Resistance Against Fluoroquinolones Mediated By Multidrug Resistance Efflux Protein NorA In Staphylococcus

NorA is a kind of major transmembran multidrug resistance efflux protein in Staphylococci.It mediates resistance against hydropilic fluoroquinolones and many other drugs of similar formation or without relation in formation,such as ethidium bromide and rhodamine.The overexpression of NorA caused intrinsic resistance against fluoroquinolones in wild Staphylococci.The protein is encoded by norA gene.Staphylococcus aureus and S.epidermidis isolated from human being and animals and their corresponding resistanct strains induced by fluoroquinolones and sodium salicylate and reserpine artificially were studied,NorA protein and norA gene were studied together.The result of resistance detecting showed that the resistance against 13 antibiotics and fluoroquinolones in Staphylococcus aureus and S.epidermidis isolated from human being and animals is serious and multidrug resistant and cross resistanct.The activities of hydrophobic fluoroquinolones is stronger than hydropilic fluoroquinolones. Inducing of resistance is succeed.Inhibiting and inducing of resistance in clinical Staphylococci isolates had been done by sodium salicylate and reserpine. Sodium salicylate made resistance stronger against fluoroquinolones in clinical Staphylococcus aureus isolates partly.The inhibitor of efflux reserpine had heavy effect on the antibiotic activities of three kinds of hydropilic fluoroquinolones.but the antibiotic activities of is lower than origin in some isolates.The fragment of norA gene (1155bp) came from 10 Staphylococcus aureus andS.epidermidis respectively was cloned into PMD-18T vector.The results of sequencingshowed that there were two amino changes in one Staphylococcus aureus isolate and one base change and no amino change in one S.epidermidis isolate.Prokaryotic expression vector pET-28a (+) - nor A was achieved and nor A was expressed successfully .Purified expressed protein was used to immune habbits.The results of ELISA and Western blotting showed that polyclonal antibody was gotten. Western blotting was used to detect the expression levels of NorA protein in E.coli BL21 in \\hich pET-28a (+) - nor A was expressed acted by sodium salicylate and reserpine and in 7 Staphylococcus aureus isolates and 5 S.epidermidis isolates.The results showed that both sodium salicylate and reserpine made the expression level of NorA protein higher in E.coli BL21.The expression levels of NorA protein is higher in stronger resistanct isolates.The expression level of NorA protein is lower and near in susceptible strains,and .but, there was no correlation between the expression level of NorA protein and MIC in some isolates.Internal standard DNA of quantu RT-PCR was constructed by gene synthesis. The level of norA mRNA were detected in 12 clinical Staphylococcus aureus and S.epidermidis isolates and two Staphylococcus aureus that was induced by fluoroquinolones and acted by sodium salicylate and reserpine.The relative expression levels of norA mRNA of one strain to the control strain and the relative expression level of NorA protein detected formly was consistent.Sodium salicylate made amount of norA mRNA a little higher in one strain,but reserpine made amount of norA mRNA a little lower in one strain.Intaping of ciprofloxacin was detected by improved CCCP-fluorospectrophotometry in 3 wild Staphylococcus aureus isolates and 1 Staphylococcus aureus control.Dose of intaping of ciprofloxacin in stonger resistant isolates is lower than weaker resistant isolates. CCCP did made the intaping of ciprofloxacin much more in all strains.