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Introduction: Respiratory infections in preterm infants contribute to the development of chronic lung disease of prematurity, also known as bronchopulmonary dysplasia. Next generation sequencing of microbial genes could replace traditional culture-based methods. Aims: We used next generation sequencing to identify microbial communities in tracheal aspirates (TA) obtained from ventilated preterm infants. Methods: TA samples were obtained daily in the first week of life then twice weekly until day 28 or until extubation, from 47 ventilated preterm infants at two UK neonatal units. Total bacterial load was estimated by qPCR of the 16S bacterial rRNA gene from DNA extracted from TA cell pellet. Next generation paired end sequencing of the V3-V4 region identified the microbes present. IL6 and IL8 concentration were measured in TA samples. Results: Sequencing results were positive in 47 of 276 samples (17%). Positive TAs were associated with higher microbial load and higher IL6 and IL8 concentrations, suggesting an infective process. Serial TA samples showed episodic peaks of microbial load. 93.6% of positive TAs contained a dominant operational taxonomic unit (>50% of reads) suggesting 28 infective episodes in 20 infants. The most commonly identified dominant genera was Staphylococci. Sequencing depth using this methodology was unable to identify organisms to a species level. Conclusion: Microbial DNA was not detectable in TA samples of preterm infants in the absence of infection most likely due to absence of microbes or below detection levels. 16S rRNA sequencing identified dominant genera suggesting respiratory infections, as evidenced by an increase in IL-6 and IL-8.

Introduction: Early life bacterial colonisation has been shown to affect short and long-term health outcomes. Lungs of healthy adults are colonised by low levels of microorganisms. However, it is unclear when such colonisation occurs in preterm babies especially those who develop chronic lung disease of prematurity (CLD). Aims: We, therefore, serially studied the microbial lung colonisation of intubated preterm infants at risk of developing CLD, associating the findings with pro-inflammatory cytokines. Methods: Non-bronchoscopic bronchoalveolar lavage (BAL) samples were obtained from intubated preterm infants (of ≤32 weeks’ gestation) daily for the first week of life, then twice weekly until 28 days of age or until extubation. DNA was extracted from BAL cell pellets and the V3-V4 region of the 16S bacterial RNA gene sequenced. BAL IL6 and IL8 were measured by ELISA. Results: 89 BAL serial samples were obtained from 19 preterm infants. Sequencing data were possible from 34 samples. Fewer samples were positive when the infants were on antibiotics and positive samples were associated with higher BAL fluid IL6 and IL8 concentrations, suggesting association with an infective process. Most positive samples revealed a dominant operational taxonomic unit (OTU) with dominant genus (commonly Acinetobacter , Enterobacter , Ureaplasma and Staphylococcus ) varying between samples obtained from the same infant. Conclusions: The microbial load in many samples was below the limit of detection in the lungs of preterm infants especially in infants receiving antibiotics. The early colonisation of preterm lungs is characterised by recurrent episodes of bacterial infection associated with rises inflammatory activity within the lung.