| BackgroundBacterial infection has become the second leading cause of death in the world,posing a huge threat to human health and bringing a heavy burden on medical resource as well as social economy.Skin and soft tissue infections(SSTIs)are infectious diseases most commonly seen in clinic,especially in immunocompromised and critical patients.Although the remarkable invention of antibiotics represents a tremendous stride forward in the antiinfection field,a new battle against multi-drug resistant bacteria has been brought into being,following the abuse of antibiotics.Infections caused by multi-drug resistant bacteria remain a challenge in treatment and have been listed as one of the top ten public health threats to mankind by the World Health Organization.Hence,there is an urgent need worldwide for more efficacious infection control by novel antimicrobial agents and therapies.Antimicrobial photodynamic therapy(aPDT)is a novel non-invasive treatment based on photochemical reactions induced by photosensitizers activated by light of specific wavelengths.It inactivates bacteria through bringing damage to the structure and function of bacterial cells by generating a large amount of highly active reactive oxygen species.Previous researches have verified the killing effect of aPDT on pathogenic microorganisms such as bacteria,fungi and viruses.Antimicrobial photodynamic therapy is rapid,effective,non-toxic and not likely to induce drug resistance.It is therefore considered to have great potential for development and application as a new anti-infection strategy.Staphylococcus aureus is the most frequently isolated bacteria from chronic wound infection and also the leading cause of nosocomial infections.methicillin-resistant Staphylococcus aureus(MRSA)is one of the most common multi-drug resistant bacteria in the world and a main cause of SSTIs.MRSA is capable of employing various virulence factors to form strengthened community,increase pathogenicity,promote occurrence of infection,disturb wound healing and to induce drug resistance,which consequently results in treatment failure.Bacterial virulence is composed of invasiveness and toxins,such as protein A,hemolysin and enterotoxin of Staphylococcus aureus.These virulence factors participate in important biological processes including adhesion,colonization,inflammation induction,tissue damage and immune escape,acting as major determinants of pathogenicity.Biofilm is a bacterial community structure wrapped by biomolecules formed on organic or inorganic surface of substances which protects bacteria against external environment and plays a crucial role in virulence.Efflux pump is a class of proteins located on cell membrane of bacteria,which expels intracellular toxic and antibacterial drugs to extracellular environment,thus inducing antibiotic resistance.It has been shown that efflux pumps interact with biofilm formation,while efflux pump inhibitors can interfere with biofilm formation.Quorum sensing(QS)system is an inter-cell communication system when bacterial population reaches a certain density and is responsible for regulating gene expressions required for survival,growth and virulence,as a strategy to adapt to environmental changes.Studies have shown that life process and biological character stics of various pathogens are regulated through QS system,including virulence such as toxin production and biofilm formation,thus increasing the invasion of host immunity and improve pathogenicity.It is believed that by inhibiting QS system,both repression of bacterial growth and reduction of bacterial virulence could be achieved.Therefore,QS system and biofilm of pathogenic bacteria could be regarded as potential antimicrobial targets.ObjectiveThis study was proposed to observe the inactivating effect of toluidine blue O-mediated antimicrobial photodynamic therapy(TBO-aPDT)on Staphylococcus aureus in both planktonic as well as biofilm state and to reveal the dose-effect relationship;to explore the mechanism of inhibitory effect of sub-lethal TBO-aPDT by observing its effect on efflux pumps,biofilm formation,QS system and virulence factors.The effect of TBO-aPDT on skin infection model was also studied in order to deepen the understanding of adaptive changes of bacteria in response to photodynamic oxidative stress.Methods1.Inactivating effect of TBO-aPDT on Staphylococcus aureus1.1 Inactivation of TBO-aPDT on Staphylococcus aureus in planktonic and biofilm state:MSSA and MRSA strains were cultured in vitro in both planktonic and biofilm state and were treated with multiple combinations with different concentrations of TBO solution and doses of 635nm red light.Flow cytometry was used to assess the intracellular ROS production.The plate colony count method,XTT assay and crystal violet staining were used to evaluate the dose-dependent inactivating effect of TBO-aPDT.The dose-effect relationship curve was depicted and then the sublethal dose was selected.1.2 Effect of TBO-aPDT on morphology of Staphylococcus aureus:MSS A and MRS A strains were cultured in vitro in both planktonic and biofilm state and were treated with different doses of TBO-aPDT.Scanning electron microscopy(SEM),confocal laser scanning microscopy(CLSM)and LIVE/DEADTM staining were used to observe the morphological changes of bacterial cells and biofilm following TBO-aPDT.2.Mechanisms of inhibitory effect of TBO-aPDT on Staphylococcus aureus2.1 Effect of TBO-aPDT on efflux pumps:planktonic MSS A and MRS A was treated with sub-lethal TBO-aPDT.Ethylene diamine tetraacetic acid(EDTA)was used to observe changes in cell membrane permeability.Carbonyl cyanide 3-chlorophenylhydrazone(CCCP)was used as an efflux pump inhibitor and EtBr efflux assay was used to evaluate efflux function of Staphylococcus aureus.The transcriptional levels of efflux pump encoding genes norB,mepA,sepA and mdeA were tested using real-time quantitative polymerasechain reaction(RT-qPCR).2.2 Effect of TBO-aPDT on biofilm formation and extracellular polysaccharide production(EPS):planktonic MSSA and MRSA were treated with sub-lethal TBO-aPDT.Crystal violet staining and Congo red binding assay were performed to evaluate biofilm formation and extracellular polysaccharide production respectively.2.3 Effect of TBO-aPDT on QS system:RT-qPCR was performed to asses the transcriptional levels of QS encoding genes agrA,agrC,rnaⅢ,sarA and hld.2.4 Effect of TBO-aPDT on virulence factors:RT-qPCR was performed to assess the transcriptional levels of virulence factor encoding genes hla,srtA,psma,spa,icaA and icaD,ELISA assay was used to test the levels of α-hemolysin and enterotoxin A,and incubation of bacterial suspension with skimmed milk was used to test total protease activity.3.Effect of TBO-aPDT on skin infection caused by Staphylococcus aureus3.1 Effect of TBO-aPDT on bacterial colonization:bacterial suspension of MRSA was injected subcutaneously into Balb/C mice to establish skin infection model.The infection tissue was resected completely and was grinded.The grinding fluid was resuspended with PBS and plate colony count assay was performed to assess the bacterial load at the infection site at different observational time points.3.2 Effect of TBO-aPDT on healing of skin infection:the infection site was observed and photographed every day after the infection model was established.The infection area was delineated and healing rate was calculated using ImgaeJ software.The skin infection tissue was resected and paraffin sections were made.Hematoxylin-eosin(HE)staining and Masson trichrome staining were performed to observe the histological characteristics and collagen deposition.Collagen volume fraction was calculated.3.3 Effect of TBO-aPDT on inflammatory factors of skin infection:on the 1st,4th,7th and 14th day after the establishment of the infection model,infection tissue was resected.Immunohistochemical staining was performed to analyze the levels of interlukin-6(IL-6),tumor necrosis factor-α(TNF-α)and vascular endothelial growth factor A(VEGFA).Enzyme linked immunosorbent assay(ELISA)was employed to test IL-6 and TNF-α in the blood sampled by eyeball extirpating.Results1.Inactivating effect of TBO-aPDT on Staphylococcus aureus1.1 Inactivation of TBO-aPDT on Staphylococcus aureus in planktonic and biofilm state:MSSA and MRSA strains in both planktonic and biofilm state were effectively inactivated in a dose-dependent manner.MSSA and MRSA in planktonic state were eradicated using red light at 40 J/cm2 and TBO at 2.0 μM.MSSA and MRSA in biofilm state were eradicated using red light at 80 J/cm2 and TBO at 4.0 μM.The viable bacterial count of MRSA was higher and the biofilm of MRSA was less destroyed compared to MSSA following the same dose of TBO-aPDT.1.2 Effect of TBO-aPDT on morphology of Staphylococcus aureus:SEM showed distinct morphological changes of bacterial cells and biofilm.The cell wall of the planktonic bacteria is cracked,rough or even collapsed.The compact and uniform three-dimensional structure of the biofilm was destroyed,the extracellular matrix was reduced,and the bacterial cells were dispersed.2.Mechanisms of inhibitory effect of TBO-aPDT on Staphylococcus aureus2.1 Effect of TBO-aPDT on efflux pumps:the sub-lethal dose of TBO-aPDT was set at red light at 40 J/cm2 and TBO at 0.5 μM according to the dose-effect curve.Planktonic Staphylococcus aureus treated with sub lethal TBO-aPDT showed increased cell permeability and inhibited efflux function.The mdeA and mepA gene showed 1.77±0.48-and 1.57±0.30fold upregulation,norA,norB and sepA showed 5.44±1.29-,3.77±0.46-and 6.14±1.11fold downregulation in MSSA.The mdeA,mepA and norA gene showed 1.59±0.31-,3.96±0.86-and 2.80±0.59-fold upregulation,norB and sepA showed 3.02±0.91-and 3.47±1.47fold downregulation in MRS A.2.2 Effect of TBO-aPDT on biofilm formation and extracellular polysaccharide production(EPS):planktonic Staphylococcus aureus treated with sublethal TBO-aPDT showed inhibited biofilm formation and reduced extracellular polysaccharide production.The intensity of biofilm cultured for 24h was decreased by 17.41±4.52%and 9.52±3.51%,the EPS production was reduced by 27.17±3.77%and 19.51±2.24%,in MSSA and MRSA respectively.The structure of biofilm formed following sub-lethal TBO-aPDT was less compact and intense with fewer bacteria attached compared to control.2.3 Effect of TBO-aPDT on QS system:QS system encoding genes were down-regulated following sublethal TBO-aPDT.agrA,agrC,rnaⅢ,sarA and hld gene showed 4.69±1.30,3.48±0.78-,4.47±0.65-,4.92±0.69-and 4.92±0.76-fold downregulation in MSSA and 3.26±0.78-,2.07±0.46-,3.88±0.54-,3.92±0.70 and 3.63±0.65-fold downregulation in MRSA.2.4 Effect of TBO-aPDT on virulence factors:virulence factor encoding genes were down-regulated following sub-lethal TBO-aPDT.hla,srtA,psma,spa,icaA and icaD gene showed 6.76±0.64-,4.87±0.46-,3.31±0.54-,1.82±0.60-,1.86±0.54-,and 4.82±0.57-fold downregulation in MSSA and 4.79±0.62-,3.78±0.61-,3.05±0.24-,1.52±0.14-and 4.15±0.5fold downregulation in MRSA.Levels of α-hemolysin and enterotoxin A were decreased and total protease activity was reduced following sub-lethal TBO-aPDT.3.Effect of TBO-aPDT on skin infection caused by Staphylococcus aureus3.1 Effect of TBO-aPDT on bacterial colonization:on the 2nd day after the establishment of skin infection model,TBO-aPDT group significantly decreased the bacterial count of colonization compared with other groups.TBO-aPDT group took 12 days to eliminate viable bacteria at the infection site compared with 15 days in control group.3.2 Effect of TBO-aPDT on healing of skin infection:TBO-aPDT group showed siginifcantly higher healing rate compared with other groups.On the 4th day after the establishment o skin inection model,the healing rate was 19.03%and 58.19%for control group and TBO-aPDT group.On the 7th day after the establishment o skin inection model,the healing rate was 51.48%and 84.87%for control group and TBO-aPDT group.The average time to complete healing was 16 days and 10 days for control group and TBO-aPDT group.On the 4th,7th and 14th day after infection,the CVF for TBO-aPDT group was 51.74%,61.66%and 82.54%,which were significantly higher than control group(33.01%,44.54%and 69.43%).3.3 Effect of TBO-aPDT on inflammatory factors of skin infection:On the 4th,7th and 14th day after infection,the percentage of VEGFA for TBO-aPDT group was 4.26%,1.42%and 0.48%,which were significantly higher than control group(3.51%,2.99%and 2.41%),the percentage of TNF-α for TBO-aPDT group was 1.61%,0.76%and 0.49%,which were significantly lower than control group(2.40%,1.71%and 1.42%),the percentage of IL-6 for TBO-aPDT group was 6.45%,3.83%and 0.32%,which were significantly higher than control group(11.59%,6.5%and 0.93%).On the 4th day after infection,the levels of IL-6 and TNF-αwere 581.33 pg/mL and 18234.00 pg/mL respectively,which were significantly lower than control group(779.00 pg/mL and 20577.67 pg/mL)Conclusions1.TBO-aPDT could effectively inactivate Staphyloccocus aureus in both planktonic and biofilm state,and cause morphological changes to bacterial cell and biofilm structure.2.TBO-aPDT could inhibit the growth of Staphyloccocus aureus through various mechanisms,including increasing cell membrane permeability,inhibiting efflux function,regulating expressions of efflux pump encoding genes,suppressing biofilm formation as well as EPS production,down-regulating QS system and virulence factor genes and repressing release of virulence factors.3.TBO-aPDT could promote healing of the skin infection caused by Staphyloccocus aureus,through decreasing bacterial colonization,reducing inflammation,and increasing collagen deposition. |
