Synergy Of Ultrasound Microbubbles And Vancomycin Against Staphylococcus Epidermidis Biofilm And Underlying Mechanisms

BIOFILM FORMING CAPABILITY AND SEQUENCE TYPING OFCLINICAL STAPHYLOCOCCUS EPIDERMIDIS ISOLATESObjectives: Staphylococcus epidermidis is the predominant pathogenof nosocomial infections, especially those associated with indwellingmedical devices. This study aims to collect S. epidermidis isolates from thehospital environment and analyze the difference between biofilm-positiveand biofilm-negative isolates with regard to phenotype and genotype.Methods: Five S. epidermidis strains were isolated during the periodbetween October,2013and January,2014from blood samples andintravascular catheters of patients admitted to UniversityChildren’s HospitalWürzburg. VITEK2Compact system was used to assess the antibioticresistance profile of the clinical S. epidermidis isolates. Congo-red agarplates and crystal violet semi-quantitative adhesion test were used to detect the biofilm forming capability of bacterial strains. The expression ofbiofilm-associated genes icaA and IS256was assessed by polymerase chainreaction (PCR). Multilocus sequence typing (MLST) was employed todetermine the sequence type (ST) of the clinical isolates, with eBURSTdiagram drawn for reference.Results: Biofilm-positive strains differed from biofim-negative strainsin terms of phenotype and genotype. Compared with biofilm-negativecounterparts, biofilm positive strains were resistant to a wider range ofantibiotics and showed more adhesion to the material surface. MLSTrevealed that biofilm-positive strains and biofilm-negative strains belongedto ST2and ST10respectively, and the former demonstrated a higherexpression level of icaA and IS256than the latter. eBURST diagram showedthat ST2is the founder ST of S. epidermidis strains disseminating in theworld.Conclusions: Biofilm phenotype and the closely related geneticflexibility contributed to the evolutionary success of S. epidermidis as theleading nosocomial pathogen. PART ⅡULTRASOUND EXPOSURE SETUP FOR IN VITROEXPERIMENTSObjectives: To establish an experimental setup to facilitate ultrasoundexposure in in vitro experiments.Methods: The bacterial strain used was S. epidermidis ATCC35984(RP62A). A standard relationship between the bacterial density and opticalabsorbance was determined by serial dilutions and plate counting. Tenmillimeter of bacterial solution was injected into the OpticellTMchamber todevelop S. epidermidis biofilm, which was then confirmed by silver stainingand scanning electron microscopy (SEM). Ultrasound microbubbles (MBs)was prepared and injected into the OpticellTMchamber, which was invertedand placed in an ultrasonic water tank. The ultrasound wave was therebytransmitted through the degassed water, polystyrene membrane and themedium to reach the biofilm which was in close contact with MBs.Results: There was linear correlation between the bacterial density andthe optical absorbance, and the density of bacterial inoculum equaled2×108colony forming unit (CFU)/ml. MB density was detected to be1.2×109/mL,with MB diameter being4-6μm. The OpticellTMchamber was successfullyused to develop S. epidermidis biofilm. The self-designed ultrasonic setupfacilitated a close contact between MBs and bacteria, and was efficient in transmitting ultrasound energy, thus improving the accuracy of theexperiment.Conclusions: We successfully established S. epidermidis bioflm invitro by using the OptiCellTMchamber, which is routinely used as a cellculture system. The developed ultrasound setup could be applied for in vitroexperiments. PART ⅢSYNERGISTIC BACTERICIDAL EFFECT BETWEENULTRASOUND MICROBUBBLES AND VANCOMYCINObjectives: Antibiotic susceptibility tests were performed on S.epidermidis bioflm to confrm the synergy between MBs and vancomycin.Methods: S. epidermidis bioflms were established in OpticellTMchambers. Biofilm culturability among different treatment conditions,namely blank control, ultrasound (US) only, MBs only, US+1%MBs andUS+4%MBs, were determined by dilution plate counting technology. TheMIC for planktonic bacteria was detected by standard broth microdilution.For bacteria grown in bioflms, the bioflm inhibitory concentration (BIC)was evaluated.Results: Bacterial recovery in the control group was8.03±0.04log10CFU/cm2. In the absence of vancomycin, US+1%MBs did not affect bioflmviability (8.041±0.049log10CFU/cm2), whereas US+4%MBs reduced thebacterial counts to7.949±0.012log10CFU/cm2(P<0.05). The MIC forplanktonic S. epidermidis was2μg/ml. On the other hand, the BIC of S.epidermidis biofilm was512μg/ml. US could remarkably reduce the BIC to128μg/ml, and a further reduction of BIC to16μg/ml was achieved byUS+MBs. MB dose-dependent synergy was also observed.Conclusions: Bacteria in the biofilm mode showed significant resistance to vancomycin as compared with their planktonic counterparts.US targeted MBs could act synergistically with vancymycin to exert anantibacterial effect on S. epidermidis bioflms. PART ⅣANTIBIOFILM EFFECT OF VANCOMYIN COMBINED WITHULTRASOUND MICROBUBBLES ON S. EPIDERMIDIS BIOFILMIN VITROObjectives: To explore the effect of vancomycin combined withultrasound microbubbles on the viability, structure and morphology of S.epidermidis bioflm.Methods: OpticellTMchambers were used to develop S. epidermidisbioflms, which were then treated under eight different conditions: nottreated (control), MBs only, US only, US+MBs, vancomycin only,MBs+vancomycin, US+vancomycin and US+MBs+vancomycin. MBs wereprepared and diluted to concentrations of1%and4%(v/v). Ultrasound wasapplied for5min at300kHz and0.5W/cm2, with a50%duty cycle.Vancomycin at the peak serum concentration of32μg/ml was used onpreformed bioflms for24h. After the treatment, bioflms were subjected toplate counting, confocal laser scanning microscopy (CLSM) and scanningelectron microscopy (SEM).Results: MBs not exposed to US did not have a bactericidal effect on S.epidermidis bioflm. US application signifcantly enhanced the antibacterialaction of vancomycin by reducing the bacterial count to38.98%±3.95%ofthat in the controls (P<0.05). This effect was more pronounced in theUS+MBs+vancomycin group than in the US+vancomycin group, and bacterial recovery from bioflms exposed to4%MBs was signifcantly lessthan that from bioflms exposed to1%MBs (6.10%±1.31%versus22.29%±2.99%, P<0.05). Biofilms treated by US+MBs showed moremicropores and a greater reduction in biofilm thickness and structuralcomplexity than those in other groups (P<0.05). Micrographs of SEMdemonstrated that US+MBs could result in bacterial damage, and the amountof cell debris was positively related to MB concentrations.Conclusions: US targeted MBs could exert a destructive effect on thestructure and morphology of S. epidermidis bioflms, thereby enhancing thebactericidal effect of vancomycin. PART ⅤPHYSICAL AND BIOCHEMICAL MECHANISMS OF THEANTIBIOFILM EFFECT OF VANCOMYCIN COMBINED WITHULTRASOUND MICROBUBBLESObjectives: To explore the underlying physical and biochemicalmechanisms of the bioeffect of ultrasound microbubbles.Methods: S. epidermidis bioflms were developed in OpticellTMchambers, and confocal laser scanning microscopy (CLSM) was used tovisualize the intracellular transportation of fluorescent dyes upon thetreatment of US+MBs. The uptake of extracellular macromolecules can beused to reflect the permeability of bacterial membrane. Diffusion test wasapplied to delineate the12-h temporal trend of vancomycin penetrationthrough the extracellular matrix of S. epidermidis bioflms, which weretreated under different conditions. After treatment with eight conditions,namely blank control, US only, US+1%MBs, US+4%MBs, vancomycinonly, US+vancomycin, US+1%MBs+vancomycin and US+4%MBs+vancomycin, the real-time polymerase chain reaction (RT-PCR) was utilizedto evaluate the expression of agrB, RNAⅢ and icaA, which are importantgenes involved in BF regulation systems.Results: Normally the bacterial membrane is impermeable tomacromolecules, as evidenced by faint fluorescence in the control group.US+MBs facilitated a significant amount of fluorescence to be transported intracellularly, and the uptake of fluorescent dyes was MB dose-dependent.The result of diffusion test showed that the amount of vancomycinpenetrated through the polystyrene membrane with or without BF was41.47%±1.6%and64.57%±4.19%of the load, respectively (P<0.05),suggesting that BF was the rate-limiting factor of vancomycin penetration.US signifcantly increased bioflm permeability to vancomycin (P<0.05),and US+MBs facilitated more vancomycin to be transported through thebioflm than US did at all time intervals. The result of RT-PCR showed thatthe expression levels of icaA, agrB and RNAⅢ in vancomycin-alone groupwere0.87,1.12and1.25times of those in the control, respectively. Amongall treatment groups, US+MBs+vancomycin had the most significant impacton gene expression. The expression levels of agrB and RNAⅢ wereincreased to1.40and1.35times of those in the control (P<0.05), while theexpression of icaA was decreased to0.67of that in the control (P<0.05).Conclusions: Ultrasound microbubbles could enhance the permeabilityof bacterial membrane and the extracellular matrix of biofilm tovancomycin molecules. Moreover, the treatment of US+MBs had an impacton the gene regulation of biofilm. The expression levels of agrB andRNAⅢ were found to be upregulated, while the expression of icaA wasdownregulated. Our results demonstrated that physical and biochemicalmechanisms could work simultaneously to contribute to the bioeffect ofultrasound microbubbles. PART ⅥANTIBIOFILM EFFECT OF VANCOMYCIN COMBINED WITHULTRASOUND MICROBUBBLES ON S. EPIDERMIDIS BIOFILMIN VIVOObjectives: To establish a rabbit model of implant-associated biofilminfection and explore the synergistic effect of vancomycin and ultrasoundmicrobubbles on S. epidermidis bioflm in vivo.Methods: Two polyurethane catheters with preformed S. epidermidisbioflm were implanted subcutaneously in a rabbit, with one of the twocatheters positioned on either side of the spine. Animals were randomizedinto different treatment groups, with each rabbit serving as its own controland treatment. After treatment, namely blank control, US only, US+MBs,vancomycin only, US+vancomycin and US+MBs+vancomycin, animalswere euthanized and biofilm-covered catheters were removed for scanningelectron microscopy (SEM) examination and dilution plate counting.Additionally, the subcutaneous tissue at the implantation site was sent forhistopathological examinations.Results: Prior to implantation the viability of BF was7.284±0.032log10CFU/catheter. The number of viable bacteria in blank control, US-alonegroup, US+MBs group, vancomycin-alone group, US+vancomycin andUS+MBs+vancomycin group was6.436±0.033log10CFU/catheter,6.464±0.066log10CFU/catheter,6.440±0.048log10CFU/catheter,5.395± 0.032log10CFU/catheter,5.152±0.035log10CFU/catheter and3.493±0.021log10CFU/catheter respectively. The antibacterial effect of US+MBs+vancomycin was statistically significant than that of US+vancomycin(P<0.05). As shown in SEM, the application of systematic vancomycinloosened BF structure and reduced the bacterial aggregation. US actedsynergistically with vancomycin, resulting in small clusters of bacteria in thevisual field. US+MBs further enhanced the effect of vancomycin, and only afew bacteria were seen scattered on the catheter surface. Histopathologicalexamination showed that vancomycin combined with ultrasoundmicrobubbles could attenuate the inflammation effect surrounding theimplanted catheter, without causing obvious damage to the subcutaneoustissues.Conclusions: Ultrasound microbubbles enhanced the antibiofilm effectof vancomycin in an in vivo rabbit model of subcutaneousimplant-associated BF infection, and also exerted an anti-inflammationeffect at the implantation site.