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

<jats:title>Abstract</jats:title><jats:sec><jats:title>Aim</jats:title><jats:p>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.</jats:p></jats:sec><jats:sec><jats:title>Methodology</jats:title><jats:p>Nutrient‐stressed biofilms (<jats:italic>Propionibacterium acnes</jats:italic>,<jats:italic> Staphylococcus epidermidis</jats:italic>,<jats:italic> Actinomyces radicidentis</jats:italic>,<jats:italic> Streptococcus mitis</jats:italic> and <jats:italic>Enterococcus faecalis </jats:italic><jats:styled-content style="fixed-case">OMGS</jats:styled-content> 3202) were grown in prepared root canals of single‐rooted teeth. The treatment groups included 1% trypsin and 2% chlorhexidine (<jats:styled-content style="fixed-case">CHX</jats:styled-content>), 1% proteinase K and 2% <jats:styled-content style="fixed-case">CHX</jats:styled-content> (with and without ultrasonics). 2% <jats:styled-content style="fixed-case">CHX</jats:styled-content> 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 (<jats:styled-content style="fixed-case">CLSM</jats:styled-content>) with live/dead staining and quantitative microbial culture.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>The trypsin and <jats:styled-content style="fixed-case">CHX</jats:styled-content> group with ultrasonics was significantly more effective in reducing viable counts and the <jats:italic>substratum</jats:italic> coverage than those of all other groups (<jats:italic>P</jats:italic> &lt; 0.05). The viable counts of the proteinase K and <jats:styled-content style="fixed-case">CHX</jats:styled-content> group used with (4.26 ± 0.58 log<jats:sub>10</jats:sub> cfu mL<jats:sup>−1</jats:sup>) or without ultrasonics (5.05 ± 1.36 log<jats:sub>10</jats:sub> cfu mL<jats:sup>−1</jats:sup>) were significantly reduced (<jats:italic>P</jats:italic> &lt; 0.05) as compared with the untreated control (7.67 ± 0.84 log<jats:sub>10</jats:sub> cfu mL<jats:sup>−1</jats:sup>) and saline groups used with (6.57 ± 0.73 log<jats:sub>10</jats:sub> cfu mL<jats:sup>−1</jats:sup>) and without ultrasonics (6.74 ± 0.10 log<jats:sub>10</jats:sub> cfu mL<jats:sup>−1</jats:sup>). The <jats:styled-content style="fixed-case">CHX</jats:styled-content> group was less effective in biofilm disruption compared to when used in combination with trypsin and proteinase K.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>The trypsin and <jats:styled-content style="fixed-case">CHX</jats:styled-content> group with ultrasonics was significantly more effective at reducing bacterial viable counts and disrupting biofilm.</jats:p></jats:sec>