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 beta and gamma, but not the alpha, 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.

FIG. 1.

SEM of DT lumen surface shown at ×2,500. Cocci and rods are shown in a dense biofilm matrix.

FIG. 2.

Phylogram of 16S rDNA gene sequenced from 165 subcloned 16S fragments from community DNA without cultivation.

FIG. 3.

Phylogram of 16S rDNA gene sequenced from 55 cultured isolates.

FIG. 4.

Distribution of the number of genera detected as a function of the number of sequences analyzed. The dotted line represents the cultivated isolates, showing 55 sequences representing 13 genera, and the solid line represents the uncultured community, in which 165 sequences revealed 40 genera. Culture-independent methods found new genera more efficiently than culture-dependent methods.