Michael J. Cox

Reagent and laboratory contamination can critically impact sequence-based microbiome analyses

BackgroundThe study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents.ResultsIn this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination.ConclusionsThese results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised.

Outgrowth of the Bacterial Airway Microbiome after Rhinovirus Exacerbation of Chronic Obstructive Pulmonary Disease

RATIONALE Rhinovirus infection is followed by significantly increased frequencies of positive, potentially pathogenic sputum cultures in chronic obstructive pulmonary disease (COPD). However, it remains unclear whether these represent de novo infections or an increased load of organisms from the complex microbial communities (microbiome) in the lower airways. OBJECTIVES To investigate the effect of rhinovirus infection on the airway bacterial microbiome. METHODS Subjects with COPD (n = 14) and healthy control subjects with normal lung function (n = 17) were infected with rhinovirus. Induced sputum was collected at baseline before rhinovirus inoculation and again on Days 5, 15, and 42 after rhinovirus infection and DNA was extracted. The V3-V5 region of the bacterial 16S ribosomal RNA gene was amplified and pyrosequenced, resulting in 370,849 high-quality reads from 112 of the possible 124 time points. MEASUREMENTS AND MAIN RESULTS At 15 days after rhinovirus infection, there was a sixfold increase in 16S copy number (P = 0.007) and a 16% rise in numbers of proteobacterial sequences, most notably in potentially pathogenic Haemophilus influenzae (P = 2.7 × 10(-20)), from a preexisting community. These changes occurred only in the sputum microbiome of subjects with COPD and were still evident 42 days after infection. This was in contrast to the temporal stability demonstrated in the microbiome of healthy smokers and nonsmokers. CONCLUSIONS After rhinovirus infection, there is a rise in bacterial burden and a significant outgrowth of Haemophilus influenzae from the existing microbiota of subjects with COPD. This is not observed in healthy individuals. Our findings suggest that rhinovirus infection in COPD alters the respiratory microbiome and may precipitate secondary bacterial infections.

The Role of Bacteria in the Pathogenesis and Progression of Idiopathic Pulmonary Fibrosis

RATIONALE Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease of unknown cause that leads to respiratory failure and death within 5 years of diagnosis. Overt respiratory infection and immunosuppression carry a high morbidity and mortality, and polymorphisms in genes related to epithelial integrity and host defense predispose to IPF. OBJECTIVES To investigate the role of bacteria in the pathogenesis and progression of IPF. METHODS We prospectively enrolled patients diagnosed with IPF according to international criteria together with healthy smokers, nonsmokers, and subjects with moderate chronic obstructive pulmonary disease as control subjects. Subjects underwent bronchoalveolar lavage (BAL), from which genomic DNA was isolated. The V3-V5 region of the bacterial 16S rRNA gene was amplified, allowing quantification of bacterial load and identification of communities by 16S rRNA quantitative polymerase chain reaction and pyrosequencing. MEASUREMENTS AND MAIN RESULTS Sixty-five patients with IPF had double the burden of bacteria in BAL fluid compared with 44 control subjects. Baseline bacterial burden predicted the rate of decline in lung volume and risk of death and associated independently with the rs35705950 polymorphism of the MUC5B mucin gene, a proven host susceptibility factor for IPF. Sequencing yielded 912,883 high-quality reads from all subjects. We identified Haemophilus, Streptococcus, Neisseria, and Veillonella spp. to be more abundant in cases than control subjects. Regression analyses indicated that these specific operational taxonomic units as well as bacterial burden associated independently with IPF. CONCLUSIONS IPF is characterized by an increased bacterial burden in BAL that predicts decline in lung function and death. Trials of antimicrobial therapy are needed to determine if microbial burden is pathogenic in the disease.

Isolation of viruses responsible for the demise of an Emiliania huxleyi bloom in the English Channel

This study used analytical flow cytometry (AFC) to monitor the abundance of phytoplankton, coccoliths, bacteria and viruses in a transect that crossed a high reflectance area in the western English Channel. The high reflectance area, observed by satellite, was caused by the demise of an Emiliania huxleyi bloom. Water samples were collected from depth profiles at four stations, one station outside and three stations inside the high reflectance area. Plots of transect data revealed very obvious differences between Station 1, outside, and Stations 2–4, inside the high reflectance area. Inside, concentrations of viruses were higher; E. huxleyi cells were lower; coccoliths were higher; bacteria were higher and virus:bacteria ratio was lower than at Station 1, outside the high reflectance area. This data can simply be interpreted as virus-induced lysis of E. huxleyi cells in the bloom causing large concentrations of coccoliths to detach, resulting in the high reflectance observed by satellite imagery. This interpretation was supported by the isolation of two viruses, Eh V84 and Eh V86, from the high reflectance area that lysed cultures of E. huxleyi host strain CCMP1516. Basic characterization revealed that they were lytic viruses approximately 170 nm–190 nm in diameter with an icosahedral symmetry. Taken together, transect and isolation data suggest that viruses were the major contributor to the demise of the E. huxleyi population in the high reflectance area. Close coupling between microalgae, bacteria and viruses contributed to a large organic carbon input. Consequent cycling influenced the succession of an E. huxleyi -dominated population to a more characteristic mixed summer phytoplankton community.

Stable-isotope probing implicates Methylophaga spp and novel Gammaproteobacteria in marine methanol and methylamine metabolism

The metabolism of one-carbon (C1) compounds in the marine environment affects global warming, seawater ecology and atmospheric chemistry. Despite their global significance, marine microorganisms that consume C1 compounds in situ remain poorly characterized. Stable-isotope probing (SIP) is an ideal tool for linking the function and phylogeny of methylotrophic organisms by the metabolism and incorporation of stable-isotope-labelled substrates into nucleic acids. By combining DNA-SIP and time-series sampling, we characterized the organisms involved in the assimilation of methanol and methylamine in coastal sea water (Plymouth, UK). Labelled nucleic acids were analysed by denaturing gradient gel electrophoresis (DGGE) and clone libraries of 16S rRNA genes. In addition, we characterized the functional gene complement of labelled nucleic acids with an improved primer set targeting methanol dehydrogenase (mxaF) and newly designed primers for methylamine dehydrogenase (mauA). Predominant DGGE phylotypes, 16S rRNA, methanol and methylamine dehydrogenase gene sequences, and cultured isolates all implicated Methylophaga spp, moderately halophilic marine methylotrophs, in the consumption of both methanol and methylamine. Additionally, an mxaF sequence obtained from DNA extracted from sea water clustered with those detected in 13C-DNA, suggesting a predominance of Methylophaga spp among marine methylotrophs. Unexpectedly, most predominant 16S rRNA and functional gene sequences from 13C-DNA were clustered in distinct substrate-specific clades, with 16S rRNA genes clustering with sequences from the Gammaproteobacteria. These clades have no cultured representatives and reveal an ecological adaptation of particular uncultured methylotrophs to specific C1 compounds in the coastal marine environment.

Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing

The 16S rRNA gene is conserved across all bacteria and as such is routinely targeted in PCR surveys of bacterial diversity. PCR primer design aims to amplify as many different 16S rRNA gene sequences from as wide a range of organisms as possible, though there are no suitable 100% conserved regions of the gene, leading to bias. In the gastrointestinal tract, bifidobacteria are a key genus, but are often under-represented in 16S rRNA surveys of diversity. We have designed modified, ‘bifidobacteria-optimised’ universal primers, which we have demonstrated detection of bifidobacterial sequence present in DNA mixtures at 2% abundance, the lowest proportion tested. Optimisation did not compromise the detection of other organisms in infant faecal samples. Separate validation using fluorescence in situ hybridisation (FISH) shows that the proportions of bifidobacteria detected in faecal samples were in agreement with those obtained using 16S rRNA based pyrosequencing. For future studies looking at faecal microbiota, careful selection of primers will be key in order to ensure effective detection of bifidobacteria.

Sequencing the human microbiome in health and disease

Molecular techniques have revolutionized the practice of standard microbiology. In particular, 16S rRNA sequencing, whole microbial genome sequencing and metagenomics are revealing the extraordinary diversity of microorganisms on Earth and their vast genetic and metabolic repertoire. The increase in length, accuracy and number of reads generated by high-throughput sequencing has coincided with a surge of interest in the human microbiota, the totality of bacteria associated with the human body, in both health and disease. Traditional views of host/pathogen interactions are being challenged as the human microbiota are being revealed to be important in normal immune system function, to diseases not previously thought to have a microbial component and to infectious diseases with unknown aetiology. In this review, we introduce the nature of the human microbiota and application of these three key sequencing techniques for its study, highlighting both advances and challenges in the field. We go on to discuss how further adoption of additional techniques, also originally developed in environmental microbiology, will allow the establishment of disease causality against a background of numerous, complex and interacting microorganisms within the human host.

Dysbiosis Anticipating Necrotizing Enterocolitis in Very Premature Infants

Using 16S rRNA gene sequencing and targeted culture, we compared microbiota in fecal samples from infants with necrotizing enterocolitis (NEC) and controls. Two significant signatures were associated with NEC: 1 with dominant Clostridium perfringens and 1 with dominant Enterobacteriaceae.

Bacterial microbiota of the upper respiratory tract and childhood asthma

Background: Patients with asthma and healthy controls differ in bacterial colonization of the respiratory tract. The upper airways have been shown to reflect colonization of the lower airways, the actual site of inflammation in asthma, which is hardly accessible in population studies. Objective: We sought to characterize the bacterial communities at 2 sites of the upper respiratory tract obtained from children from a rural area and to relate these to asthma. Methods: The microbiota of 327 throat and 68 nasal samples from school‐age farm and nonfarm children were analyzed by 454‐pyrosequencing of the bacterial 16S ribosomal RNA gene. Results: Alterations in nasal microbiota but not of throat microbiota were associated with asthma. Children with asthma had lower &agr;‐ and &bgr;‐diversity of the nasal microbiota as compared with healthy control children. Furthermore, asthma presence was positively associated with a specific operational taxonomic unit from the genus Moraxella in children not exposed to farming, whereas in farm children Moraxella colonization was unrelated to asthma. In nonfarm children, Moraxella colonization explained the association between bacterial diversity and asthma to a large extent. Conclusions: Asthma was mainly associated with an altered nasal microbiota characterized by lower diversity and Moraxella abundance. Children living on farms might not be susceptible to the disadvantageous effect of Moraxella. Prospective studies may clarify whether Moraxella outgrowth is a cause or a consequence of loss in diversity.

Airway Microbiota in Severe Asthma and Relationship to Asthma Severity and Phenotypes.

Background The lower airways harbor a community of bacterial species which is altered in asthma. Objectives We examined whether the lower airway microbiota were related to measures of asthma severity. Methods We prospectively recruited 26 severe asthma, 18 non-severe asthma and 12 healthy subjects. DNA was extracted from induced sputum and PCR amplification of the V3-V5 region of bacterial 16S rRNA gene was performed. Results We obtained 138,218 high quality sequences which were rarefied at 133 sequences/sample. Twenty OTUs had sequences ≥1% of total. There were marked differences in the distribution of Phyla between groups (P = 2.8x10-118). Bacteroidetes and Fusobacteria were reduced in non-severe and severe asthmatic groups. Proteobacteria were more common in non-severe asthmatics compared to controls (OR = 2.26; 95% CI = 1.94–2.64) and Firmicutes were increased in severe asthmatics compared to controls (OR = 2.15; 95%CI = 1.89–2.45). Streptococcal OTUs amongst the Firmicutes were associated with recent onset asthma, rhinosinusitis and sputum eosinophilia. Conclusions Sputum microbiota in severe asthma differs from healthy controls and non-severe asthmatics, and is characterized by the presence of Streptococcus spp with eosinophilia. Whether these organisms are causative for the pathophysiology of asthma remains to be determined.