454 sequencing of 16S rRNA genes from endotracheal biofilms. human lung metagenome

Ventilator-associated pneumonia (VAP) is a common nosocomial infection in mechanically ventilated patients. Biofilm formation is one of the mechanisms through which the endotracheal tube (ET) facilitates contamination of the lower airways with bacterial pathogens. In the present study, we analysed the composition of the ET biofilm flora by means of culture dependent and culture independent (16S rRNA gene clone libraries and pyrosequencing) approaches. Overall, the microbial diversity was high and members of different phylogenetic lineages were detected (e.g. Actinobacteria, beta-Proteobacteria, Candida spp., Clostridia, epsilon-Proteobacteria, Eubacteriaceae, Firmicutes, Fusobacteria and gamma-Proteobacteria). Culture dependent analysis, based on the use of selective growth media and conventional microbiological tests, resulted in the identification of typical nosocomial pathogens which are known to play a role in the development of VAP, e.g. Staphylococcus aureus and Pseudomonas aeruginosa. Other opportunistic pathogens were also identified, including Staphylococcus epidermidis and Kocuria spp. In general, there was little correlation between the results obtained by sequencing 16S rRNA gene clone libraries and the results of cultivation, indicating the value of using a combination of culture dependent and culture independent approaches. Pyrosequencing of PCR amplified 16S rRNA genes of four selected samples resulted in the identification of a much wider variety of bacteria. The results from the pyrosequencing analysis suggest that these four samples were dominated by members of the normal oral flora such as Prevotella spp., Peptostreptococcus spp. and lactic acid bacteria. A combination of methods is recommended to obtain a complete picture of the microbial diversity of the ET biofilm flora.