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

Protease inhibitor cocktails are routinely added to clinical samples used for proteomic studies to inactivate proteases. As these same samples are often used for microbial studies, we determined whether the 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 sodium dodecyl sulfate polyacrylamide gel electrophoresis 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.