Activity and mechanism of artesunate against multidrug resistant Mycobacterium tuberculosis
Objective To explore the antibacterial activity of artesunate alone or in combination with anti-tuberculosis drugs rifampicin and isoniazid against multidrug resistant Mycobacterium tuberculosis(MDR-TB),and the mechanism of artesunate combined with anti-tuberculosis drugs on MDR-TB.Methods A total of 20 cases of confirmed multidrug resistant Mycobacterium tuberculosis strains stored in the Changsha Central Hospital from January 2021 to May 2022 were selected for drug sensitivity test in vitro.According to the different drug treatment,they were divided into drug free control group,artesunate group,rifampin group,isoniazid group,artesunate combined with rifampin group,and artesunate combined with isoniazid group,and their minimum inhibitory concentrations(MIC)were measured.Gene chip method was used to detect the mutation site analysis of MDR-TB resistance gene of artesunate;real-time fluorescence quantitative PCR was used to detect the relationship between artesunate improving the effect of anti-tuberculosis drugs on MDR-TB and efflux pump,and scanning electron microscopy and transmission electron microscopy were used to observe the changes of bacterial cells before and after drug treatment.Results The 20 drug-resistant strains were all identified as MDR-TB,and the resistance genes were rpoB531C-T mutation,rpoB526C-T mutation and rpoB516A-T mutation,katG315G-A mutation and katG315G-C mutation;among them,10 strains were rpoB531C-T mutation and katG315G-A mutation;6 strains were rpoB531C-T mutation and katG315G-C mutation;4 strains were rpoB516A-T mutation and katG315G-A mutation.Strains No.3 and 8 were randomly selected.Their MDR-TB resistance sites were rpoB531C-T mutation and KatG315G-C mutation.After treatment with artesunate for 24 hours,rpoB531C-T site mutation and KatG315G-C site mutation were detected.None had been changed.When MDR-TB was treated by artesunate alone,the MIC was higher;when treated with artesunate combined with rifampicin and isoniazid,the MIC value was significantly lower,the differences were statistically significant(all P＜0.001).Among the 20 MDR-TB strains,only 2 strains in the artesunate group could significantly inhibit the expression of efflux pump genes efpA and RV1250 mRNA,and the differences were statistically significant(t=6.635,16.520;both P＜0.05).There was no statistically significant difference in the expression of efpA and RV1250 mRNA among the other strains(all P＞0.05).Scanning electron microscopy results showed that compared with the control group,after adding artesunate,the bacterial cells dried up and shrank;transmission electron microscopy results showed that after artesunate treatment,lipids fell off and the bacterial cells died.Conclusions Artesunate alone did not show anti-tuberculosis activity against MDR-TB,but when combined with anti-tuberculosis drugs,it could improve the drug sensitivity of anti-tuberculosis drugs against MDR-TB.The drug efflux pump efpA and RV1250 genes might not be the main mechanism by which artesunate exerted its effects.Artesunate could destroy the integrity of Mycobacterium tuberculosis cell structure,and its mechanism might be related to artesunate increasing the accumulation of anti-tuberculosis drugs in Mycobacterium tuberculosis.
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