Utility of Conventional Culture and MALDI-TOF MS for Identification of Microbial Communities in Bronchoalveolar Lavage Fluid in Comparison with the GS Junior Next Generation Sequencing System
1
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
Background: Diverse microbiota exist in the lower respiratory tract. Although next generation sequencing (NGS) is the most widely used microbiome analysis technique, it is difficult to implement NGS in clinical microbiology laboratories. Therefore, we evaluated the performance of conventional culture methods together with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying microbiota in bronchoalveolar lavage (BAL) fluid. Methods: BAL fluid samples (n=27) were obtained from patients undergoing diagnostic bronchoscopy for lung mass evaluation. Bacterial and fungal culture was performed with conventional media used in clinical microbiology laboratories. On an average, 20 isolated colonies were picked from each agar plate and identified by MALDI-TOF MS. Microbiome analysis using 16S rRNA NGS was conducted for comparison. Results: Streptococcus spp. and Neisseria spp. were most frequently cultured from the BAL fluid samples. In two samples, Enterobacteriaceae grew predominantly on MacConkey agar. Actinomyces and Veillonella spp. were commonly identified anaerobes; gut bacteria, such as Lactobacillus, Bifidobacterium, and Clostridium, and fungi were also isolated. NGS revealed more diverse bacterial communities than culture, and Prevotella spp. were mainly identified solely by NGS. Some bacteria, such as Staphylococcus spp., Clostridium spp., and Bifidobacterium spp., were identified solely by culture, indicating that culture may be more sensitive for detecting certain bacteria. Conclusions: Culture and NGS of BAL fluid samples revealed common bacteria with some different microbial communities. Despite some limitations, culture combined with MALDI-TOF MS might play a complementary role in microbiome analysis using 16S rRNA NGS.
Abstract
Background: Diverse microbiota exist in the lower respiratory tract. Although next generation sequencing (NGS) is the most widely used microbiome analysis technique, it is difficult to implement NGS in clinical microbiology laboratories. Therefore, we evaluated the performance of conventional culture methods together with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying microbiota in bronchoalveolar lavage (BAL) fluid. Methods: BAL fluid samples (n=27) were obtained from patients undergoing diagnostic bronchoscopy for lung mass evaluation. Bacterial and fungal culture was performed with conventional media used in clinical microbiology laboratories. On an average, 20 isolated colonies were picked from each agar plate and identified by MALDI-TOF MS. Microbiome analysis using 16S rRNA NGS was conducted for comparison. Results: Streptococcus spp. and Neisseria spp. were most frequently cultured from the BAL fluid samples. In two samples, Enterobacteriaceae grew predominantly on MacConkey agar. Actinomyces and Veillonella spp. were commonly identified anaerobes; gut bacteria, such as Lactobacillus, Bifidobacterium, and Clostridium, and fungi were also isolated. NGS revealed more diverse bacterial communities than culture, and Prevotella spp. were mainly identified solely by NGS. Some bacteria, such as Staphylococcus spp., Clostridium spp., and Bifidobacterium spp., were identified solely by culture, indicating that culture may be more sensitive for detecting certain bacteria. Conclusions: Culture and NGS of BAL fluid samples revealed common bacteria with some different microbial communities. Despite some limitations, culture combined with MALDI-TOF MS might play a complementary role in microbiome analysis using 16S rRNA NGS.
참고문헌 (34)
- Twigg HL 3rd, "Use of bronchoalveolar lavage to assess the respiratory microbiome: signal in the noise", Lancet Respir Med, 1, pp.354~356 (2013)[DOI]
- Charlson ES, "Topographical continuity of bacterial populations in the healthy human respiratory tract", Am J Respir Crit Care Med, 184, pp.957~963 (2011)[DOI]
- Erb-Downward JR, "Analysis of the lung microbiome in the healthy smoker and in COPD", PLoS One, 6, pp.e16384~ (2011)[DOI]
- Sze MA, "The lung tissue microbiome in chronic obstructive pulmonary disease", Am J Respir Crit Care Med, 185, pp.1073~1080 (2012)[DOI]
- Morris A, "Comparison of the respiratory microbiome in healthy nonsmokers and smokers", Am J Respir Crit Care Med, 187, pp.1067~1075 (2013)[DOI]
- Hamady M, "Microbial community profiling for human microbiome projects: tools, techniques, and challenges", Genome Res, 19, pp.1141~1152 (2009)[DOI]
- Allen-Vercoe E, "Bringing the gut microbiota into focus through microbial culture: recent progress and future perspective", Curr Opin Microbiol, 16, pp.625~629 (2013)[DOI]
- Sommer MO, "Advancing gut microbiome research using cultivation", Curr Opin Microbiol, 27, pp.127~132 (2015)[DOI]
- Goodman AL, "Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice", Proc Natl Acad Sci U S A, 108, pp.6252~6257 (2011)[DOI]
- Lagier JC, "Microbial culturomics: paradigm shift in the human gut microbiome study", Clin Microbiol Infect, 18, pp.1185~1193 (2012)[DOI]
- Rettedal EA, "Cultivation-based multiplex phenotyping of human gut microbiota allows targeted recovery of previously uncultured bacteria", Nat Commun, 5, pp.4714~ (2014)[DOI]
- Brady C, "Phylogeny and identification of Pantoea species associated with plants, humans and the natural environment based on multilocus sequence analysis (MLSA)", Syst Appl Microbiol, 31, pp.447~460 (2008)[DOI]
- Heikens E, "Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci", J Clin Microbiol, 43, pp.2286~2290 (2005)[DOI]
- Neuberger A, "Clinical impact of a PCR assay for rapid identification of Klebsiella pneumoniae in blood cultures", J Clin Microbiol, 46, pp.377~379 (2008)[DOI]
- Liu Z, "Short pyrosequencing reads suffice for accurate microbial community analysis", Nucleic Acids Res, 35, pp.e120~ (2007)[DOI]
- Edgar RC, "UCHIME improves sensitivity and speed of chimera detection", Bioinformatics, 27, pp.2194~2200 (2011)[DOI]
- Kim OS, "Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species", Int J Syst Evol Microbiol, 62, pp.716~721 (2012)[DOI]
- Charlson ES, "Assessing bacterial populations in the lung by replicate analysis of samples from the upper and lower respiratory tracts", PLoS One, 7, pp.e42786~ (2012)[DOI]
- Bassis CM, "Analysis of the upper respiratory tract microbiotas as the source of the lung and gastric microbiotas in healthy individuals", MBio, 6, pp.e00037~ (2015)
- Salter SJ, "Reagent and laboratory contamination can critically impact sequence-based microbiome analyses", BMC Biol, 12, pp.87~ (2014)[DOI]
- Walker AW, "16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice", Microbiome, 3, pp.26~ (2015)[DOI]
- Davis IJ, "The Unculturables: targeted isolation of bacterial species associated with canine periodontal health or disease from dental plaque", BMC Microbiol, 14, pp.196~ (2014)[DOI]
- Ohno M, "Establishing the independent culture of a strictly symbiotic bacterium Symbiobacterium thermophilum from its supporting Bacillus strain", Biosci Biotechnol Biochem, 63, pp.1083~1090 (1999)[DOI]
- Hiergeist A, "Analyses of Intestinal Microbiota: Culture versus Sequencing", ILAR J, 56, pp.228~240 (2015)[DOI]
- Maukonen J, "The currently used commercial DNA-extraction methods give different results of clostridial and actinobacterial populations derived from human fecal samples", FEMS Microbiol Ecol, 79, pp.697~708 (2012)[DOI]
- Willner D, "Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples", PLoS One, 7, pp.e34605~ (2012)[DOI]
- Kennedy NA, "The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing", PLoS One, 9, pp.e88982~ (2014)[DOI]
- Beck JM, "ABCs of the lung microbiome", Ann Am Thorac Soc, 11(Suppl 1), pp.S3~S6 (2014)[DOI]
- Eckburg PB, "Diversity of the human intestinal microbial flora", Science, 308, pp.1635~1638 (2005)[DOI]
- Zoetendal EG, "A microbial world within us", Mol Microbiol, 59, pp.1639~1650 (2006)[DOI]
- Fournier PE, "From culturomics to taxonomogenomics: a need to change the taxonomy of prokaryotes in clinical microbiology", Anaerobe, 36, pp.73~78 (2015)[DOI]
- Lagier JC, "The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota", Clin Microbiol Rev, 28, pp.237~264 (2015)[DOI]
- Lagier JC, "Human gut microbiota: repertoire and variations", Front Cell Infect Microbiol, 2, pp.136~ (2012)
- Samb-Ba B, "MALDI-TOF Identification of the Human Gut Microbiome in People with and without Diarrhea in Senegal", Plos One, 9, pp.e87419~ (2014)[DOI]
