Mechnism Of Multi-resistance Mediated By Porin F(OmpF) And Its Regulatory Gene Of MicF In Shigella Strains

Bacterial dysentery,also known as Shigella disease,is one of the most common infectious diarrhea in our country,living in the third highest incidence of statutory infectious diseases at present.The pathogen of Shigella is a class of highly contagious,generally susceptible and serious harmful intestinal infectious diseases.In recent years,due to the widespread use of antibiotics,the phenomenon of antibiotic resistance commonly occuranced in Shigella,even showing multiple drug resistance trend.The change of cell permeability which was mediated by outer membrane protein is one of important mechanisms of bacterial multiple drug-resistance.The outer membrane protein is the only way to enter the cell for some antibiotics to produce effective.Therefore,the structure of the outer membrane protein and the change of the factors which regulating protein expression can cause the change of drug resistance.Objective:To investigate the relative expression of ompF and micF gene at transcriptional level,exploring the relationship between outer membrane porin OmpF and drug resistance,micF regulation of ompF expression,and whether micF regulates cell membrane resistance of Shigella.Materials and methods:1.There were 13 clinical isolates of Shigella from the stool samples of diarrhea patients who presented to the Second Hospital of Tianjin Medical University from May to October in 2015,and the other number of Shigella is 8 strains,which were preserved by the Institute of Infectious Diseases,Second Hospital of TianJin Medical Univercity.The serotypes were determined by serum agglutination and routine biochemical identification.2.The antibiotic susceptibility test was performed,such as ciprofloxacin,ceftriaxone and ampicillin,and the K-B paper diffusion method were used to screen out the multidrug-resistant and non-multidrug-resistant strains.3.The ompF gene was amplified by PCR,detectded whether the ompF gene absence or not.4.Total RNA was extracted from Shigella.The micF and ompF genes at transcriptional levels in the two group of strains were detected by quantitation real-time PCR.And SPSS20.0 software was used to analyze the results.5.The standard concentration curve of ciprofloxacin was established.Intracellular concentrations of ciprofloxacin in the two groups of Shigella strains were measured by automatic microplate reader.Results:1.21 strains were all Shigella by biochemical identification and antibiotic susceptibility test.Among them,8 strains of Shigella flexneri,Shigella boydii and Shigella sonnei were 6 strains,Shigella dysenteria only 1 strain.2.According to the result of antibiotic susceptibility test,13 strains that were resistant to 3 or more than 3 antibiotics were classified into the multidrug-resistant group,while the other 8 strains that were sensitive to all antibiotics used in this study or only resistant to 1 or 2 antibiotics were classified into the non-multidrug-resistant group.3.All of the 21 Shigella strains carried the ompF gene.4.Compared with the non-multidrug-resistant strains,the multidrug-resistant strains showed higher expression of micF gene,but lower expression of ompF gene.The differences in micF and ompF genes between the two groups were statistically significant.The result of correlation analysis suggested that there was a negative correlation between micF and ompF genes(r =-0.244).5.The intracellular concentrations of ciprofloxacin in multidrug-resistant strains were lower than those in the non-multidrug-resistant strains(P<0.05).Conclusion:1.The decreased expression of OmpF was one of the possible mechanisms of multidrug-resistance2.The micF gene maybe negatively related to the expression of OmpF.3.The decreased intracellular concentrations of ciprofloxacin in multidrug-resistant strains might be related to the decreased expression of OmpF.