Molecular analysis of microbial communities in endotracheal tube biofilms

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. The 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 2.1×10(8) colony forming units (cfu)/cm(2) of endotracheal tube (mean 1.4×10(7) cfu/cm(2)). 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. Molecular 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.