Molecular Analysis of Microbial Communities in Endotracheal Tube Biofilms

Niyaz Ahmed,Scott Cairns,Melanie Julia Wilson,Paul John Frost,Michael Alexander Oxenham Lewis,John Gilbert Thomas,Matthew Peter Wise,Samuel James Hooper,David Wynne Williams

doi:10.1371/journal.pone.0014759

Niyaz Ahmed, Scott Cairns + Show 7 more

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https://doi.org/10.1371/journal.pone.0014759

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Abstract

BackgroundVentilator-associated pneumonia is the most prevalent acquired infection of patients on intensive care units and is associated with considerable morbidity and mortality. Evidence suggests that an improved understanding of the composition of the biofilm communities that form on endotracheal tubes may result in the development of improved preventative strategies for ventilator-associated pneumonia.Methodology/Principal FindingsThe aim of this study was to characterise microbial biofilms on the inner luminal surface of extubated endotracheal tubes from ICU patients using PCR and molecular profiling. Twenty-four endotracheal tubes were obtained from twenty mechanically ventilated patients. Denaturing gradient gel electrophoresis (DGGE) profiling of 16S rRNA gene amplicons was used to assess the diversity of the bacterial population, together with species specific PCR of key marker oral microorganisms and a quantitative assessment of culturable aerobic bacteria. Analysis of culturable aerobic bacteria revealed a range of colonisation from no growth to 2.1×108 colony forming units (cfu)/cm2 of endotracheal tube (mean 1.4×107 cfu/cm2). PCR targeting of specific bacterial species detected the oral bacteria Streptococcus mutans (n = 5) and Porphyromonas gingivalis (n = 5). DGGE profiling of the endotracheal biofilms revealed complex banding patterns containing between 3 and 22 (mean 6) bands per tube, thus demonstrating the marked complexity of the constituent biofilms. Significant inter-patient diversity was evident. The number of DGGE bands detected was not related to total viable microbial counts or the duration of intubation.Conclusions/SignificanceMolecular profiling using DGGE demonstrated considerable biofilm compositional complexity and inter-patient diversity and provides a rapid method for the further study of biofilm composition in longitudinal and interventional studies. The presence of oral microorganisms in endotracheal tube biofilms suggests that these may be important in biofilm development and may provide a therapeutic target for the prevention of ventilator-associated pneumonia.

Highlights

Ventilator-associated pneumonia (VAP) is the most frequent nosocomial infection in the intensive care unit (ICU) occurring in 8–28% of mechanically ventilated patients [1], [2]
Sottile and colleagues first suggested a link between the endotracheal biofilm and pulmonary infection [32], and this was supported by identical pathogenic bacteria being present in the lung of patients with VAP and the endotracheal biofilm [8]
Improved oral hygiene has proved to be an effective strategy for reducing VAP [9,10,11,12] and since potential respiratory pathogens are isolated from dental plaque of mechanically ventilated patients [13], [14], we hypothesised that microbes which constitute the normal oral flora might be present in the endotracheal biofilm

Summary

Introduction

Ventilator-associated pneumonia (VAP) is the most frequent nosocomial infection in the intensive care unit (ICU) occurring in 8–28% of mechanically ventilated patients [1], [2]. The presence of an endotracheal tube is an independent risk factor for developing VAP and whilst tracheal intubation is necessary to facilitate mechanical ventilation, it circumvents elements of patients’ innate immunity. The endotracheal tube disrupts the cough reflex, promotes accumulation of tracheobronchial secretions and mucus, and provides a direct conduit for pathogenic microorganisms to reach the lower respiratory tract, increasing the risk of infection [5]. Ventilator-associated pneumonia is the most prevalent acquired infection of patients on intensive care units and is associated with considerable morbidity and mortality. Evidence suggests that an improved understanding of the composition of the biofilm communities that form on endotracheal tubes may result in the development of improved preventative strategies for ventilator-associated pneumonia

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