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 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.

FIG 1

Reproducibility of pyrosequencing protocol. Positive control mixtures of fixed amounts and types of bacterial 16S rRNA gene amplicons (3.3 ng of total DNA comprising equal parts from each listed plasmid) were serially resequenced using the same GS Junior system. The run corresponding to the current study is identified (run 10).

FIG 2

Detection of bacteria in BAL fluid specimens by pyrosequencing and conventional culture techniques. BAL fluid specimens with bacteria detected via culture (any culture growth) are divided into those with specific species identified and reported (bacterial species reported) and those for which only oral flora was reported (“oral flora” only). Proportions compared using Fisher's exact test.

FIG 3

Culture-independent analysis of BAL fluid specimens according to culture results. BAL fluid specimens were separated and analyzed according to their culture results: oral Flora only, no growth via culture, and bacterial species identified and reported. Specimens were compared for (A) bacterial DNA burden, (B) Shannon diversity index, and relative abundance of bacterial phyla for (C) Proteobacteria and (D) Bacteroidetes. Group means compared using unpaired ANOVA with Tukey's multiple-comparison test. NS, not significant; LOD, limit of detection.

FIG 4

Case illustrations of the limitations of bacterial culture. (A) Two years after bilateral lung transplant for pulmonary fibrosis, a 52-year-old man developed cough and decreased lung function. Computed tomography (CT) scan revealed basilar ground-glass opacities. Bronchoscopy was performed (BAL 1). Bacterial culture revealed no bacterial growth, but subsequent analysis with pyrosequencing revealed overwhelming abundance of Escherichia sp. In following weeks, treatment with a fluoroquinolone for an unrelated indication resulted in improved respiratory symptoms. A repeat BAL fluid specimen obtained 1 month later (BAL 2) resulted in oral flora reported via culture; subsequent pyrosequencing revealing increased community diversity and near absence of Escherichia sp. (B) Five years after bilateral lung transplant for COPD, a 59-year-old woman developed dyspnea, cough, and sputum production. CT scan revealed multifocal infiltrates. Bronchoscopy revealed purulent secretions with a predominantly neutrophilic BAL fluid cell count, but culture results were reported only as oral flora. Subsequent analysis of the BAL fluid via pyrosequencing revealed overwhelming abundance of Corynebacterium sp., with low community diversity and high bacterial DNA burden.

FIG 5

Culture-independent analysis of quantitative BAL fluid cultures. BAL fluid specimens were divided into three groups based upon quantitative culture results as follows: P. aeruginosa ≥10⁴ CFU/ml, P. aeruginosa <10⁴ CFU/ml, and no bacterial growth. Specimens were compared by (A) bacterial DNA burden, (B) Shannon diversity index, and (C) relative abundance of OTU 1053 (P. aeruginosa). Group means compared by unpaired ANOVA with Tukey's multiple-comparison test.