Efficacy Of Linezolid Combined With Fosfomycin In The Treatment Of Methicillin-resistant Staphylococcus Aureus USA300-infected Murine Model With Pneumonia

Objectives:The current clinical treatment of methicillin-resistant Staphylococcus-induced pneumonia is single,and the proliferation of drug resistance has led to a surge in public health pressure,so it is urgent to find new treatment options.In this experiment,we investigated the effect of linezolid combined with fosfomycin in the treatment of MRSAinfected pneumonia mice,and revealed the mechanism of action of the combined drug through the host immunity and bacterial virulence,which provided a new idea for the clinical treatment of MRSA-infected pneumonia.Methods:1.Susceptibility testing and checkerboard assay were used to evaluate the synergistic effect of linezolid combined with Fosfomycin on USA300 in vitro.2.Through MRSA pneumonia murine model,the efficacy of linezolid combined with fosfomycin in vivo was verified by survival curve,bacterial load count,lung HE,and lung dry-wet weight.3.By qRT-PCR and ELISA assays to study the release levels of pulmonary inflammatory factors IL-1β,TNF-α and chemokine MIP-2 in the treatment group.4.Collect and extract bacterial RNA in vitro and in vivo with the drugs alone or in combination,and detect changes in transcript levels of MRSA-releasing virulence factors PSM,HLα,and Spa by q RT-PCR.5.Flow cytometry was used to detect the number of neutrophils and macrophages in bronchoalveolar lavage fluid,MPO activity measurement and immunohistochemistry to quantify the number of neutrophils in the lungs to observe the effect of the combination on immune regulation.Results:1.USA300 was highly sensitive to linezolid and fosfomycin with MICs of 2,8 mg/L,respectively,and showed synergistic effects of the combination combined drugs on USA300.2.In vivo,linezolid combined with fosfomycin significantly reduced the mortality rate of infected mice,and the combination was able to achieve 1.63 log reduction from the best corresponding single drug group by counting the lung bacterial load,and the reduced bacterial load in the single-agent group and the combination group were 1.21 log,1.40 log,and 3.03 log,respectively.The wet and dry lung specific gravity data showed that the combined group was the most effective in suppressing pulmonary edema with values of 10.49 and 4.03 in the infected and combined groups,respectively,while HE further showed that the high-dose linezolid group and the combination group could effectively protect the lungs from invasion.3.All treatment groups were able to regulate the immune status of the body,downregulate the release of pro-inflammatory factors TNF-α,IL-1β and chemokine MIP-2,among which the inhibitory effect of 40mg/kg linezolid group was weak,after increasing the dose of 80mg/kg linezolid significantly inhibited the release of related inflammatory factors,the fosfomycin group was similar to the combination group and was at a moderate inhibitory level.4.The study of bacterial virulence factor transcription levels by q RT-PCR showed that the inhibitory effect in the combination group was best in vitro by the 1/2×MIC drug combination,and the in vivo data further showed that the combination group could inhibit the release of pneumonia-related virulence factors to a very low level,which was significantly different from the corresponding single-agent group(P<0.05).5.The MPO data initially showed differences in expression in different treatment groups and subsequently flow cytometry was used to detect specific differences in macrophages and neutrophils in the Bronchoalveolar lavage fluid of the treatment groups.It showed excessive infiltration of neutrophils with a small number of macrophages in the low-dose linezolid group;the number of the high-dose linezolid group significantly decreased;the fosfomycin group was at the middle level;and the combination group showed a decrease compared with the corresponding single-agent group,but higher than that of the high-dose linezolid group.Subsequent analysis of lung histochemistry data showed similar results to flow cytometry.Conclusions:Our experiment explored the synergistic effect of linezolid combined with fosfomycin in the treatment of MRSA USA300 pneumonia mice for the first time.The results showed that the combined administration could not only effectively inhibit the proliferation of bacteria,but also effectively mobilize the organism to exert significant immune effects in the lung while inhibiting the virulence of bacteria,providing new ideas for the clinical treatment of MRSA-infected pneumonia.