no adverse events reported (n = 7). We provide the first report of a detailed manufacturing protocol and specification for an encapsulated lyophilised formulation of FMT. As clinical trials into intestinal microbiota interventions proceed, it is important to use a well-controlled investigational medicinal product in the studies so that any beneficial results can be replicated in clinical practice.

Rifaximin-α reduces gut-derived inflammation and mucin degradation in cirrhosis and encephalopathy: RIFSYS randomised controlled trial.

BACKGROUND & AIMS: Rifaximin-α is efficacious for the prevention of recurrent hepatic encephalopathy (HE), but its mechanism of action remains unclear. We postulated that rifaximin-α reduces gut microbiota-derived endotoxemia and systemic inflammation, a known driver of HE. METHODS: In a placebo-controlled, double-blind, mechanistic study, 38 patients with cirrhosis and HE were randomised 1:1 to receive either rifaximin-α (550 mg BID) or placebo for 90 days. PRIMARY OUTCOME: 50% reduction in neutrophil oxidative burst (OB) at 30 days. SECONDARY OUTCOMES: changes in psychometric hepatic encephalopathy score (PHES) and neurocognitive functioning, shotgun metagenomic sequencing of saliva and faeces, plasma and faecal metabolic profiling, whole blood bacterial DNA quantification, neutrophil toll-like receptor (TLR)-2/4/9 expression and plasma/faecal cytokine analysis. RESULTS: Patients were well-matched: median MELD (11 rifaximin-α vs. 10 placebo). Rifaximin-α did not lead to a 50% reduction in spontaneous neutrophil OB at 30 days compared to baseline (p = 0.48). However, HE grade normalised (p = 0.014) and PHES improved (p = 0.009) after 30 days on rifaximin-α. Rifaximin-α reduced circulating neutrophil TLR-4 expression on day 30 (p = 0.021) and plasma tumour necrosis factor-α (TNF-α) (p <0.001). Rifaximin-α suppressed oralisation of the gut, reducing levels of mucin-degrading sialidase-rich species, Streptococcus spp, Veillonella atypica and parvula, Akkermansia and Hungatella. Rifaximin-α promoted a TNF-α- and interleukin-17E-enriched intestinal microenvironment, augmenting antibacterial responses to invading pathobionts and promoting gut barrier repair. Those on rifaximin-α were less likely to develop infection (odds ratio 0.21; 95% CI 0.05-0.96). CONCLUSION: Rifaximin-α led to resolution of overt and covert HE, reduced the likelihood of infection, reduced oralisation of the gut and attenuated systemic inflammation. Rifaximin-α plays a role in gut barrier repair, which could be the mechanism by which it ameliorates bacterial translocation and systemic endotoxemia in cirrhosis. CLINICAL TRIAL NUMBER: ClinicalTrials.gov NCT02019784. LAY SUMMARY: In this clinical trial, we examined the underlying mechanism of action of an antibiotic called rifaximin-α which has been shown to be an effective treatment for a complication of chronic liver disease which effects the brain (termed encephalopathy). We show that rifaximin-α suppresses gut bacteria that translocate from the mouth to the intestine and cause the intestinal wall to become leaky by breaking down the protective mucus barrier. This suppression resolves encephalopathy and reduces inflammation in the blood, preventing the development of infection.

Novel detection of specific bacterial quorum sensing molecules in saliva: Potential non-invasive biomarkers for pulmonary Pseudomonas aeruginosa in cystic fibrosis.

Pseudomonas aeruginosa produces specific signalling molecules, 2-alkyl-4-quinolones (AQs) that are detectable in the sputum of adults with cystic fibrosis (CF) and who have pulmonary infection with this opportunistic pathogen. This study aimed to determine whether AQs could be detected in saliva of patients with CF and known infection with Pseudomonas aeruginosa. Saliva and sputum samples were obtained from 89 adults with CF and analyzed using liquid chromatography-tandem mass spectrometry. AQs were detected in 39/89 (43.8%) saliva samples and 70/77(90.9%) sputum samples. Salivary AQs had a sensitivity of 50% (95%CI; 37.8; 62.2), specificity of 100% (95%CI; 47.8; 100), when compared to a molecular microbiological measure of P. aeruginosa in sputum as measured using polymerase chain reaction. Specific AQs produced by P. aeruginosa can be detected in the saliva and warrant investigation as potential non-invasive biomarkers of pulmonary P. aeruginosa.

2-Alkyl-4-quinolone quorum sensing molecules are biomarkers for culture-independent Pseudomonas aeruginosa burden in adults with cystic fibrosis.

Introduction. Pseudomonas aeruginosa produces quorum sensing signalling molecules including 2-alkyl-4-quinolones (AQs), which regulate virulence factor production in the cystic fibrosis (CF) airways.Hypothesis/Gap statement. Culture can lead to condition-dependent artefacts which may limit the potential insights and applications of AQs as minimally-invasive biomarkers of bacterial load.Aim. We aimed to use culture-independent methods to explore the correlations between AQ levels and live P. aeruginosa load in adults with CF.Methodology. Seventy-five sputum samples at clinical stability and 48 paired sputum samples obtained at the beginning and end of IV antibiotics for a pulmonary exacerbation in adults with CF were processed using a viable cell separation technique followed by quantitative P. aeruginosa polymerase chain reaction (qPCR). Live P. aeruginosa qPCR load was compared with the concentrations of three AQs (HHQ, NHQ and HQNO) detected in sputum, plasma and urine.Results. At clinical stability and the beginning of IV antibiotics for pulmonary exacerbation, HHQ, NHQ and HQNO measured in sputum, plasma and urine were consistently positively correlated with live P. aeruginosa qPCR load in sputum, compared to culture. Following systemic antibiotics live P. aeruginosa qPCR load decreased significantly (P<0.001) and was correlated with a reduction in plasma NHQ (plasma: r=0.463, P=0.003).Conclusion. In adults with CF, AQ concentrations correlated more strongly with live P. aeruginosa bacterial load measured by qPCR compared to traditional culture. Prospective studies are required to assess the potential of systemic AQs as biomarkers of P. aeruginosa bacterial burden.

Gut microbiota development during infancy: Impact of introducing allergenic foods.

BACKGROUND: The gut microbiota potentially plays an important role in the immunologic education of the host during early infancy. OBJECTIVE: We sought to determine how the infant gut microbiota evolve during infancy, particularly in relation to hygiene-related environmental factors, atopic disorders, and a randomized introduction of allergenic solids. METHODS: A total of 1303 exclusively breast-fed infants were enrolled in a dietary randomized controlled trial (Enquiring About Tolerance study) from 3 months of age. In this nested longitudinal study, fecal samples were collected at baseline, with additional sampling of selected cases and controls at 6 and 12 months to study the evolution of their gut microbiota, using 16S ribosomal RNA gene-targeted amplicon sequencing. RESULTS: In the 288 baseline samples from exclusively breast-fed infant at 3 months, the gut microbiota was highly heterogeneous, forming 3 distinct clusters: Bifidobacterium-rich, Bacteroides-rich, and Escherichia/Shigella-rich. Mode of delivery was the major discriminating factor. Increased Clostridium sensu stricto relative abundance at 3 months was associated with presence of atopic dermatitis on examination at age 3 and 12 months. From the selected cases and controls with longitudinal samples (n = 70), transition to Bacteroides-rich communities and influx of adult-specific microbes were observed during the first year of life. The introduction of allergenic solids promoted a significant increase in Shannon diversity and representation of specific microbes, such as genera belonging to Prevotellaceae and Proteobacteria (eg, Escherichia/Shigella), as compared with infants recommended to exclusively breast-feed. CONCLUSIONS: Specific gut microbiota characteristics of samples from 3-month-old breast-fed infants were associated with cesarean birth, and greater Clostridium sensu stricto abundance was associated with atopic dermatitis. The randomized introduction of allergenic solids from age 3 months alongside breast-feeding was associated with differential dynamics of maturation of the gut microbial communities.

The airways microbiome of individuals with asthma treated with high and low doses of inhaled corticosteroids.

BACKGROUND: Inhaled corticosteroids (ICS) are the mainstay of asthma treatment, but evidence suggests a link between ICS usage and increased rates of respiratory infections. We assessed the composition of the asthmatic airways microbiome in asthma patients taking low and high dose ICS and the stability of the microbiome over a 2 week period. METHODS: We prospectively recruited 55 individuals with asthma. Of these, 22 were on low-dose ICS and 33 on high-dose ICS (16 on budesonide, 17 on fluticasone propionate). Sputum from each subject underwent DNA extraction, amplification and 16S rRNA gene sequencing of the bacterial component of the microbiome. 19 subjects returned for further sputum induction after 24 h and 2 weeks. RESULTS: A total of 5,615,037 sequencing reads revealed 167 bacterial taxa in the asthmatic airway samples, with the most abundant being Streptococcus spp. No significant differences in sputum bacterial load or overall community composition were seen between the low- and high-dose ICS groups. However, Streptococcus spp. showed significantly higher relative abundance in subjects taking low-dose ICS (p = 0.002). Haemophilus parainfluenzae was significantly more abundant in subjects on high-dose fluticasone propionate than those on high-dose budesonide (p = 0.047). There were no statistically significant changes in microbiota composition over a 2-week period. DISCUSSION: Whilst no significant differences were observed between the low- and high-dose ICS groups, increased abundance of the potential pathogen H. parainfluenzae was observed in patients taking high-dose fluticasone propionate compared to those taking high-dose budesonide. The microbiota were stable over fourteen days, providing novel evidence of the established community of bacteria in the asthmatic airways. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT02671773.

Antibiotic-in-Cyclodextrin-in-Liposomes: Formulation Development and Interactions with Model Bacterial Membranes.

Gram-negative bacteria possess numerous defenses against antibiotics, due to the intrinsic permeability barrier of their outer membrane (OM), explaining the recalcitrance of some common and life-threatening infections. We report the formulation of a new drug, PPA148, which shows promising activity against all Gram-negative bacteria included in the ESKAPEE pathogens. PPA148 was solubilized by inclusion complexation with cyclodextrin followed by encapsulation in liposomes. The complex and liposomal formulation presented increased activity against E. coli compared to the pure drug when assessed with the Kirby Bauer assay. The novel formulation containing 1 µg PPA148 reached similar efficacy levels equivalent to those of 30 µg of pure rifampicin. A range of biophysical techniques was used to explore the mechanism of drug uptake. Langmuir trough (LT) and neutron reflectivity (NR) techniques were employed to monitor the interactions between the drug and the formulation with model membranes. We found evidence for liposome fusion with the model Gram-negative outer membrane and for cyclodextrins acting as inner membrane (IM) permeation enhancers without presenting intrinsic antimicrobial activity. An antibiotic-in-cyclodextrin-in-liposomes (ACL) formulation was developed, which targets both the bacterial OM and IM, and offers promise as a means to breach the Gram-negative cell envelope.

Lung function and microbiota diversity in cystic fibrosis.

BACKGROUND: Chronic infection and concomitant airway inflammation is the leading cause of morbidity and mortality for people living with cystic fibrosis (CF). Although chronic infection in CF is undeniably polymicrobial, involving a lung microbiota, infection surveillance and control approaches remain underpinned by classical aerobic culture-based microbiology. How to use microbiomics to direct clinical management of CF airway infections remains a crucial challenge. A pivotal step towards leveraging microbiome approaches in CF clinical care is to understand the ecology of the CF lung microbiome and identify ecological patterns of CF microbiota across a wide spectrum of lung disease. Assessing sputum samples from 299 patients attending 13 CF centres in Europe and the USA, we determined whether the emerging relationship of decreasing microbiota diversity with worsening lung function could be considered a generalised pattern of CF lung microbiota and explored its potential as an informative indicator of lung disease state in CF. RESULTS: We tested and found decreasing microbiota diversity with a reduction in lung function to be a significant ecological pattern. Moreover, the loss of diversity was accompanied by an increase in microbiota dominance. Subsequently, we stratified patients into lung disease categories of increasing disease severity to further investigate relationships between microbiota characteristics and lung function, and the factors contributing to microbiota variance. Core taxa group composition became highly conserved within the severe disease category, while the rarer satellite taxa underpinned the high variability observed in the microbiota diversity. Further, the lung microbiota of individual patient were increasingly dominated by recognised CF pathogens as lung function decreased. Conversely, other bacteria, especially obligate anaerobes, increasingly dominated in those with better lung function. Ordination analyses revealed lung function and antibiotics to be main explanators of compositional variance in the microbiota and the core and satellite taxa. Biogeography was found to influence acquisition of the rarer satellite taxa. CONCLUSIONS: Our findings demonstrate that microbiota diversity and dominance, as well as the identity of the dominant bacterial species, in combination with measures of lung function, can be used as informative indicators of disease state in CF. Video Abstract.

Selection and assembly of indigenous bacteria and methanogens from spent metalworking fluids and their potential as a starting culture in a fluidized bed reactor.

Waste metalworking fluids (MWFs) are highly biocidal resulting in real difficulties in the, otherwise favoured, bioremediation of these high chemical oxygen deman (COD) wastes anaerobically in bioreactors. We have shown, as a proof of concept, that it is possible to establish an anaerobic starter culture using strains isolated from spent MWFs which are capable of reducing COD or, most significantly, methanogenesis in this biocidal waste stream. Bacterial strains (n = 99) and archaeal methanogens (n = 28) were isolated from spent MWFs. The most common bacterial strains were Clostridium species (n = 45). All methanogens were identified as Methanosarcina mazei. Using a random partitions design (RPD) mesocosm experiment, we found that bacterial diversity and species-species interactions had significant effects on COD reduction but that bacterial composition did not. The RPD study showed similar effects on methanogenesis, except that composition was also significant. We identified bacterial species with positive and negative effects on methane production. A consortium of 16 bacterial species and three methanogens was used to initiate a fluidized bed bioreactor (FBR), in batch mode. COD reduction and methane production were variable, and the reactor was oscillated between continuous and batch feeds. In both microcosm and FBR experiments, periodic inconsistencies in bacterial reduction in fermentative products to formic and acetic acids were identified as a key issue.

Circadian leaf movements facilitate overtopping of neighbors.

Many plants exhibit circadian clock-driven leaf movements whereby the leaves are raised during the day to achieve a relatively high angle during the evening, before lowering late in the night. Such leaf movements were first recorded over 2000 years ago but there is still much debate as to their purpose. We investigated whether such leaf movements within Arabidopsis, a ruderal rosette plant, can aid in overtopping leaves of neighboring plants. Wild type and circadian clock mutant plants were grown in an alternating grid system so that their leaves would meet as the plants grew. Experiments were performed using day lengths that matched the endogenous rhythm of either wild type or mutant. Plants grown in a day length shorter than their endogenous rhythm were consistently overtopped by plants which were in synchrony with the day night cycle, demonstrating a clear overtopping advantage resulting from circadian leaf movement rhythms. Furthermore, we found that this leaf overtopping as a result of correctly synchronized circadian leaf movements is additive to leaf overtopping due to shade avoidance. Curiously, this did not apply to plants grown in a day length longer than their endogenous period. Plants grown in a day length longer than their endogenous period were able to adapt their leaf rhythms and suffered no overtopping disadvantage. Crucially, our results show that, in a context-dependent manner, circadian clock-driven leaf movements in resonance with the external light/dark cycle can facilitate overtopping of the leaves of neighboring plants.

Clinical and symptom scores are significantly correlated with fecal microbiota features in patients with symptomatic uncomplicated diverticular disease: a pilot study.

BACKGROUND: There is growing consensus that symptomatic uncomplicated diverticular disease is a chronic inflammatory condition, and that alterations in the fecal microbiota may contribute to its pathogenesis. OBJECTIVE: The aim of this study was to relate the fecal microbiota composition in symptomatic uncomplicated diverticular disease to measures of inflammation, symptoms, and history of previous acute diverticulitis. PARTICIPANTS AND METHODS: Fecal microbiota composition in 28 individuals with symptomatic uncomplicated diverticular disease was characterized by 16S RNA gene amplicon sequencing. Microbiota composition was related to clinical history, symptom and inflammation measures, and demographic variables. RESULTS: Previous acute diverticulitis was associated with higher relative abundance of Pseudobutyrivibrio, Bifidobacterium, Christensenellaceae family, and Mollicutes RF9 order (P=0.004, 0.006, 0.010, and 0.019, respectively), but not microbiota alpha or beta diversity. A higher bloating severity score was significantly correlated with a higher relative abundance of Ruminococcus (P=0.032), and significantly inversely correlated with the relative abundance of the Roseburia (P=0.002). Fecal calprotectin levels were positively correlated with alpha diversity (Shannon index, P=0.005) and the relative abundance of Lactobacillus (P=0.004). Pain score was positively correlated with the relative abundance of Cyanobacterium (adjusted P=0.032). CONCLUSION: Patient symptoms in symptomatic diverticular disease are significantly correlated with features of the fecal microbiota. Our findings suggest the potential utility of therapies that target intestinal microbiology, such as dietary prebiotic supplements.

How can the cystic fibrosis respiratory microbiome influence our clinical decision-making?

PURPOSE OF REVIEW: Almost 15 years have now passed since bacterial community profiling techniques were first used to analyse respiratory samples from people with cystic fibrosis. Since then, many different analytical approaches have been used to try to better understand the contribution of the cystic fibrosis lung microbiota to disease, with varying degrees of success. We examine the extent to which cystic fibrosis respiratory microbiome research has been successful in informing clinical decision-making, and highlight areas that we believe have the potential to yield important insight. RECENT FINDINGS: Recent research on the cystic fibrosis lung microbiome can be broadly divided into efforts to better characterize microbiota composition, particularly relative to key clinical events, and attempts to understand the cystic fibrosis lung microbiology as an interactive microbial system. The latter, in particular, has led to the development of a number of models in which microbiome-mediated processes precipitate clinical events. SUMMARY: Growing technological sophistication is enabling increasingly detailed microbiological data to be generated from cystic fibrosis respiratory samples. However, translating these data into clinically useful measures that accurately predict outcomes and guide treatments remains a formidable challenge. The development of systems biology approaches that enable the integration of complex microbiome and host-derived data provide an exciting opportunity to address this goal.

Contributions of Composition and Interactions to Bacterial Respiration Are Reliant on the Phylogenetic Similarity of the Measured Community.

Bacterial diversity underpins many ecosystem functions; however, the impact of within-species variation on the relationship between diversity and function remains unclear. Processes involving strain differentiation, such as niche radiation, are often overlooked in studies that focus on phylogenetic variation. This study used bacterial isolates assembled in two comparable microcosm experiments to test how species variation affected ecosystem function. We compared the relationship between diversity and activity (CO2 production) in increasingly diverse multispecies microcosms and with multiple ecotypes of a single species. The bacteria used were isolated from a low-diversity environment and are species of potential clinical significance such as Pseudomonas aeruginosa. All isolates were profiled for single carbon source utilisation. These data showed an increased breadth of resource use in the multiple ecotypes when compared to the mixed-species. The study observed significantly increasing respiration in more complex mixed-species assemblages, which was not observed when ecotypes of a single species were combined. We further demonstrate that the variation observed in the bacterial activity was due to the roles of each of the constituent isolates; between different species, the interactions between the isolates drove the variation in activity, whilst in single species, assemblage variation was due to which isolates were present. We conclude that both between- and within-species variations play different roles in community function, although through different mechanisms, and should be included in models of changing diversity and ecosystem functioning.

Divergent Relationships between Fecal Microbiota and Metabolome following Distinct Antibiotic-Induced Disruptions.

The intestinal microbiome plays an essential role in regulating many aspects of host physiology, and its disruption through antibiotic exposure has been implicated in the development of a range of serious pathologies. The complex metabolic relationships that exist between members of the intestinal microbiota and the potential redundancy in functional pathways mean that an integrative analysis of changes in both structure and function are needed to understand the impact of antibiotic exposure. We used a combination of next-generation sequencing and nuclear magnetic resonance (NMR) metabolomics to characterize the effects of two clinically important antibiotic treatments, ciprofloxacin and vancomycin-imipenem, on the intestinal microbiomes of female C57BL/6 mice. This assessment was performed longitudinally and encompassed both antibiotic challenge and subsequent microbiome reestablishment. Both antibiotic treatments significantly altered the microbiota and metabolite compositions of fecal pellets during challenge and recovery. Spearman's correlation analysis of microbiota and NMR data revealed that, while some metabolites could be correlated with individual operational taxonomic units (OTUs), frequently multiple OTUs were associated with a significant change in a given metabolite. Furthermore, one metabolite, arginine, can be associated with increases/decreases in different sets of OTUs under differing conditions. Taken together, these findings indicate that reliance on shifts in one data set alone will generate an incomplete picture of the functional effect of antibiotic intervention. A full mechanistic understanding will require knowledge of the baseline microbiota composition, combined with both a comparison and an integration of microbiota, metabolomics, and phenotypic data. IMPORTANCE Despite the fundamental importance of antibiotic therapies to human health, their functional impact on the intestinal microbiome and its subsequent ability to recover are poorly understood. Much research in this area has focused on changes in microbiota composition, despite the interdependency and overlapping functions of many members of the microbial community. These relationships make prediction of the functional impact of microbiota-level changes difficult, while analyses based on the metabolome alone provide relatively little insight into the taxon-level changes that underpin changes in metabolite levels. Here, we used combined microbiota and metabolome profiling to characterize changes associated with clinically important antibiotic combinations with distinct effects on the gut. Correlation analysis of changes in the metabolome and microbiota indicate that a combined approach will be essential for a mechanistic understanding of the functional impact of distinct antibiotic classes.

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

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.

Republished: Respiratory microbiota: addressing clinical questions, informing clinical practice.

Over the last decade, technological advances have revolutionised efforts to understand the role played by microbes in airways disease. With the application of ever more sophisticated techniques, the literature has become increasingly inaccessible to the non-specialist reader, potentially hampering the translation of these gains into improvements in patient care. In this article, we set out the key principles underpinning microbiota research in respiratory contexts and provide practical guidance on how best such studies can be designed, executed and interpreted. We examine how an understanding of the respiratory microbiota both challenges fundamental assumptions and provides novel clinical insights into lung disease, and we set out a number of important targets for ongoing research.

Respiratory microbiota: addressing clinical questions, informing clinical practice.

Over the last decade, technological advances have revolutionised efforts to understand the role played by microbes in airways disease. With the application of ever more sophisticated techniques, the literature has become increasingly inaccessible to the non-specialist reader, potentially hampering the translation of these gains into improvements in patient care. In this article, we set out the key principles underpinning microbiota research in respiratory contexts and provide practical guidance on how best such studies can be designed, executed and interpreted. We examine how an understanding of the respiratory microbiota both challenges fundamental assumptions and provides novel clinical insights into lung disease, and we set out a number of important targets for ongoing research.

Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses.

BACKGROUND: Best practice when performing culture-independent microbiological analysis of sputum samples involves their rapid freezing and storage at -80°C. However, accessing biobanked collections can mean that material has been passed through repeated freeze-thaw cycles. The aim of this study was to determine the impact of these cycles on microbial community profiles. METHODS: Sputum was collected from eight adults with cystic fibrosis, and each sample was subjected to six freeze-thaw cycles. Following each cycle, an aliquot was removed and treated with propidium monoazide (PMA) prior to DNA extraction and 16S rRNA gene pyrosequencing. RESULTS: The impact of freeze-thaw cycles was greatest on rare members of the microbiota, with variation beyond that detected with within-sample repeat analysis observed after three cycles. CONCLUSION: Four or more freeze thaw cycles result in a significant distortion of microbiota profiles from CF sputum.

Environmentally determined differences in the murine lung microbiota and their relation to alveolar architecture.

Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect on tissue homeostasis and host defense. In contrast to the upper respiratory tract, the lower respiratory tract of healthy individuals has largely been considered free of microorganisms. To understand airway micro-ecology we studied microbiota of sterilely excised lungs from mice of different origin including outbred wild mice caught in the natural environment or kept under non-specific-pathogen-free (SPF) conditions as well as inbred mice maintained in non-SPF, SPF or germ-free (GF) facilities. High-throughput pyrosequencing of reverse transcribed 16S rRNA revealed metabolically active murine lung microbiota in all but GF mice. The overall composition across samples was similar at the phylum and family level. However, species richness was significantly different between lung microbiota from SPF and non-SPF mice. Non-cultivatable Betaproteobacteria such as Ralstonia spp. made up the major constituents and were also confirmed by 16S rRNA gene cloning analysis. Additionally, Pasteurellaceae, Enterobacteria and Firmicutes were isolated from lungs of non-SPF mice. Bacterial communities were detectable by fluorescent in situ hybridization (FISH) at alveolar epithelia in the absence of inflammation. Notably, higher bacterial abundance in non-SPF mice correlated with more and smaller size alveolae, which was corroborated by transplanting Lactobacillus spp. lung isolates into GF mice. Our data indicate a common microbial composition of murine lungs, which is diversified through different environmental conditions and affects lung architecture. Identification of the microbiota of murine lungs will pave the path to study their influence on pulmonary immunity to infection and allergens using mouse models.