Intubation and mechanical ventilation lead to an extensive expansion of VAP-associated bacterial species in respiratory secretions of ventilated pigs

Abstract

Ventilator associated pneumonia (VAP) remains one of the most critical nosocomial infections in intensive care medicine. Between 20-50% of ventilated patients develop VAP during treatment, often leading to long term sequelae and death, despite successful therapy of their underlying disease. VAP is typically caused by Gram-negative bacteria that can previously be detected on the mucosal membranes of the oral and upper respiratory tract. To assess and quantify bacterial colonization of the respiratory tract following intubation and ventilation a well-established pig model was used. Methods: After induction of anaesthesia and endotracheal intubation pigs underwent mechanical ventilation continuously for up to three days. To assess bacterial colonization of the upper respiratory tract, samples of respiratory secretions were taken at day 0, 1 and 2 directly above the cuff outside of the endotracheal tube (ETT) and by aspiration of tracheal secretions through the ETT. Quantification of bacteria was carried out by serial dilutions and CFU/ml determinations on sheep blood agar and MacConkey agar plates. Species identification was achieved by 16S-RNA gene sequencing. Results: Respiratory samples of 19 pigs were analysed and showed a sharp increase of predominantly Gram-negative organisms in the respiratory material sampled above the cuff from day 0 to 1. Starting at a median of 7.3 x 103 immediately after intubation numbers increased to a median of 2.0 x 107 Gram-negative bacteria at day 1 and 3.2 x 107 at day 2. Gram-negative species that were detected included Pseudomonas aeruginosa, Acinetobacter baumanii, Enterobacter aerogenes, Klebsiella pneumoniae and other Enterobacteriaceae. Tracheal secretions contained much less bacteria. While no Gram-negative bacteria could be cultured at the beginning of the experiment in 18 of 19 animals, colonization with Gram-negative bacteria gradually increased and was found in 7 of 19 and 9 of 14 animals on day 1 and day 2 respectively. Interestingly, in the vast majority of colonized tracheal secretions the identical Gram-negative species had previously been detected in secretions collected > 24h earlier at the cuff. Conclusion: A massive increase of the concentration of Gram-negative bacteria occurred at 24h of ventilation in mucosal secretions above the cuff. Detected species were the same Gram-negative bacteria that are frequently responsible for human infections, suggesting that especially the accumulation of bacteria in secretions above the cuff may be crucial in the development of VAP.