| Object: Before the study,it was found that the subinhibitory concentration pretreatment of gentamicin could inhibit the swarming of Escherichia coli standard strains,which may be related to the inhibiting the expression of succinic dehydrogenase(SDH)and the reducing the production of fumaric acid,suggesting that gentamicin has its own advantages in the treatment of Escherichia coli infection.In this study,gentamicin pretreatment was performed on clinical isolates and standard isolates,combined in vitro and in vivo experiments,and fluorescence real-time quantitative PCR was performed to verify that gentamicin at subinhibitory concentration could reduce bacterial virulence and its related mechanisms.Methods: 1 Using the in vitro experiment gentamicin in clinical strains of swarming inhibition,broth dilution method is used to test the clinical strains of minimum bacteriostatic concentration(MIC),and then use 1/2 MIC gentamicin processing strains,untreated as control group,two groups of 2 of the ul microbial inoculation in cluster move(swarm)tablet and swimming(swim)tablet,incubation at room temperature,measuring the colony diameter.2 In vivo experiments further verified that 1/2 mic gentamicin can reduce bacterial toxicity.3 The motility of escherichia coli mainly depends on flagellum,and the genes involved in flagellum movement have been reported at present,such as motA,motB,etc.,and fliA,as a secondary regulatory system,also participates in flagellum synthesis.The gene expression of fliA before and after drug treatment was observed by RT-PCR,and it was indirectly inferred that it was related to the mechanism of reducing virulence,which provided a theoretical basis for the follow-up study on the action mechanism of sdhfumarate-fliA signaling pathway in inhibiting the dynamic virulence reduction of escherichia coli in gentamicin.Results: 1 In vitro verification confirmed that the swarming of clinical strains was significantly inhibited after gentamicin treatment at sub-inhibitory concentration.Each time,three experiments were conducted experiment repeated three times.2 In vivo tests,buy 20 g around kunming male mice,according to the standard 968 strains ATCC25922 and clinical strains(separation in urine specimens)group,one of the drug treatment for experimental group,untreated as control group,mice abdominal cavity injection,the results showed that each group of the experimental group and the control group(n = 10)injection of 0.5 0.5 x 107 x 108,e.coli,the 1/2 mic gentamicin escherichia coli virulence decreased obviously after the pretreatment,mice mortality within 48 hr 108 cfu/ml,The standard strain decreased from 100% to 50%,and the clinical strain decreased from 100% to 70%.At 107cfu/ml,the standard strain decreased from 50% to 0,and the clinical strain decreased from 30% to 0.Due to the limited test time,the number of experimental animals can be further expanded in the later stage for further verification.3 RT-PCR,gentamicin pretreatment for the experimental group,gentamicin and fumaric acid treatment as well as the experimental group,don't do processing to the control group,by real-time fluorescent quantitative PCR,it is concluded that use gentamicin fliA expression quantity cut processing,gentamicin and fumaric acid treatment at the same time because of fumaric acid concentration fliA expression quantity is different,the PCR results for our proposed hypotheses:Gentamicin inhibits SDH transcription,resulting in reduced synthesis of fumaric acid.Fumaric acid,as an intracellular signaling molecule,triggers the downregulation of fliA(28),a flagella-related transcription factor,which in turn leads to the downregulation of some downstream flagellar genes,and ultimately reduces the motility of e.coli.Conclusion: 1 Gentamicin at subinhibitory concentration has the effect of inhibiting the pleximosity and thus reducing the virulence of escherichia coli.2 Gentamicin at subinhibitory concentration can inhibit the motility of bacterial flagellum by reducing the expression of relevant genes. |
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