Effect Of Ambroxol Combined With Vancomycin On Bacterial Killing Of Staphylococcus Epidermidis Biofilms And Their Regulatory Genes

PART IEffect of ambroxol combined with vancomycin on bacterial killing ofStaphylococcus epidermidis biofilms【Objective】 To establish mature Staphylococcus epidermidisRP62A strain’s biofilm modal on Polyethylene disc in vitro. To investigatethe effect of ambroxol combined with vancomycin on bacterial killing ofStaphylococcus epidermidis biofilms (BF).【Methods】In vitro models of24h RP62A BF were established, anddivided into four groups: the blank control group, vancomycin group,ambroxol group, ambroxol and vancomycin group; After treatment，Themetabolic activity of the bacteria inside BF was measured by tetrazoliumsalt (XTT) reduction assay.【Result】The mature Staphylococcus epidermidis RP62A strain’sbiofilm was observed by microscope. Bacteria adhered to each other and gathered by groups，connected by silk like Extracellular polysaccharides（EPS）.Results of XTT assays showed that the OD450values of blankcontrol group, vancomycin group, ambroxol group, ambroxol combinedwith vancomycin group were2.867±0.425,2.869±0.325,1.812±0.210and0.875±0.191, vancomycin group compared with blank control group haveno significant difference, P>0.05,ambroxol group, ambroxol andvancomycin group compared with blank control group have significantdifference, P﹤0.05, ambroxol&vancomycin group compared withambroxol group have significant difference,P﹤0.05.【Conclusion】Vancomycin alone had no effect on biofilms，whenambroxo（l1.875mg/ml）showed a significant effect on biofilms. ambroxolcombined with vancomycin could killed the bacterium in Staphylococcusepidermidis biofilms (BF). Ambroxol had the synergistic effect withvancomycin, have significant role in the killing of Staphylococcusepidermidis BF.PART ⅡEffect of ambroxol combined with vancomycin on structures ofStaphylococcus epidermidis biofilms【Objective】 To investigate the effect of ambroxol combined withvancomycin on structures of Staphylococcus epidermidis biofilms. Toinvestigate the ambroxol had the synergistic effect with vancomycin.【Methods】In vitro models of24h RP62A BF were established, and divided into four groups: the blank control group, vancomycin group,ambroxol group, ambroxol and vancomycin group; After treatment，BFwas stained by SYTO9/PI, monitored by confocal laser scanningmicroscopy(CLSM), quantification of the effect be calculated by imagestructure analyzer (ISA) software.【Result】CLSM images showed that the control group was major oflive bacteria with green fluorescence,mixed with few red fluorescence ofdead bacteria. Vancomycin group was similar with the control group.ambroxol group showed that the distribution of green fluorescencedecreased with red fluorescence of dead bacteria increased. Andambroxol and vancomycin group had a better effect compared withambroxol group. From the data of ISA softwe,we found that, ambroxolgroup, ambroxol&vancomycin group compared with blank control group,the thickness, average diffusion distance(ADD), textual entropy(TE) wasreduced（P﹤0.05）and areal porosity(AP) was increased（P﹤0.05）.Inaddition ambroxol and vancomycin group had more pronounced effectcompared with ambroxol group.【Conclusion】Vancomycin alone had no effect on structures ofStaphylococcus epidermidis biofilms.，when ambroxol（1.875mg/ml）showed a significant effect on structures of biofilms. Ambroxol had thesynergistic effect with vancomycin, ambroxol combined with vancomycincould destroyed the structures of Staphylococcus epidermidis biofilms (BF), made them loose, simple and monolayer, have significant role in thekilling of Staphylococcus epidermidis BF.PART ⅢEffect of ambroxol combined with vancomycin on the expression ofregulatory genes of Staphylococcus epidermidis biofilms【Objective】 To investigate the effect of ambroxol combined withvancomycin on the expression of regulatory genes (agr icaR icaA) ofStaphylococcus epidermidis biofilms【Methods】In vitro models of24h RP62A BF were established, anddivided into four groups: the blank control group, vancomycin group,ambroxol group, ambroxol and vancomycin group; After treatment，theexpression of agr icaR icaA was observed by reverse transcriptionRT-PCR.【Result】Results of RT-PCR showed that the expression of agricaR icaA genes of vancomycin group compared with blank controlgroup have no significant difference, P>0.05. Ambroxol group, ambroxol&vancomycin group compared with blank control group, the expressionof agr icaR was increased（P﹤0.05）, in addition ambroxol andvancomycin group had more pronounced effect compared with ambroxolgroup. Ambroxol group, ambroxol&vancomycin group compared withblank control group, the expression of icaA was decreased（P﹤0.05）, inaddition ambroxol and vancomycin group had more pronounced effect compared with ambroxol group.【Conclusion】Ambroxol had effect on the expression of agr icaRicaA genes of Staphylococcus epidermidis biofilms, and ambroxolcombined with vancomycin had more pronounced effect compared withambroxol group, thus inhibiting Staphylococcus epidermidis biofilms.