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Ventilator-associated pneumonia (VAP) is the most common nosocomial infections in patients admitted to the ICU. The adapted island model predicts several changes in the respiratory microbiome during intubation and mechanical ventilation.We hypothesised that mechanical ventilation and antibiotic administration decrease the diversity of the respiratory microbiome and that these changes are more profound in patients who develop VAP.Intubated and mechanically ventilated ICU-patients were included. Tracheal aspirates were obtained three times a week. 16S rRNA gene sequencing with the Roche 454 platform was used to measure the composition of the respiratory microbiome. Associations were tested with linear mixed model analysis and principal coordinate analysis.111 tracheal aspirates were obtained from 35 patients; 11 had VAP, 18 did not have VAP. Six additional patients developed pneumonia within the first 48 hours after intubation. Duration of mechanical ventilation was associated with a decrease in α diversity (Shannon index; fixed-effect regression coefficient (β): −0.03 (95% CI −0.05 to −0.005)), but the administration of antibiotic therapy was not (fixed-effect β: 0.06; 95% CI −0.17 to 0.30). There was a significant difference in change of β diversity between patients who developed VAP and control patients for Bray-Curtis distances (p=0.03) and for Manhattan distances (p=0.04). Burkholderia, Bacillales and, to a lesser extent, Pseudomonadales positively correlated with the change in β diversity.Mechanical ventilation, but not antibiotic administration, was associated with changes in the respiratory microbiome. Dysbiosis of microbial communities in the respiratory tract was most profound in patients who developed VAP.