In Vitro Antibacterial Study Of Platelet-rich Plasma On MRSA

Objective:PRP（platelet-rich plasma）and its activated products PRG（Platelet-rich gel）are highly concentrated products of blood.They are widely used in various kinds of soft tissue repair because of their abundant growth factors.Studies have shown that platelets can exert anti-infective effects in many ways.However,in many in vitro studies,we found that the antimicrobial effects of PRP on some common bacteria（e.g.E.coli,Pseudomonas aeruginosa,Enterococcus faecalis and Klebsiella pneumoniae）were inconsistent.Recent studies have suggested that PRP also has antimicrobial effects on methicillin-resistant Staphylococcus aureus（MRSA）.The purpose of this study is to further clarify whether PRP and its derivative PRG prepared from whole blood of healthy volunteers have antimicrobial effect on MRSA clinical strains in vitro,and to preliminarily understand the application value of PRP in MRSA infection in order to guide clinical treatment.Methods:Clinical strains of MRSA were obtained and inoculated into Colombian blood agar plate to activate and select single colony in the nutritious broth,put it in the constant temperature oscillator to add bacteria and reserve.The blue cap containing sodium citrate was used to collect 9 ml of blood from the anterior elbow vein of six healthy volunteers at one time.PRP was prepared by manual twice centrifugation at room temperature（the first 300×g 10 min,the second 700×g 10 min）.The platelets in the whole blood and PRP were counted by automatic blood cell analyzer after full shaking.The OD₆₀₀ value of bacterial suspension after enrichment was determined by spectrophotometer.The concentration of bacteria was adjusted according to OD₆₀₀ value.Twenty bacterial plates were prepared by pre-adding bacterial liquid pouring plate method.The plates were mixed with MRSA by MH agar medium（Mueller-Hinton Ager）cooled to 45℃.Each plate was divided into four regions.The Oxford cup punching method was used for each plate.In vitro bacteriostasis experiment was carried out after drilling in the center of the region and selecting the medium in the hole.Four holes in each dish were divided into four groups:A,B,C and D.The positive control group in group A was treated with 50ul gentamicin sulfate solution,the PRP group in group B was treated with 50ul PRP,the PRG group was treated with 50ul PRP+5ul calcium gluconate-thrombin solution（pre-use configuration）,and the negative control group was treated with50ul sterilized pure water.The Petri dishes were placed on the front side of the Petri dish and cultured in the incubator at 37℃for 12 hours.The size of the bacteriostatic zone was observed and measured by vernier caliper cross method.Statistical analysis was carried out by SPSS 18.0 statistical software.The measurements were expressed by mean（±standard deviation）.There was statistical significance in P<0.05 among the groups.Results:The number of platelets in whole blood and PRP of 6 healthy volunteers were（161±19.2）×10⁹/L and（876.3±73.3）×10⁹/L,The average platelet enrichment factor in PRP was 5.4 times,which met the PRP preparation standard.The OD₆₀₀ value of MRSA bacterial suspension was 1.375 after adding bacteria to nutritive broth.The OD₆₀₀ value of bacterial suspension was 0.534after multiple dilution.The bacterial concentration of MRSA in diluted broth was1.76×109 CFU/ml after coating counting.The bacterial concentration of MRSA in corresponding MH agar plate was 1.76×10⁶ CFU/ml.After 12 hours of co-culture,the bacteria in the culture medium were evenly distributed,the edge of the bacteriostasis circle was regular,the boundary was clear and the recognition degree was high.In the gentamicin sulfate solution group,significant inhibitory circles were observed.The average diameter of inhibitory circles was（17.15±0.96）mm.In the PRP group,PRG group and sterilized water group,the diameter of inhibitory circles was 0 mm.Conclusion:1.It is a feasible,simple,practical and reproducible bacteriostasis experiment method to combine perforation with pre-added liquid pouring plate method in PRP bacteriostasis experiment.2.PRP and PRG prepared from whole blood of healthy volunteers have no antimicrobial effect on clinical strains of MRSA in this experiment.Although it is believed that PRP can inhibit the growth of many bacteria,in clinical practice,for some drug-resistant strains,this effect may be very weak.We should not exaggerate the anti-infective effect of PRP and neglect the treatment of some infectious diseases themselves.