Molecular analysis of biofilms on the surface of neonatal endotracheal tubes based on 16S rRNA PCR-DGGE and species-specific PCR.

Ventilator-associated pneumonia (VAP) results in considerable morbidity and mortality in neonatal intensive care units. VAP is associated with polymicrobial biofilms that form on endotracheal tubes (ETTs). We aimed to evaluate the diversity and the bacterial community in biofilms on ETTs extubated from mechanically ventilated newborns. ETTs (N = 29) and aerobic sputum cultures were obtained from 20 mechanically ventilated newborns. Denaturing gradient gel electrophoresis (DGGE) was used to characterize the bacterial species in the biofilms on the ETTs. Species-specific PCR was used to detect common oropharyngeal Streptococcus species and known ETT-associated pathogens. DGGE profiling of ETT biofilms showed multiple banding patterns indicating a diverse bacterial community. The dominant bacterial species were Klebsiella spp. (29/29), Streptococcus spp. (27/29), and Pseudomonas spp. (24/29). The most frequently occurring Streptococcus species was Streptococcus mitis (N = 18). Oropharyngeal bacteria were present in 25 of 29 ETT specimens. Streptococcus spp. often co-existed with K. pneumoniae and/or P. aeruginosa. In contrast, only one bacterial species was isolated from each sputum culture, K. pneumoniae or Acinetobacter baumannii. Our results demonstrated that Klebsiella spp., Streptococcus spp., and Pseudomonas spp. were the most frequent microbes on the surface of neonatal ETTs. The co-existence of oral commensals and pathogenic bacteria on the same tubes may play a crucial role for biofilm formation.