Synergistic effect of 2% chlorhexidine combined with proteolytic enzymes on biofilm disruption and killing

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To investigate the dynamics of a disinfection regimen using 1% trypsin and 1% proteinase K in combination with 2% chlorhexidine (with or without ultrasonics) using a nutrient-stressed endodontic multispecies model biofilm.


Nutrient-stressed biofilms (Propionibacterium acnes, Staphylococcus epidermidis, Actinomyces radicidentis, Streptococcus mitis and Enterococcus faecalis OMGS 3202) were grown in prepared root canals of single-rooted teeth. The treatment groups included 1% trypsin and 2% chlorhexidine (CHX), 1% proteinase K and 2% CHX (with and without ultrasonics). 2% CHX was the positive control and untreated group, and sterile saline (with and without ultrasonics) was the negative control. The biofilms were investigated using confocal laser scanning microscopy (CLSM) with live/dead staining and quantitative microbial culture.


The trypsin and CHX group with ultrasonics was significantly more effective in reducing viable counts and the substratum coverage than those of all other groups (P < 0.05). The viable counts of the proteinase K and CHX group used with (4.26 ± 0.58 log10 cfu mL−1) or without ultrasonics (5.05 ± 1.36 log10 cfu mL−1) were significantly reduced (P < 0.05) as compared with the untreated control (7.67 ± 0.84 log10 cfu mL−1) and saline groups used with (6.57 ± 0.73 log10 cfu mL−1) and without ultrasonics (6.74 ± 0.10 log10 cfu mL−1). The CHX group was less effective in biofilm disruption compared to when used in combination with trypsin and proteinase K.


The trypsin and CHX group with ultrasonics was significantly more effective at reducing bacterial viable counts and disrupting biofilm.

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