Next-Generation Sequencing of Culture Negative Bronchoalveolar Lavage Reveals the Presence of Potentially Pathogenic Microorganisms

Patients undergoing mechanical ventilation are at increased risk for developing nosocomial pneumonia. Traditionally, the diagnosis of pneumonia has relied on the identification of an etiologic agent by the hospital pathology lab via quantitative culture. A problem arises when a patient experiences clinical signs and symptoms of pneumonia, but culturing of bronchoalveolar lavage (BAL) fluid reveals only normal “respiratory tract flora” or results in “no growth”. I hypothesize that culture-negative, presumptive positive BAL is infected with pathogenic bacteria that are not being cultivated on traditional culture media. To investigate this hypothesis, culture-independent techniques were chosen to examine culture-positive and culture-negative BAL. Sanger and Ion Torrent Sequencing were used to verify that molecular techniques are able to identify the same pathogens the hospital lab finds within culture-positive BAL. Ion Torrent sequencing was then used to characterize the microbial community within culture-negative BAL. Cytokine assays were used to determine if culture-positive and culture-negative patients mount a similar immune response. By sequencing colonies picked from the same culture plates used by the hospital lab, I confirmed the presence of the same pathogens identified by the hospital. Ion Torrent sequencing was able to identify hundreds of genera in both the culture-positive and culture-negative BAL samples. However, no difference in 16S copy number was found between the groups. A group of culture-negative BAL with high diversity and similar bacterial communities were observed. These samples clustered together upon principal coordinates analysis, I believe this grouping may represent a core microbiome. Production of pro-inflammatory cytokines were found to be increased in samples that were dominated by a particular pathogen/pathogens as opposed to those that were more diverse and had lower cytokine measurements. This work highlights the advantages associated with using culture-independent techniques for the diagnosis of pneumonia specifically when traditional culturing techniques fail to identify an etiologic agent.