Analysis of Culture-Dependent versus Culture-Independent Techniques for Identification of Bacteria in Clinically Obtained Bronchoalveolar Lavage Fluid

Abstract

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 highthroughput 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. Pneumonia remains a leading cause of death in the United States (1), and respiratory infections are responsible for a greater global burden of disease than malignancy, ischemic heart disease, or diabetes mellitus (2). The diagnosis and management of pneumonia are limited by the use of conventional culturebased techniques (3). In recent years, novel culture-independent techniques of microbial identification have revealed that bronchoalveolar lavage (BAL) fluid specimens contain diverse communities of bacteria previously undetected via culture-based approaches (4-6). These techniques, while promising, have not been systematically compared to conventional culture-based approaches, including quantitative BAL fluid cultures (7).In this study, we compared conventional BAL fluid cultures (which were optimized to identify acute infection) with a culture-independent research technique, pyrosequencing (which is designed to identi...

The dynamics of the pulmonary microbiome during mechanical ventilation in the intensive care unit and the association with occurrence of pneumonia

“…Indeed, ETA cultures have a limited predictive value for VAP compared with BAL 35. This might also explain that the sequencing results did not correspond to the culture results, although this was observed previously in other studies that used BAL fluid as well 25 36. In the adapted island model, the trachea is seen as the first island close to the continent (the mouth) and every species present further down should pass here 7.…”

Spatial Variation in the Healthy Human Lung Microbiome and the Adapted Island Model of Lung Biogeography

“…When compared with conventional, culture-based approaches, sequencing-based techniques reveal increased diversity of the respiratory microbiome and are not limited by microbe viability or the ability to grow in limited in vitro culture conditions (19,21). We have previously described our methods of genomic DNA extraction and amplification (21,22). We amplified the V4 region of the 16s rRNA gene from each sample using published primers (23) and the dualindexing sequencing strategy developed by the laboratory of Patrick D. Schloss (24).…”

Intraalveolar Catecholamines and the Human Lung Microbiome

“…Increased intraalveolar catecholamine concentrations were strongly associated with decreased community diversity in the bacterial lung microbiome, a feature of pneumonia (8). As measured by the Shannon diversity index, diversity was negatively correlated with norepinephrine and epinephrine concentrations (P < 0.05 for each) and combined catecholamine concentration (P = 0.009).…”

“…All patients were receiving both systemic immunosuppression and Pneumocystis jirovecii prophylaxis at the time of bronchoscopy; additional antibiotic exposure in both groups has been previously reported (7). Patient characteristics, methods of bronchoscopy, DNA isolation and amplification, pyrosequencing, and analysis have previously been published (7,8). Catecholamines were quantified using 2-CAT (A-N) Research ELISA (Rocky Mountain Diagnostics, Colorado Springs, CO).…”