Molecular Analysis for Bacterial Contamination in Dental Unit Water Line

Objectives: The contamination of opportunistic and antibiotic-resistant pathogens in dental unit water lines (DUWL) is a recent increasing concern in dentistry. The aim of this study was to monitor and evaluate the bacterial contamination in DUWL by molecular biological techniques in addition to usual culture method. Methods: DUWL samples (n=10) and hand wash units (HWU) samples (n=2) were collected. 1) Culture method; The water samples were plated on R2A agar for heterotrophic bacteria and WYO-alfa agar for Legionella pneumophila, and incubated (25^oC and 37^oC, 7d) respectively, to obtain total bacterial counts in colony forming units(CFU) per millilitre. 2) Molecular biological methods; Bacterial community structure forming biofilm in the DUWL were analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) and following the 16S rRNA genes were sequenced. Antibiotic-resistant pathogens, MRSA, multi-drug resistant Pseudomonas and ESBL-producing bacteria, in the DUWL were identified by PCR to amplify the selected bacterial antibiotic-resistant genes, mecA, blaIMP, blaVIM and blaTEM,respectively. Contaminations of L. pneumophila were also checked by using PCR for the 16S rRNA gene. Results: Colony counts in R2A agar from water samples of five DUWL and all HWU were below 500-CFU/ml. PCR-DGGE analysis revealed that Novosphingobium sp. shown to behave as opportunistic pathogens were most prevalent and dominant contaminated bacteria in DUWL. Dechoromonas sp., Blastobacter sp. and Sphingomonadaceae sp. were also identified as minor contaminants in DUWL. No bacterial antibiotic-resistant genes and L. pneumophila 16S rRNA gene was detected in all tested DUWL and HWU samples. Conclusion: Bacteria in aquatic environment are somewhat fastidious being difficult to isolate and culture. PCR-DGGE was shown to be a potentially useful molecular analytical tool for monitoring DUWL bacterial contamination. Conventional PCR for antibiotic-resistant gene could to be applied for rapid monitoring and tracing the source of nosocomial infection through DUWL.