Effect of protease inhibitors on the quantitative and qualitative assessment of oral microorganisms

Abstract

Protease inhibitor cocktails are routinely added to clinical samples used for proteomic studies to inactivate proteases. Since these same samples are often used for microbial studies, we determined whether addition of protease inhibitors could affect the quantitative or qualitative assessment of microbial profiles. Twenty-two saliva samples were collected and processed immediately with or without the addition of a protease inhibitor cocktail. Conventional cultivation methods were used to evaluate total bacterial growth. Total genomic DNA was isolated and a specific 16S rRNA gene-targeted region was PCR-amplified and separated by denaturing gradient gel electrophoresis. A combination of 1D SDS-PAGE and LC-MS/MS methods was used to determine the effect of the protease inhibitors on the integrity of salivary proteins and peptides. Interestingly, no significant differences were observed in either the bacterial growth and composition or the integrity of salivary proteins between the two groups. Correlation coefficients between the paired samples for total cultivable microbiota (r2=0.847), total mutans streptococci (r2=0.898), total oral lactobacilli (r2=0.933), and total Streptococcus mutans (r2=0.870) also exceeded expected values. The results suggest that the addition of a protease inhibitor cocktail in saliva samples does not impact the growth of oral microbiota or compromise the ability to characterize its composition.

Baseline burden and antimicrobial susceptibility of pathogenic bacteria recovered from oral lesions of patients with HIV/AIDS in South‐Western Uganda

“…The candle glows till extinction to create an atmosphere containing reduced oxygen and increased carbon dioxide to support the growth of fastidious aero‐tolerant oral pathogenic bacteria species. Overnight broth suspensions were plated out on a BHI agar supplemented with 5% sheep blood to get a blood agar, which was then incubated overnight on reduced oxygen tension as mentioned above [–].…”

“…The candle glows till extinction to create an atmosphere containing reduced oxygen and increased carbon dioxide to support the growth of fastidious aero-tolerant oral pathogenic bacteria species. Overnight broth suspensions were plated out on a BHI agar supplemented with 5% sheep blood to get a blood agar, which was then incubated overnight on reduced oxygen tension as mentioned above [26][27][28]. Inoculation of samples onto prepared media was done aseptically, and after an 18-48-h incubation, suspect pathogenic bacteria species of clinical significance (>25 discrete colonies per culture plate) were picked for further identification.…”

HIV Infection Affects Streptococcus mutans Levels, but Not Genotypes

“…Detailed procedures for oral bacterial cultivation, S. mutans identification, chromosomal DNA isolation, and genotyping have been described elsewhere (Li and Caufield, 1995;Liu et al, 2010;Appendix). Briefly, after a 72-hour anaerobic incubation (85% N 2 , 10% CO 2 , and 5% H 2 ) at 37°C, the colony-forming units (CFU) were counted for evaluation of S. mutans levels.…”

HIV Infection and Microbial Diversity in Saliva

“…Whole-saliva samples (1 ml) were used for bacterial genomic DNA extraction, as described previously (34,35). Briefly, each saliva sample was centrifuged (18,000 ϫ g for 3 min), the supernatant was discarded, and total bacterial genomic DNA was extracted from the pellet using a DNA purification kit (MasterPure; Epicentre, Madison, WI).…”