Clinical trials evaluating the efficacy of nonoxynol-9 (N-9) as a topical microbicide concluded that N-9 offers no in vivo protection against human immunodeficiency virus type 1 (HIV-1) infection, despite demonstrated in vitro inactivation of HIV-1 by N-9. These trials emphasize the need for better model systems to determine candidate microbicide effectiveness and safety in a preclinical setting. To that end, time-dependent in vitro cytotoxicity, as well as in vivo toxicity and inflammation, associated with N-9 exposure were characterized with the goal of validating a mouse model of microbicide toxicity. In vitro studies using submerged cell cultures indicated that human cervical epithelial cells were inherently more sensitive to N-9-mediated damage than human vaginal epithelial cells. These results correlated with in vivo findings obtained by using Swiss Webster mice in which intravaginal inoculation of 1% N-9 or Conceptrol gel (containing 4% N-9) resulted in selective and acute disruption of the cervical columnar epithelial cells 2 h postapplication accompanied by intense inflammatory infiltrates within the lamina propria. Although damage to the cervical epithelium was apparent out to 8 h postapplication, these tissues resembled control tissue by 24 h postapplication. In contrast, minimal damage and infiltration were associated with both short-and long-term exposure of the vaginal mucosa to either N-9 or Conceptrol. These analyses were extended to examine the relative toxicity of polyethylene hexamethylene biguanide (PEHMB), a polybiguanide compound under evaluation as a candidate topical microbicide. In similar studies, in vivo exposure to 1% PEHMB caused minimal damage and inflammation of the genital mucosa, a finding consistent with the demonstration that PEHMB was >350-fold less cytotoxic than N-9 in vitro. Collectively, these studies highlight the murine model of toxicity as a valuable tool for the preclinical assessment of toxicity and inflammation associated with exposure to candidate topical microbicides.
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.
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