| ObjectiveThis paper aims to investigate the therapeutic effect of allicin combined with vancomycin on mouse pneumonia infected with Methicillin resistant Staphylococcus aureus(MRSA).Method(1)In vitro study:The minimum inhibitory concentration(MIC)of allicin against clinical MRSA isolate was determined by broth turbidimetric observation method and broth enzyme marker detection method.Different dose combinations between allicin and vancomycin were designed to observe the inhibitory effect of on MRSA biofilm in vitro experiment.(2)In vivo study:Pulmonary infection models were created by tracheal dripping MRSA suspension 6-8 weeks SPF KM mice.The modelling mice were administrated allicin as single drug,and allicin combined with vancomycin at different doses through endotracheal drip method.The effect of drug combination against mouse pulmonary infection was analyzed and evaluated.The behaviour of modelling mice was observed during 4 days successive administration.The survival and death of mice after administration were recorded.The colony number of mice lung homogenate was counted.The pathological changes of the lungs of each administrated group were observed by routine HE staining.RNA was extracted from administrated mouse lung tissues,and the expression levels of biofilm-related genes in each lung sample were determined quantitatively by qRT-PCR.Results(1)The results of minimum inhibitory concentration test showed that the growth of clinical MRSA isolates used in this study was inhibited when the allicin concentration was 15.75μg/mL.(2)Congo Red experiment showed that high-dose allicin combined with vancomycin had the smallest effect on the biofilm community of Staphylococcus aureus.(3)Compared with the control group,the diet and mental state of the mice treated with allicin alone and vancomycin were improved,while the diet and mental state of the mice treated with allicin combined with vancomycin were significantly improved.(4)The results of qRT-PCR showed that the IcaA in the lung of MRSA model mice was decreased by 48.3%,24.7% and 11%,and the expression of AgrA was decreased by24.9%,20.2% and 14.9%,with statistical significance(P<0.01).The expression levels of IcaA and AgrA were decreased by 58.4% and 81.9% respectively when high dose of allicin and vancomycin were combined,and the results were statistically significant(P<0.05).(5)The results of lung colony count of mice showed that the high-dose allicin combined with vancomycin group had the best effect on the number of infected bacteria,and allicin or vancomycin alone could inhibit the lung MASA colony.Compared with the group treated alone,allicin combined with vancomycin had a better inhibitory effect on the number of MRSA-infected bacterial colonies in mouse lungs.(6)In the pathological changes and H&E staining of mouse lung tissue,high-dose allicin combined with vancomycin had the best recovery of lung inflammation.Score analysis showed that the pathological difference of high-dose allicin combined group was extremely significant(P<0.01).and the pathological difference of medium-and low-dose allicin combined group was statistically significant(P<0.05)The survival rate of mice was determined after 4 days of administration,and medium-high dose allicin combined with vancomycin was preserved(P <0.05)。ConclusionIn this study,during the same duration of MRSA infection,compared with allicin or vancomycin alone,allicin combined with vancomycin treatment significantly improved the clinical manifestations of MRSA resistant mouse pneumonia model,more effectively reduced the number of lung tissue cells in MRSA resistant mice,and alleviated the pathological symptoms of pneumonia in mice.It can significantly reduce the expression of IcaA and AgrA genes related to resistance of Staphylococcus aureus biofilm,and significantly reduce the number of bacterial cells in lung tissue.It significantly increased the survival rate of mice infected with methicillin resistant Staphylococcus aureus. |
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