Molecular Identification of Bacteria in Tracheal Aspirate Fluid from Mechanically Ventilated Preterm Infants

BACKGROUND Despite strong evidence linking infections to the pathogenesis of bronchopulmonary dysplasia (BPD), limitations of bacterial culture methods have precluded systematic studies of airway organisms relative to disease outcomes. Application of molecular bacterial identification strategies may provide new insight into the role of bacterial acquisition in the airways of preterm infants at risk for BPD. METHODS Serial (within 72 hours, 7, 14, and 21 days of life) tracheal aspirate samples were collected from 10 preterm infants with gestational age ≤34 weeks at birth, and birth weight of 500-1250 g who required mechanical ventilation for at least 21 days. Samples were analyzed by quantitative real time PCR assays for total bacterial load and by pyrosequencing for bacterial identification. RESULTS Subjects were diagnosed with mild (1), moderate (3), or severe (5) BPD. One patient died prior to determination of disease severity. 107,487 sequences were analyzed, with mean of 3,359 (range 1,724-4,915) per sample. 2 of 10 samples collected <72 hours of life contained adequate bacterial DNA for successful sequence analysis, one of which was from a subject exposed to chorioamnionitis. All other samples exhibited bacterial loads >70copies/reaction. 72 organisms were observed in total. Seven organisms represented the dominant organism (>50% of total sequences) in 31/32 samples with positive sequences. A dominant organism represented>90% of total sequences in 13 samples. Staphylococcus, Ureaplasmaparvum, and Ureaplasmaurealyticum were the most frequently identified dominant organisms, but Pseudomonas, Enterococcus, and Escherichia were also identified. CONCLUSIONS Early bacterial colonization with diverse species occursafter the first 3 days of life in the airways of intubated preterm infants, and can be characterized by bacterial load and marked species diversity. Molecular identification of bacteria in the lower airways of preterm infants has the potential to yield further insight into the pathogenesis of BPD.