| Part Ⅰ Study on the biological characteristics of linezolid and PA-824 dual-resistant Mycobacterium tuberculosis strainsObjective Mycobacterium tuberculosis(MTB)strains that are resistant to both linezolid(LZD)and PA-824 were induced in vitro by a two fold increase in concentration.After phenotypic and genotype identification,we investigated the biological characteristics of LZD and PA-824 dual drug-resistant MTB strains,including the growth characteristics,ultrastructure,and virulence of these strains.Methods Continuously subculture LZD or PA-824 drug-resistant strains on drugcontaining(LZD and PA-824)solid medium with 2-fold increasing concentration,and gradually induce MTB single drug-resistant strains to be resistant to LZD and PA-824 simultaneously(that is,dual drug-resistant MTB strains),and then identify MTB resistant strains by phenotype and genotype,including the detection of LZD resistancerelated genes(rpl C,rrl)and PA-824 resistance-related genes(ddn and fgd1).In addition,H37 Rv was used as a control strain,and six MTB resistant strains were selected as representative strains to investigate the biological characteristics of different MTB resistant strains,including LZD resistant strains(L1 and L3),PA-824 resistant strains(P1 and P3)and LZD and PA-824 dual-resistant MTB strains(LPDR: LP11 and PL7).By culturing LZD and PA-824 single or dual drug-resistant strains in vitro to investigate their growth characteristics,observe the ultrastructure of LZD and PA-824 single or double drug-resistant strains by transmission electron microscopy to assess their cell wall thickness and structural characteristics.Our study infected mice with the above 7 MTB strains,and observed the survival time of mice,changes in body weight,spleen and lung specific gravity,spleen and lung bacterial load,and histopathology were used to investigate the virulence characteristics of each MTB resistant strain.Results For the 7 LZD resistant strains obtained from H37 Rv induction,the MIC values for LZD were 5.99~18.28 μg/ml,which were more than 8 times before induction(0.25 μg / ml),and 7 resistant strains were detected T460 C mutation in rpl C gene.For the five PA-824 resistant strains obtained from H37 Rv induction,their MIC values(> 20 μg / ml)for PA-824 were more than 8 times that before induction(0.07 μg / ml),and sequencing results showed that the mutation of fgd1 gene did not occur,but ddn gene mutation was detected in all five strains.LZD resistant strains were subcultured on solid medium containing PA-824 with a 2-fold increase in concentration,resulting in 19 LPDR strains(labeled as LP strains;LP1-LP19);PA-824 resistant strains were through 5 generations of subculture on a solid medium containing LZD with a 2-fold increase in concentration,7 LPDR strains(labeled as PL strains;PL1-PL7)were finally obtained.For 26 LPDR strains(LP and PL strains),their MIC values for LZD and PA-824 are more than 8 times before induction,and mutations of resistance-related genes(ddn and rpl C genes)of some strains(such as LP1 and PL7)were occurred at the same time and successfully obtained strains with dual resistance to LZD and PA-824.In the LPDR strains marked as LP,the mutation frequencies of rpl C,ddn and fgd1 genes were 100%,37% and 11%,respectively;in the LPDR strains marked as PL,the mutation frequencies of rpl C,ddn and fgd1 genes were 86 %,100% and 14%,respectively.Compared with the H37 Rv,the growth curve and cell wall thickness of LZD and PA-824 single-or double-resistant MTB strains were not significantly different.After mice were infected with different MTB resistant strains(H37Rv,L1,L3,P1,P3,LP11 and PL7),the median survival time of the mice in each infection group was 27.5,more than 85,83,63.5,67,68.5,and 57 days,respectively.The survival time of mice infected with LPDR strain and PA-824 resistant strain was similar,while the survival time of mice infected with LZD resistant strain was significantly longer than that of mice infected with LPDR strain and PA-824 resistant strain,and there were statistical differences;The spleen and lung of mice infected with MTB resistant strains were roughly the same in bacterial load;pathological histology observed atypical inflammatory lesions in lung tissues,and the infection of LZD resistant strains was relatively mild.Conclusion Through the double-increasing induction in vitro,MTB strains with dual resistance to LZD and PA-824(LPDR strains)can be obtained,and it is easier to induce PA-824 resistant strains in vitro than LZD resistant strains;the C154 R mutation of the rpl C gene may be used as a marker of the LPDR strain;the LPDR strain is similar to the PA-824 resistant strain and is more virulent than the LZD resistant strain.Part Ⅱ Study on drug resistance mechanism of linezolid and PA-824 single or dual-drug resistant Mycobacterium tuberculosis strainsObjective Using linezolid(LZD)and PA-824 single or simultaneous resistant Mycobacterium tuberculosis(MTB)strains induced in vitro to explore the resistance mechanism of MTB to further clarify the related potential new drug resistance mechanisms of MTB resistant strains.Methods 38 strains of LZD and PA-824 single-resistant or double-resistant MTB strains preserved in our laboratory were chosen as the research object,we compared and analyzed the genetic sequences of these resistant strains and MTB standard strain H37 Rv by whole genome sequencing technology to find the differential genes.Then,using a suitable screening strategy,a part of the differential genes that may not be closely related to drug resistance were initially screened out.Finally,two more likely differential genes(Rv1144 and Rv2983)were selected from the screened differential genes for knockout verification.After phage-mediated homologous recombination technology was used to knock out the Rv1144 and Rv2983 genes of H37 Rv strain,respectively,MTB Rv1144 and Rv2983 gene knockout strains(Δ1144 and Δ2983)were obtained.Then determine the minimum inhibitory concentration(MIC)of LZD and PA-824 on Δ1144 and Δ2983 strains,and select MTB gene knockout strains with significantly higher MIC values(such as> 4 times MIC)for knockout genes(Rv1144 or Rv2983)backfill verification.Results Through the whole genome sequencing analysis technology,after comparing 38 LZD and PA-824 single-resistant or dual-resistant MTB strains with the standard sequence of MTB standard strain H37 Rv,5 genes were initially screened for possible resistance to MTB against LZD and PA-824 single-resistance or the dual resistance genes,which were mbt F,Rv0051,Rv1954 c,Rv1144 and Rv2983 genes.The drug susceptibility results showed that the MIC values of LZD and PA-824 on Δ1144 knockout strains were not significantly changed;however,the MIC values of PA-824 on Δ2983 knockout strains were> 20 μg/ml(The MIC value of PA-824 for H37 Rv is 0.07 μg/ml).The empty plasmid(p MV361 vector)and the replacement plasmid(p MV361-Rv2983)were transformed into H37 Rv strain by electric shock.The Δ2983 knockout strain(Δ2983-C0)and the Δ2983 knockout strain(Δ2983-C)were obtained.).The drug sensitivity results showed that the MIC value of PA-824 on Δ2983-C strain was 0.04 μg / ml,and the MIC value of Δ2983-C0 strain was> 20 μg/ml.Conclusion The mutation of Rv2983 gene is closely related to the resistance of MTB to PA-824.Part Ⅲ Induction of linezolid and PA-824 dual-resistant Mycobacterium tuberculosis strains in vitroObjective A multidrug-resistant(MDR)Mycobacterium tuberculosis(MTB)strain was used as the parent MTB strain,and linezolid(LZD)and PA-824-resistant MTB were obtained through induction in vitro.Strains will lay the foundation for screening new compounds with good antituberculosis activity and exploring the potential drug resistance mechanism of MTB dual drug resistant strains.Methods One clinical isolate MDR strain(No.16030)was selected and continuously subcultured on a drug-containing(LZD or PA-824)medium with a doubled concentration to gradually induce resistance of the 16030 strain to LZD or PA-824.The obtained LZD or PA-824 resistant strains are then continuously subcultured on a drugcontaining(LZD and PA-824)medium with a doubling concentration to gradually induce MTB mono-resistant strains to be resistant to both drug of LZD and PA-824(ie,LZD and PA-824 dual drug-resistant MDR strains,LPDR-MDR strains),and finally measured by microplate Alamar Blue assay(MABA)method.The minimum inhibitory concentration(MIC)of some anti-tuberculosis drugs on LPDR-MDR strains,that is,the phenotypic identification of LPDR-MDR strains.Results Using the 16030 strain as the parent MDR strain,two-way induction was performed by a two-fold increment method.Finally,17 LPDR-MDR strains were obtained on a medium containing 8 times MIC of LZD and 16 times MIC of PA-824.LPDR-MDR strains(labeled as LPM or PLM strains respectively),that is,LPM1 to LPM10 strains and PLM1 to PLM7 strains,their MIC values for LZD and PA-824 were more than 8 times higher than before induction.Conclusion MDR strains can obtain MDR strains(LPDR-MDR strains)that are resistant to both LZD and PA-824 through in vitro induction tests,and it is easier to induce PA-824 resistant strains in vitro than LZD resistant strains. |
PDF Full Text Request