Molecular analysis of oral and respiratory bacterial species associated with ventilator-associated pneumonia

Abstract

Trauma intensive care unit (TICU) patients requiring mechanical respiratory support frequently develop ventilator-associated pneumonia (VAP). Oral and oropharyngeal bacteria are believed to be responsible for many cases of VAP, but definitive evidence of this relationship is lacking. Earlier studies used conventional culture-based methods for identification of bacterial pathogens, but these methods are insufficient, as some bacteria may be uncultivable or difficult to grow. The purpose of this study was to use a culture-independent molecular approach to analyze and compare the bacterial species colonizing the oral cavity and the lungs of TICU patients who developed VAP. Bacterial samples were acquired from the dorsal tongue and bronchoalveolar lavage fluid of 16 patients. Bacterial DNA was extracted, and the 16S rRNA genes were PCR amplified, cloned into Escherichia coli, and sequenced. The sequencing data revealed the following: (i) a wide diversity of bacterial species in both the oral and pulmonary sites, some of them novel; (ii) known and putative respiratory pathogens colonizing both the oral cavity and lungs of 14 patients; and (iii) a number of bacterial pathogens (e.g., Dialister pneumosintes, Haemophilus segnis, Gemella morbillorum, and Pseudomonas fluorescens) in lung samples that had not been reported previously at this site when culture-based methods were used. Our data indicate that the dorsal surface of the tongue serves as a potential reservoir for bacterial species involved in VAP. Furthermore, it is clear that the diversity of bacterial pathogens for VAP is far more complex than the current literature suggests.

FIG. 1.

Phylogenetic tree of bacterial species or phylotypes identified in BAL samples. Bacterial species or phylotype clones and GenBank accession numbers are shown. Species in blue are bacterial species previously unreported from BAL samples. Species in red are novel phylotypes identified in this study. Novel phylotypes are defined as those clones with 16S rRNA sequences having less than 99% similarity to the sequences of their closest relatives. Colored boxes: black, species or phylotype detected in BAL samples; gray, BAL species or phylotype detected in the oral cavity. The bar represents a 5% difference in nucleotide sequences. Pt, patient.