Research Of Low-frequency Ultrasound Combined With Ambroxol Hydrochloride On Staphylococcus Epidermidis Biofilms:Structural Changes And Bactericidal Effects

Part I： Bactericidal effects of low-frequency ultrasoundcombined with ambroxol hydrochloride on staphylococcusepidermidis biofilms【Objective】 To establish mature Staphylococcus EpidermidisRP62A strain’s biofilm （BF） modal on Polyethylene disc in vitro. ToQuantitatively Analyze the enhanced bactericidal effects of low-frequencyultrasound combined with ambroxol hydrochloride on the established S.epidermidis biofilms.【Methods】The mature Staphylococcus Epidermidis RP62Astrain’sbiofilm （BF） modal was established on Polyethylene disc in vitro. Thestructures of biofilms were observed and identified by silver staining. Aftermature biofilms were established, they were divided into seven groups withdifferent treatments, the blank control group, group treated withlow-frequency ultrasound alone for1h, group treated with ambroxol hydrochloride （concertration of0.938,1.875mg/ml） alone for24h, grouptreated with ambroxol hydrochloride （concertration of0.938,1.875mg/ml）combined with low-frequency ultrasound, group treated with ambroxolhydrochloride （concertration of1.875mg/ml） combined with low-frequencyultrasound for0.5h. After treatments, the metabolic activity of bacteriainside bioflims was quantified using the mean Optical Density （OD） at450nm wavelength in a multifunctional microplate reader by tetrazolium dye,XTT assay.【Results】 The mature Staphylococcus Epidermidis RP62A strain’sbiofilm was observed by microscope. Bacteria adhered to each other andgatherred by goups, connected by silk like Extracellular polysaccharides（EPS）. Results of XTT assays showed that the OD₄₅₀values of controlgroup, low-frequency ultrasound group were1.895±0.146、1.932±0.077（P>0.05）, when ambroxol reached a concerntration of1.875mg/ml, theOD₄₅₀ value was0.472±0.056, compared with control group, P<0.05;theOD₄₅₀ values of Ambroxol（1.875mg/ml） combined with low-frequencyultrasound（0.5h,1h） were0.104±0.052、0.064±0.048, compared with theother groups （P<0.05）.【Conclusion】 Low-frequency ultrasound alone had no effect onbiofilms, when ambroxol reached a concerntration of1.875mg/ml, itshowed a significant effect on biodilms. Low-frequency ultrasound can hadan enhanced bctericidal effects on biofilms when combined with ambroxol hydrochloride.Part II: Effects of low-frequency ultrasound combined withambroxol hydrochloride on structures of StaphylococcusEpidermidis biofilms【Objective】 To verify the enhanced effects of low-frequencyultrasound with ambroxol on the structures of the mature biofilms ofStaphylococcus Epidermidis on polyethylene disc in vitro.【Methods】The mature Staphylococcus Epidermidis RP62Astrain’sbiofilms were established on polyethylene disc in vitro. After maturebiofilms were established, they were divided into five groups with differenttreatments, the blank control group, group treated with low-frequencyultrasound alone for1h, group treated with ambroxol hydrochloride（concertration of1.875mg/ml） alone for24h, group treated with ambroxolhydrochloride （concertration of1.875mg/ml） combined with low-frequencyultrasound for1h, group treated with ambroxol hydrochloride（concertration of1.875mg/ml） combined with low-frequency ultrasound for0.5h. After treatments, they were stained with SYT09/PI and observed byConfocal Laser Scanning Microscopy, CLSM. Image Structure Analyzer，ISA was used to analyse paremeters of biofilm structures to investigate theeffects of low-frequency ultrasound combined with ambroxol on thestructures of biofilms. Changes of structures of biofims were observed by Scanning Electron Microscope, SEM.【Results】 CLSM images showed that the control group was majorof live bacteria with green flourescence, mixed with few red flourescenceof dead bacteria. Group of low-frequency ultrasound treated alone wassimilar with the control group. Group of ambroxol（1.875mg/mL） treatedalone showed that the distribution of green flourescence decreasedevidently with red flourescence of dead bacteria incresed, demonstratedthat ambroxol alone had effects on the biofilms. When combined withambroxol,the low-frequency ultrasound manifested an obvious enhancedroles on the biofilms with the green flourescence declined sharply and redflourescence fullfill the field demonstrated that the biofilms were full ofdead bacteria. Quantitative analysis of parameters of structures of biofilmswith ISA software showed that the thickness, average diffusiondistance（ADD）, textual entropy（TE） and areal porosity（AP） of the controlgroup was respectively25.25±3.39μm,2.61±0.34,7.88±0.09,0.65±0.03.Group of low-frequency ultrasound showed no significant difference withcontrol group （P>0.05）, group of ambroxol （1.875mg/mL） treated showedsignificant differencewith thickness, AP, ADD, TE of15.45±2.48,0.75±0.09,2.02±0.06,7.19±0.19,（P<0.05）. Group of ultrasound combinedwith ambroxol for different time（1h and0.5h） showed statistic significantdifference compared with group of ambroxol （1.875mg/mL） treated,（P<0.05）, the former two group showed no significant difference （P>0.05）. SEM showed in control group, group of low-frequency ultrasoundtreated alone, the biolfilms were complex with multiplayers, bacteria wereadhered to each other and gatherred. Group with ambroxol （1.875mg/mL）treated showed that the layers of biofilms decreased, group of ultrasoundcombined with ambroxol for different time（1h and0.5h）, the structures ofbiofilm were simple and monolayer, and became more loose. The biofilmswere destroyed obviously，became loose and thin,only small amount ofbacteria Scattered.【conclusion】 Low-frequency ultrasound could enhanced the effectsof ambroxol at concentration of1.875mg/ml on the structures of biofilmsand make them loose, simple and monolayer.Part III: Effects of low-frequency ultrasound combined withambroxol hydrochloride on Extracellular Polysaccharides ofStaphylococcus epidermidis biofilms【Objective】 To verify the enhanced effects of low-frequencyultrasound with ambroxol on Extracellular Polysaccharides of the maturebiofilms of Staphylococcus epidermidis on polyethylene disc in vitro.【Methods】The mature Staphylococcus Epidermidis RP62A strain’sbiofilms were established on polyethylene disc in vitro. After maturebiofilms were established, they were divided into five groups with differenttreatments, the blank control group, group treated with low-frequency ultrasound alone for1h, group treated with ambroxol hydrochloride（concertration of1.875mg/ml） alone for24h, group treated with ambroxolhydrochloride （concertration of1.875mg/ml） combined with low-frequencyultrasound for1h, group treated with ambroxol hydrochloride（concertration of1.875mg/ml） combined with low-frequency ultrasound for0.5h. After treatments, the EPS of bilfilms were stained by fluoresceinisothiocyanate conjugated concanavalin A, FITC-ConA and observed byfluorescence microscopy.【Results】 After stained, EPS showed green flourescence observedby fluorecent microscope. The highlight green flourescence spots werebacteria, surrounded by cloud-like materia which were the extracellularmatrix called extracellular polysaccharides （EPS）. Group of ultrasoundtreated was similar to the control group. The highlight green spots andcloud-like EPS decreased in group of ambroxol （1.875mg/mL） treated.Combined with ultrasound （1h and0.5h）, the green flourescence weaken,cloud-like EPS flourescence rarely saw, demonstrated that low-frequencyultrsound could significantly promote ambroxol to clear EPS.【Conclusion】 Low-frequency ultrasound could enhanced the effectsof ambroxol at concentration of1.875mg/ml on EPS of biofilms and makebiofilms loose.