Analysis of culture-dependent versus culture-independent techniques for identification of bacteria in clinically obtained bronchoalveolar lavage fluid
Open accessAbstract
The diagnosis and management of pneumonia are limited by the use of culture-based techniques of microbial identification, which may fail to identify unculturable, fastidious, and metabolically active viable but unculturable bacteria. Novel high-throughput culture-independent techniques hold promise but have not been systematically compared to conventional culture. We analyzed 46 clinically obtained bronchoalveolar lavage (BAL) fluid specimens from symptomatic and asymptomatic lung transplant recipients both by culture (using a clinical microbiology laboratory protocol) and by bacterial 16S rRNA gene pyrosequencing. Bacteria were identified in 44 of 46 (95.7%) BAL fluid specimens by culture-independent sequencing, significantly more than the number of specimens in which bacteria were detected (37 of 46, 80.4%, P ≤ 0.05) or "pathogen" species reported (18 of 46, 39.1%, P ≤ 0.0001) via culture. Identification of bacteria by culture was positively associated with culture-independent indices of infection (total bacterial DNA burden and low bacterial community diversity) (P ≤ 0.01). In BAL fluid specimens with no culture growth, the amount of bacterial DNA was greater than that in reagent and rinse controls, and communities were markedly dominated by select Gammaproteobacteria, notably Escherichia species and Pseudomonas fluorescens. Culture growth above the threshold of 10(4) CFU/ml was correlated with increased bacterial DNA burden (P < 0.01), decreased community diversity (P < 0.05), and increased relative abundance of Pseudomonas aeruginosa (P < 0.001). We present two case studies in which culture-independent techniques identified a respiratory pathogen missed by culture and clarified whether a cultured "oral flora" species represented a state of acute infection. In summary, we found that bacterial culture of BAL fluid is largely effective in discriminating acute infection from its absence and identified some specific limitations of BAL fluid culture in the diagnosis of pneumonia. We report the first correlation of quantitative BAL fluid culture results with culture-independent evidence of infection.
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Bacterial pneumonia is characterised by increased bacterial burden and low community diversity, generally one dominant pathogen.
“Bacterial pneumonia, a true lung infection, exemplifies these features: it is characterised by increased bacterial burden and low community diversity (generally one dominant pathogen).”
From The microbiome and critical illness (2016) · cites this paper as [130]
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- Authors
- Dickson, R. P., Erb-Downward, J. R., Prescott, H. C., Martinez, F. J., Curtis, J. L., Lama, V. N., & Huffnagle, G. B.
- Affiliations
- University of Michigan (US)
- Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA
- Department of Internal Medicine, Weill Cornell Medical College, New York, New York, USA
- Venue
- Journal of Clinical Microbiology
- Publisher
- American Society for Microbiology
- Published
- 2014-10
- Volume
- 52
- Issue
- 10
- Pages
- 3605-3613
- ISSN
- 0095-1137 (ISSN-L)
- 1098-660X
- PMC
- PMC4187760
- OpenAlex
- W1974429915
- Cited by
- 176
- References
- 27
- Citations by year
- 2026: 12 · 2025: 9 · 2024: 18 · 2023: 12 · 2022: 13 · 2021: 25 · 2020: 24 · 2019: 11 · 2018: 21 · 2017: 12 · 2016: 7 · 2015: 12
- FWCI
- 7.12
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Journal of Clinical Microbiology (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- University of Pennsylvania
- University of Michigan
- National Heart, Lung, and Blood Institute (U01HL098961, R01HL094622, L30 HL120241, T32HL00774921, R01HL114447, T32HL007749)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Bacterial Identification and Susceptibility Testing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
- Keywords
- bronchoalveolar lavage fluid, bacterial identification, culture-independent methods, 16S rRNA gene pyrosequencing, lung transplant recipients, pneumonia diagnosis, bacterial community diversity, Pseudomonas aeruginosa, Escherichia, Pseudomonas fluorescens, Gammaproteobacteria
- MeSH terms
- Bacteria, Bacteriological Techniques, Bronchoalveolar Lavage Fluid, DNA, Bacterial, DNA, Ribosomal, Female, Humans, Male, Middle Aged, RNA, Ribosomal, 16S, Lung Transplantation, Sequence Analysis, DNA, Pneumonia, Bacterial, Molecular Diagnostic Techniques, Transplant Recipients
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