(248e) Bacterial Colonization of Nanomodified ETT In a Bench Top Airway Model

Introduction: Ventilator associated pneumonia (VAP) is a serious and costly clinical problem. Specifically, receiving mechanical ventilation over a 48 hour time period increases the risk of VAP and is associated with high morbidity, mortality and medical costs. Endotracheal tubes (ETTs) are essential to the process of mechanical ventilation. Endotracheal tubes present a special concern to clinicians because they are often colonized by oropharyngeal bacteria during long-term mechanical ventilation.  Cost effective ETTs that are resistant to bacterial infection would be essential tools in the prevention of VAP. The objective of this study was two fold, first to develop strategies to decrease bacterial adhesion on ETTs using nanotechnology and secondly to develop better methods to assess in vitro bacterial adhesion and biofilm formation on ETTs using a bench top experimental model and computer simulations of air flow.

Materials and Methods: Nanoroughened ETT were created by exposing polyvinyl chloride (PVC) ETTs (Sheridan^®) to a 0.1% mass solution of R. arrhizus lipase dissolved in a potassium phosphate buffer.  The samples were gently agitated for a total of 48 hours at 37° C and lipase media was replaced every 24 hrs. The tubes were then analyzed using scanning electron microscopy and atomic force microscopy. Static studies were performed to analyze a bacterium commonly found in VAP, Staphylococcus aureus (ATCC #25923).  These two bacterial strains were inoculated into trypticase soy broth (TSB) media. PVC pieces were then immersed into the one of the inoculated media and into control containing media without bacteria. Bacterial growth on the surface of the PVC was assessed at 4, 12, 24, and 72 hour time points for TSB media.  The bacteria found on these samples were stained with crystal violet.  Biofilm formation was then analyzed using optical density. Nanomodified ETTs were tested in a custom made bench top model airway. Endotracheal tubes placed in the system were modified on both the inner and outer surfaces. Sterilization tests were performed routinely to detect any cross contamination. For each run, the system was inoculated with 10³S. aureus.  To quantify the number of bacteria within the system, 1 mL samples of the fluid in the oropharynx chamber and the lung chamber were extracted every twelve hours. The samples were then diluted, plated, and incubated for 48 hours. At the end of each trial ETT were cut into ten 1.5 cm pieces.   These tubes were either stained with crystal violet and analyzed with optical density or processed using a vortexing method to determine the number of bacteria on the surface of the tube. Fluid analysis of the tubes consisted of an analysis of the curvature of the tubes and the creation of a 3D finite element model to simulate flow within our system.

 Results and Discussion: Results showed that nanomodified PVC ETTs were effective at reducing bacterial colonization in static studies. Reduced S. aureus was observed on the nanomodified ETTs at the 12 and 24 hr time points in TSB media. Twenty-four hour studies performed in the dynamic flow chamber showed a marked difference in the biofilm formation on different areas of untreated tubes.   Areas of tube curvature, such as at the entrance to the mouth and the connection between the oropharynx and the larynx, were correlated with larger amounts of bacteria on the untreated tubes. Notably, the dynamic values for biofilm density along many areas of the tube were substantially less than the static values obtained in our stagnant media samples illustrating the importance of dynamic media analysis of ETT. Our computational model shows skewing of the velocity profile at both curves in the ETT, with consequent variations in the wall shear rates along the tube.  Areas of low wall shear created by this secondary motion in the curved portion of the tube, correspond to experimental areas of high bacterial density.

Conclusions:  Both static and dynamic studies show lipase etching can create nano-rough surface features on PVC ETTs that suppresses S. aureus growth.  Additionally, flow conditions within the ETT influenced both the location and concentration of bacterial growth on the ETT.  The differences in both bacterial number and optical density recorded along the length of the tube suggest that wall shear stress plays a significant role in bacterial colonization. The results of both the static and dynamic models suggest that nanomodified tubes could provide clinicians with an effective and inexpensive tool to combat hospital acquired infections like VAP, and should be studied in greater depth.

Acknowledgements: The authors would like to thank the Hermann Foundation for funding.