BACKGROUND: Pseudomonas aeruginosa frequently colonises intubated patients and causes life-threatening ventilator-associated pneumonia (VAP). The role of quorum sensing (QS), regulating virulence in this pathogen, during colonisation and development of VAP is unknown. METHODS: P aeruginosa isolates and tracheal aspirates were prospectively collected from intubated patients. Genotypes and QS-independent virulence traits (exoU, exoS, PAPI-1 and PAPI-2) harboured by colonising isolates were identified in vitro with the CLONDIAG array. The production of elastase and rhamnolipids was measured to assess QS-dependent virulence. To monitor QS activity 'in patient', total RNA was extracted directly from tracheal aspirates and expression of QS genes was measured. RESULTS: 320 P aeruginosa isolates and tracheal aspirates were obtained from 29 patients of whom 6 developed VAP (20%). Seven patients (24%) were initially colonised by QS-proficient isolates; 57% of them developed VAP as compared with 9% of patients colonised by QS-deficient isolates (p=0.018). Of all tested virulence traits from the initial colonising isolates, only rhamnolipids were associated with development of VAP (p=0.003). VAP occurred more frequently in patients colonised during the entire observation period by isolates producing high levels of rhamnolipids (p=0.001). 'In patient' monitoring of QS genes showed non-induced expression profiles in patients without VAP. In contrast, exponential induction of QS circuit and target gene expression was observed for two patients with VAP, and an 'in patient' QS gene expression profile and hierarchy similar to those in vitro was measured for one patient with VAP. CONCLUSIONS: Production of the QS-dependent virulence factor rhamnolipids by colonising P aeruginosa isolates is associated with development of VAP.