scholarly journals Repeated bronchoscopy in health and obstructive lung disease: is the airway microbiome stable?

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Rune Nielsen ◽  
Yaxin Xue ◽  
Inge Jonassen ◽  
Ingvild Haaland ◽  
Øyvind Kommedal ◽  
...  
Keyword(s):  
Beta Diversity ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Lower Airway ◽  
Trend Test ◽  
Rrna Gene ◽  
Core Microbiome ◽  
Airway Microbiome ◽  
The Stability ◽  
Paired End Sequencing

Abstract Objective Little is known concerning the stability of the lower airway microbiome. We have compared the microbiota identified by repeated bronchoscopy in healthy subjects and patients with ostructive lung diseaseases (OLD). Methods 21 healthy controls and 41 patients with OLD completed two bronchoscopies. In addition to negative controls (NCS) and oral wash (OW) samples, we gathered protected bronchoalveolar lavage in two fractions (PBAL1 and PBAL2) and protected specimen brushes (PSB). After DNA extraction, we amplified the V3V4 region of the 16S rRNA gene, and performed paired-end sequencing (Illumina MiSeq). Initial bioinformatic processing was carried out in the QIIME-2 pipeline, identifying amplicon sequence variants (ASVs) with the DADA2 algorithm. Potentially contaminating ASVs were identified and removed using the decontam package in R and the sequenced NCS. Results A final table of 551 ASVs consisted of 19 × 106 sequences. Alpha diversity was lower in the second exam for OW samples, and borderline lower for PBAL1, with larger differences in subjects not having received intercurrent antibiotics. Permutational tests of beta diversity indicated that within-individual changes were significantly lower than between-individual changes. A non-parametric trend test showed that differences in composition between the two exams (beta diversity) were largest in the PSBs, and that these differences followed a pattern of PSB > PBAL2 > PBAL1 > OW. Time between procedures was not associated with increased diversity. Conclusion The airways microbiota varied between examinations. However, there is compositional microbiota stability within a person, beyond that of chance, supporting the notion of a transient airways microbiota with a possibly more stable individual core microbiome.

scholarly journals Effect of Bovine MFGM and LF in Infant Formula on Gut Microbiome and Metabolome at Four Months of Age

2021 ◽  
Author(s):  
Maciej Chichlowski ◽  
Nicholas Bokulich ◽  
Cheryl L Harris ◽  
Jennifer L Wampler ◽  
Fei Li ◽  
...  
Keyword(s):  
Infant Formula ◽  
Beta Diversity ◽  
Milk Fat ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Term Infants ◽  
Linear Discriminant ◽  
Alpha And Beta Diversity ◽  
Metabolite Profiles

Abstract Background Milk fat globule membrane (MFGM) and lactoferrin (LF) are human milk bioactive components demonstrated to support gastrointestinal (GI) and immune development. Significantly fewer diarrhea and respiratory-associated adverse events through 18 months of age were previously reported in healthy term infants fed a cow's milk-based infant formula with added source of bovine MFGM and bovine LF through 12 months of age. Objectives To compare microbiota and metabolite profiles in a subset of study participants. Methods Stool samples were collected at Baseline (10–14 days of age) and Day 120 (MFGM + LF: 26, Control: 33). Bacterial community profiling was performed via16S rRNA gene sequencing (Illumina MiSeq) and alpha and beta diversity were analyzed (QIIME 2). Differentially abundant taxa were determined using Linear discriminant analysis effect size (LefSE) and visualized (Metacoder). Untargeted stool metabolites were analyzed (HPLC/mass spectroscopy) and expressed as the fold-change between group means (Control: MFGM + LF ratio). Results Alpha diversity increased significantly in both groups from baseline to 4 months. Subtle group differences in beta diversity were demonstrated at 4 months (Jaccard distance; R2 = 0.01, P = 0.042). Specifically, Bacteroides uniformis and Bacteroides plebeius were more abundant in the MFGM + LF group at 4 months. Metabolite profile differences for MFGM + LF vs Control included: lower fecal medium chain fatty acids, deoxycarnitine, and glycochenodeoxycholate, and some higher fecal carbohydrates and steroids (P < 0.05). After applying multiple test correction, the differences in stool metabolomics were not significant. Conclusions Addition of bovine MFGM and LF in infant formula was associated with subtle differences in stool microbiome and metabolome by four months of age, including increased prevalence of Bacteroides species. Stool metabolite profiles may be consistent with altered microbial metabolism. Trial registration:  https://clinicaltrials.gov/ct2/show/NCT02274883).

scholarly journals Resident microbes of lactation rooms and daycares

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e8168
Author(s):  
Diana H. Taft ◽  
Samir Akre ◽  
Nicolas Madrid ◽  
Andre Knoesen ◽  
David A. Mills ◽  
...  
Keyword(s):  
Beta Diversity ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Shannon Index ◽  
Rrna Gene ◽  
Sample Collection ◽  
Unifrac Distance ◽  
Modern Development ◽  
Temperature And Humidity ◽  
The Impact

Dedicated lactation rooms are a modern development as mothers return to work while still providing breastmilk to their absent infants. This study describes the built environment microbiome of lactation rooms and daycares, and explores the influence of temperature and humidity on the microbiome of lactation rooms. Sterile swabs were used to collect samples from five different sites in lactation rooms at University of California, Davis and from five different sites in daycares located in Davis, California. DNA from the swabs was extracted and the V4 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Temperature and relative humidity data were collected on a subset of the lactation rooms. Sampled lactation rooms could be either dedicated lactation rooms or could also serve other functions (e.g., combined lactation room and restroom lounge). The majority of sequence reads were identified as belonging to family Moraxellaceae, with 73% of all reads included in analysis identified as an unknown species of Acinetobacter. Alpha diversity was analyzed using the Shannon index, while beta diversity was analyzed using unweighted and weighted UniFrac distance. The Jaccard distance was used to measure amount of change at sampling locations between time points for analysis of the impact of temperature and humidity on the microbiome. There were significant differences in the beta diversity of the microbiome of lactation rooms by room type. There were also significant differences in the beta diversity of the microbiome by sample collection location. There were no significant differences in either alpha or beta diversity associated with room temperature or humidity. Additional studies are needed to understand if the differences in lactation room type may result in differences in the breastmilk microbiome of milk collected in those rooms, and to what extent any such differences may influence the infant microbiome.

scholarly journals The Lung Microbiome in Young Children with Cystic Fibrosis: A Prospective Cohort Study

2021 ◽  
Vol 9 (3) ◽  
pp. 492
Author(s):  
Barry Linnane ◽  
Aaron M. Walsh ◽  
Calum J. Walsh ◽  
Fiona Crispie ◽  
Orla O’Sullivan ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Demographic Data ◽  
Alpha Diversity ◽  
16S Rrna Gene Sequencing ◽  
Illumina Miseq ◽  
Lower Airway ◽  
Rrna Gene ◽  
Lung Microbiome ◽  
Microbiome Diversity ◽  
R Project

The cystic fibrosis (CF) lung harbours a diverse microbiome and reduced diversity in the CF lung has been associated with advancing age, increased inflammation and poorer lung function. Data suggest that the window for intervention is early in CF, yet there is a paucity of studies on the lung microbiome in children with CF. The objective of this study was to thoroughly characterise the lower airway microbiome in pre-school children with CF. Bronchoalveolar lavage (BAL) samples were collected annually from children attending the three clinical centres. Clinical and demographic data were collated on all subjects alongside BAL inflammatory markers. 16S rRNA gene sequencing was performed on the Illumina MiSeq platform. Bioinformatics and data analysis were performed using Qiime and R project software. Data on 292 sequenced BALs from 101 children with CF and 51 without CF show the CF lung microbiome, while broadly similar to that in non-CF children, is distinct. Alpha diversity between the two cohorts was indistinguishable at this early age. The CF diagnosis explained only 1.1% of the variation between the cohort microbiomes. However, several key genera were significantly differentially abundant between the groups. While the non-CF lung microbiome diversity increased with age, diversity reduced in CF with age. Pseudomonas and Staphylococcus were more abundant with age, while genera such as Streptococcus, Porphyromonas and Veillonella were less abundant with age. There was a negative correlation between alpha diversity and interleukin-8 and neutrophil elastase in the CF population. Neither current flucloxacillin or azithromycin prophylaxis, nor previous oral or IV antibiotic exposure, was correlated with microbiome diversity. Consecutive annual BAL samples over 5 years from a subgroup of children demonstrated diverse patterns of development in the first years of life.

scholarly journals Study on the Diversity of Epiphytic Bacteria on Corn and Alfalfa using Illumina MiSeq/NovaSeq High-throughput Sequencing System

2021 ◽  
Author(s):  
Mei-Xiao Wu ◽  
hui Tang
Keyword(s):  
High Throughput ◽  
Beta Diversity ◽  
High Throughput Sequencing ◽  
Raw Materials ◽  
Diversity Index ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Diversity Analysis ◽  
Epiphytic Bacteria ◽  
Paired End Sequencing

Abstract PurposeTo investigate the diversity of the epiphytic bacteria on corn and alfalfa collected in Hengshui city and Xingtai city, hebei province, China.MethodsIllumina MiSeq/NovaSeq High-throughput sequencing systerm was used to conduct Paired-end sequencing of community DNA fragments from surface of corn and alfalfa in Hengshui and Xingtai. QIIME2 and R language were employed to sort and calculate the number of sequences and taxonomic units for each sample. Thereafter, the abundance, distribution, alpha diversity index of species, beta diversity and the differences of abundance among the samples were analyzed. ResultAt phylum level, the advantage bacterium group are Proteobacteria (70%), Firmicutes (13%), Actinobacteria (9%) and Bacteroidetes (7%). The dominant genera are Pseudomonas (8%), Acinetobacter (4%), Chryseobacterium (3%), Hymenobacter (1%). Enterobacteriaceae (24%) are the most predominant bacteria on both corn and alfalfa samples. Alpha diversity analysis and beta diversity analysis showed that the diversity of epiphytic microbial community was significantly affected by plant species, but not by region. The diversity and richness of epiphytic bacterial community of alfalfa were significantly higher than that of corn, yet corn had more LAB than alfalfa samples. ConclusionThis study contributes to expanding our understanding of the diversity of epiphytic microorganisms in corn and alfalfa silage and provide a basis for the selection of raw materials.

scholarly journals Study on the diversity of epiphytic bacteria on corn and alfalfa using Illumina MiSeq/NovaSeq high-throughput sequencing system

2021 ◽  
Vol 71 (1) ◽  
Author(s):  
Meixiao Wu ◽  
Yuehua Wang ◽  
Yijing Wang ◽  
Xuefei Wang ◽  
Ming Yu ◽  
...  
Keyword(s):  
High Throughput ◽  
Beta Diversity ◽  
High Throughput Sequencing ◽  
Raw Materials ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Diversity Indices ◽  
Epiphytic Bacteria ◽  
Alpha And Beta Diversity ◽  
Paired End Sequencing

Abstract Purpose To investigate the diversity of the epiphytic bacteria on corn (Zea mays) and alfalfa (Medicago sativa) collected in Hengshui City and Xingtai City, Hebei Province, China, and explore crops suitable for natural silage. Methods The Illumina MiSeq/NovaSeq high-throughput sequencing system was used to conduct paired-end sequencing of the community DNA fragments from the surface of corn and alfalfa collected in Hengshui and Xingtai. QIIME2 and R software were used to sort and calculate the number of sequences and taxonomic units for each sample. Thereafter, the alpha and beta diversity indices at of species level were calculated, and the abundance and distribution of taxa were analyzed and compared between samples. Result At phylum level, the dominant groups were Proteobacteria (70%), Firmicutes (13%), Actinobacteria (9%), and Bacteroidetes (7%). Meanwhile, the dominant genera were Pseudomonas (8%), Acinetobacter (4%), Chryseobacterium (3%), and Hymenobacter (1%). Enterobacteriaceae (24%) were the most predominant bacteria in both the corn and alfalfa samples. Alpha diversity analysis and beta diversity indices revealed that the diversity of epiphytic microbial communities was significantly affected by plant species but not by region. The diversity and richness of the epiphytic bacterial community of alfalfa were significantly higher than those of corn. Conclusion This study contributes to the expanding knowledge on the diversity of epiphytic bacteria in corn and alfalfa silage and provides a basis for the selection of raw materials.

scholarly journals The Fecal Bacterial Microbiota in Horses with Equine Recurrent Uveitis

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 745
Author(s):  
Michelle Martin de Bustamante ◽  
Diego Gomez ◽  
Jennifer MacNicol ◽  
Ralph Hamor ◽  
Caryn Plummer
Keyword(s):  
Beta Diversity ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Diversity Indices ◽  
Rrna Gene ◽  
Linear Discriminant ◽  
Alpha And Beta Diversity ◽  
Bacterial Microbiota ◽  
Healthy Control ◽  
Equine Recurrent Uveitis

The objective of this study was to describe and compare the fecal bacterial microbiota of horses with equine recurrent uveitis (ERU) and healthy horses using next-generation sequencing techniques. Fecal samples were collected from 15 client-owned horses previously diagnosed with ERU on complete ophthalmic examination. For each fecal sample obtained from a horse with ERU, a sample was collected from an environmentally matched healthy control with no evidence of ocular disease. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene. The relative abundance of predominant taxa, and alpha and beta diversity indices were calculated and compared between groups. The phyla Firmicutes, Bacteroidetes, Verrucomicrobia, and Proteobacteria predominated in both ERU and control horses, accounting for greater than 60% of sequences. Based on linear discriminant analysis effect size (LEfSe), no taxa were found to be enriched in either group. No significant differences were observed in alpha and beta diversity indices between groups (p > 0.05 for all tests). Equine recurrent uveitis is not associated with alteration of the gastrointestinal bacterial microbiota when compared with healthy controls.

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

BMC Genomics ◽  
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.

scholarly journals AB1232 ORAL DYSBIOSIS REFLECTS THE IMMUNOLOGICAL ALTERATION OF RA REGARDING TO ACPA AND HLA DRB1*SE: NAGASAKI ISLAND STUDY

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1907.2-1907
Author(s):  
Y. Tsuji ◽  
M. Tamai ◽  
S. Morimoto ◽  
D. Sasaki ◽  
M. Nagayoshi ◽  
...  
Keyword(s):  
Beta Diversity ◽  
Healthy Subjects ◽  
Microbial Community Composition ◽  
Alpha Diversity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rrna Gene ◽  
Oral Microbiota ◽  
Unifrac Distance ◽  
Alpha And Beta Diversity ◽  
The Difference

Background:Anti-citrullinated protein antibody (ACPA) production is observed in several organs even prior to the onset of rheumatoid arthritis (RA), and oral mucosa is considered to be one of the important tissues. The presence of HLA-DRB1*SE closely associates with ACPA production. Saliva is considered to reflect the oral microbiota including periodontal disease. Alteration of oral microbiota of RA becomes to be normalized by DMARDs treatment, however, the interaction of HLA-DRB1*SE, ACPA and oral microbiota of RA patients remains to be elucidated.Objectives:The Nagasaki Island Study, which had started in 2014 collaborating with Goto City, is intended for research of the preclinical stage of RA, including ACPA/HLA genotype screening and ultrasound and magnetic resonance imaging examinations in high-risk subjects. Using the samples accumulated in this cohort, we have tried to investigate the difference of oral microbiota among RA patients and healthy subjects regarding to ACPA and HLA-DRB1*SE.Methods:Blood and salivary samples were obtained from 1422 subjects out of 4276 who have participated in the Nagasaki Island Study from 2016 to 2018. ACPA positivity was 1.7 % in total. Some of RA patients resided in Goto City participated in the Nagasaki Island Study. At this point, we selected 291 subjects, who were ACPA positive non-RA healthy subjects (n=22) and patients with RA (n=33, 11 subjects were ACPA positive and 22 ACPA negative respectively) as the case, age and gender matched ACPA negative non-RA healthy subjects (n=236) as the control. ACPA was measured by an enzyme-linked immunosorbent assay, and HLA genotyping was quantified by next-generation sequencing (Ref.1). The operational taxonomic unit (OUT) analysis using 16S rRNA gene sequencing were performed. The richness of microbial diversity within-subject (alpha diversity) was scaled via Shannon entropy. The dissimilarity between microbial community composition was calculated using Bray-Curtis distance as a scale, and differences between groups (beta diversity) were tested by permutational multivariate analysis of variance (PERMANOVA). In addition, UniFrac distance calculated in consideration of the distance on the phylogenetic tree were performed.Results:Median age 70 y.o., % Female 58.8 %. Among RA and non-RA subjects, not alpha diversity but beta diversity was statistically significance (p=0.022, small in RA). In RA subjects, both alpha and beta diversity is small (p<0.0001), especially significant in ACPA positive RA (Figure 1). Amongt RA subjects, presence of HLA-DRB1*SE did not show the difference but the tendency of being small of alpha diversity (p=0.29).Conclusion:Our study has suggested for the first time the association of oral microbiota alteration with the presence of ACPA and HLA-DRB1*SE. Oral dysbiosis may reflect the immunological status of patients with RA.References:[1]Kawaguchi S, et al. Methods Mol Biol 2018;1802: 22Disclosure of Interests:None declared

scholarly journals Association of Broiler Litter Microbiome Composition and Campylobacter Isolation

2021 ◽  
Vol 8 ◽  
Author(s):  
Robert Valeris-Chacin ◽  
Maria Pieters ◽  
Haejin Hwang ◽  
Timothy J. Johnson ◽  
Randall S. Singer
Keyword(s):  
Beta Diversity ◽  
Conditional Logistic Regression ◽  
Statistical Significance ◽  
Alpha Diversity ◽  
Rrna Gene ◽  
Campylobacter Coli ◽  
Broiler Litter ◽  
Logistic Regression Models ◽  
Microbiome Composition ◽  
Broiler House

Infection with Campylobacter species is one of the leading causes of bacterial diarrhea in humans in the US. Chickens, which become colonized on the farm, are important reservoirs of this bacterium. Campylobacter can establish itself in the broiler house via a variety of sources, can survive in the litter of the house, and possibly persist over successive flock cycles. However, the role of the broiler litter microbiome on Campylobacter persistence is not clear. A matched case-control study was conducted to determine whether the broiler litter microbiome composition was associated with Campylobacter isolation within the broiler house. Flocks were classified as cases when either Campylobacter jejuni or Campylobacter coli was isolated in boot sock samples, or as controls otherwise. Case and control flocks were matched at the broiler house level. Composite broiler litter samples were collected and used for DNA extraction and 16S rRNA gene V4 region sequencing. Reads were processed using the DADA2 pipeline to obtain a table of amplicon sequence variants. Alpha diversity and differential bacterial relative abundance were used as predictors of Campylobacter isolation status in conditional logistic regression models adjusting for flock age and sampling season. Beta diversity distances were used as regressors in stratified PERMANOVA with Campylobacter isolation status as predictor, and broiler house as stratum. When Campylobacter was isolated in boot socks, broiler litter microbiome richness and evenness were lower and higher, respectively, without reaching statistical significance. Campylobacter isolation status significantly explained a small proportion of the beta diversity (genus-level Aitchison dissimilarity distance). Clostridium and Anaerostipes were positively associated with Campylobacter isolation status, whereas Bifidobacterium, Anaerosporobacter, and Stenotrophomonas were negatively associated. Our results suggest the presence of bacterial interactions between Campylobacter and the broiler litter microbiome. The negative association of Campylobacter with Bifidobacterium, Anaerosporobacter, and Stenotrophomonas in litter could be potentially exploited as a pre-harvest control strategy.

scholarly journals Impact of Early Life Antibiotic Exposure and Neonatal Hyperoxia on the Murine Microbiome and Lung Injury

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Melissa H. Althouse ◽  
Christopher Stewart ◽  
Weiwu Jiang ◽  
Bhagavatula Moorthy ◽  
Krithika Lingappan
Keyword(s):  
Lung Injury ◽  
Beta Diversity ◽  
Alpha Diversity ◽  
Intestinal Microbiome ◽  
Rrna Gene ◽  
Antibiotic Exposure ◽  
Neonatal Mice ◽  
Intestinal Dysbiosis ◽  
Neonatal Hyperoxia

Abstract Cross talk between the intestinal microbiome and the lung and its role in lung health remains unknown. Perinatal exposure to antibiotics disrupts the neonatal microbiome and may have an impact on the preterm lung. We hypothesized that perinatal antibiotic exposure leads to long-term intestinal dysbiosis and increased alveolar simplification in a murine hyperoxia model. Pregnant C57BL/6 wild type dams and neonatal mice were treated with antibiotics before and/or immediately after delivery. Control mice received phosphate-buffered saline (PBS). Neonatal mice were exposed to 95% oxygen for 4 days or room air. Microbiome analysis was performed using 16S rRNA gene sequencing. Pulmonary alveolarization and vascularization were analyzed at postnatal day (PND) 21. Perinatal antibiotic exposure modified intestinal beta diversity but not alpha diversity in neonatal mice. Neonatal hyperoxia exposure altered intestinal beta diversity and relative abundance of commensal bacteria in antibiotic treated mice. Hyperoxia disrupted pulmonary alveolarization and vascularization at PND 21; however, there were no differences in the degree of lung injury in antibiotic treated mice compared to vehicle treated controls. Our study suggests that exposure to both hyperoxia and antibiotics early in life may cause long-term alterations in the intestinal microbiome, but intestinal dysbiosis may not significantly influence neonatal hyperoxic lung injury.

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