An In Vitro Study Of The Antibacterial Effects Of High-purity Nisin Z Alone And In Combination With Sodium Hypochlorite Against Enterococcus Faecalis

Objectives: 1.To explore the effectiveness of high-purity Nisin Z(Nisin ZP)alone and in combination with sodium hypochlorite on planktonic and biofilm populations of Enterococcus faecalis in vitro.2.To provide the theoretical basis for the application of Nisin Z in the root canal therapy.Methods: 1.The minimal inhibitory concentration(MIC)and minimal bactericidal concentration(MBC)of Nisin Z or Na Cl O against planktonic populations of Enterococcus faecalis were determined using microdilution method according to the Clinical and Laboratory Standards Institute(CLSI)standard M07-A9.2.The microdilution chequerboard assay was used to evaluate synergistic,additional,indifferent or antagonistic interactions between Nisin Z and Na Cl O according to the fractional inhibitory concentration index(FICI)of the two drugs in combination.The FICI was calculated according to the following formula.FICI=(MICa in combination/MICa alone)+(MICb in combination/MICb alone).Synergy was defined when FICI≤0.5 and antagonism when FICI>4.The FICI between 0.5 and 1 was interpreted as addition and between 1 and 4 as indifference.3.The Time-Kill curves were tested for obtaining information about the dynamic interaction between Nisin Z and Na Cl O against Enterococcus faecalis strain.This procedure has been well described in CLSI M26-A document.4.Enterococcus faecalis biofilms grown in wells of the 96-well cell culture plates for 24 hours were randomly diveded into different groups and treated for 24 hours with various concentrations of the Nisin Z solution,with B1(256×MIC),B2(64×MIC),B3(16×MIC),B4(4×MIC),B5(1×MIC)and B6(sterile three-steamed water)as negative control or B7(2.5% Na Cl O)as positive control.The MTT assay was then used to compare the antimicrobial activities of different groups according to the optical density.The same volume of Nisin Z and various concentrations of Na Cl Owere mixed and divided as C1(1×MIC Nisin Z+1% Na Cl O),C2(1×MIC Nisin Z+0.5% Na Cl O)and C3(1×MIC Nisin Z+0.25% Na Cl O).Biofilms were randomly diveded into different groups and treated for 24 hours with different concentrations of C1(1×MIC Nisin Z+1% Na Cl O),C2(1×MIC Nisin Z+0.5% Na Cl O),C3(1×MIC Nisin Z+0.25% Na Cl O)and C4(sterile three-steamed water)as negative control or C5(2.5% Na Cl O)as positive control.The MTT method was conducted as above.5.Enterococcus faecalis biofilms formed on glass slides in wells of the 6-well cell culture plates for 24 hours were randomly diveded into different groups and treated for 24 hours with Nisin Z or Nisin Z-Na Cl O combinations,with D1(1×MIC),D2(4×MIC),D3(16×MIC),D4(64×MIC),D5(256×MIC),D6(1×MIC Nisin Z+0.25% Na Cl O),D7(1×MIC Nisin Z+0.5% Na Cl O),D8(1×MIC Nisin Z+1% Na Cl O)and D9(2.5% Na Cl O)as positive control or D10(sterile three-steamed water)as negative control.The treated biofilms were stained by SYTO-9/PI and examined through CLSM to observe the bacteria staining and the disruption of the E.f biofilms.Results: 1.The MIC of Nisin Z was observed at 6μg/ml,with the MBC of 15μg/ml.The MIC and MBC of Na Cl O were 0.0625% and 0.25%.2.The minimal inhibitory concentration was obtained when Nisin Z of 0.023μg/ml(1/256×MIC)was combined with Na Cl O of 0.03125%(1/2×MIC).The FICI was 0.504,indicating the interaction between Nisin Z and Na Cl O was additional effect.3.The 24-hours Time-Kill curves showed that Nisin Z or Na Cl O decreased the growth of planktonic E.faecalis in a dose-dependent manner and showed noticeable antibacterial effects from 2×MIC on and bactericidal activities with 4×MIC,when the two tested drugs used alone.Furthermore,when used together under the same concentrations,they showed bactericidal effect in shorter time.4.There were differences in the OD values of the treated biofilms when groups of B1(256×MIC),B2(64×MIC),B3(16×MIC),B4(4×MIC)and B5(1×MIC)compared with B6(NC)(P<0.05),or groups of B1(256×MIC),B2(64×MIC),B3(16×MIC),B4(4×MIC)and B5(1×MIC)compared with B7(PC)(P<0.05).As to pairwise comparisons in groups of B1(256×MIC),B2(64×MIC),B3(16×MIC),B4(4×MIC)and B5(1×MIC),there were no statistically significant differences between B2(64×MIC)and B3(16×MIC)(P>0.05)and there were statistically significant differences between any two groups in the rest(P<0.05).There were significant differences in the OD values of the treated biofilms when groups of C1(1×MIC Nisin Z+1% Na Cl O),C2(1×MIC Nisin Z+0.5% Na Cl O)and C3(1×MIC Nisin Z+0.25% Na Cl O)compared with C4(NC)(P<0.05),or groups of C1(1×MIC Nisin Z+1% Na Cl O),C2(1×MIC Nisin Z+0.5% Na Cl O)and C3(1×MIC Nisin Z+0.25% Na Cl O)compared with C5(PC)(P<0.05).As to pairwise comparisons in groups of C1(1×MIC Nisin Z+1% Na Cl O),C2(1×MIC Nisin Z+0.5% Na Cl O)and C3(1×MIC Nisin Z+0.25% Na Cl O),there were no statistically significant differences between C1(1×MIC Nisin Z+1% Na Cl O)and C2(1×MIC Nisin Z+0.5% Na Cl O)(P>0.05)and there were statistically significant differences between C1(1×MIC Nisin Z+1% Na Cl O)and C3(1×MIC Nisin Z+0.25% Na Cl O)(P<0.05)or between C2(1×MIC Nisin Z+0.5% Na Cl O)and C3(1×MIC Nisin Z+0.25% Na Cl O)(P<0.05).5.The images of the biofilms treated by various concentrations of Nisin Z showed that the proportion of green fluorescence decreased while the red fluorescence increased and the structure thinned out as the concentration of Nisin Z increased in groups of D1(1×MIC),D2(4×MIC),D3(16×MIC),D4(64×MIC)and D5(256×MIC).The vast majority of the microscope fields were red stained bacteria in Nisin Z-Na Cl O combination groups of D6(1×MIC Nisin Z+0.25% Na Cl O),D7(1×MIC Nisin Z+0.5% Na Cl O)and D8(1×MIC Nisin Z+1% Na Cl O),leading to the superposition of the overlayed views being red or saffron yellow.With the concentration of Na Cl O increasing,the number of the whole bacteria in the biofilms decreased,and the biofilms were significantly disrupted,becoming few and scattered.After treated with D9(PC),there were sparse biofilms left,with the overall fluorescence,mainly red,diminishing.It could be seen in the D10 group(NC)that the biofilm bacteria were dense and layered.Most of the bacteria were stained green with scattered red bacteria and the superposition graph was mainly green.Conclusions: 1.Nisin Z exerts good antibacterial effect against planktonic Enterococcus faecalis.2.Nisin Z or Na Cl O inhibits E.faecalis dose-dependently.In vitro Nisin Z has an additional effect on the activity of sodium hypochlorite against Enterococcus faecalis,with the two drugs combined to reduce the using concentrations and shorter the working time.3.Nisin Z has a certain inhibitory effect on the biofilms of Enterococcus faecalis,with enhanced final anti-biofilm activity when combined with Na Cl O.