Microbial Diversity of Biofilms in Dental Unit Water Systems

Abstract

We investigated the microbial diversity of biofilms found in dental unit water systems (DUWS) by three methods. The first was microscopic examination by scanning electron microscopy (SEM), acridine orange staining, and fluorescent in situ hybridization (FISH). Most bacteria present in the biofilm were viable. FISH detected the ␤ and ␥, but not the ␣, subclasses of Proteobacteria. In the second method, 55 cultivated biofilm isolates were identified with the Biolog system, fatty acid analysis, and 16S ribosomal DNA (rDNA) sequencing. Only 16S identified all 55 isolates, which represented 13 genera. The most common organisms, as shown by analyses of 16S rDNA, belonged to the genera Afipia (28%) and Sphingomonas (16%). The third method was a culture-independent direct amplification and sequencing of 165 subclones from community biofilm 16S rDNA. This method revealed 40 genera: the most common ones included Leptospira (20%), Sphingomonas (14%), Bacillus (7%), Escherichia (6%), Geobacter (5%), and Pseudomonas (5%). Some of these organisms may be opportunistic pathogens. Our results have demonstrated that a biofilm in a health care setting may harbor a vast diversity of organisms. The results also reflect the limitations of culture-based techniques to detect and identify bacteria. Although this is the greatest diversity reported in DUWS biofilms, other genera may have been missed. Using a technique based on jackknife subsampling, we projected that a 25-fold increase in the number of subclones sequenced would approximately double the number of genera observed, reflecting the richness and high diversity of microbial communities in these biofilms.

Novel Model for Multispecies Biofilms That Uses Rigid Gas-Permeable Lenses

“…An important aspect of this approach was a requirement for the bacteria to continue to survive and replicate following staining by the dyes. Traditional nucleic acid stains, such as acridine orange, hexidium iodide, and Hoechst 33342, have been used to stain multiple species of planktonic and biofilm bacteria for visualization using standard fluorescent microscopy (21,46) and confocal laser scanning microscopy (10,27). However, generally these have been used as terminal analytical approaches for bacterial counts and/or viability (30,45) or to provide an evaluation of bacterial aggregation onto solid surfaces (10, 27) with minimal assessment for visualizing and maintaining cell viability.…”

Evaluation of Bacterial Contamination in Dental Unit Waterlines of Qazvin’ Dental School ,Iran

“…In a study by Singh et al (10) in 2003, the main techniques used was advanced fluorescent in situ hybridization (FISH) and electron microscopy. They investigated the biofilm obtained from dental unit water lines (DUWL) and reported that the high contamination prevalence in dental unit water samples could be the result of high amounts of microbial biofilms enriched with bacteria along the narrow waterlines of these units.…”

The microbial profiles of dental unit waterlines in a dental school clinic

“…These findings are similar to previous studies. 22,23 Sphingomonas paucimobilis was reported to cause outbreaks of bacteremia among immunocompromised patients in hematology and oncology units due to bacterial contamination of hospital water systems. 24 It is now emerging as an opportunistic pathogen that is frequently reported in clinical settings.…”