We performed molecular identification of bacteria in serial tracheal aspirate samples from intubated preterm infants by isolating rRNA genes harvested by PCR to determine the composition of bacterial communities and the timing of acquisition in the airways of these infants. We found that during the first 72 hours after birth, the majority of tracheal aspirate samples had low or undetectable bacterial loads. However, within 7 days of life, sustained bacterial colonization with diverse species is present in the airways of intubated preterm infants. 96.9% of samples with bacterial detection exhibited a dominant organism, with Staphylococcus and Ureaplasma species most frequently identified as the dominant organisms. Molecular methods identified all species detected by traditional clinical cultures, but also identified potential pathogens, including Ureaplasma, that were not detected by standard bacterial culture methods. Thus, improved resolution of bacterial detection with regard to overall bacterial load, species diversity within the microbiome or the identification of specific organisms for directed therapy, has the potential to yield further insights into the pathogenesis of BPD and late respiratory outcomes of preterm newborns.
These findings are important because early respiratory infection and inflammation have been shown to impair alveolar development 
and contribute to the development of BPD 
. Inflammatory injury disrupts lung growth in part by production of pro-inflammatory cytokines 
. Although pro-inflammatory cytokines have been associated with the development of BPD and adverse outcomes 
, variable assessments of the pro-inflammatory response, and lack of context specificity (identification of all aspects of the perinatal milieu contributing to disease) have precluded the use of cytokines as adequate biomarkers for BPD. While antenatal and postnatal infections, hyperoxia, and mechanical ventilation have garnered much attention as inflammation provoking stimuli, the possible impact of airway colonization on the development of BPD is less clear and has generated contradictory results 
. The presence of Ureaplasma
in the respiratory tract of preterm infants, even without signs of infection, has been associated with a pro-inflammatory response and an increased risk for BPD 
. Although earlier trials of erythromycin treatment for the treatment of Ureaplasma
in preterm infants failed to show benefit 
, a recent trial of azithromycin treatment revealed a significant reduction in the endpoint of BPD or death in the subgroup of patients who were found to be colonized or infected with Ureaplasma 
. Thus, Ureaplasma remains a plausible but still unconfirmed contributor to BPD. Ureaplasma
was the dominant organism in at least one sample for 6 of the 10 infants in our study. Ureaplasma
was found to be the dominant organism in late samples even when not detected in initial samples, suggesting the possibility of post-natal acquisition and/or the presence of environmental factors, such as antibiotic administration, that encourage selection of this genus. The association between antibiotic administration, Ureaplasma
colonization, and risk for BPD should be explored further in future studies. Exposure to colonizing bacteria may impact the preterm infant's immune response and subsequent lung development. Improved resolution of bacterial detection techniques as demonstrated here in conjunction with assessments of inflammation could improve our understanding of the developing host immune response in premature infants and its relationship to pathogen exposure.
Molecular methods have contradicted the traditional theory that human lower airways are sterile by demonstrating that the lungs of healthy older children and adults are rich with microbial flora 
, and that traditional culture methods are not adequate to investigate the contribution of microbial communities to disease. Our results, together with a previous report 
suggest that microbial communities are present early in the lower airways of intubated preterm infants. Rapidly accumulating evidence demonstrates that microbial communities play an important role in human physiology 
. Several diseases have been associated with changes in composition and diversity of these microbial communities 
. Some have shown that microbiota of limited diversity and relative absence of commensal organisms are associated with increased inflammation, barrier permeability, and disease status 
. Early evaluations of the respiratory tract microbiota in pulmonary diseases such as cystic fibrosis, asthma, and COPD 
also reveal shifts in microbial composition compared to healthy patients, suggesting the composition of the respiratory tract microbiota can play a direct role in pathophysiology of lung disease. Neonatal oropharyngeal colonization with Streptococcus pneumoniae
, Moraxella catarrhalis
, or a combination of these organisms, but not colonization with Staphylococcusaureus
, was significantly associated with persistent wheeze in childhood 
. Children living on farms are exposed to a wider range of microbes than were children living in urban environments, and these children appear relatively protected from asthma and atopy 
. Together, these studies suggest the respiratory tract microbiota early in life can also modulate the host immune response and future lung disease.
Only 2of 10 samples collected within 72 hours after birth exhibited sufficient bacterial load for sequence analysis, suggesting the lower airways of preterm infants typically have comparatively low bacterial loads. One of these two samples was obtained from an infant exposed to chorioamnionitis and revealed Ureaplasmaparvum
, a known cause of chorioamnionitis, as the dominant organism. Although a diverse array of bacteria were detected within the samples of these 10 infants, the diversity of organisms within samples was far less than detected in respiratory samples of older children and adults 
. Further, bacterial diversity was not associated with age of the infant or BPD severity.
The use of antibiotics administered to treat confirmed or potential infections may have long term effects on the composition of the airway microbial community. Potential negative consequences include the elimination of commensal bacteria and selection for other pathogenic bacteria, especially those that are not readily detected by clinical culture techniques and that promote chronic airway inflammation. Further studies will need to investigate the potential role of antibiotics on the composition of the airway microbiota and the impact on early lung development.
There are several limitations to our study. First, the small sample size in this pilot study precludes rigorous evaluation of the presence of specific bacteria, bacterial loads, or species diversity to the risk of BPD. All of the infants included in this study were less than 28 weeks gestation and required mechanical ventilation for at least 21 days, which represents a very high risk population for severe BPD. Serial evaluation of the airway microbiome in preterm infants who do not develop BPD will be important to determine how the composition of the airway microbiome is related to risk of BPD. Since preterm infants who do not develop BPD often do not require long periods of mechanical ventilation support, noninvasive methods of airway sampling will be required. Second, all infants in this study were treated with systemic antibiotics which are likely to impact the evolution of microbial composition, limiting the ability to ascertain the natural acquisition of airway colonization after birth. The presence of endotracheal tubes also impacts the access of microbes to lower airways. Although we attempted to compare molecular detection methods to clinical culture results, the samples were not simultaneously obtained. Therefore, the discrepancies between the two results cannot be accounted for by methodology alone. In addition, sampling of tracheal aspirates may not adequately reflect bacterial exposure at the alveolar level. Simultaneous sampling of the upper airway, lower airways and alveolar samples are required to provide a more complete assessment of bacterial colonization. Finally, future studies are needed to more directly link the presence of specific airway organisms, airway bacterial load, and diversity of the microbiota with altered innate immunity and inflammatory cytokines to better understand how early colonization impacts lung inflammation.
In summary, early bacterial colonization with diverse species are present in the airways of intubated preterm infants, and can be characterized by bacterial load and species diversity. Molecular detection of Ureaplasma species in the airways of preterm infants late in the course of mechanical ventilation provides further support for the concept that Ureaplasma may be involved in the pathogenesis of BPD and requires further investigation. Molecular identification of bacteria in the lower airways of preterm infants has the potential to yield further insight into the pathogenesis of BPD and to identify novel therapeutic targets.