[Show abstract][Hide abstract] ABSTRACT: The 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.
[Show abstract][Hide abstract] ABSTRACT: Rationale: 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, while polymorphisms in genes related to epithelial integrity and host defence 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, non-smokers and subjects with moderate Chronic Obstructive Pulmonary Disease (COPD) as controls. 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 qPCR and pyrosequencing. Measurements and Main Results: Sixty five IPF patients had double the burden of bacteria in Bronchoalveolar lavage (BAL) fluid compared to 44 controls. Baseline bacterial burden predicted the rate of decline in lung volume, 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 controls. Regression analyses indicated that these specific OTUs as well as bacterial burden associated independently with IPF. Conclusions: IPF is characterised 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.
American Journal of Respiratory and Critical Care Medicine 09/2014; · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rationale: Rhinovirus (RV) infection is followed by significantly increased frequencies of positive, potentially pathogenic, sputum cultures in chronic obstructive pulmonary disease (COPD). It however 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 RV infection on the airway bacterial microbiome. Methods: Subjects with COPD (n=14) and healthy controls with normal lung function (n=17) were infected with rhinovirus. Induced sputum was collected at baseline prior to rhinovirus inoculation and again at days 5, 15 and 42 post rhinovirus infection and DNA extracted. The V3-V5 region of the bacterial 16S rRNA gene was amplified and pyrosequenced resulting in 370,849 high quality reads from 112 of the possible 124 time points. Measurements and Main Results: Fifteen days post rhinovirus infection there was a six-fold increase in 16S copy number (P=0.007) and a 16% rise in numbers of Proteobacterial sequences, in particular potentially pathogenic Haemophilus influenzae (P=2.7e-20), from a pre-existing community. These changes only occurred in the sputum microbiome of COPD subjects and were still evident 42 days post infection. This was in contrast to the temporal stability demonstrated in the microbiome of healthy smokers and non-smokers. Conclusions: Following rhinovirus infection there is a rise in bacterial burden and a significant outgrowth of Haemophilus influenzae from the existing microbiota of COPD subjects. This is not observed in healthy individuals. Our findings suggest that RV infection in COPD alters the respiratory microbiome and may precipitate secondary bacterial infections.
American Journal of Respiratory and Critical Care Medicine 08/2013; · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular techniques have revolutionised the practise 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 in 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.
Human Molecular Genetics 08/2013; · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Correspondence noise is a major factor limiting direction discrimination performance in random-dot kinematograms . In the current study we investigated the influence of correspondence noise on Dmax, which is the upper limit for the spatial displacement of the dots for which coherent motion is still perceived. Human direction discrimination performance was measured, using 2-frame kinematograms having leftward/rightward motion, over a 200-fold range of dot-densities and a four-fold range of dot displacements. From this data Dmax was estimated for the different dot densities tested. A model was proposed to evaluate the correspondence noise in the stimulus. This model summed the outputs of a set of elementary Reichardt-type local detectors that had receptive fields tiling the stimulus and were tuned to the two directions of motion in the stimulus. A key assumption of the model was that the local detectors would have the radius of their catchment areas scaled with the displacement that they were tuned to detect; the scaling factor k linking the radius to the displacement was the only free parameter in the model and a single value of k was used to fit all of the psychophysical data collected. This minimal, correspondence-noise based model was able to account for 91% of the variability in the human performance across all of the conditions tested. The results highlight the importance of correspondence noise in constraining the largest displacement that can be detected.
PLoS ONE 10/2012; 7(10):e42995. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Observations that the airway microbiome is disturbed in asthma may be confounded by the widespread use of antibiotics and inhaled steroids. We have therefore examined the oropharyngeal microbiome in early onset wheezing infants from a rural area of tropical Ecuador where antibiotic usage is minimal and glucocorticoid usage is absent.
We performed pyrosequencing of amplicons of the polymorphic bacterial 16S rRNA gene from oropharyngeal samples from 24 infants with non-infectious early onset wheezing and 24 healthy controls (average age 10.2 months). We analyzed microbial community structure and differences between cases and controls by QIIME software.
We obtained 76,627 high quality sequences classified into 182 operational taxonomic units (OTUs). Firmicutes was the most common and diverse phylum (71.22% of sequences) with Streptococcus being the most common genus (49.72%). Known pathogens were found significantly more often in cases of infantile wheeze compared to controls, exemplified by Haemophilus spp. (OR = 2.12, 95% Confidence Interval (CI) 1.82-2.47; P = 5.46×10(-23)) and Staphylococcus spp. (OR = 124.1, 95%CI 59.0-261.2; P = 1.87×10(-241)). Other OTUs were less common in cases than controls, notably Veillonella spp. (OR = 0.59, 95%CI = 0.56-0.62; P = 8.06×10(-86)).
The airway microbiota appeared to contain many more Streptococci than found in Western Europe and the USA. Comparisons between healthy and wheezing infants revealed a significant difference in several bacterial phylotypes that were not confounded by antibiotics or use of inhaled steroids. The increased prevalence of pathogens such as Haemophilus and Staphylococcus spp. in cases may contribute to wheezing illnesses in this age group.
PLoS ONE 10/2012; 7(10):e46803. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Methyl halides have a significant impact on atmospheric chemistry, particularly in the degradation of stratospheric ozone. Bacteria are known to contribute to the degradation of methyl halides in the oceans and marine bacteria capable of using methyl bromide and methyl chloride as sole carbon and energy source have been isolated. A genetic marker for microbial degradation of methyl bromide ( cmuA ) was used to examine the distribution and diversity of these organisms in the marine environment. Three novel marine clades of cmuA were identified in unamended seawater and in marine enrichment cultures degrading methyl halides. Two of these cmuA clades are not represented in extant bacteria, demonstrating the utility of this molecular marker in identifying uncultivated marine methyl halide-degrading bacteria. The detection of populations of marine bacteria containing cmuA genes suggests that marine bacteria employing the CmuA enzyme contribute to methyl halide cycling in the ocean.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 03/2012; 7(3):e32543. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic rhinosinusitis (CRS) is a common disease with a complex pathophysiology involving a microbial component. Culture-independent molecular analysis represents a promising new approach to clarify the microbiology of CRS, but standardized, optimized sampling methods still have not been defined. This study was designed to compare nucleic acid extraction rates and recovery of bacteria for two methods of sampling the maxillary sinus, mucosal biopsy, and brushing.
Samples were obtained from 20 patients undergoing maxillary sinus surgery. Total extracted nucleic acid concentration and bacterial burden were compared between sample types.
Total nucleic acid concentration varied across patients. No statistically significant difference in mean total DNA concentration from mucosal biopsy specimens or brushings was observed. However, compared with biopsy specimens, brush samples possessed a significant (p < 0.035) increase in bacterial copy number.
Endoscopically directed mucosal brushings of the maxillary sinus provide equivalent concentrations of total DNA to mucosal biopsy specimens but possess greater concentrations of bacterial DNA, likely because of the greater surface area sampled by this method. Given the additional advantage of lower risk associated with obtaining brush samples, we suggest they represent the preferred sampling method for future genomic sinus studies.
American Journal of Rhinology and Allergy 07/2010; 24(4):263-5.
[Show abstract][Hide abstract] ABSTRACT: Bacterial communities in the airways of cystic fibrosis (CF) patients are, as in other ecological niches, influenced by autogenic and allogenic factors. However, our understanding of microbial colonization in younger versus older CF airways and the association with pulmonary function is rudimentary at best. Using a phylogenetic microarray, we examine the airway microbiota in age stratified CF patients ranging from neonates (9 months) to adults (72 years). From a cohort of clinically stable patients, we demonstrate that older CF patients who exhibit poorer pulmonary function possess more uneven, phylogenetically-clustered airway communities, compared to younger patients. Using longitudinal samples collected form a subset of these patients a pattern of initial bacterial community diversification was observed in younger patients compared with a progressive loss of diversity over time in older patients. We describe in detail the distinct bacterial community profiles associated with young and old CF patients with a particular focus on the differences between respective "early" and "late" colonizing organisms. Finally we assess the influence of Cystic Fibrosis Transmembrane Regulator (CFTR) mutation on bacterial abundance and identify genotype-specific communities involving members of the Pseudomonadaceae, Xanthomonadaceae, Moraxellaceae and Enterobacteriaceae amongst others. Data presented here provides insights into the CF airway microbiota, including initial diversification events in younger patients and establishment of specialized communities of pathogens associated with poor pulmonary function in older patient populations.
PLoS ONE 06/2010; 5(6):e11044. · 3.53 Impact Factor