Parallel Analysis of Cystic Fibrosis Sputum and Saliva Reveals Overlapping Communities and an Opportunity for Sample Decontamination

ABSTRACT Culture-independent studies of the cystic fibrosis (CF) airway microbiome typically rely on expectorated sputum to assess the microbial makeup of lower airways. These studies have revealed rich bacterial communities. There is often considerable overlap between taxa observed in sputum and those observed in saliva, raising questions about the reliability of expectorated sputum as a sample representing lower airway microbiota. These concerns prompted us to compare pairs of sputum and saliva samples from 10 persons with CF. Using 16S rRNA gene sequencing and droplet digital PCR (ddPCR), we analyzed 37 pairs of sputum and saliva samples, each collected from the same person on the same day. We developed an in silico postsequencing decontamination procedure to remove from sputum the fraction of DNA reads estimated to have been contributed by saliva during expectoration. We demonstrate that while there was often sizeable overlap in community membership between sample types, expectorated sputum typically contains a higher bacterial load and a less diverse community compared to saliva. The differences in diversity between sputum and saliva were more pronounced in advanced disease stage, owing to increased relative abundance of the dominant taxa in sputum. Our effort to model saliva contamination of sputum in silico revealed generally minor effects on community structure after removal of contaminating reads. Despite considerable overlap in taxa observed between expectorated sputum and saliva samples, the impact of saliva contamination on measures of lower airway bacterial community composition in CF using expectorated sputum appears to be minimal. IMPORTANCE Cystic fibrosis is an inherited disease characterized by chronic respiratory tract infection and progressive lung disease. Studies of cystic fibrosis lung microbiology often rely on expectorated sputum to reflect the microbiota present in the lower airways. Passage of sputum through the oropharynx during collection, however, contributes microbes present in saliva to the sample, which could confound interpretation of results. Using culture-independent DNA sequencing-based analyses, we characterized the bacterial communities in pairs of expectorated sputum and saliva samples to generate a model for “decontaminating” sputum in silico. Our results demonstrate that salivary contamination of expectorated sputum does not have a large effect on most sputum samples and that observations of high bacterial diversity likely accurately reflect taxa present in cystic fibrosis lower airways.

Download Full-text

16S rRNA gene sequencing reveals site-specific signatures of the upper and lower airways of cystic fibrosis patients

10.1101/125187 ◽

2017 ◽

Author(s):

Sarah K. Lucas ◽

Robert Yang ◽

Jordan M. Dunitz ◽

Holly C. Boyer ◽

Ryan C. Hunter

Keyword(s):

Cystic Fibrosis ◽

Bacterial Communities ◽

Gene Sequencing ◽

Microbial Colonization ◽

Sinus Surgery ◽

Lower Airway ◽

Rrna Gene ◽

Inflammatory Disorder ◽

Airway Infections ◽

Lower Airways

ABSTRACTRationaleChronic rhinosinusitis (CRS) is an inflammatory disorder of the sinonasal mucosa associated with microbial colonization. Metastasis of sinus microbiota into the lower airways is thought have significant implications in the development of chronic respiratory disease. However, this dynamic has not been thoroughly investigated in cystic fibrosis (CF) patients, where lower airway infections are the primary driver of patient mortality. Given the high prevalence of CRS in CF patients and the proposed infection dynamic between the upper and lower airways, a better understanding of sinus-lung continuum is warranted.ObjectiveTo compare the microbiome of matched sinus mucus and lung sputum samples from CF subjects undergoing functional endoscopic sinus surgery (FESS) for treatment of CRS.MethodsMucus was isolated from the sinuses and lungs of twelve CF patients undergoing FESS. 16S ribosomal RNA gene sequencing was then performed to compare bacterial communities of the CF lung and sinus niches. Finally, functional profiling was performed to predict bacterial metagenomes from the 16S dataset, and was used to compare pathogenic bacterial phenotypes between the upper and lower airways.Measurements and Main ResultsBacterial richness was comparable between airway sites, though sinus and lung environments differed in community evenness, with the sinuses harboring a higher prevalence of dominant microorganisms. Beta diversity metrics also revealed that samples clustered more consistently by airway niche rather than by individual. Finally, predicted metagenomes showed that anaerobic metabolism was enriched in the lung environment, while genes associated with both biofilm formation and Gram identity were not variable between sites.ConclusionsSinus and lung microbiomes are distinct with respect to richness and evenness, while sinus communities have a higher incidence of a dominant taxon. Additionally, ordination analyses point to sinus and lung environments as being stronger determinants of microbial community structure than the individual patient. Finally, BugBase-predicted metagenomes revealed anaerobic phenotypes to be in higher abundance in the lung relative to the sinuses. Our findings indicate that while the paranasal sinuses and lungs may still comprise a unified airway in which lower airways are seeded by sinus microbiota, these discrete airway microenvironments harbor distinct bacterial communities.

Download Full-text

Divergence of bacterial communities in the lower airways of CF patients in early childhood

PLoS ONE ◽

10.1371/journal.pone.0257838 ◽

2021 ◽

Vol 16 (10) ◽

pp. e0257838

Author(s):

John B. O’Connor ◽

Madison M. Mottlowitz ◽

Brandie D. Wagner ◽

Kathleen L. Boyne ◽

Mark J. Stevens ◽

...

Keyword(s):

Cystic Fibrosis ◽

Early Childhood ◽

Bacterial Communities ◽

Quantitative Polymerase Chain Reaction ◽

Bacterial Load ◽

Negative Relationship ◽

Lower Airway ◽

Rrna Gene ◽

Valuable Insight ◽

Neutrophilic Inflammation

Rationale Chronic airway infection and inflammation resulting in progressive, obstructive lung disease is the leading cause of morbidity and mortality in cystic fibrosis. Understanding the lower airway microbiota across the ages can provide valuable insight and potential therapeutic targets. Objectives To characterize and compare the lower airway microbiota in cystic fibrosis and disease control subjects across the pediatric age spectrum. Methods Bronchoalveolar lavage fluid samples from 191 subjects (63 with cystic fibrosis) aged 0 to 21 years were collected along with relevant clinical data. We measured total bacterial load using quantitative polymerase chain reaction and performed 16S rRNA gene sequencing to characterize bacterial communities with species-level sensitivity for select genera. Clinical comparisons were investigated. Measurements and main results Cystic fibrosis samples had higher total bacterial load and lower microbial diversity, with a divergence from disease controls around 2–5 years of age, as well as higher neutrophilic inflammation relative to bacterial burden. Cystic fibrosis samples had increased abundance of traditional cystic fibrosis pathogens and decreased abundance of the Streptococcus mitis species group in older subjects. Interestingly, increased diversity in the heterogeneous disease controls was independent of diagnosis and indication. Sequencing was more sensitive than culture, and antibiotic exposure was more common in disease controls, which showed a negative relationship with load and neutrophilic inflammation. Conclusions Analysis of lower airway samples from people with cystic fibrosis and disease controls across the ages revealed key differences in airway microbiota and inflammation. The divergence in subjects during early childhood may represent a window of opportunity for intervention and additional study.

Download Full-text

Marine cyanolichens from different littoral zones are associated with distinct bacterial communities

PeerJ ◽

10.7717/peerj.5208 ◽

2018 ◽

Vol 6 ◽

pp. e5208 ◽

Cited By ~ 6

Author(s):

Nyree J. West ◽

Delphine Parrot ◽

Claire Fayet ◽

Martin Grube ◽

Sophie Tomasi ◽

...

Keyword(s):

Bacterial Communities ◽

Littoral Zone ◽

Resistant Bacteria ◽

Rrna Gene ◽

Moderately Thermophilic ◽

Culture Independent ◽

Radiation Resistant ◽

Bacteroidetes Phylum ◽

And Function ◽

The Impact

The microbial diversity and function of terrestrial lichens have been well studied, but knowledge about the non-photosynthetic bacteria associated with marine lichens is still scarce. 16S rRNA gene Illumina sequencing was used to assess the culture-independent bacterial diversity in the strictly marine cyanolichen speciesLichina pygmaeaandLichina confinis, and the maritime chlorolichen speciesXanthoria aureolawhich occupy different areas on the littoral zone. Inland terrestrial cyanolichens from Austria were also analysed as for the marine lichens to examine further the impact of habitat/lichen species on the associated bacterial communities. TheL. confinisandL. pygmaeacommunities were significantly different from those of the maritimeXanthoria aureolalichen found higher up on the littoral zone and these latter communities were more similar to those of the inland terrestrial lichens. The strictly marine lichens were dominated by the Bacteroidetes phylum accounting for 50% of the sequences, whereas Alphaproteobacteria, notablySphingomonas, dominated the maritime and the inland terrestrial lichens. Bacterial communities associated with the twoLichinaspecies were significantly different sharing only 33 core OTUs, half of which were affiliated to the Bacteroidetes generaRubricoccus,TunicatimonasandLewinella, suggesting an important role of these species in the marineLichinalichen symbiosis. Marine cyanolichens showed a higher abundance of OTUs likely affiliated to moderately thermophilic and/or radiation resistant bacteria belonging to the Phyla Chloroflexi, Thermi, and the families Rhodothermaceae and Rubrobacteraceae when compared to those of inland terrestrial lichens. This most likely reflects the exposed and highly variable conditions to which they are subjected daily.

Download Full-text

Pulmonary Bacterial Communities in Surgically Resected Noncystic Fibrosis Bronchiectasis Lungs Are Similar to Those in Cystic Fibrosis

Pulmonary Medicine ◽

10.1155/2012/746358 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 11

Author(s):

Heather Maughan ◽

Kristopher S. Cunningham ◽

Pauline W. Wang ◽

Yu Zhang ◽

Marcelo Cypel ◽

...

Keyword(s):

Cystic Fibrosis ◽

Bacterial Communities ◽

Bacterial Infections ◽

Rrna Gene ◽

Community Characteristics ◽

Library Community ◽

Wide Range ◽

Gene Libraries ◽

Culture Independent ◽

Common Genus

Background. Recurrent bacterial infections play a key role in the pathogenesis of bronchiectasis, but conventional microbiologic methods may fail to identify pathogens in many cases. We characterized and compared the pulmonary bacterial communities of cystic fibrosis (CF) and non-CF bronchiectasis patients using a culture-independent molecular approach.Methods. Bacterial 16S rRNA gene libraries were constructed from lung tissue of 10 non-CF bronchiectasis and 21 CF patients, followed by DNA sequencing of isolates from each library. Community characteristics were analyzed and compared between the two groups.Results. A wide range of bacterial diversity was detected in both groups, with between 1 and 21 bacterial taxa found in each patient.Pseudomonaswas the most common genus in both groups, comprising 49% of sequences detected and dominating numerically in 13 patients. AlthoughPseudomonasappeared to be dominant more often in CF patients than in non-CF patients, analysis of entire bacterial communities did not identify significant differences between these two groups.Conclusions. Our data indicate significant diversity in the pulmonary bacterial community of both CF and non-CF bronchiectasis patients and suggest that this community is similar in surgically resected lungs of CF and non-CF bronchiectasis patients.

Download Full-text

Marine cyanolichens from different littoral zones are associated with distinct bacterial communities

10.1101/209320 ◽

2017 ◽

Author(s):

Nyree J. West ◽

Delphine Parrot ◽

Claire Fayet ◽

Martin Grube ◽

Sophie Tomasi ◽

...

Keyword(s):

Bacterial Communities ◽

Littoral Zone ◽

Resistant Bacteria ◽

Rrna Gene ◽

Moderately Thermophilic ◽

Culture Independent ◽

Radiation Resistant ◽

Bacteroidetes Phylum ◽

And Function ◽

The Impact

AbstractThe microbial diversity and function of terrestrial lichens has been well studied, but knowledge about the non-photosynthetic bacteria associated with marine lichens is still scarce. 16S rRNA gene Illumina sequencing was used to assess the culture-independent bacterial diversity in the strictly marine cyanolichen speciesLichina pygmaeaandLichina confinis, and the maritime chlorolichen speciesXanthoria aureolawhich occupy different areas on the littoral zone. Inland terrestrial cyanolichens from Austria were also analysed as for the marine lichens to examine further the impact of habitat/lichen species on the associated bacterial communities. TheL. confinisandL. pygmaeacommunities were significantly different from those of the maritimeXanthoria aureolalichen found higher up on the littoral zone and these latter communities were more similar to those of the inland terrestrial lichens. The strictly marine lichens were dominated by the Bacteroidetes phylum accounting for 50% of the sequences, whereas Alphaproteobacteria, notablySphingomonas, dominated the maritime and the inland terrestrial lichens. Bacterial communities associated with the twoLichinaspecies were significantly different sharing only 33 core OTUs, half of which were affiliated to the Bacteroidetes generaRubricoccus, TunicatimonasandLewinella, suggesting an important role of these species in the marineLichinalichen symbiosis. Marine cyanolichens showed a higher abundance of OTUs likely affiliated to moderately thermophilic and/or radiation resistant bacteria belonging to the Phyla Chloroflexi, Thermi, and the families Rhodothermaceae and Rubrobacteraceae when compared to those of inland terrestrial lichens. This most likely reflects the exposed and highly variable conditions to which they are subjected daily.

Download Full-text

Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4

BMC Genomics ◽

10.1186/s12864-020-07252-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Christine Drengenes ◽

Tomas M. L. Eagan ◽

Ingvild Haaland ◽

Harald G. Wiker ◽

Rune Nielsen

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Bacterial Load ◽

Marker Gene ◽

Gene Region ◽

Lower Airway ◽

Rrna Gene ◽

Wide Range ◽

Airway Microbiome ◽

The Impact

Abstract Background Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls. Results The number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups. Conclusions Differences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

Download Full-text

The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure

Microorganisms ◽

10.3390/microorganisms8010131 ◽

2020 ◽

Vol 8 (1) ◽

pp. 131 ◽

Cited By ~ 6

Author(s):

Leonardo Mancabelli ◽

Christian Milani ◽

Gabriele Andrea Lugli ◽

Federico Fontana ◽

Francesca Turroni ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

In Silico ◽

In Silico Analysis ◽

Amplification Efficiency ◽

Rrna Gene ◽

Test Case ◽

Bacterial Populations ◽

Taxonomic Marker ◽

The Impact

Next Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.

Download Full-text

Culture-Independent Characterization of Bacterial Communities Associated with the Cold-Water Coral Lophelia pertusa in the Northeastern Gulf of Mexico

Applied and Environmental Microbiology ◽

10.1128/aem.02357-08 ◽

2009 ◽

Vol 75 (8) ◽

pp. 2294-2303 ◽

Cited By ~ 65

Author(s):

Christina A. Kellogg ◽

John T. Lisle ◽

Julia P. Galkiewicz

Keyword(s):

Gulf Of Mexico ◽

Shallow Water ◽

Bacterial Communities ◽

Cold Water ◽

Environmental Gradients ◽

Rrna Gene ◽

Lophelia Pertusa ◽

Culture Independent ◽

Northeastern Gulf Of Mexico ◽

Study Sites

ABSTRACT Bacteria are recognized as an important part of the total biology of shallow-water corals. Studies of shallow-water corals suggest that associated bacteria may benefit the corals by cycling carbon, fixing nitrogen, chelating iron, and producing antibiotics that protect the coral from other microbes. Cold-water or deep-sea corals have a fundamentally different ecology due to their adaptation to cold, dark, high-pressure environments and as such have novel microbiota. The goal of this study was to characterize the microbial associates of Lophelia pertusa in the northeastern Gulf of Mexico. This is the first study to collect the coral samples in individual insulated containers and to preserve coral samples at depth in an effort to minimize thermal shock and evaluate the effects of environmental gradients on the microbial diversity of samples. Molecular analysis of bacterial diversity showed a marked difference between the two study sites, Viosca Knoll 906/862 (VK906/862) and Viosca Knoll 826 (VK826). The bacterial communities from VK826 were dominated by a variety of unknown mycoplasmal members of the Tenericutes and Bacteroidetes, whereas the libraries from VK906/862 were dominated by members of the Proteobacteria. In addition to novel sequences, the 16S rRNA gene clone libraries revealed many bacterial sequences in common between Gulf of Mexico Lophelia corals and Norwegian fjord Lophelia corals, as well as shallow-water corals. Two Lophelia-specific bacterial groups were identified: a cluster of gammaproteobacteria related to sulfide-oxidizing gill symbionts of seep clams and a group of Mycoplasma spp. The presence of these groups in both Gulf and Norwegian Lophelia corals indicates that in spite of the geographic heterogeneity observed in Lophelia-associated bacterial communities, there are Lophelia-specific microbes.

Download Full-text

Dichotomy between regulation of coral bacterial communities and calcification physiology under ocean acidification conditions

Applied and Environmental Microbiology ◽

10.1128/aem.02189-20 ◽

2021 ◽

Author(s):

A. Shore ◽

R. D. Day ◽

J. A. Stewart ◽

C.A. Burge

Keyword(s):

Ocean Acidification ◽

Bacterial Communities ◽

16S Rrna Gene Sequencing ◽

Ph Gradient ◽

Rrna Gene ◽

Seawater Ph ◽

Coral Health ◽

And Function ◽

The Impact

Ocean acidification (OA) threatens the growth and function of coral reef ecosystems. A key component to coral health is the microbiome, but little is known about the impact of OA on coral microbiomes. A submarine CO2 vent at Maug Island in the Northern Marianas Islands provides a natural pH gradient to investigate coral responses to long-term OA conditions. Three coral species (Pocillopora eydouxi, Porites lobata, and Porites rus) were sampled from three sites where mean seawater pH is 8.04, 7.98, and 7.94. We characterized coral bacterial communities (using 16S rRNA gene sequencing) and determined pH of the extracellular calcifying fluid (ECF) (using skeletal boron isotopes) across the seawater pH gradient. Bacterial communities of both Porites species stabilized (decreases in community dispersion) with decreased seawater pH, coupled with large increases in the abundance of Endozoicomonas, an endosymbiont. P. lobata experienced a significant decrease in ECF pH near the vent, whereas P. rus experienced a trending decrease in ECF pH near the vent. By contrast, Pocillopora exhibited bacterial community destabilization (increases in community dispersion), with significant decreases in Endozoicomonas abundance, while its ECF pH remained unchanged across the pH gradient. Our study shows that OA has multiple consequences on Endozoicomonas abundance and suggests that Endozoicomonas abundance may be an indicator of coral response to OA. We reveal an interesting dichotomy between two facets of coral physiology (regulation of bacterial communities and regulation of calcification), highlighting the importance of multidisciplinary approaches to understanding coral health and function in a changing ocean. IMPORTANCE Ocean acidification (OA) is a consequence of anthropogenic CO2 emissions that is negatively impacting marine ecosystems such as coral reefs. OA affects many aspects of coral physiology, including growth (i.e. calcification) and disrupting associated bacterial communities. Coral-associated bacteria are important for host health, but it remains unclear how coral-associated bacterial communities will respond to future OA conditions. We document changes in coral-associated bacterial communities and changes to calcification physiology with long-term exposure to decreases in seawater pH that are environmentally relevant under mid-range IPCC emission scenarios (0.1 pH units). We also find species-specific responses that may reflect different responses to long-term OA. In Pocillopora, calcification physiology was highly regulated despite changing seawater conditions. In Porites spp., changes in bacterial communities do not reflect a breakdown of coral-bacterial symbiosis. Insights into calcification and host-microbe interactions are critical to predicting the health and function of different coral taxa to future OA conditions.

Download Full-text

One versus Many: Polymicrobial Communities and the Cystic Fibrosis Airway

mBio ◽

10.1128/mbio.00006-21 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Fabrice Jean-Pierre ◽

Arsh Vyas ◽

Thomas H. Hampton ◽

Michael A. Henson ◽

George A. O’Toole

Keyword(s):

Cystic Fibrosis ◽

Species Interactions ◽

Rrna Gene ◽

Microbial Composition ◽

Community Function ◽

Cystic Fibrosis Airway ◽

Airway Infections ◽

Culture Independent ◽

Polymicrobial Communities

ABSTRACT Culture-independent studies have revealed that chronic lung infections in persons with cystic fibrosis (pwCF) are rarely limited to one microbial species. Interactions among bacterial members of these polymicrobial communities in the airways of pwCF have been reported to modulate clinically relevant phenotypes. Furthermore, it is clear that a single polymicrobial community in the context of CF airway infections cannot explain the diversity of clinical outcomes. While large 16S rRNA gene-based studies have allowed us to gain insight into the microbial composition and predicted functional capacities of communities found in the CF lung, here we argue that in silico approaches can help build clinically relevant in vitro models of polymicrobial communities that can in turn be used to experimentally test and validate computationally generated hypotheses. Furthermore, we posit that combining computational and experimental approaches will enhance our understanding of mechanisms that drive microbial community function and identify new therapeutics to target polymicrobial infections.

Download Full-text