Effects Of Sub-Inhibitory Concentration Of Oxacillin On Extracellular Vesicles Secretion Of OS-MRSA And Its Molecular Regulatory Mechanism

Objective:To analyze the drug resistance and molecular epidemiological characteristics of oxacillin-sensitive MRSA（OS-MRSA）clinical strains isolated from inpatients in Inner Mongolia.And to explore subinhibitory concentration of oxacillin in OS-MRSA extracellular vesicle（OS-MRSA EV）secretion effect and the influence of the molecular mechanisms.Methods:1.Screening of methicillin-resistant Staphylococcus aureus（MRSA）sensitive to oxacillin by broth microdilution method and disk diffusion method.And the mec A gene of Staphylococcus aureus was detected by PCR to confirm the OS-MRSA strain.2.The sensitivity of OS-MRSA strains to commonly used antibiotics in vitro was detected by instrumental method.3.MLST and Spa methods were used to identify the dominant genotypes among OS-MRSA isolates.4.The virulence gene（fnbA,clfA,clfB,cna,sdrC,sdrD,sdrE,ebp S,edin,icaA,icaD,icaB,sas G,sasC,bap,sasX,bbp,eno,ebp S,tsst,eta,etb,pvl,luk DE,luk M,hla,hlb,hld,hlg,hlg-2）of OS-MRSA isolates were detected by PCR method.5.The biofilm forming abilities of OS-MRSA isolates were detected by crystal violet staining method.6.One strain of OS-MRSA（OS-200）was selected as the research objects and cultured in vitro.Different concentrations of oxacillin（1/2MIC,1/8MIC,control）were added to the medium,and the extracellular vesicles（EV）were isolated and concentrated by ultra-high speed centrifugation.7.Transmssion Electron Microscope（TEM）,Nanoparticle Tracking Analysis（NTA）and protein content detection methods were used to analyze and compare the morphological characteristics,particle size distribution,particle density and protein content of OS-200 isolate in different treatment groups.8.Tandem Mass spectrometry（TMT）was used to screen the differential proteins of 3 groups of EV(EV_1/2MIC,EV_1/8MICand EV_Control).Multiple Reaction Monitoring（MRM）was used to verify differential protein expression.9.Cell proliferation assay,intracellular tracer assay and erythrocyte lysis assay were used to observe the difference of EV biological activity in different treatment groups.10.Crystal violet staining was used to observe the effect of EV on bacterial biofilm formation in different treatment groups.Results:1.From January 2011 to August 2022,37 strains of OS-MRSA（1.6%,37/2375）were isolated from 2 hospitals in Inner Mongolia.All the 37 strains of OS-MRSA carried mec A gene（100%,37/37）,of which 27 strains were sensitive to oxacillin and cefoxitin（72.97%,27/37）,and 10 strains were sensitive to oxacillin but resistant to cefoxitin（27.03%,10/37）.The isolation rate of OS-MRSA in children（0-12 years old）was 21.62%（8/37）.2.The molecular typing of 37 strains of OS-MRSA showed diverse characteristics.ST6-t701,ST22-t309,ST25-t078 were the dominant gene clones,accounting for 8.11%（3/37）.3.The carrying rates of virulence genes in 37 OS-MRSA strains:sdrC,icaA,icaD,sasC,eno,ebp S,hla,hld,hlg,hlg-2were 100%,and the carrying rates of other virulence genes were significantly different.4.The film-producing ability of the 37 strains of OS-MRSA:the proportion of strong film-producing strains was 13.51%（5/37）,that of medium film-producing strains was 51.35%（19/37）,and that of weak film-producing strains was 35.14%（13/37）.5.The three groups of OS-200 EV(EV_control,EV_1/8MICand EV_1/2MIC)obtained by ultra-high speed centrifugation showed disc-shaped vesicles with clear membrane structure under TEM.6.The results of NTA showed that the average particle density and particle size range of OS-200 EV_controlgroup（2.03 E+10 Particles/m L;173.1±89nm）were smaller than OS-200 EV_1/8MICgroup（3.07E+10 Particles/m L,p=0.016;183.7±152.5nm,p=0.0015）and OS-200 EV_1/2MICgroup（27E+10 Particles/m L,p=0.003;220.4±194.8nm,p<0.001）.Compared with the OS-200 EV_control（84.1-262.1nm）,the OS-200 EV_1/8MICgroup and OS-200 EV_1/2MICgroup had a wide range of particle diameter（31.2-336.2nm;25.6-415.2nm）.7.Bradford assay showed that the protein concentration of EV_1/8MICgroup and EV_1/2MICgroup was significantly higher than that of EV_controlgroup（0.37±0.02 vs 0.58±0.04,t=-7.938,p=0.016;0.37±0.02 vs 0.80±0.05,t=-14.626,p=0.005）.8.Proteomic results showed that 586proteomic numbers were identified in 3 groups of OS-200 EV(EV_control,EV_1/8MICand EV_1/2MIC)samples.In EV_1/2MICvs EV_controlgroup,109 differential proteins were detected,74 up-regulated proteins and 35 down-regulated proteins.In EV_1/8MICvs EV_controlgroup,75 differential proteins were detected,31 up-regulated proteins and 44down-regulated proteins.EV_1/2MICvs EV_1/8MICgroup detected 57 differential proteins,39 up-regulated proteins and 18 down-regulated proteins.9.Bioinformatics analysis results showed that,GO enrichment analysis suggested that differential proteins were mainly enriched in material transport,physiological metabolism,nucleic acid modification,Staphylococcus aureus infection and other processes.It mainly plays a role in pathogenicity and membrane permeability-related protein activity.It is mainly located in cell membrane,plasma membrane and extracellular region.KEGG pathway analysis showed that the pathway classification and proportion of differential proteins in the three comparison groups(EV_1/2MICand EV_controlgroup,EV_1/8MICand EV_controlgroup,EV_1/2MICand EV_1/8MICgroup):（1）metabolism of new town（75%,36/48;69%,25/36;77%,27/35）,including microbial metabolism in different environments,the synthesis of secondary metabolic organisms,glycolysis,gluconeogenesis,carbon metabolism,etc.（2）human diseases（4%,2/48;6%,2/36;0%,20/35）,includingβ-lactam resistance and Staphylococcus aureus infection;（3）genetic information processing（10%,5/48;17%,6/36;11%,4/35）,including ribosomes,RNA degradation,protein export,aminoacyl-trna biosynthesis,etc.（4）Environmental information processing（8%,4/48;6%,2/36;9%,3/35）,including ABC transporter two-component system;（5）cellular processes（2%,1/48;3%,1/36;2%,1/35）,including quorum sensing.10.The results of cell proliferation assay showed that there was no significant difference in the proliferation of A549 cells after co-incubation with different treatment groups of OS-200 EV for 24 hours（p>0.05）.For the same group of EV,the survival rate of A549 cells in the high concentration EV group（30ug/ml）was lower than that in the low concentration EV group（10ug/ml）,but the difference was not statistically significant（p>0.05）.11.Three groups of OS-200 EV(EV_control,EV_1/8MICand EV_1/2MIC)labeled with PKH67 were taken up by A549 cells.It was obvious that OS-200 EV(EV_1/8MICand EV_1/2MIC)adhered more to the surface of A549 cells under the treatment of oxacillin at sub-inhibitory concentration,which further proved that oxacillin at sub-inhibitory concentration could enhance the adhesion ability of S.aureus to host cells through EV.12.The results of red blood cell lysis test showed that after co-incubation of red blood cells with OS-200 EV of different treatment groups for 2 hours,the cell survival rate of each group was significantly different（p<0.05）.Compared with the control group（100%）,the cell survival rate of 2%Ra E+EV_1/2MICgroup（49.25%）decreased most significantly（p<0.05）,followed by 2%Ra E+EV_controlgroup（88.30%）（p<0.05）,and 2%Ra E+EV_1/8MICgroup（98.78%）had no significant difference（p>0.05）.Compared with2%Ra E+EV_controlgroup（88.30%）,the cell survival rate of 2%Ra E+EV_1/2MICgroup（49.25%）was significantly decreased（p<0.05）,and 2%Ra E+EV_1/8MICgroup（98.78%）was significantly increased（p<0.05）.13.The results of biofilm formation index（BFI）experiment showed that the BFI value of the control group（0.125±0.008）with weak film producing strain（OS-200）compared with BFI values in EV_controlgroup,EV_1/8MICgroup and EV_1/2MICgroup（0.126±0.012,t=-0.182,p=0.872;0.107±0.018,t=3.051,p=0.093;0.230±0.028,t=-9.032,p=0.012）,and the trend of BFI values in EV_1/2MICgroup was the most obvious,and the difference was statistically significant.The BFI value of the control group（0.595±0.087）with strong film producing strain（OS-3445）compared with BFI values in EV_controlgroup,EV_1/8MICgroup and EV_1/2MICgroup（0.361±0.093,t=1.658,p=0.239;0.384±0.072,t=1.557,p=0.26;0.595±0.087,t=-3.327,p=0.08）,and the BFI value of EV_1/2MICgroup showed an increasing trend.But the difference was not statistically significant.Conclusions:1.In this study,the isolation rate of OS-MRSA was 1.6%（37/2375）,and the proportion of patients sensitive to both oxacillin and cefoxitin was relatively high.The OS-MRSA strains were easily misidentified as MSSA by instrument method,and should be detected by mec A gene detection method in order to reduce the missed detection rate of OS-MRSA.2.A high proportion of OS-MRSA strains carried a variety of adherence and virulence related genes,including sdr C、ica A、ica D、sas C、eno、ebp S、hla、hld、hlg、hlg-2.3.Sub-mic oxacillin can significantly promote the secretion of OS-MRSA EV,enhance the content of EV protein,and significantly affect the distribution characteristics of EV particle size（particle size range and average particle size）.4.OS-MRSA EV contained a variety of pathogenic（Hld,Hlg,HlgB,PSM,Beta-channel forming cytolysin,CtaB,QoxB,Lipoprotein,LcpA,SortaseA,Deg P/HtrA,Enolase）and drug resistance（Bla Z,FemA,Tca R,Hsd M,Lcp A）related active proteins.The sub-inhibitory concentration of oxacillin can affect the contents of various active proteins in EV.5.OS-MRSA EV could inhibit the proliferation of A549 cells in a concentration-dependent manner,while oxacillin exposure had a concentration-dependent effect on the proliferation of OS-MRSA EV.6.The EV of OS-MRSA strain induced by high concentration of oxacillin showed obvious erythrocyte hemolytic activity,which might be related to virulence proteins such as Hlg,HlgB,Beta-channel forming cytolysin,CtaB,QoxB,Lipoprotein,and Lcp A.7.Sub-mic oxacillin can improve the biofilm formation ability of OS-MRSA by affecting the content of EV adhesion related proteins.The reason may be that oxacillin at sub-inhibitory concentration promotes the levels of heme synthesis-related proteins（CtaB,Qox B）and adhesion proteins（Enolase,Sortase A,Deg P/Htr ）in OS-MRSA EV...