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Applied and Environmental Microbiology, June 2003, p. 3412-3420, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3412-3420.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Microbial Diversity of Biofilms in Dental Unit Water Systems

Ruby Singh,¹ O. Colin Stine,² David L. Smith,² John K. Spitznagel Jr.,³ Mohamed E. Labib,⁴ and Henry N. Williams^1*

Department of Oral and Craniofacial Biological Sciences,¹ Department of Epidemiology and Preventive Medicine,² Department of Periodontics, University of Maryland, Baltimore, Maryland 21201,³ Novaflux Technologies, Princeton, New Jersey 08540⁴

Received 4 September 2002/ Accepted 20 February 2003

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 ß 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.

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* Corresponding author. Mailing address: Department of Oral & Craniofacial Biological Sciences, 666 West Baltimore St., Dental School, University of Maryland, Baltimore, MD 21201. Phone: (410) 706-7211. Fax: (410) 706-0963. E-mail: hnw001{at}dental.umaryland.edu.

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Applied and Environmental Microbiology, June 2003, p. 3412-3420, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3412-3420.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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