Assessment of microbial diversity in biofilms recovered from endotracheal tubes using culture dependent and independent approaches.

Sarah K Highlander,Ilse Vandecandelaere,Dieter Deforce,Peter Vosters,Pieter Depuydt,Nele Matthijs,Tom Coenye,Filip Van Nieuwerburgh,Hans J Nelis,Liesbet De Bus

doi:10.1371/journal.pone.0038401

Sarah K Highlander, Ilse Vandecandelaere + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0038401

Copy DOI

Journal: PloS one	Publication Date: Jun 5, 2012
Citations: 120	License type: CC BY 4.0

Affiliation: Ghent University, Ghent University Hospital

Abstract

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 bacterial contamination of the lower airways. In the present study, we analyzed the composition of the ET biofilm flora by means of culture dependent and culture independent (16 S rRNA gene clone libraries and pyrosequencing) approaches. Overall, the microbial diversity was high and members of different phylogenetic lineages were detected (Actinobacteria, beta-Proteobacteria, Candida spp., Clostridia, epsilon-Proteobacteria, 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 aerobic 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 varians. In general, there was little correlation between the results obtained by sequencing 16 S rRNA gene clone libraries and by cultivation. Pyrosequencing of PCR amplified 16 S 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.

Highlights

Nosocomial pneumonia or health-care associated pneumonia is one of the most common life-threatening infections in hospitals and long-term care facilities, with an incidence ranging from 4 to 50 cases per 1000 admissions [1]
71 coagulase/DNase positive isolates were identified as S. aureus (12 endotracheal tube (ET)). 237 isolates were coagulase/DNase negative and 40 of them were identified as Micrococcus luteus (16 ETs) and 3 as Kocuria varians (3 ETs)
S. aureus was found in 22% of ET biofilms; it is frequently involved in the development of late-onset ventilatorassociated pneumonia (VAP) [23]

Summary

Introduction

Nosocomial pneumonia or health-care associated pneumonia is one of the most common life-threatening infections in hospitals and long-term care facilities, with an incidence ranging from 4 to 50 cases per 1000 admissions [1]. The endotracheal tube (ET) itself is a pivotal element in the pathogenesis of VAP as it allows the direct entry of (oropharyngeal) microorganisms in the lower airways [1,2,3,4]. This ET is rapidly (within hours after its insertion) colonized by microorganisms that form a biofilm on its surface [2,5,6,7]. Several mechanisms by which ET biofilm bacteria can infect the lungs were already suggested: biofilm pieces might be dispersed and passively moved towards the lungs [9], biofilms cells can be aerosolized and aspirated into the lungs and individual cells in contact with liquids can be transferred deeply into the lungs [10]

Methods

Results

Discussion

Conclusion