| Objective:Vancomycin-resistance associated system VraSR（TCS-VraSR）plays an important role in biofilm formation,cell wall synthesis,programmed cell death,and other biological functions of Staphylococcus epidermidis.In this thesis,we explored the mechanism by which VraSR regulates persister formation of S.epidermidis,then it lay a foundation for clinically effective prevention and treatment of persistent infection and research on drug targets against persister in S.epidermidis.Methods:1.Detection of susceptibility test and growth curve of SE1457:To determine the drug concentration and growth phase selected for persisters assay in S.epidermidis,the MIC values of SE1457 against three antibiotics with different mechanisms of action（vancomycin,ciprofloxacin,and gentamicin）were performed by tube dilution method,and the growth curve of SE1457 was detected.2.Persisters assay of SE1457:The above three antibiotics（10×,20×,30×,40×,and50×MIC value）were added in three different growth phases of the growth curve of SE1457（Log phase,stationary phase,and decline phase）,and continuously cultured for84hrs.The bacteria were taken once every 12hrs,and washed with NS,then CFU counting was performed.Biphasic killing kinetics of bactericidal antibiotic treatment occurred indicating persisters were formed.To elucidate the persister formation（proportion and conditions）of SE1457 different growth phases,and treated with the above drugs.3.Differences persister formation of SE1457 and its isogenic vraSR deletion mutant:The wild strain of SE1457,?vraSR mutant,?vraSR（p CN51-vraSR）complementary strain,?vraSR（p CN51）empty vector strain were selected as experimental strains.Staphylococci grown in different growth phases（Log phase,stationary phase,decline phase）and treated with drugs（vancomycin,ciprofloxacin,gentamicin,respectively）were cultured for 84hrs,and CFU counting was performed every 12hrs.The persister formation of SE1457 and its isogenic vraSR deletion mutants were analyzed.4.RNA samples of persisters of SE1457 andΔvraSR in the stationary phase treated with vancomycin were extracted and transcriptome sequencing（RNA-seq）was performed.Differentially expressed genes（DEGs）were verified using q RT-PCR,and the activity of some DEG-related proteins was detected.Results:1.The sensitivity（MIC values）of SE1457 to vancomycin,ciprofloxacin,and gentamicin were 4μg/m L,0.25μg/m L,and 0.5μg/m L,respectively.2.Staphylococci grown in different growth phases treated with above mentioned antibiotics（10×,20×,30×,40×,and 50×MIC values）for a period of time were taken for CFU counting.Persisters of SE1457 were formed as follows:1)In log phase,persisters of SE1457 strain treated with vancomycin for 60 hrs were formed,and the levels of persister formed SE1457 upon the above five concentrations were 7.04×10-6,5.45×10-6,5.31×10-7,4.95×10-7and 4.22×10-7,respectively.Persisters of SE1457 were formed treated with ciprofloxacin for 36hrs,and the proportions of persisters were 4.72×10-5,2.54×10-5,2.43×10-5,1.88×10-5 and 1.70×10-5,respectively.Persisters of SE1457 were formed treated with gentamicin for 60hrs,,and the proportions of persisters were 6.40×10-7,6.36×10-7,5.43×10-7,5.56×10-7 and 4.02×10-7,respectively.2)In stationary phase,persisters of SE1457 strain treated with vancomycin for 60hrs were formed,and the levels of persister formed SE1457 upon the above five concentrations were 1.5×10-3,1.4×10-3,1.01×10-4,1.0×10-4 and 6.8×10-5,respectively.Persisters of SE1457 were formed treated with ciprofloxacin for 36hrs,and the proportions of persisters were 1.0×10-2,4.82×10-3,6.29×10-3,6.57×10-3 and 7.92×10-3,respectively.Persisters of SE1457 were formed treated with gentamicin for 60hrs,,and the proportions of persisters were 3.7×10-8,1.66×10-8,4.75×10-9,3.82×10-9 and 3.08×10-9,respectively.3)In decline phase,persisters of SE1457 strain treated with vancomycin for 36 hrs were formed,and the levels of persister formed SE1457 upon the above five concentrations were 5.42×10-3,3.79×10-3,7.85×10-4,7.0×10-4and 3.14×10-4,respectively.Persisters of SE1457 were formed treated with ciprofloxacin for 12hrs,and the proportions of persisters were 3.25×10-4,2.80×10-4,2.24×10-4,1.80×10-4and 1.34×10-4,respectively.Persisters of SE1457 were formed treated with gentamicin for 48hrs,and the proportions of persisters were 7.09×10-10,4.32×10-10,5.54×10-10,1.63×10-10and5.74×10-11,respectively.4)Pyruvate levels decreased significantly in SE1457 persisters:The intracellular pyruvate content of SE1457 persister formed during the stationary phase under the influence of three drugs decreased significantly compared to the original bacteria,with contents of 57%,53%,and 51%of the original bacteria content,respectively.3.Differences persister formation of SE1457 and its isogenic vraSR deletion mutant:1)In log phase,persisters of SE1457 and its isogenic vraSR deletion mutant treated with 200μg/m L vancomycin（Van+）for 60hrs were formed,and the proportion of persisters formed byΔvraSR mutant(8.42×10-8)was 10 times lower than that of SE1457(5.93×10-7).?vraSR（p CN51-vraSR）strain persister formation ratio(4.6×10-7)returns to the level of SE1457,?vraSR（p CN51）strain persister formation ratio(9.07×10-8)was similar to?vraSR.Persisters of SE1457,?vraSR,?vraSR（PCN51-vraSR）and?vraSR（p CN51）were formed treated with 12.5μg/m L ciprofloxacin（Cip+）for 36hrs,and the proportions of persisters were 1.45×10-5,8.07×10-6,4.08×10-5,and 8.25×10-6,respectively.Persisters of SE1457 and?vraSR（PCN51-vraSR）were formed treated with10μg/m L gentamicin（Gen+）for 60hrs,and the proportions of persisters were 3.62×10-8and 6.31×10-7,while?vraSR and?vraSR（p CN51）did not form persisters.2)In stationary phase,persisters of SE1457 and its isogenic vraSR deletion mutant treated with Van+for 60hrs were formed,the proportion of persister formed byΔvraSR mutant(7.04×10-8)was nearly 100 times lower than that of SE1457(7.81×10-6).?vraSR（p CN51-vraSR）strain persisters formation ratio(8.47×10-6)returns to the level of SE1457,?vraSR（p CN51）strain persisters formation ratio(6.15×10-8)was similar to?vraSR.Cip+36hrs,the proportions of SE1457,?vraSR,?vraSR（p CN51-vraSR）and?vraSR（p CN51）persisters were 2.78×10-3,3.65×10-3,3.29×10-3 and 3.62×10-3,respectively.Gen+60hrs,the proportions of SE1457 and?vraSR（p CN51-vraSR）persisters were 1.13×10-8 and2.46×10-8,while?vraSR and?vraSR（p CN51）did not form persisters.3)In decline phase,persisters of SE1457 and its isogenic vraSR deletion mutant treated with Van+for 36hrs were formed,the proportion of persister formed byΔvraSR mutant(1.33×10-7)was nearly 1000 times lower than that of SE1457(3.17×10-4).?vraSR（p CN51-vraSR）strain persister formation ratio(5.18×10-4)returns to the level of SE1457,?vraSR（p CN51）strain persister formation ratio(1.53×10-7)was similar to?vraSR.Cip+12hrs,the proportions of SE1457,?vraSR,?vraSR（p CN51-vraSR）and?vraSR（p CN51）persisters were 1.7×10-4,2.37×10-5,1.46×10-4 and 2.45×10-5,respectively.Gen+48h,the proportions of SE1457 and?vraSR（p CN51-vraSR）persisters were 6.69×10-10 and5.87×10-10,while?vraSR and?vraSR（p CN51）did not form persisters.4)Transcriptome sequencing（RNA-seq）and q RT-PCR analysis of SE1457 andΔvraSR mutant persisters:RNA samples of persisters of SE1457 andΔvraSR in the stationary phase treated with vancomycin were extracted and transcriptome sequencing（RNA-seq）was performed.There were 440 genes differentially expressed between theΔvraSR mutant and the SE1457 parent strain,among which 106 genes were upregulated and 334 genes were downregulated in theΔvraSR mutant.The transcription levels of differentially expressed genes related to drug tolerance（serp1412）,amino acid synthesis metabolism（trp A）,ABC transporter（serp1395）,cell wall（serp0305）,ATP synthesis（atp A）,ribosomal protein（rpl L）and other functions changed.Additionally,the SDH activity（23.33±3.70 U/mg prot）and NOX activity（366.72±44.71U/mg prot）ofΔvraSR persisters were significantly higher than those of SE1457（11.58±2.02,240.97±56.86U/mg prot）.Conclusion:1.SE1457 can form persisters with vancomycin,ciprofloxacin,and gentamicin.Under the action of vancomycin,the proportion of SE1457 persisters gradually increased from the log phase to the decline phase.In contrast,the proportion of persisters gradually decreased under gentamicin.Under ciprofloxacin,the highest proportion of persisters was in the stationary phase,and the lowest was in the log phase.2.Compared with SE1457,under the action of vancomycin from the log phase to the decline phase,the proportion of persister formed byΔvraSR deletion mutant was significantly reduced,and the degree of reduction increased exponentially;under the action of gentamicin,ΔvraSR deletion mutant could not form persisters;compared with SE1457,the metabolic activity of persister formed byΔvraSR deletion mutant increased,the cell wall became thinner,and the number of bacterial lysis increased.3.TCS-VraSR may regulate the formation of S.epidermidis persisters through non-TA pathways,such as regulating cell respiration and energy metabolism,drug efflux pumps,cell wall synthesis,amino acid synthesis and metabolism,TCA cycle pathways,etc.4.TCS-VraSR can be a potential drug target for forming S.epidermidis persisters under gentamicin stress. |
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