A Study On Genotyping Of Mycobacterium Tuberculosis Clinical Isolates And Confirmation Of A Newly-detected Mutation Conferring Resistance To Isoniazid

ObjectivesIn addition to the widespread implementation of STOP TB program, we must establish a complete and accurate epidemiological surveillance system for TB control, undertake research on the mechanism of drug resistance of Mycobacterium tuberculosis (MTB) and the detection of drug-resistant TB. In order to understand the prevalence and distribution of genotypes of the circulating MTB strains in Fuzhou area and evalulate the effect of genotyping, we genotyped the clinical isolates that had been identified in recent years. The evidences of a newly-detected mutation Asn329Val in katG conferring Isoniazid resistance were presented here to elucidate the mechanism of drug resistance. Reverse dot blotting was applied to examine the mutations in the genes associated with drug resistance as a preliminary study for establishing a rapid detection of drug resistant TB strains.Methods1. A total of 245 clinical isolates of Mycobacterium tuberculosis from Fuzhou area were genotyped by standard Spoligotyping, and the patterns were compared with the international Spoligotyping database named SpolDB4. Statistical analysis was done to clarify the association between Beijing family genotype and age, gender and drug resistance.2. All of the above isolates were also categorized using the 15 loci MLVA typing method. Then the BioNumerics software (Version 5.0) was used to cluster the genotypes. The clustering rates of MTB among the different group patients were compared to explore the population characteristics that facilitate the spread of TB.3. Discriminatory power of the two genotyping methods was evaluated with HDGI (the Hunter-Gaston discriminatory index).4. Three MTB strains were recruited for the drug resistance mechanism study: a target strain FJ05122 harboring the katG with a newly-detected mutation Asn329Val, a control strain with the katG Ser315Thr and the reference strain H37Rv harboring wild-type katG gene. Expression clones and recombinant shuttle plasmids of different katG gene harboring one of the above mutations or Arg463Leu alone, or combined mutations of one of the above mutation with Arg463Leu, or wild type katG were constructed.5. The recombinant KatGs were expressed by pProEx HTb plasmids and purified to a high purity. A double staining was used to show the difference of enzymatic activities of the recombinant KatGs. The catalase and peroxidase activities were also measured and compared.6. The different katG genes were also cloned into the E. coli-mycobacterial shuttle plasmid pMV361 and then electro-transformed into Mycobacterium smegmatis strains with a low or high resistant level to INH. MIC (minimum inhibitory concentration) to isoniazid of the transformants harboring different katG genes and the parent strains were determined to detect the difference of isoniazid resistance.7. The rpoB and katG gene fragments containing the hotspot zone for mutations were amplified and sequenced to characterize the gene mutations in drug resistant isolates in Fuzhou area.8. Probes were designed to detect gene mutations in the drug resistant isolates. The sensitivity and specificity of the reverse DNA blotting assay were evaluated in comparison to DNA sequencing. Results1. Of 245 isolates spoligotyped, 223(91.02%)displayed the known spoligotypes, while 22(8.98%) were not recorded in the database. Major spoligotypes identified in Fuzhou area were Beijing plus Beijing-like lineages (53.06%, 130/245), followed by H3 lineages (14.69%, 36/245) and T1 lineages (9.80%, 24/245).2. When spoligotyping was employed alone, 70 patterns were identified, including 16 clusters by 191 isolates and 54 unique patterns. The largest cluster comprising 116 isolates was the Beijing family. The HGDI was 76.78%. The clustering rate of the isolates was 77.96%. Beijing family strains were not statistically associated with age, gender and drug resistance phenotypes. Neither were the different spoligotypes associated with drug resistance.3. When MLVA was employed alone, 163 patterns were identified, including 27 clusters by 109 isolates and 136 unique patterns. The HGDI was 98.28%. The clustering rate of the isolates was 44.49%. The 15 loci showed a variation of diversity between 0.040~0.661, among which ETR-E,MIRU26,MIRU10 were highly discriminant (h>0.58) and MIRU23 was poorly discriminant(h<0.10).4. The combination of spoligotyping and MLVA revealed 182 patterns, including 24 clusters by 87 isolates and 158 unique patterns. The HGDI was improved to 98.65%. The largest cluster comprising 116 isolates from the Beijing family could be further categorized by MLVA into 65 patterns, including 14 clusters and 51 unique patterns.5.Recombinant KatGs of high purity were obtained. Compared with those of wild type KatG, the catalase activities of recombinant KatGs with mutant Asn329val were completely depleted, while its peroxidase activities were reduced by about 30%. The recombinant KatG with Ser315Thr decreased its catalase activities and peroxidase activities by 60% and 30% respectively. 6. When transformed into the Mycobacterium Smegmatis, the mutant Asn329val katG and Ser315Thr katG had an effect of increasing the isoniazid MIC of the host from 16 to 1024, while the wild type katG lowered the MIC from 16 to 4. These effects were consistent whether in Mycobacterium smegmatis strains with low or high resistant level to INH.7. The most frequent mutations in the rpoB gene in drug-resistant isolates from Fuzhou occurred at codon Ser531, His526 and Asp516, with the mutation rates of 57.12%, 17.24% and 8.05%, respectively. The most common mutation of katG was Ser315Thr with a mutation rate of 46.91%.8. Reversed dot blotting was employed to detect mutations in drug-resistant strains. In a panel of 97 MTB isolates including 79 MDR and 18 all-sensitive ones, compared with the sequencing, for rpoB mutation detection, the sensitivity, specitivity and concordance were 87.69%, 96.88%, 90.22%; for katG S315T detection, the sensitivity, specitivity and concordance were 94.44%, 90.16%, 91.75%;Conclusions:1. Beijing family is the major genotype in Fuzhou area, with a prevalence rate of 53.06%. Beijing family strains were not statistically associated with age, gender and drug resistance phenotypes. Twenty-one new genotypes not recorded in the SpolDB4 were identified in this study.2. Spoligotyping and MLVA are simple, practical genotyping methods that can be used in TB epidemiological surveillance. The discriminatory power of MLVA is much higher than that of Spoligotyping, but spoligotyping can easily identify Beijing family. Combination of the two methods can improve the efficiency of typing.3. The diversities of MLVA loci varied greatly. MLVA discriminatory power could be improved by replacing the poorly discriminant loci with the highly discriminant ones. 4. The mechanism of Asn329Val katG conferring isoniazid resistance was that the mutant KatG lost its catalase activities completely. The mutant katG gene increased the MIC of the transformants. It is evident that the Asn329Val mutation causes the INH resistance directly.5. The reverse dot blotting can detect the gene mutations rapidly and simply.