Microbiota analysis optimization for human bronchoalveolar lavage fluid

Schneeberger, Pierre H. H.; Prescod, Janice; Levy, Liran; Hwang, David; Martinu, T.; Coburn, Bryan

doi:10.1186/s40168-019-0755-x

Cited by 31 publications

(35 citation statements)

References 36 publications

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“…Absolute quantification of the bacterial DNA present in our extracts suggested that the contrasting OTU profiles were likely due to differences in the recovery of bacterial DNA associated with each extraction method. In light of these results we agree with recent global recommendation that, when possible, bacterial DNA quantification should be performed to discern true biological variation from environmental noise ( Schneeberger et al, 2019 ).…”

Section: Discussionsupporting

confidence: 91%

“…Several methods have been described in the literature to isolate bacterial DNA from BALF ( Supplementary Table 1 ; Charlson et al, 2011 ; Erb-Downward et al, 2011 ; Pragman et al, 2012 ; Willner et al, 2012 ; Borewicz et al, 2013 ; Han et al, 2014 ; Renwick et al, 2014 ; May et al, 2015 ; Bernasconi et al, 2016 ; Dickson et al, 2016a , b ; Laguna et al, 2016 ; Marsh et al, 2016 ; Wen et al, 2016 ; Frayman et al, 2017 ; Zemanick et al, 2017 ; Ahmed et al, 2018 ; Kloepfer et al, 2018 ; Pattaroni et al, 2018 ; Esther et al, 2019 ; Gomes et al, 2019 ; Jorth et al, 2019 ; Kyo et al, 2019 ; Schneeberger et al, 2019 ; Tong et al, 2019 ; Wang et al, 2019 ). In some instances, successful recovery of DNA has been associated with the utilization of large volumes of starting material.…”

Section: Resultsmentioning

confidence: 99%

“…Previous studies using BALF samples have quantified bacterial load through qPCR ( Erb-Downward et al, 2011 ; May et al, 2015 ; Bernasconi et al, 2016 ; Laguna et al, 2016 ; Marsh et al, 2016 ; Zemanick et al, 2017 ; Pattaroni et al, 2018 ; Esther et al, 2019 ; Jorth et al, 2019 ; Schneeberger et al, 2019 ). In these reports, authors targeted DNA fragments of variable length ranging from 180 to 590 bp ( Erb-Downward et al, 2011 ; May et al, 2015 ; Bernasconi et al, 2016 ; Laguna et al, 2016 ; Marsh et al, 2016 ; Zemanick et al, 2017 ; Pattaroni et al, 2018 ; Esther et al, 2019 ; Jorth et al, 2019 ; Schneeberger et al, 2019 ). Except for those primers used by Bernasconi et al (2016) (180 bp), this amplicon length range is far from that recommended for qPCR (50–150 bp), because longer products show suboptimal amplification efficiencies ( Debode et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

“…Different DNA extraction techniques have been used to profile microbial communities from BALF samples; commercial silica column-based kits ( Charlson et al, 2011 ; Willner et al, 2012 ; Han et al, 2014 ; Renwick et al, 2014 ; May et al, 2015 ; Dickson et al, 2016b ; Laguna et al, 2016 ; Marsh et al, 2016 ; Wen et al, 2016 ; Zemanick et al, 2017 ; Ahmed et al, 2018 ; Pattaroni et al, 2018 ; Kyo et al, 2019 ; Schneeberger et al, 2019 ; Tong et al, 2019 ; Wang et al, 2019 ), magnetic beads-based procedures ( Erb-Downward et al, 2011 ; Pragman et al, 2012 ; Bernasconi et al, 2016 ; Frayman et al, 2017 ; Kloepfer et al, 2018 ; Esther et al, 2019 ; Gomes et al, 2019 ), homemade protocols ( Borewicz et al, 2013 ; Jorth et al, 2019 ), salt-extraction approaches or the use of cationic detergents in combination with the classical phenol-chloroform extraction technique ( Willner et al, 2012 ). However, not many studies have compared the efficiency of these protocols, particularly using samples from young children ( Willner et al, 2012 ).…”

Section: Introductionmentioning

confidence: 99%

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Microbiomic Analysis on Low Abundant Respiratory Biomass Samples; Improved Recovery of Microbial DNA From Bronchoalveolar Lavage Fluid

et al. 2020

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In recent years the study of the commensal microbiota is driving a remarkable paradigm shift in our understanding of human physiology. However, intrinsic technical difficulties associated with investigating the Microbiomics of some body niches are hampering the development of new knowledge. This is particularly the case when investigating the functional role played by the human microbiota in modulating the physiology of key organ systems. A major hurdle in investigating specific Microbiome communities is linked to low bacterial density and susceptibility to bias caused by environmental contamination. To prevent such inaccuracies due to background processing noise, harmonized tools for Microbiomic and bioinformatics practices have been recommended globally. The fact that the impact of this undesirable variability is negatively correlated with the DNA concentration in the sample highlights the necessity to improve existing DNA isolation protocols. In this report, we developed and tested a protocol to more efficiently recover bacterial DNA from low volumes of bronchoalveolar lavage fluid obtained from infants and adults. We have compared the efficiency of the described method with that of a commercially available kit for microbiome analysis in body fluids. We show that this new methodological approach performs better in terms of extraction efficiency. As opposed to commercial kits, the DNA extracts obtained with this new protocol were clearly distinguishable from the negative extraction controls in terms of 16S copy number and Microbiome community profiles. Altogether, we described a cost-efficient protocol that can facilitate microbiome research in low-biomass human niches.

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Section: Discussionsupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Microbiomic Analysis on Low Abundant Respiratory Biomass Samples; Improved Recovery of Microbial DNA From Bronchoalveolar Lavage Fluid

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Despite a low microbial diversity, absolute quantification of the bacteria present in the BAL fluid samples analyzed revealed a very high bacterial biomass during infection (up to 10 11 bacteria per ml of BAL fluid) due to the high abundance of the pathogen. Thus, although the possibility of contamination derived from bronchoscopy and the saline solution cannot be excluded in this study, such contamination, if present, would significantly affect the results only for BAL fluid samples with lower bacterial loads (less than 8Eϩ04 16S rRNA copies/ml), which was not the case in our study (33).…”

Section: Discussionmentioning

confidence: 65%

Persistent Legionnaires’ Disease and Associated Antibiotic Treatment Engender a Highly Disturbed Pulmonary Microbiome Enriched in Opportunistic Microorganisms

Pérez‐Cobas

Ginevra

Rusniok

et al. 2020

mBio

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Despite the importance of pneumonia to public health, little is known about the composition of the lung microbiome during infectious diseases, such as pneumonia, and how it evolves during antibiotic therapy. To study the possible relation of the pulmonary microbiome to the severity and outcome of this respiratory disease, we analyzed the dynamics of the pathogen and the human lung microbiome during persistent infections caused by the bacterium Legionella pneumophila and their evolution during antimicrobial treatment. We collected 10 bronchoalveolar lavage fluid samples from three patients during long-term hospitalization due to pneumonia and performed a unique longitudinal study of the interkingdom microbiome, analyzing the samples for presence of bacteria, archaea, fungi, and protozoa by high-throughput Illumina sequencing of marker genes. The lung microbiome of the patients was characterized by a strong predominance of the pathogen, a low diversity of the bacterial fraction, and an increased presence of opportunistic microorganisms. The fungal fraction was more stable than the bacterial fraction. During long-term treatment, no genomic changes or antibiotic resistance-associated mutations that could explain the persistent infection occurred, according to whole-genome sequencing analyses of the pathogen. After antibiotic treatment, the microbiome did not recover rapidly but was mainly constituted of antibiotic-resistant species and enriched in bacteria, archaea, fungi, or protozoa associated with pathogenicity. The lung microbiome seems to contribute to nonresolving Legionella pneumonia, as it is strongly disturbed during infection and enriched in opportunistic and/or antibiotic-resistant bacteria and microorganisms, including fungi, archaea, and protozoa that are often associated with infections. IMPORTANCE The composition and dynamics of the lung microbiome during pneumonia are not known, although the lung microbiome might influence the severity and outcome of this infectious disease, similar to what was shown for the microbiome at other body sites. Here we report the findings of a comprehensive analysis of the lung microbiome composition of three patients with long-term pneumonia due to L. pneumophila and its evolution during antibiotic treatment. This work adds to our understanding of how the microbiome changes during disease and antibiotic treatment and points to microorganisms and their interactions that might be beneficial. In addition to bacteria and fungi, our analyses included archaea and eukaryotes (protozoa), showing that both are present in the pulmonary microbiota and that they might also play a role in the response to the microbiome disturbance.

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Differential Oral Microbial Input Determines Two Microbiota Pneumo‐Types Associated with Health Status

Zhang

Liu

et al. 2022

Advanced Science

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The oral and upper respiratory tracts are closely linked anatomically and physiologically with the lower respiratory tract and lungs, and the influence of oral and upper respiratory microbes on the lung microbiota is increasingly being recognized. However, the ecological process and individual heterogeneity of the oral and upper respiratory tract microbes shaping the lung microbiota remain unclear owing to the lack of controlled analyses with sufficient sample sizes. Here, the microbiomes of saliva, nasal cavity, oropharyngeal area, and bronchoalveolar lavage samples are profiled and the shaping process of multisource microbes on the lung microbiota is measured. It is found that oral and nasal microbial inputs jointly shape the lung microbiota by occupying different ecological niches. It is also observed that the spread of oral microbes to the lungs is heterogeneous, with more oral microbes entering the lungs being associated with decreased lung function and increased lung proinflammatory cytokines. These results depict the external shaping process of lung microbiota and indicate the great value of oral samples, such as saliva, in monitoring and assessing lung microbiota status in clinical settings.

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Microbiota analysis optimization for human bronchoalveolar lavage fluid

Cited by 31 publications

References 36 publications

Microbiomic Analysis on Low Abundant Respiratory Biomass Samples; Improved Recovery of Microbial DNA From Bronchoalveolar Lavage Fluid

Microbiomic Analysis on Low Abundant Respiratory Biomass Samples; Improved Recovery of Microbial DNA From Bronchoalveolar Lavage Fluid

Persistent Legionnaires’ Disease and Associated Antibiotic Treatment Engender a Highly Disturbed Pulmonary Microbiome Enriched in Opportunistic Microorganisms

Differential Oral Microbial Input Determines Two Microbiota Pneumo‐Types Associated with Health Status

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