Limited effects of azithromycin on the oropharyngeal microbiome in children with CF and early pseudomonas infection.

BACKGROUND: Tobramycin inhalation solution (TIS) and chronic azithromycin (AZ) have known clinical benefits for children with CF, likely due to antimicrobial and anti-inflammatory activity. The effects of chronic AZ in combination with TIS on the airway microbiome have not been extensively investigated. Oropharyngeal swab samples were collected in the OPTIMIZE multicenter, randomized, placebo-controlled trial examining the addition of AZ to TIS in 198 children with CF and early P. aeruginosa infection. Bacterial small subunit rRNA gene community profiles were determined. The effects of TIS and AZ were assessed on oropharyngeal microbial diversity and composition to uncover whether effects on the bacterial community may be a mechanism of action related to the observed changes in clinical outcomes. RESULTS: Substantial changes in bacterial communities (total bacterial load, diversity and relative abundance of specific taxa) were observed by week 3 of TIS treatment for both the AZ and placebo groups. On average, these shifts were due to changes in non-traditional CF taxa that were not sustained at the later study visits (weeks 13 and 26). Bacterial community measures did not differ between the AZ and placebo groups. CONCLUSIONS: This study provides further evidence that the mechanism for AZ's effect on clinical outcomes is not due solely to action on airway microbial composition.

Mucosal Microbiota Associated With Eosinophilic Esophagitis and Eosinophilic Gastritis.

OBJECTIVE: The aim of the study was to determine the mucosal microbiota associated with eosinophilic esophagitis (EoE) and eosinophilic gastritis (EoG) in a geographically diverse cohort of patients compared to controls. METHODS: We conducted a prospective study of individuals with eosinophilic gastrointestinal disease (EGID) in the Consortium of Eosinophilic Gastrointestinal Disease Researchers, including pediatric and adult tertiary care centers. Eligible individuals had clinical data, mucosal biopsies, and stool collected. Total bacterial load was determined from mucosal biopsy samples by quantitative polymerase chain reaction (PCR). Community composition was determined by small subunit rRNA gene amplicons. RESULTS: One hundred thirty-nine mucosal biopsies were evaluated corresponding to 93 EoE, 17 EoG, and 29 control specimens (18 esophageal) from 10 sites across the United States. Dominant community members across disease activity differed significantly. When comparing EoE and EoG with controls, the dominant taxa in individuals with EGIDs was increased ( Streptococcus in esophagus; Prevotella in stomach). Specific taxa were associated with active disease for both EoE ( Streptococcus , Gemella ) and EoG ( Leptotrichia ), although highly individualized communities likely impacted statistical testing. Alpha diversity metrics were similar across groups, but with high variability among individuals. Stool analyses did not correlate with bacterial communities found in mucosal biopsy samples and was similar in patients and controls. CONCLUSIONS: Dominant community members ( Streptococcus for EoE, Prevotella for EoG) were different in the mucosal biopsies but not stool of individuals with EGIDs compared to controls; taxa associated with EGIDs were highly variable across individuals. Further study is needed to determine if therapeutic interventions contribute to the observed community differences.

Temporal airway microbiome changes related to ventilator-associated pneumonia in children.

We sought to determine whether temporal changes in the lower airway microbiome are associated with ventilator-associated pneumonia (VAP) in children.Using a multicentre prospective study of children aged 31 days to 18 years requiring mechanical ventilation support for >72 h, daily tracheal aspirates were collected and analysed by sequencing of the 16S rRNA gene. VAP was assessed using 2008 Centers for Disease Control and Prevention paediatric criteria. The association between microbial factors and VAP was evaluated using joint longitudinal time-to-event modelling, matched case-control comparisons and unsupervised clustering.Out of 366 eligible subjects, 66 (15%) developed VAP at a median of 5 (interquartile range 3-5) days post intubation. At intubation, there was no difference in total bacterial load (TBL), but Shannon diversity and the relative abundance of Streptococcus, Lactobacillales and Prevotella were lower for VAP subjects versus non-VAP subjects. However, higher TBL on each sequential day was associated with a lower hazard (hazard ratio 0.39, 95% CI 0.23-0.64) for developing VAP, but sequential values of diversity were not associated with VAP. Similar findings were observed from the matched analysis and unsupervised clustering. The most common dominant VAP pathogens included Prevotella species (19%), Pseudomonas aeruginosa (14%) and Streptococcus mitis/pneumoniae (10%). Mycoplasma and Ureaplasma were also identified as dominant organisms in several subjects.In mechanically ventilated children, changes over time in microbial factors were marginally associated with VAP risk, although these changes were not suitable for predicting VAP in individual patients. These findings suggest that focusing exclusively on pathogen burden may not adequately inform VAP diagnosis.

Microbial and Biogeochemical Indicators of Methane in Groundwater Aquifers of the Denver Basin, Colorado.

The presence of methane and other hydrocarbons in domestic-use groundwater aquifers poses significant environmental and human health concerns. Isotopic measurements are often relied upon as indicators of groundwater aquifer contamination with methane. While these parameters are used to infer microbial metabolisms, there is growing evidence that isotopes present an incomplete picture of subsurface microbial processes. This study examined the relationships between microbiology and chemistry in groundwater wells located in the Denver-Julesburg Basin of Colorado, a rapidly urbanizing area with active oil and gas development. A primary goal was to determine if microbial data can reliably indicate the quantities and sources of groundwater methane. Comprehensive chemical and molecular analyses were performed on 39 groundwater well samples from five aquifers. Elevated methane concentrations were found in only one aquifer, and both isotopic and microbial data support a microbial origin. Microbial parameters had similar explanatory power as chemical parameters for predicting sample methane concentrations. Furthermore, a subset of samples with unique microbiology corresponded with unique chemical signatures that may be useful indicators of methane gas migration, potentially from nearby coal seams interacting with the aquifer. Microbial data may allow for more accurate determination of groundwater contamination and improved long-term water quality monitoring compared solely to isotopic and chemical data in areas with microbial methane.

Influence of Acid Blockade on the Aerodigestive Tract Microbiome in Children With Cystic Fibrosis.

BACKGROUND: Acid blockade is commonly prescribed in patients with cystic fibrosis (CF). Growing concerns, however, exist about its possible role in the pathophysiology of pulmonary infections. We aimed to investigate if acid blockade alters esophageal and respiratory microbiota leading to dysbiosis and inflammation. METHODS: We performed a cross sectional study of children with CF who were either prescribed acid blockade or not. Samples from the gastrointestinal and respiratory tracts were obtained and microbiome analyzed. Mixed effect models were used to compare outcomes between cohorts and across sampling sites. A random subject intercept was included to account for the multiple sampling sites per individual. RESULTS: A cohort of 25 individuals, 44% girls with median age of 13.8 years [IQR 11.2--14.8] were enrolled. Alpha diversity, total bacterial load, and beta diversity were similar across anatomic compartments, across the upper gastrointestinal tract, and in respiratory samples. Similar alpha diversity, total bacterial load, and beta diversity results were also observed when comparing individuals on versus those off acid blockade. IL-8 was elevated in the distal versus proximal esophagus in the whole cohort (P < 0.01). IL-8 concentrations were similar in the distal esophagus in patients on and off acid blockade, but significantly greater in the proximal esophagus of subjects on treatment (P < 0.01). CONCLUSIONS: On the basis of these data, acid blockade use does not appear to influence the microbiome of the aerodigestive tract in children with cystic fibrosis suggesting a complex interplay between these medications and the bacterial composition of the esophagus and lung.

Nanodiagnostics to monitor biofilm oxygen metabolism for antibiotic susceptibility testing.

In clinical environments, many serious antibiotic-resistant infections are caused by biofilm-forming species. This presents issues when attempting to determine antimicrobial dosing as traditional antibiotic susceptibility tests (ASTs) are typically designed around planktonic bacteria and thus offer information that is not relevant to the biofilm phenotype present in the patient. Even the popular Calgary biofilm device may provide inaccurate minimum biofilm inhibitory concentrations (MBICs) and can be time- and material-intensive. In this work, we present a method utilizing oxygen-sensitive nanosensor technology to monitor the oxygen consumption dynamics of living biofilms as they are exposed to antibiotics. We incorporated our nanosensors into biofilms grown from P. aeruginosa strains of varying sensitivity to traditional classes of antibiotics. Through measuring nanosensor response under antibiotic administration we determined the concentrations able to cease biofilm metabolism. This method provides information on the MBIC as well as kinetic response information in a manner that requires fewer materials and is more reflective of biofilm behavior than a traditional AST.

Pulmonary Aptamer Signatures in Children's Interstitial and Diffuse Lung Disease.

Rationale: Biomarker signatures are needed in children with children's interstitial and diffuse lung disease (chILD) to improve diagnostic approaches, increase our understanding of disease pathogenesis, monitor disease progression, and develop new treatment strategies. Proteomic technology using SOMAmer (Slow Off-rate Modified Aptamer) nucleic acid-based protein-binding reagents allows for biomarker discovery.Objectives: We hypothesized that proteins and protein pathways in BAL fluid (BALF) would distinguish children with neuroendocrine cell hyperplasia of infancy (NEHI), surfactant dysfunction mutations, and other chILD diagnoses and control subjects.Methods: BALF was collected for clinical indications and banked in patients with chILD and disease control subjects using standardized protocols over 10 years. BALF supernatant was analyzed using an aptamer assay to measure 1,129 protein levels. Protein levels were compared between groups using an ANOVA and adjusted for multiple comparisons using false discovery rate. Proteins were classified into pathways. Hierarchical clustering was used to define endotypes in the group of children with NEHI.Measurements and Main Results: After correcting for multiple testing, children with NEHI (n = 22) had 202 aptamers that were significantly different (P < 0.05) in BALF compared with control subjects (n = 9). Children with surfactant mutation (n = 8) had 51 aptamers significantly different (P < 0.05) in BALF compared with control subjects (n = 9). Proteins associated with pulmonary fibrosis and inflammation were associated with the surfactant dysfunction group but not the NEHI group. Using hierarchical clustering analysis, two distinct NEHI endotypes were identified.Conclusions: Distinct proteins and protein pathways can be determined from BALF of children with chILD, and these hold promise to further our understanding of chILD.

Infection Is Not Required for Mucoinflammatory Lung Disease in CFTR-Knockout Ferrets.

RATIONALE: Classical interpretation of cystic fibrosis (CF) lung disease pathogenesis suggests that infection initiates disease progression, leading to an exuberant inflammatory response, excessive mucus, and ultimately bronchiectasis. Although symptomatic antibiotic treatment controls lung infections early in disease, lifelong bacterial residence typically ensues. Processes that control the establishment of persistent bacteria in the CF lung, and the contribution of noninfectious components to disease pathogenesis, are poorly understood. OBJECTIVES: To evaluate whether continuous antibiotic therapy protects the CF lung from disease using a ferret model that rapidly acquires lethal bacterial lung infections in the absence of antibiotics. METHODS: CFTR (cystic fibrosis transmembrane conductance regulator)-knockout ferrets were treated with three antibiotics from birth to several years of age and lung disease was followed by quantitative computed tomography, BAL, and histopathology. Lung disease was compared with CFTR-knockout ferrets treated symptomatically with antibiotics. MEASUREMENTS AND MAIN RESULTS: Bronchiectasis was quantified from computed tomography images. BAL was evaluated for cellular differential and features of inflammatory cellular activation, bacteria, fungi, and quantitative proteomics. Semiquantitative histopathology was compared across experimental groups. We demonstrate that lifelong antibiotics can protect the CF ferret lung from infections for several years. Surprisingly, CF animals still developed hallmarks of structural bronchiectasis, neutrophil-mediated inflammation, and mucus accumulation, despite the lack of infection. Quantitative proteomics of BAL from CF and non-CF pairs demonstrated a mucoinflammatory signature in the CF lung dominated by Muc5B and neutrophil chemoattractants and products. CONCLUSIONS: These findings implicate mucoinflammatory processes in the CF lung as pathogenic in the absence of clinically apparent bacterial and fungal infections.

Airway microbiota across age and disease spectrum in cystic fibrosis.

Our objectives were to characterise the microbiota in cystic fibrosis (CF) bronchoalveolar lavage fluid (BALF), and determine its relationship to inflammation and disease status.BALF from paediatric and adult CF patients and paediatric disease controls undergoing clinically indicated bronchoscopy was analysed for total bacterial load and for microbiota by 16S rDNA sequencing.We examined 191 BALF samples (146 CF and 45 disease controls) from 13 CF centres. In CF patients aged <2 years, nontraditional taxa (e.gStreptococcus, Prevotella and Veillonella) constituted ∼50% of the microbiota, whereas in CF patients aged ≥6 years, traditional CF taxa (e.gPseudomonas, Staphylococcus and Stenotrophomonas) predominated. Sequencing detected a dominant taxon not traditionally associated with CF (e.gStreptococcus or Prevotella) in 20% of CF BALF and identified bacteria in 24% of culture-negative BALF. Microbial diversity and relative abundance of Streptococcus, Prevotella and Veillonella were inversely associated with airway inflammation. Microbiota communities were distinct in CF compared with disease controls, but did not differ based on pulmonary exacerbation status in CF.The CF microbiota detected in BALF differs with age. In CF patients aged <2 years, Streptococcus predominates, whereas classic CF pathogens predominate in most older children and adults.

Longitudinal and Source-to-Tap New Orleans, LA, U.S.A. Drinking Water Microbiology.

The two municipal drinking water systems of New Orleans, LA, U.S.A. were sampled to compare the microbiology of independent systems that treat the same surface water from the Mississippi River. To better understand temporal trends and sources of microbiology delivered to taps, these treatment plants and distribution systems were subjected to source-to-tap sampling over four years. Both plants employ traditional treatment by chloramination, applied during or after settling, followed by filtration before distribution in a warm, low water age system. Longitudinal samples indicated microbiology to have stability both spatially and temporally, and between treatment plants and distribution systems. Disinfection had the greatest impact on microbial composition, which was further refined by filtration and influenced by distribution and premise plumbing. Actinobacteria spp. exhibited trends with treatment. In particular, Mycobacterium spp., very low in finished waters, occurred idiosyncratically at high levels in some tap waters, indicating distribution and/or premise plumbing as main contributors of mycobacteria. Legionella spp., another genus containing potential opportunistic pathogens, also occurred ubiquitously. Source water microbiology was most divergent from tap water, and each step of treatment brought samples more closely similar to tap waters.

Defective innate immunity and hyperinflammation in newborn cystic fibrosis transmembrane conductance regulator-knockout ferret lungs.

Mucociliary clearance (MCC) and submucosal glands are major components of airway innate immunity that have impaired function in cystic fibrosis (CF). Although both of these defense systems develop postnatally in the ferret, the lungs of newborn ferrets remain sterile in the presence of a functioning cystic fibrosis transmembrane conductance regulator gene. We evaluated several components of airway innate immunity and inflammation in the early CF ferret lung. At birth, the rates of MCC did not differ between CF and non-CF animals, but the height of the airway surface liquid was significantly reduced in CF newborn ferrets. CF ferrets had impaired MCC after 7 days of age, despite normal rates of ciliogenesis. Only non-CF ferrets eradicated Pseudomonas directly introduced into the lung after birth, whereas both genotypes could eradicate Staphylococcus. CF bronchoalveolar lavage fluid (BALF) had significantly lower antimicrobial activity selectively against Pseudomonas than non-CF BALF, which was insensitive to changes in pH and bicarbonate. Liquid chromatography-tandem mass spectrometry and cytokine analysis of BALF from sterile Caesarean-sectioned and nonsterile naturally born animals demonstrated CF-associated disturbances in IL-8, TNF-α, and IL-ß, and pathways that control immunity and inflammation, including the complement system, macrophage functions, mammalian target of rapamycin signaling, and eukaryotic initiation factor 2 signaling. Interestingly, during the birth transition, IL-8 was selectively induced in CF BALF, despite no genotypic difference in bacterial load shortly after birth. These results suggest that newborn CF ferrets have defects in both innate immunity and inflammatory signaling that may be important in the early onset and progression of lung disease in these animals.

The upper respiratory tract as a microbial source for pulmonary infections in cystic fibrosis. Parallels from island biogeography.

A continuously mixed series of microbial communities inhabits various points of the respiratory tract, with community composition determined by distance from colonization sources, colonization rates, and extinction rates. Ecology and evolution theory developed in the context of biogeography is relevant to clinical microbiology and could reframe the interpretation of recent studies comparing communities from lung explant samples, sputum samples, and oropharyngeal swabs. We propose an island biogeography model of the microbial communities inhabiting different niches in human airways. Island biogeography as applied to communities separated by time and space is a useful parallel for exploring microbial colonization of healthy and diseased lungs, with the potential to inform our understanding of microbial community dynamics and the relevance of microbes detected in different sample types. In this perspective, we focus on the intermixed microbial communities inhabiting different regions of the airways of patients with cystic fibrosis.

Explicet: graphical user interface software for metadata-driven management, analysis and visualization of microbiome data.

Studies of the human microbiome, and microbial community ecology in general, have blossomed of late and are now a burgeoning source of exciting research findings. Along with the advent of next-generation sequencing platforms, which have dramatically increased the scope of microbiome-related projects, several high-performance sequence analysis pipelines (e.g. QIIME, MOTHUR, VAMPS) are now available to investigators for microbiome analysis. The subject of our manuscript, the graphical user interface-based Explicet software package, fills a previously unmet need for a robust, yet intuitive means of integrating the outputs of the software pipelines with user-specified metadata and then visualizing the combined data.

Carbon dioxide and hydrogen sulfide associations with regional bacterial diversity patterns in microbially induced concrete corrosion.

The microbial communities associated with deteriorating concrete corrosion fronts were characterized in 35 samples taken from wastewater collection and treatment systems in ten utilities. Bacterial communities were described using Illumina MiSeq sequencing of the V1V2 region of the small subunit ribosomal ribonucleic acid (SSU-rRNA) gene recovered from fresh corrosion products. Headspace gas concentrations (hydrogen sulfide, carbon dioxide, and methane), pore water pH, moisture content, and select mineralogy were tested for correlation to community outcomes and corrosion extent using pairwise linear regressions and canonical correspondence analysis. Corroding concrete was most commonly characterized by moisture contents greater than 10%, pore water pH below one, and limited richness (<10 taxa). Bacterial community composition was not correlated to geographic location when considered independently from other environmental factors. Corrosion was most severe in sites with high levels of hydrogen sulfide (>100 ppm) and carbon dioxide (>1%) gases, conditions which also were associated with low diversity biofilms dominated by members of the acidophilic sulfur-oxidizer genus Acidithiobacillus.

The effects of airway microbiome on corticosteroid responsiveness in asthma.

RATIONALE: The role of airway microbiome in corticosteroid response in asthma is unknown. OBJECTIVES: To examine airway microbiome composition in patients with corticosteroid-resistant (CR) asthma and compare it with patients with corticosteroid-sensitive (CS) asthma and normal control subjects and explore whether bacteria in the airways of subjects with asthma may direct alterations in cellular responses to corticosteroids. METHODS: 16S rRNA gene sequencing was performed on bronchoalveolar lavage (BAL) samples of 39 subjects with asthma and 12 healthy control subjects. In subjects with asthma, corticosteroid responsiveness was characterized, BAL macrophages were stimulated with pathogenic versus commensal microorganisms, and analyzed by real-time polymerase chain reaction for the expression of corticosteroid-regulated genes and cellular p38 mitogen-activated protein kinase (MAPK) activation. MEASUREMENTS AND MAIN RESULTS: Of the 39 subjects with asthma, 29 were CR and 10 were CS. BAL microbiome from subjects with CR and CS asthma did not differ in richness, evenness, diversity, and community composition at the phylum level, but did differ at the genus level, with distinct genus expansions in 14 subjects with CR asthma. Preincubation of asthmatic airway macrophages with Haemophilus parainfluenzae, a uniquely expanded potential pathogen found only in CR asthma airways, resulted in p38 MAPK activation, increased IL-8 (P < 0.01), mitogen-activated kinase phosphatase 1 mRNA (P < 0.01) expression, and inhibition of corticosteroid responses (P < 0.05). This was not observed after exposure to commensal bacterium Prevotella melaninogenica. Inhibition of transforming growth factor-ß-associated kinase-1 (TAK1), upstream activator of MAPK, but not p38 MAPK restored cellular sensitivity to corticosteroids. CONCLUSIONS: A subset of subjects with CR asthma demonstrates airway expansion of specific gram-negative bacteria, which trigger TAK1/MAPK activation and induce corticosteroid resistance. TAK1 inhibition restored cellular sensitivity to corticosteroids.

Metabolomics analysis of bronchoalveolar lavage fluid predicts unique features of the lower airway in pediatric cystic fibrosis.

BACKGROUND: Progressive, obstructive lung disease resulting from chronic infection and inflammation is the leading cause of morbidity and mortality in persons with cystic fibrosis (PWCF). Metabolomics and next -generation sequencing (NGS) of airway secretions can allow for better understanding of cystic fibrosis (CF) pathophysiology. In this study, global metabolomic profiling on bronchoalveolar lavage fluid (BALF) obtained from pediatric PWCF and disease controls (DCs) was performed and compared to lower airway microbiota, inflammation, and lung function. METHODS: BALF was collected from children undergoing flexible bronchoscopies for clinical indications. Metabolomic profiling was performed using a platform developed by Metabolon Inc. Total bacterial load (TBL) was measured using quantitative polymerase chain reaction (qPCR), and bacterial communities were characterized using 16S ribosomal RNA (rRNA) sequencing. Random Forest Analysis (RFA), principal component analysis (PCA), and hierarchical clustering analysis (HCA) were performed. RESULTS: One hundred ninety-five BALF samples were analyzed, 142 (73 %) from PWCF. Most metabolites (425/665) and summed categories (7/9) were significantly increased in PWCF. PCA of the metabolomic data revealed CF BALF exhibited more dispersed clustering compared to DC BALF. Higher metabolite concentrations correlated with increased inflammation, increased abundance of Staphylococcus, and decreased lung function. CONCLUSIONS: The lower airway metabolome of PWCF was defined by a complex expansion of metabolomic activity. These findings could be attributed to heightened inflammation in PWCF and aspects of the CF airway polymicrobial ecology. CF-specific metabolomic features are associated with the unique underlying biology of the CF airway.

An integrated metaproteomics workflow for studying host-microbe dynamics in bronchoalveolar lavage samples applied to cystic fibrosis disease.

Airway microbiota are known to contribute to lung diseases, such as cystic fibrosis (CF), but their contributions to pathogenesis are still unclear. To improve our understanding of host-microbe interactions, we have developed an integrated analytical and bioinformatic mass spectrometry (MS)-based metaproteomics workflow to analyze clinical bronchoalveolar lavage (BAL) samples from people with airway disease. Proteins from BAL cellular pellets were processed and pooled together in groups categorized by disease status (CF vs. non-CF) and bacterial diversity, based on previously performed small subunit rRNA sequencing data. Proteins from each pooled sample group were digested and subjected to liquid chromatography tandem mass spectrometry (MS/MS). MS/MS spectra were matched to human and bacterial peptide sequences leveraging a bioinformatic workflow using a metagenomics-guided protein sequence database and rigorous evaluation. Label-free quantification revealed differentially abundant human peptides from proteins with known roles in CF, like neutrophil elastase and collagenase, and proteins with lesser-known roles in CF, including apolipoproteins. Differentially abundant bacterial peptides were identified from known CF pathogens (e.g., Pseudomonas), as well as other taxa with potentially novel roles in CF. We used this host-microbe peptide panel for targeted parallel-reaction monitoring validation, demonstrating for the first time an MS-based assay effective for quantifying host-microbe protein dynamics within BAL cells from individual CF patients. Our integrated bioinformatic and analytical workflow combining discovery, verification, and validation should prove useful for diverse studies to characterize microbial contributors in airway diseases. Furthermore, we describe a promising preliminary panel of differentially abundant microbe and host peptide sequences for further study as potential markers of host-microbe relationships in CF disease pathogenesis.IMPORTANCEIdentifying microbial pathogenic contributors and dysregulated human responses in airway disease, such as CF, is critical to understanding disease progression and developing more effective treatments. To this end, characterizing the proteins expressed from bacterial microbes and human host cells during disease progression can provide valuable new insights. We describe here a new method to confidently detect and monitor abundance changes of both microbe and host proteins from challenging BAL samples commonly collected from CF patients. Our method uses both state-of-the art mass spectrometry-based instrumentation to detect proteins present in these samples and customized bioinformatic software tools to analyze the data and characterize detected proteins and their association with CF. We demonstrate the use of this method to characterize microbe and host proteins from individual BAL samples, paving the way for a new approach to understand molecular contributors to CF and other diseases of the airway.

Host DNA depletion on frozen human respiratory samples enables successful metagenomic sequencing for microbiome studies.

Background: Most respiratory microbiome studies have focused on amplicon rather than metagenomics sequencing due to high host DNA content. We evaluated efficacy of five host DNA depletion methods on previously frozen human bronchoalveolar lavage (BAL), nasal swabs, and sputum prior to metagenomic sequencing. Results: Median sequencing depth was 76.4 million reads per sample. Untreated nasal, sputum and BAL samples had 94.1%, 99.2%, and 99.7% host-reads. The effect of host depletion differed by sample type. Most treatment methods increased microbial reads, species richness and predicted functional richness; the increase in species and predicted functional richness was mediated by higher effective sequencing depth. For BAL and nasal samples, most methods did not change Morisita-Horn dissimilarity suggesting limited bias introduced by host depletion. Conclusions: Metagenomics sequencing without host depletion will underestimate microbial diversity of most respiratory samples due to shallow effective sequencing depth and is not recommended. Optimal host depletion methods vary by sample type.

Culture-independent analysis of aerosol microbiology in a metropolitan subway system.

The goal of this study was to determine the composition and diversity of microorganisms associated with bioaerosols in a heavily trafficked metropolitan subway environment. We collected bioaerosols by fluid impingement on several New York City subway platforms and associated sites in three sampling sessions over a 1.5-year period. The types and quantities of aerosolized microorganisms were determined by culture-independent phylogenetic analysis of small-subunit rRNA gene sequences by using both Sanger (universal) and pyrosequencing (bacterial) technologies. Overall, the subway bacterial composition was relatively simple; only 26 taxonomic families made up ~75% of the sequences determined. The microbiology was more or less similar throughout the system and with time and was most similar to outdoor air, consistent with highly efficient air mixing in the system. Identifiable bacterial sequences indicated that the subway aerosol assemblage was composed of a mixture of genera and species characteristic of soil, environmental water, and human skin commensal bacteria. Eukaryotic diversity was mainly fungal, dominated by organisms of types associated with wood rot. Human skin bacterial species (at 99% rRNA sequence identity) included the Staphylococcus spp. Staphylococcus epidermidis (the most abundant and prevalent commensal of the human integument), S. hominis, S. cohnii, S. caprae, and S. haemolyticus, all well-documented human commensal bacteria. We encountered no organisms of public health concern. This study is the most extensive culture-independent survey of subway microbiota so far and puts in place pre-event information required for any bioterrorism surveillance activities or monitoring of the microbiological impact of recent subway flooding events.

Toll-like receptor 4-dependent Kupffer cell activation and liver injury in a novel mouse model of parenteral nutrition and intestinal injury.

UNLABELLED: Infants with intestinal failure who are parenteral nutrition (PN)-dependent may develop cholestatic liver injury and cirrhosis (PN-associated liver injury: PNALI). The pathogenesis of PNALI remains incompletely understood. We hypothesized that intestinal injury with increased intestinal permeability combined with administration of PN promotes lipopolysaccharide (LPS)-Toll-like receptor 4 (TLR4) signaling dependent Kupffer cell (KC) activation as an early event in the pathogenesis of PNALI. We developed a mouse model in which intestinal injury and increased permeability were induced by oral treatment for 4 days with dextran sulphate sodium (DSS) followed by continuous infusion of soy lipid-based PN solution through a central venous catheter for 7 (PN7d/DSS) and 28 (PN28d/DSS) days. Purified KCs were probed for transcription of proinflammatory cytokines. PN7d/DSS mice showed increased intestinal permeability and elevated portal vein LPS levels, evidence of hepatocyte injury and cholestasis (serum aspartate aminotransferase, alanine aminotransferase, bile acids, total bilirubin), and increased KC expression of interleukin-6 (Il6), tumor necrosis factor α (Tnfα), and transforming growth factor ß (Tgfß). Markers of liver injury remained elevated in PN28d/DSS mice associated with lobular inflammation, hepatocyte apoptosis, peliosis, and KC hypertrophy and hyperplasia. PN infusion without DSS pretreatment or DSS pretreatment alone did not result in liver injury or KC activation, even though portal vein LPS levels were elevated. Suppression of the intestinal microbiota with broad spectrum antibiotics or ablation of TLR4 signaling in Tlr4 mutant mice resulted in significantly reduced KC activation and markedly attenuated liver injury in PN7d/DSS mice. CONCLUSION: These data suggest that intestinal-derived LPS activates KC through TLR4 signaling in early stages of PNALI.