Effect of Protease Inhibitors on Assessment of Oral Microbes

Commercially available protease inhibitor (PIs) cocktails have been routinely added to clinical samples for proteomic studies. However, it has been reported that several PIs are a potent inhibitor of bacterial growth and proliferation. Objectives: To determine if Halt™ Protease Inhibitor Cocktail (Thermo, Rockford) interfere quantitatively and qualitatively with the analysis of total cultivable and uncultivable bacterial colonization in the saliva. Methods: Twenty-two stimulated whole saliva samples were obtained and processed immediately with and without PIs added. Conventional cultivation method was used to evaluate cultivable bacterial growth measured by total colony-forming units (CFU) with a non-selective enrichment medium and three selective media. Meanwhile, total bacterial genomic DNA was isolated from the saliva samples; a targeted 16S rRNA fragment was amplified and separated by denaturing gradient gel electrophoresis (DGGE). Total cultivable and uncultivable bacterial composition profiles were obtained. Results: There was no significant difference in the mean CFU counts between the PIs and non-PIs groups. We also observed a high degree of correlation between the paired samples for total cultivable microbiota (r² = 0.867), total mutans streptococci (r² = 0.898), total oral lactobacilli (r² = 0.933), and total Streptococcus mutans (r² = 0.870). The PIs and non-PIs groups shared as much as 95.7% of similarity in total bacterial composition. Meanwhile, proteomic analysis of saliva also showed that commercial protease inhibitors have no significant effect on the integrity of saliva proteins. Conclusions: These results demonstrated that the addition of PIs in the saliva sample for proteomic analysis has no significant effect on the evaluation of total microbial cultivation and diversity. Supported by research grants U19 DE018385, DE013937, and DE015706 from NIH/NIDCR.