Analysis on the characteristics of rpsL gene mutations in Streptomycin resistance isolates of Mycobacterium tuberculosis
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The objective was to understand the characteristics of rpsL gene mutation in Streptomycin resistant clinical isolates of Mycobacterium tuberculosis(M.tuberculosis) in China,and evaluate the application of rapid detection of rpsL gene mutation by PCR.The 302 M.tuberculosis clinical isolates were detected by drug sensitivity test(DST),and the rpsL was detected by PCR-directly sequence(PCR-DS).Of 59 strains were pan-susceptible to 5 drugs including INH,RFP,SM,EMB and PAS,and 61 strains were sensitive to streptomycin(SM) but resistant to the other drugs.The mutation rate of SM susceptible isolates was 5.00%(6/120).In the 182 SM resistant isolates,the rpsL mutation rate was 70.33%,and the mutation rate of codon43 Lys→Arg was 52.20%;there were 4 mutation types in codon 88,and the mutation rate was 20.33%;the mutation rate of codon 86 Arg→Gln was 0.55%.The rpsL gene mutation rate of SM resistant strains was significantly higher than that of SM-sensitive strains χ2=125.04,P0.05.In conclusion,the rpsL mutation in M.tuberculosis is highly associated with Streptomycin resistance,and codon 43 Lys→Arg mutation is the main and important type.Meanwhile,the PCR-DS could be applied to rapid detection of rpsL mutation in clinical M.tuberculosis isolates for identifying SM susceptibility.Cite
[Objective] To investigate the mutations situation of streptomycin-resistant genes in M. tuberculosis, and analysis the correlation with results of traditional drug-susceptibility testing. [Methods] The traditional cultivation, drug-susceptibility testing and LiPA were performed in 50 M. tuberculosis. [Results] The traditional drug-susceptibility showed that there were 7 rifampicin resistant strains, 12 isoniazid resistant strains, 15 streptomycin resistant strains and 2 ethambutol resistant strains. 50 M. tuberculosis were detected by LiPA showed that 4 rifampicin-resistant M. tuberculosis had TCG to TTG muta- tion at codon 531 in rpoB gene, 5 Isoniazid resistant strains had AGC to ACC mutation at codon 315 in katG gene, 6 streptomycin resistant strains and 1 streptomycin-susceptible strains had AAG to AGG mutation at codon 43 in rpsL gene, 1 streptomycin resistant strains had AAG to AGG mutation at codon 88 in rpsL gene, none strains had mutation at codon 512 or 513 in rrs gene, and 5 ethambutol resistant strains had ATG to GTG mutation at codon 306 in embB gene. The results in LiPA were consistency with PCR-DS. [Conclusion] The situation is not optimistic, it’s necessary to increasing monitoring and implementing integrated intervention.
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Objective To explore the correlation between mutations in rrs and rpsl genes of Mycobacterium tuberculosis and resistance to second-line-aminoglycosides. Methods The species identification and drug susceptibility testing of 136 clinical Mycobacterium tuberculosis isolates were conducted by using the proportion method. The genomic DNA was extracted from clinical isolates which were resistant to second-line-aminoglycosides, and used for PCR amplification of DNA fragments specific for rrs and rpsl genes and sequence analysis by means of comparison with the standard laboratory strain(H37Rv). Results Out of 136 clinical isolates, 25(18.3%) were resistant to second-line-aminoglycosides, including 13(9.56%) resistant to capreomycin, 9(6.62%) resistant to kanamycin-resistant, 2(1.47%) resistant to capreomycin and kanamycin, and 1(0.73%) resistant to capreomycin, kanamycin and amikacin. By analyzing the sequencing results with the BLAST program at the NCBI website, rrs and/or rpsl gene mutations were identified in all 25 isolates(100%, 25/25), which included 3 with combined mutations in both genes: A1401G mutation in rrs and AAG43AGG and AAA121AAG mutations in rpsl(2 isolates), and A1401G mutation in rrs and AAG88AGG and AAA121AAG mutations in rpsl(1 isolate); 3 with two mutations in rpsl: AAG43AGG and AAA121AAG(2 strains), and GGT11GTT and AAA121AAG(1 strain). The newly identified mutation at codon 11 of rpsl gene(GGT→GTT) has not been reported previously; and the rest 19 isolates had the point mutation at codon 121(AAA→AAG) in rpsl gene. Conclusion This study further confirmed the correlation between mutations in rrs and rpsl genes of Mycobacterium tuberculosis and resistance to second-line-aminoglycosides. The identification of new mutation in rpsl gene and A1401G mutation in rrs gene will provide new information for further development of rapid detection methods for drug-resistant tuberculosis.
Capreomycin
Kanamycin
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The worldwide emergence and spread of drug - resistant strains of Mycobacterium tuberculosis has adverse effects on tuberculosis (TB) control programs. The goal of this paper is to describe the advances made in the understanding of the molecular basis of M. tuberculosis resistance to streptomycin and to discuss the potential of molecular methods in early diagnosis of streptomycin resistant TB.Molecular methods such as DNA sequencing, polymerase chain reaction have been used to identify/detect mutations in rpsL gene-encoding proteins.Of the 77 SM resistant isolates, 22 (28.6%) exhibited mutation at codon 43 Lys→Arg, 11 (14.3%) isolates exhibited mutation at codon 83 Arg→Gln, 9(%) isolates exhibited mutation at codon 95 Tyr→His,whereas remaining 23 (40.8%) SM resistant isolates showed no mutation in rpsL gene. Among the 77 SM resistant isolates, 31(40.2%) were SM mono resistant and 46(59.8%) strains were poly resistant. All the 23 SM susceptible isolates as well as reference strain M.tuberculosis H37Rv exhibited wild type sequences of rpsL gene. Molecular methods to detect the most frequent mutations in the gene encoding functions that are targets for streptomycin drug have provided encouraging results for early diagnosis of resistance nature.
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Objective To establish PCR-SSCP for rapid analyzing resistant gene in multiple drug resistance of mycobacterium tuberculosis and to evaluate its use in clinics. Method 30 mycobacterium tuberculosis strains in multiple drug resistance were analyzed by PCR-SSCP and classic drug susceptibility tests. The fragments of rpoB, rpsL and katG were analyzed by single-stranded conformation polymorphism (SSCP). Results The gene mutation rate of rpoB, rpsL and katG resistance of isolated strains were 90% (27/30)?53% (16/30)?63%(19/30) Respectively. There are total 8(26.7%) strains with three genes mutation and 18(60%) strains with two genes mutation. Among the MDR-TB strains,there are 2 strains with single gene mutation and 2 strains without gene mutation. Conclusion Alterations in rpoB, rpsL, katG gene may be the important mechanisms of mycobacterium tuberculosis resistance to rifampin, streptomycin and isoniazid. The PCR-SSCP is an accurate and stable method to identify rpoB rpsL, katG showed mutation of mycobacterium tuberculosis, and it is useful for quickly identifying the MDR-TB strains in clinics.
rpoB
Single-strand conformation polymorphism
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Abstract Objective To study the molecular mechanisms of drug resistance in Mycobacterium (M) tuberculosis , to evaluate the value of the β subunit of RNA polymerase (rpoB), the ribosomal siz protein (rpsL), 16Sr RNA (rrs), catalase peroxidase gene (katG) genes, and inhA regulatory sequence as genetic markers for rifampin (RFP), streptomycin (SM), isoniazid (INH) resistance, and to develop new methods for detecting the drug resistance Method The rpoB, rpsL, rrs, katG genes, and inhA regulatory sequence in 85 M tuberculosis isolates were analyzed with polymerase chain reaction (PCR), PCR single stranded conformation polymorphism analyses (SSCP), PCR nucleotide sequence analyses (NS) and PCR restriction fragment length polymorphism (RFLP) Results The sensitivity of amplifying the drug resistant genes with PCR was 1-10 pg DNA Twenty eight drug sensitive strains had no alterations in the rpoB, rpsL, rrs, katG genes, and inhA regulatory sequences 93 3% of 45 M tuberculosis RFP resistant (RFP r) isolates had rpoB mutations Codon 531 and 526 of the rpoB are the most common sites of nucleotide substitutions 72 5% of 40 SM resistant (SM r) isolates had an identical mutation at codon 43 of the rpsL gene No isolates had a mutation at codon 88 of the rpsL Only 7 5% of these SM r isolates had A to C transversions at position 513 of the rrs gene Of 34 INH resistant (INH r) isolates, 11 8% had complete katG deletions, 55 9% had mutations in the selected region of katG Only 8 8% had alterations in the inhA regulatory sequences 60 9% of RFP r, INH r, and SM r isolates had mutations in genetic markers for these drug resistance Conclusions Most drug resistance in M tuberculosis was due to simple mutations occurring in chromosomally encoded genes Alterations in rpoB, rpsL and katG gene may be the important mechanism of M tuberculosis resistance to RFP, SM, and INH PCR, PCR SSCP, PCR NS, and PCR RFLP are going to become the simple, rapid and reliable diagnostic tests for drug resistance in M tuberculosis n polymorphism · nucleotide sequence analyses · restriction fragment length polymorphism · Mycobacterium tuberculosis
rpoB
INHA
Single-strand conformation polymorphism
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Objective To investigate possible association between the gene mutations and the development of rifampin (RFP),isoniazid (INH),streptomycin (SM),and ethambutol (EMB) resistance of Mycobacterium tuberculosis isolated from Shenzhen.Methods 182 MTB clinical isolates from Shenzhen were extensively analyzed for their rpoB,katG,inhA,rpsL and embB mutations using the reverse dot blot hybridization assay (RDBHA).L-J slant proportion method was used to evaluate the susceptibility of the isolates to INH,RFP,SM,and EMB.Results The overall frequency of gene mutation was 34.62% (63/182),and rpoB had the highest mutation frequency (24.17%,44/182).The frequency of MDR-TB was 17.03% (31/182) among the 182 isolates.The sensitivity to rpoB,katG/inhA,rpsL,and embB was 88.89%,67.50%,81.82% and 64.29%,respectively;and the specificity was 97.08%,94.37%,97.99% and 92.86%,respectively.Conclusions Mutation of ropB,especially the S531L mutation,is commonly found in the isolates.Most of the isolate were resistant to the four first-line anti-tuberculosis drugs.RDBHA can be used for fast detection of drug resistance.
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INHA
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Objective To study the molecular mechanism of Mycobacterium tuberculosis resistance to streptomycin,and establish a rapid detective method of rpsL gene mutation in M.tuberculosis.Methods The rpsL genes of 80 mycobac- terium tuberculosis clinical isolates were detected by PCR-SSCP and PCR-RFLP.Results Strain H37Rv was used as the control.In 80 mycobacterium tuberculosis clinical isolates,1 of 21(4.76%)drug-susceptible isolates and 36 of 59 (61.02 %)streptomycin-resistant isolates showed rpsL gene mutation by SSCP and RFLP.Conclusion Drug resistance of M.tuberculosis to streptomycin is related to rpsL gene mutation,PCR-SSCP and PCR-RFLP might become a rapid de- tecting method of streptomycin-resistance of M.tuberculosis.
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