PCR DGGE to Unmask Streptococcus mutans in Endotrach Biofilms

OBJECTIVE: Mounting evidence is highlighting the importance of oral flora as a reservoir of pathogens historically associated with ventilator-associated pneumonia (VAP). Here, using PCR and DGGE (Denaturing Gradient Gel Electrophoresis), we wanted to unmask the genetic diversity in the plaque-like matrix, we called Bio-Plaque, while incorporating non-culture technique to better define the Viable, But Non-Cultivable (VBNC) flora. It was our hypothesis that Streptococcus mutans would act as an early colonizer forming the initial step in the co-habitation of other oral flora in the ETT.

METHODS: 20 endotrachs were recovered from ICU patients at the University Hospital of Wales, Cardiff. Sampling included the physical removal of the biofilm within a defined area of the endotrach lumen. Quantification of cultable organisms (aerobic, anaerobic and yeast) was achieved using a Spiral Plating System while molecular detection was accomplished using DGGE, following 16S RNA amplification of a 200 base pair target. DGGE marker controls included: dental plaque, Strep mutans, Staph aureus and P. gingivalis. Eighteen clinical parameters were recorded.

RESULTS: Traditional culture yielded two/three organisms/ETT bioburden/cm2 independent of Early (<5 days) vs. Late (>5 days) in length of mechanical ventilator (MV). One-third yielded Candida independent of LOS. PCR-DGGE yielded a minimum of three recognized strains and a maximum of 17, also, independent of length of MV. However, Strep mutans colonization was detected in 8/14 Early ETT MV while only 2/6 Late MV by DGGE; none by culture. Staph aureus, E. corredens and P. gingivalis were the other viable isolates.

CONCLUSION: Strep mutans is a frequent organism in the diverse microbiota of colonized luminal endotrachs (Bio-Plaque) suggesting significant importance in Early co-colonization of oral origin .