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Abstract

Introduction: Current culture-based techniques may be inadequate for identifying causative agents in patients with ventilator-associated pneumonia (VAP). We used a culture-independent molecular approach to assess the bacterial species colonizing the aerodigestive tract of mechanically ventilated children. Hypothesis: We hypothesized that culture-independent methods would identify phylotypes and difficult-to-grow species that are not detected by routine culture. These data may shed new light on the differences in the oral microbiome between age groups and might uncover novel VAP-associated pathogens. Methods: Oral swabs as well as tracheal and gastric aspirates were obtained sequentially from mechanically ventilated children (<18 years) in the ICU following elective surgery. Samples were sent for routine bacterial culture and DNA isolation for PCR amplification of 16S rRNA genes followed by analysis using a previously validated microarray (HOMIM). Results: We analyzed 97 clinical samples from 14 subjects (50% female, mean 5.6 years) following cardiac (n=10) and spinal fusion surgery (n=4). Routine cultures revealed 4 potential bacterial pathogens in 6 samples from 2 subjects. PCR amplification of 16S rRNA genes was successful in 28 samples (29%). Over 100 distinct taxa were detected on microarray analysis. The oral microbiome burden was significantly higher in older children compared to infants <12 months (p<0.05 by Wilcoxon rank sum test). Microbial profiles were also distinct between the 2 groups. One subject had VAP, and growth of Pseudomonas aeruginosa from tracheal aspirate was mirrored by molecular detection of the organism first in oral and then tracheal samples during the study. Conclusions: Culture-independent molecular methods using 16S rRNA gene sequences allowed identification of known VAP-associated pathogens and a variety of unculturable microbes in oral, tracheal and gastric samples. The clinical significance of these taxa warrants further investigation. Edentulate infants have a lower microbiome burden and distinct profile compared to older children. Microbiome identification may guide bedside practices in the ICU, such as oral hygiene, and enhance our understanding of VAP pathogenesis.