| Escherichia coli（E.coli）is both a commensal and a common conditional pathogen,widely present in the gastrointestinal tract of livestock and in the natural environment.The E.coli disease it causes is not only a health hazard for livestock and poultry,but also an economic loss to the farming industry.In particular,with the growing problem of bacterial drug resistance,New Delhi Metallo-β-lactamase-1（NDM-1）,a so-called"superbug",has been created,which not only stably and efficiently hydrolyses almost allβ-lactam antibiotics,but also The production of NDM-1 not only stabilises and efficiently hydrolyses almost allβ-lactam antibiotics,but is not inhibited by the currently clinically used inhibitors of serine-β-lactamases（SBLs）.E.coli carrying the NDM-1 gene has a wide spectrum of drug resistance,many variants and other characteristics that are easily transmitted,posing new challenges to veterinary clinicians.Ceftiofur（EFT）is a broad-spectrumβ-lactam antibiotic for livestock and poultry,widely used in veterinary practice to treat infections caused by sensitive bacteria and is a common first-line therapeutic agent;Meropenem（MEM）is an antibiotic of the carbapenem family and is not yet approved for use in veterinary practice.Vidofludimus（VFM）is an NDM-1 inhibitor developed in our laboratory.Previous studies have demonstrated that VFM inhibits the enzymatic activity of NDM-1 and has a good synergistic effect in combination with meropenem,but the effect of combining with EFT has not been reported.Therefore,this study investigated the in vitro antibacterial activity of EFT in combination with VFM and its therapeutic effect on NDM-1-positive E.coli infection in mice.The results of the study provide a theoretical basis and new strategies for further development of new agents and effective clinical treatment of NDM-1-positive E.coli infection.In this study,morphology,Gram stain microscopy,bacterial biochemical tests and PCR biological identification of NDM-1-positive E.coli of porcine origin preserved in the laboratory were used to determine that the tested strains matched the characteristics of NDM-1-positive E.coli.The results of minimum inhibitory concentration（MIC）and graded inhibitory concentration index（FICI）showed that the combination of NDM-1 inhibitor VFM with EFT or MEM could reduce the MIC value of EFT and MEM against NDM-1 positive E.coli by 4 times,and the FICI was 0.5 and0.375 respectively,which had a synergistic effect;By plotting growth curves and time-kill curve tests,the results showed that the inhibitor VFM itself had no significant effect on the growth of NDM-1-positive E.coli.Because in vitro synergy does not determine the efficacy of the combination in vivo.Therefore,based on the establishment of a mouse NDM-1 positive E.coli systemic infection model,mice were divided into nine groups,namely,blank control group（CG group）,positive control group（AC group）,EFT（50 mg/kg）treatment group（EFT group）,MEM（10 mg/kg）treatment group（MEM group）,VFM（50 mg/kg）treatment group（VFM group）,EFT+VFM（H）high dose treatment group（50 mg/kg+100 mg/kg）,EFT+VFM（M）medium dose treatment group（50 mg/kg+50 mg/kg）,EFT+VFM（L）low dose treatment group（50 mg/kg+25 mg/kg）,MEM+VFM combination treatment group（10 mg/kg+10 mg/kg）,the in vivo antimicrobial efficacy of the different treatments was evaluated by a combination of clinical symptoms,survival rate determination,hematological physiological and biochemical index tests,serum inflammatory factors IL-1β,TNF-α,INF-γ),histopathology（organ index,tissue bacterial load,HE sections）,and the expression levels of relevant inflammatory factors in serum and tissues（liver,spleen,small intestine）.The results showed that the optimal attack concentration for NDM-1 positive E.coli was determined to be 1 x 107.5 CFU/m L by infection modeling;The survival rate test showed that the survival rate of mice in the AC group was 10%,which was extremely significantly lower than that of the CG group（P<0.01）,while the survival rate of mice in the EFT+VFM（H）group was 90%,which was extremely significantly higher than that of the AC group,（P<0.01）and comparable to that of the MEM+VFM group（P>0.05）;The results of blood physiological indexes showed that compared with the CG group,the WBC and LYMPH levels in the AC group were significantly increased（P<0.01）,while the RBC,HGB,HCT,MCV and MCH levels were significantly decreased（P<0.01）;compared with the AC group,the abnormal blood physiological indexes in the EFT+VFM（H）and EFT+VFM（M）groups were highly significantly improved（P<0.01）.（P<0.01）,but not significantly different from the MEM+VFM and CG groups（P>0.05）;The results of blood biochemical indexes showed that compared with the CG group,the levels of ALT,AST,ALP,TG,BUN and CREA in the AC group were highly significantly increased（P<0.01）and the levels of TP,ALB and TC were highly significantly decreased（P<0.01）,and the blood biochemical indexes of mice in the EFT+VFM（H）and EFT+VFM（M）groups were highly significantly improved（P<0.01）,which were not significantly different from the MEM+VFM and CG groups（P>0.05）;The results of serum inflammatory factor index test showed that compared with the CG group,the levels of serum inflammatory factors IL-1β,TNF-αand INF-γin mice in the AC group were highly significantly increased（P<0.01）,compared with the AC group,the abnormally elevated serum inflammatory factors IL-1β,TNF-αand INF-γindexes in mice in the EFT+VFM（H）and EFT+VFM（M）groups were highly significantly lower（P<0.01）and not significantly different from the MEM+VFM and CG groups（P>0.05）;Organ index results showed that liver index,spleen index and thymus index were highly significant lower in the EFT+VFM（H）and EFT+VFM（M）groups than in the AC group（P<0.01）,and not significantly different from the MEM+VFM and AC groups（P>0.05）;The tissue load results showed that the number of colonies in the liver,spleen and small intestine of mice in the EFT+VFM（H）group was highly significant（P<0.01）compared to the AC group,and the MEM was significantly lower in line with the VFM group（P>0.05）;HE section staining showed that EFT+VFM（H）and EFT+VFM（M）were able to reduce pathological damage to the liver,spleen and small intestine organs caused by NDM-1-positive E.coli infection very significantly compared to mice in the AC group（P<0.01）,consistent with the effect of the combination of MEM+VFM（P>0.05）;RT-PCR results showed that compared with the CG group,the m RNA expression of pro-inflammatory factors content IL-1β,IL-6,IL-8,TNF-αand INF-γin liver,spleen and small intestine of mice in the AC group was highly significantly increased（P<0.01）,and the expression of the anti-inflammatory factor IL-10 was highly significantly decreased（P<0.01）;and,EFT+VFM（H）was highly significantly decreased The gene expression of inflammatory factors IL-1β,IL-6,IL-8,TNF-αand INF-γin the liver,spleen and small intestine of infected mice（P<0.01）,while the m RNA expression of inflammatory factor IL-10 in the liver,spleen and small intestine of infected mice was highly significantly increased（P<0.01）,consistent with the trend in the MEM+VFM group mice（P>0.05）.In summary,the combination of EFT or MEM with the inhibitor VFM had synergistic antibacterial effects in vitro,and the combination of EFT and MEM with VFM,respectively,for the treatment of NDM-1-positive E.coli-infected mice significantly increased the survival rate of infected mice,improved the abnormal physiological and biochemical status of blood cells,inhibited the bacterial-induced inflammatory response and organ load,and reduced histopathological damage in the liver,spleen and small intestine.The high dose combination of EFT（50 mg/kg）and VFM（100 mg/kg）is comparable to the combination of MEM（10 mg/kg）and VFM（10 mg/kg）and can be used as a recommended dose for later clinical trials.The combination of EFT（50 mg/kg）and VFM（50 mg/kg）at medium doses was not as effective as the combination at high doses;the results of the study provide a theoretical basis for the development of effective therapeutic drugs and new treatment strategies for NDM-1 positive E.coli infections in livestock. |
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