DEEP SEQUENCING OF CULTIVATION-NEGATIVE BRONCHOAVEOLAR LAVAGE SAMPLES FROM MECHANICALLY VENTILATED TRAUMA PATIENTS REVEALS DEFICITS IN TRADITIONAL CLINICAL PROTOCOLS

Patients undergoing mechanical ventilation are at risk for infections such as ventilator-acquired pneumonia. Standard clinical protocols call for pathogen screening on culture plates which can only detect a limited number of bacteria. We sampled the lungs of mechanically ventilated trauma patients, and by using molecular-based deep sequencing techniques we found several potentially pathogenic bacteria in samples that traditional techniques labeled as “no growth” or normal “respiratory tract flora." Purpose (a): Bronchoaveolar lavage (BAL) is a method of screening mechanically ventilated patients for potentially pathogenic microogranisms. Traditional culture-based techniques used in clinical laboratories detect a limited number of known bacteria; consequently, some samples may be falsely reported as “cultivation-negative” or possessing only “normal respiratory tract flora.” To ascertain the potential for false negative results, we performed next generation DNA sequencing on BAL samples previously determined to be “cultivation negative” or “respiratory tract flora” by standard plate methods. Methods (b): Nine samples were taken from mechanically ventilated trauma patients in the Surgical Intensive Care Unit (SICU) subjected to BAL as part of the standard of care. DNA was extracted from the samples and the 16S rRNA subunit was amplified and sequenced using the Ion Torrent Personal Genome Machine. Sequences were analyzed using Mothur data-analysis pipeline to identify the appropriate taxonomic designation. Results (c): Results indicated that the majority (>77%) were dominated by a single organism. One third (3/9) of the samples analyzed were dominated by Neisseria spp. Other potential pathogens found to be dominant within the BAL samples included Streptococcus, Haemophilus, Aeromonas, and Rothia spp. Contamination from the oral cavity were also likely in two samples as evidenced by the identification of Porphyromonas and Prevotella spp. Conclusions (d): Our study demonstrates potential benefits of using next-generation sequencing to supplement the current culture-dependent clinical diagnostic methods. The knowledge gained from analyzing the lung microbiome of “culture-negative” and “respiratory tract flora” may be an important tool for identifying difficult to cultivate species associated with lung infections in mechanically ventilated patients.