Molecular Analysis of Microbial Communities in Endotracheal Tube Biofilms

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.

Microbial investigations in throat swab and tracheal aspirate specimens are beneficial to predict the corresponding endotracheal tube biofilm flora among intubated neonates with ventilator-associated pneumonia

“…The ET was collected immediately following extubation and stored in sterile PBS at 4˚C. Vortex and sonication were conducted to release biofilms from the inner surface of the endotracheal tube as previously described (9). Prior to extubation, throat swabs and tracheal aspirates were collected and processed as previously described (14).…”

“…Furthermore, due to concerns for patient safety and the fact that reintubation itself carries a significant risk of neonatal VAP (3), ETs are not able to be extubated during intubation. The majority of ET samples are only available at the time of extubation (9)(10)(11), which means that the microbial signatures of ET biofilms prior to extubation are unknown. Therefore, identifying substituent samples to investigate microbiota in ET biofilms is beneficial to identify the floral risk factors associated with VAP.…”

“…Denaturing gradient gel electrophoresis (DGGE) is a culture-independent method that facilitates rapid analyses and comparisons of microbial communities (17), and is simpler and less expensive than other culture-independent methods. DGGE has been widely applied in microbiological studies (9,17,18), such as a recent study among intubated adults, which reported a considerable biofilm compositional complexity based on DGGE (9). However, to the best of our knowledge similar studies among neonatal patients are scarce, particularly those with VAP.…”

Novel Preventive Strategies for Ventilator-associated Pneumonia

“…With time and despite routine suctioning, the inner surface of the ETT becomes covered by a layer of mucus and cells soon after intubation, which increases with duration of ETT use. Th is layer of 'biofi lm' is an optimal environment for growth of a wide range of bacterial species [14], through which antibiotics penetrate with diffi culty. Biofi lm forms on both the internal and the external surface of the ETT.…”

Factors associated with oral biofilm in ICU patients with infectious diseases

“…However, other pathogens are often present in biofilm and may constitute vehicles for the development of other systemic diseases. Indeed, previous studies have reported the presence of Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumonia and Escherichia coli, bacteria that are responsible for hospital-acquired infections and outbreaks, in oral biofilm of intubated patients 8,9 .…”