Molecular identification of bacteria in tracheal aspirate fluid from mechanically ventilated preterm infants.

Peter M Mourani,J Kirk Harris,Charles E Robertson,Steven H Abman,Marci K Sontag,Josef Neu

doi:10.1371/journal.pone.0025959

Abstract

BackgroundDespite 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.MethodsSerial (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.ResultsSubjects 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.ConclusionsEarly 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.

Highlights

Bronchopulmonary dysplasia (BPD) was initially attributed to mechanical ventilation and oxygen induced lung injury in infants born preterm, as first described by Northway and colleagues in 1967 [1]
Recent studies have shown that airway colonization with specific organisms during the neonatal period was associated with persistent wheeze in childhood [8], suggesting that early specific bacterial exposure and/or the host response contribute to future disease
All samples collected from day 7–21 of life contained detectable bacterial load (.70 copies/ reaction), and each sample was successfully amplified for bacterial identification

Summary

Introduction

Bronchopulmonary dysplasia (BPD) was initially attributed to mechanical ventilation and oxygen induced lung injury in infants born preterm, as first described by Northway and colleagues in 1967 [1]. Inflammation of the developing lung from these interventions and other causes is a predominate theory underlying thepathogenesisof BPD [2,3]. Both prenatal and postnatal sources of inflammation, including infection, may directly disrupt lung growth [4] and predispose infants to BPD [5,6]. Little is known about the timing of bacterial exposure and acquisition in the airways of preterm infants, mechanisms that promote or impede colonization of specific organisms, or the inflammatory responses to colonizing bacteria in relation to injury to the developing lung.

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