katG Mutations Research Articles

Cross-resistance of isoniazid, para-aminosalicylic acid and pasiniazid against isoniazid-resistant Mycobacterium tuberculosis isolates in China.

Pasiniazid is a chemical complex of isoniazid (INH) and para-aminosalicylic acid (PAS). The aim of this study was to explore the cross-resistance of INH, PAS and pasiniazid against INH-resistant Mycobacterium tuberculosis isolates in China. A Microplate alamarBlue® Assay was performed to determine the minimum inhibitory concentrations (MICs) of INH, PAS and pasiniazid against 109 INH-resistant M. tuberculosis isolates. A statistical analysis of the relationship between different genotypes, gene mutations, and INH, PAS or pasiniazid susceptibility was then performed. Among the 109 INH-resistant isolates, 13 (11.9%) and 21 (19.3%) showed resistance to PAS and pasiniazid, respectively. Among the 13 PAS-resistant M. tuberculosis isolates, 11 remained susceptible to pasiniazid. Of 63 INH-resistant isolates harbouring mutations in katG, the inhA promoter or the oxyR-ahpC intergenic region, 52 remained susceptible to pasiniazid. Moreover, 11 of 13 pasiniazid-resistant isolates carried mutations in katG, the inhA promoter or the oxyR-ahpC intergenic region. Taken together, these results demonstrate that PAS resistance and mutations in thekatG gene, inhA promoter or oxyR-ahpC intergenic region in INH-resistant M. tuberculosis have little effect on pasiniazid susceptibility.

Change in prevalence and molecular characteristics of isoniazid-resistant tuberculosis over a 10-year period in China

BackgroundIsoniazid (INH) represents the cornerstone for the treatment of cases infected with Mycobacterium tuberculosis (MTB) strains. Several molecular mechanisms have been shown to be the major causes for INH resistance, while the dynamic change of mutations conferring INH resistance among MTB strains during the past decade is still unknown in China.MethodsIn this study, we carried out a comparative analysis of the INH minimal inhibitory concentration (MIC) distribution, and investigate the dynamic change of molecular characteristics among INH-resistant MTB strains between 2005 and 2015.ResultsThe proportion of INH resistance (39.0%, 105/269) in 2015 was significantly higher than in 2005 (30.0%, 82/273; P = 0.03). Among 269 isolates collected in 2015, 76 (28.3%, 76/269) exhibited high-level INH-resistance (MIC≥32 mg/L), which was significantly higher than that in 2005 (20.5%, 56/273, P = 0.04). In addition, a significantly higher percentage of INH-resistant isolates carried inhA promoter mutations in 2015 (26.7%) versus that in 2005 (14.6%, P = 0.04), while no significant difference was observed in the rates of isolates containing katG mutations between 2005 (76.8%) and 2015 (70.5%, P = 0.33). Notably, the proportion of MTB isolates with inhA mutations (26.7%, 28/105) for patients who had previous exposure to protionamide (PTH) was higher than that for patients who had no previous exposure to PTH (21.4%, 6/28).ConclusionsIn conclusion, our results demonstrated that the proportion of INH-resistant MTB isolates significantly increased during the last decade, which was mainly attributed to an increase of high-level INH-resistant MTB. In addition, prior exposure to PTH may be associated with the increased frequency of INH-resistant tuberculosis strains with inhA mutations in China.

Use of nanotechnology for infectious disease diagnostics: application in drug resistant tuberculosis

BackgroundThe increased transmission of multidrug-resistant (MDR) tuberculosis (TB) poses a challenge to tuberculosis prevention and control in Sri Lanka. Isoniazid (INH) is a key element of the first line anti tuberculosis treatment regimen. Resistance to INH may lead to development of MDR TB. Therefore, early detection of INH resistance is important to curb spread of resistance. Due to the limited availability of rapid molecular methods for detection of drug resistance in Sri Lanka, this study was aimed at developing a simple and rapid gold nanoparticle (AuNP) based lateral flow strip for the simultaneous detection of the most common INH resistance mutation (katG S315 T, 78.6%) and Mycobacterium tuberculosis (MTb).MethodsLateral flow strip was designed on an inert plastic backing layer containing a sample pad, nitrocellulose membrane and an absorption pad. Biotin labeled 4 capture probes which separately conjugated with streptavidin were immobilized on the nitrocellulose. The test sample was prepared by multiplex PCR using primers to amplify codon 315 region of the katG gene and MTb specific IS6110 region. The two detection probes complementary to the 5′ end of each amplified fragment was conjugated with gold nanoparticles (20 nm) and coupled with the above amplified PCR products were applied on the sample pad. The hybridization of the amplified target regions to the respective capture probes takes place when the sample moves towards the absorption pad. Positive hybridization is indicated by red colour lines.ResultsThe three immobilized capture probes on the strip (for the detection of TB, katG wild type and mutation) were 100 and 96.6% specific and 100 and 92.1% sensitive respectively.ConclusionThe AuNP based lateral flow assay was capable of differentiating the specific mutation and the wild type along with MTb identification within 3 h.

Computational approach identifies protein off-targets for Isoniazid-NAD adduct: hypothesizing a possible drug resistance mechanism in Mycobacterium tuberculosis

Isoniazid is an important antitubercular molecule identified as a drug of choice in tuberculosis treatment. As such, INH is an inactive prodrug; it acquires an active conformation by forming an adduct with NAD. The adduct targets inhA protein, a reductase responsible for fatty acid chain elongation in the cell wall of Mycobacterium tuberculosis. Resistance to INH is majorly contributed by mutations in inhA, katG and geneic and non-geneic regions associated with efflux genes. Despite being widespread, the mechanism of resistance remains unknown in ∼15% of INH-resistant strains. Studies report that an intracellular increase in NADH concentration prevents inhA inhibition, leading to INH resistance. In the pursuit of finding possible resistance mechanisms, we set out to find NAD binding proteins to explore similarities in structure and NAD binding property of these proteins with that of inhA. We identified 172 NAD binding proteins, of which 53 were identified to have sequence or structural similarity to inhA. By performing docking analysis on selected proteins, we identified INH-adduct to have good binding affinity despite very minimal structural similarity to inhA. This analysis was further supported by principal component analysis, which identified 65 proteins with NAD binding conformation similar to that of inhA. These findings prompt us to hypothesize that upon exposure to INH, bacteria tries to reduce inhA susceptibility by inducing expression of these NAD binding proteins through increase in NADH concentration. This in turn favours off-target binding and leads to decreased binding and potency of INH, thus contributing indirectly to INH resistance.Communicated by Ramaswamy H. Sarma

RpoB, katG and inhA mutations in multi-drug resistant strains of Mycobacterium tuberculosis clinical isolates from southeast Mexico

IntroductionPrevious knowledge of molecular mechanisms related with multi-drug resistances in tuberculosis is important if molecular diagnostic procedures want to be used in specific geographical regions. For that reason, the aim of this study was to investigate the mutations at rpoB, katG and inhA in multi-drug resistant tuberculosis isolates from Southeast Mexico. MethodsIsolates of tuberculosis with a confirmed resistance against rifampicin and isoniazid were collected and sequencing analysis was performed of the rpoB rifampicin resistance-determining region, the katG and the encoding region of inhA. ResultOf 74 isolates with multidrug resistance, 34 (46%) presented six mutations in katG; the most abundant was katG315 in 29 (39%) isolates. At inhA, nine (11%) isolates presented three mutations; the most frequent was inhA21, located in five (6%) strains. Eleven polymorphisms were observed at rpoB in 61 (82%) isolates, prevailing rpoB531 and rpoB 526 in 48 (64%) and ten (12%) isolates, respectively. Eleven double combinations were observed in 39 (52%) isolates, the most common of which was rpoB531+katG315, found in 22 (29%) strains. ConclusionThis study provides valuable information on the diversity of polymorphisms in genes related to multidrug-resistant tuberculosis, as well as the presence of new mutations not previously described; this information should be considered in the implementation of molecular diagnostic tests.

Role of line probe assay in diagnosis and detection of drug resistance in Mycobacterium tuberculosis

Tuberculosis caused by Mycobacterium tuberculosis has remained a major global health problem worldwide. TB requires prolonged period of time for isolation by conventional culture methods. The emergence and spread of multi drug resistant (MDR-TB) poses great threats and challenges in controlling the infection. MDR-TB is resistant to both first line drugs rifampicin and isoniazid. PCR tests are based on targeting the mutation in rpoB, katG and inhA genes which can detect resistance to these drugs. To compare microscopy, conventional culture and Line probe assay for the detection of M. tuberculosis & detect rifampicin and isoniazid resistance using Lineprobe assay in various clinical samples. A total of 347 suspected patients of tuberculosis were included in the study. Demographic details & clinical presentation was noted. Various samples were received & processed for ZN staining, culture on LJ media and Line probe assay. Out of 347 cases, majority of cases were in the age group of 51-60 years (18.4%). Majority of the population was males (65.1%). Among suspected tuberculosis patients, cough with expectoration (55.9%) was the commonest complaint. Microscopy was positive in 17.3%, conventional culture was positive in 16.1% and line probe assay was positive in 26.2%. Out of 347, 91 were diagnosed with MTB, out of which 85.7% were sensitive to both rifampicin and isoniazid whereas 14.3% showed resistance to either rifampicin / isoniazid or both. LPA & direct microscopy are a good screening method for early diagnosis and detection of drug resistance but are not a complete replacement of conventional culture which is still a gold standard.

Capacity of Abbott RealTime MTB RIF/INH to detect rifampicin- and isoniazid-resistant tuberculosis.

<sec id="st1"> <title>BACKGROUND</title> Abbott RealTime MTB RIF/INH Resistance (RT RIF/INH) is a new assay for the detection of resistance to rifampicin (RIF) and isoniazid (INH) in tuberculosis (TB) patients. </sec> <sec id="st2"> <title>OBJECTIVE</title> To evaluate the capacity of RT RIF/INH to detect resistance-associated mutations in target genes. </sec> <sec id="st3"> <title>METHODS</title> A total of 311 Mycobacterium tuberculosis strains that had been pre-characterised using genotypic methods (GenoType® MTBDRplus, Sanger sequencing) and phenotypic drug susceptibility testing were subjected to DNA extraction on Abbott m2000sp and analysed using RT RIF/INH. Detection of heteroresistant mutations was studied with artificial mixtures of wild-type and mutant DNA. </sec> <sec id="st4"> <title>RESULTS</title> Overall sensitivity and specificity values of RT RIF/INH to detect resistance were respectively 87.2% and 98.4% for RIF and respectively 90.1% and 99.2% for INH. The capacity of RT RIF/INH to detect specific mutations was 100% for katG, inhA and frequent rpoB mutations, and 76% for rare rpoB mutations. Among the latter, two rare mutations were not consistently detected. With heteroresistant samples, RT RIF/INH reported resistance if samples contained at least 75-90% of mutant DNA. </sec> <sec id="st5"> <title>CONCLUSION</title> RT RIF/INH is a reliable high-throughput assay for the detection of RIF and INH resistance markers. The ability to detect INH resistance also may be of benefit in areas with high rates of INH-resistant, non-multidrug-resistant TB. </sec>.

Characterisation of drug resistance-associated mutations among clinical multidrug-resistant Mycobacterium tuberculosis isolates from Hebei Province, China.

Multidrug-resistant tuberculosis (MDR-TB) is a major public-health problem in China. However, there is little information on the molecular characterisation of clinical MDR-TB isolates in Hebei Province. In this study, 123 MDR-TB isolates were identified in sputum cultures using traditional drug susceptibility testing. The isolates were analysed for mutations in seven genes associated with resistance to antituberculous four drugs: katG and inhA promoter for isoniazid (INH); rpoB for rifampicin (RIF); gyrA and gyrB for ofloxacin (OFLX); and rrs and eis promoter for kanamycin (KAN). All strains were genotyped by spoligotyping and 15-loci MIRU-VNTR analysis. A total of 39 distinct mutations were found at the seven loci in 114/123 (92.7%) MDR-TB isolates. Frequencies of INH, RIF, OFLX and KAN resistance-associated mutations were 82.1% (101/123), 83.7% (103/123), 92.1% (35/38) and 76.2% (16/21), respectively. The most prevalent mutations involved in resistance were: Ser315Thr in katG (70/123; 56.9%) and C(-15)T in inhA (15/123; 12.2%) for INH; Ser531Leu in rpoB (72/123; 58.5%) for RIF; Asp94Gly in gyrA (10/38; 26.3%) for OFLX; and A1401G in rrs (12/21; 57.1%) for KAN. Four novel gyrB mutants (Leu442Leu, Ser447Phe, Asn499Thr and Ala504Val) were identified. Mutations in katG, rpoB (or both) and the inhA promoter showed a sensitivity of 75.6% and specificity of 97.0% for detection of MDR-TB. DNA sequencing of the seven loci was 57.1% sensitive and 91.0% specific for prediction of XDR-TB isolates. These results may be of value in rapid molecular detection of MDR- and XDR-TB isolates in clinical samples in Hebei Province.

Prevalence and detection of drug resistant mutations in Mycobacterium tuberculosis among drug na\xefve patients in Nairobi, Kenya

BackgroundTuberculosis (TB), an ancient scourge of humanity known for several thousands of years, is still a significant public health challenge in many countries today even though some progress has been made in recent years in controlling the disease. The study’s aim was to determine the prevalence of mutations responsible for drug resistance in Mycobacterium tuberculosis among patients visiting selected health centers in Nairobi, Kenya.MethodsThe cross-sectional study involved 132 TB positive patients visiting Mbagathi and Chandaria hospitals between September 2015 and August 2016. Sputum samples were collected from the participants and handled in a biosafety level 3 laboratory at the Kenya Medical Research Institute (KEMRI). Samples were decontaminated using N-Acetyl-L-Cysteine (NALC) – Sodium Hydroxide (NALC-NaOH), stained using Zeihl–Neelsen (ZN), and cultured in Mycobacterium Growth Indicator Tube (MGIT). DNA extracted from cultured isolates using Genolyse™ technique was subjected to Multiplex PCR amplification and reverse hybridization for detection of drug resistance mutations on rpoB, katG, inhA, gyrA, gyrB, rrs and eis genes using Hain Genotype MTBDRplus and MTBDRsl.ResultsAll 132 (100%) patients included in the study were culture positive for M. tuberculosis. Among them, 72 (54%) were male while the remaining 60 (46%) were female. The mean age of the patients was 26.4 ± 19.4 (SD) with a range of 18 to 60 years. Overall, the prevalence of the resistance to first and second-line TB drugs was 1.5% (2/132). Resistance to isoniazid (INH) was observed in 1 of 132 patients (0.8%), as was multi-drug resistant tuberculosis (MDR-TB), also at 0.8%. No resistance to fluoroquinolones (FQ) or kanamycin (KAN) was observed. The INH resistant strain had the katG mutations S315 T, while mutations detected for the MDR-TB were katG S513 T for INH, rpoB S531 L for rifampicin (RIF) and rrs G1484 T for cross-resistance to aminoglycosides/capreomycin (AG/CP).ConclusionsMolecular analysis confirms transmission of the drug-resistant M. tuberculosis strains. The data suggested that there is homogeneity when it comes to the type of drug resistance and mutation that occurs in the region. This calls for intensified drug resistance surveillance and drug adherence among patients infected with TB.

Molecular epidemiology and whole genome sequencing analysis of clinical Mycobacterium bovis from Ghana.

BackgroundBovine tuberculosis (bTB) caused by Mycobacterium bovis is a re-emerging problem in both livestock and humans. The association of some M. bovis strains with hyper-virulence, MDR-TB and disseminated disease makes it imperative to understand the biology of the pathogen.MethodsMycobacterium bovis (15) among 1755 M. tuberculosis complex (MTBC) isolated between 2012 and 2014 were characterized and analyzed for associated patient demography and other risk factors. Five of the M. bovis isolates were whole-genome sequenced and comparatively analyzed against a global collection of published M. bovis genomes.ResultsMycobacterium bovis was isolated from 3/560(0.5%) females and 12/1195(1.0%) males with pulmonary TB. The average age of M. bovis infected cases was 46.8 years (7-72years). TB patients from the Northern region of Ghana (1.9%;4/212) had a higher rate of infection with M. bovis (OR = 2.7,p = 0.0968) compared to those from the Greater Accra region (0.7%;11/1543). Among TB patients with available HIV status, the odds of isolating M. bovis from HIV patients (2/119) was 3.3 higher relative to non-HIV patients (4/774). Direct contact with livestock or their unpasteurized products was significantly associated with bTB (p<0.0001, OR = 124.4,95% CI = 30.1–508.3). Two (13.3%) of the M. bovis isolates were INH resistant due to the S315T mutation in katG whereas one (6.7%) was RIF resistant with Q432P and I1491S mutations in rpoB. M. bovis from Ghana resolved as mono-phyletic branch among mostly M. bovis from Africa irrespective of the host and were closest to the root of the global M. bovis phylogeny. M. bovis-specific amino acid mutations were detected among MTBC core genes such as mce1A, mmpL1, pks6, phoT, pstB, glgP and Rv2955c. Additional mutations P6T in chaA, G187E in mgtC, T35A in Rv1979c, S387A in narK1, L400F in fas and A563T in eccA1 were restricted to the 5 clinical M. bovis from Ghana.ConclusionOur data indicate potential zoonotic transmission of bTB in Ghana and hence calls for intensified public education on bTB, especially among risk groups.

Genetic diversity and drug resistance of Mycobacterium tuberculosis in Ecuador.

The genetic diversity of Mycobacterium tuberculosis in Quito, Ecuador is not well known. To investigate mutations related to drug resistance and bacterial genotypes in M. tuberculosis strains in Ecuador. This was a retrospective study of M. tuberculosis isolates from 104 patients. Isolates were phenotypically resistant to rifampicin (RMP) and/or isoniazid (INH). The genotype was determined using 24-locus mycobacterial interspersed repetitive units-variable-number tandem repeats (MIRU-VNTR). Isolates showed mutations in the rpoB and katG genes, and the inhA promoter. In rpoB, we found 13 genetic alterations at codons 511, 513, 514, 515, 516, 526 and 531. Forty-six (44.2%) RMP-resistant isolates belonged to codon 531. In katG, there were nine genetic alterations at codons 296, 312, 314, 315, 322, 324 and 351. Fifty-three (51%) INH-resistant isolates belonged to codon 315. Five mutations not previously described were identified in katG: Thr324Ser, Thr314Ala, Ala312Pro, Trp351Stop and deleted G at 296 codon. The Latin American Mediterranean (LAM) (33.7%) and Ghana (30.8%) lineages presented most of the main mutations observed. This is the first report from Ecuador; it describes five new mutations in katG and indicates that LAM is the most prevalent lineage.

РАСПРОСТРАНЕННОСТЬ МУТАЦИЙ В ГЕНАХ МИКОБАКТЕРИЙ ТУБЕРКУЛЕЗА, КОДИРУЮЩИХ ЛЕКАРСТВЕННУЮ УСТОЙЧИВОСТЬ К ИЗОНИАЗИДУ И РИФАМПИЦИНУ, У БОЛЬНЫХ ТУБЕРКУЛЕЗОМ В РАЗНЫХ ВОЗРАСТНЫХ ГРУППАХ

The objective : to study the prevalence and ranges of mutations in katG, inhA, and ahpC genes coding resistance to isoniazid, and in rpoB gene coding resistance to rifampicin in patients with pulmonary tuberculosis from different age groups. Subjects and methods . 253 patients were examined; based on their age they were divided into 3 groups (Group 1 – from 18 to 30 years, Group 2 – from 31 to 60 years, group 3 – from 61 to 80 years). Biological microchips with TB-Biochip reagent kit (Russia) were used. Results . In Groups 1 and 2, compared to Group 3, mutations were recorded more often simultaneously in katG, inhA, ahpC genes and rpoB genes which meant that multiple drug resistance (MDR) prevailed (32.4 and 36.1% against 13.2%, respectively). In general, mutations in katG gene (including MDR + isolated isoniazid resistance) were also confidently more frequent in Groups 1 and 2 (30.9 and 32.7%) versus Group 3 (10.5%). Similar changes were found in the prevalence of the adverse type of ser 315-> Thr mutation of katG gene, which was registered in 23.5% of cases in Group 1, in 21.8% of cases in Group 2, and in 7.9% – in Group 3. In Group 3, mutations in rpoB gene (including MDR) were less frequently recorded and made 26.5% versus 45.5% in Group 1 and 46.9% in Group 2. There were no confident differences in ranges of mutations in rpoB gene between the examined groups of patients. Thus, a significantly lower level of resistance to isoniazid and rifampicin in elderly and senile tuberculosis patients was found compared to young and middle-aged patients, which was registered at the level of genetic mutations.

Detection of Genetic mutations in inhA and katG for Isoniazid and its Association with Rifampicin Resistance in Tuberculosis Confirmed by Line Probe Assay-its Rationality or Isoniazid Prophylaxis Empirical or DST Guided

Out of 442 cases of rifampicin resistant MTB strains 410 cases showed Isoniazid resistance. Out of which exclusive involvement with inhA was seen in 32 cases. 19 cases showed involvement of both inhA as well as katG resistance 359 cases accounting to 87.5% showed exclusive involvement of katG thereby conferring high degree of resistance. The higher percentage of katG induced Isoniazid resistance among the Rifampicin resistant strains depicts the spread of higher degree of Isoniazid resistance. The question remains how effective would be the Isoniazid prophylaxis in pediatric age group in this scenario. The analysis has shown that only around 14.93% cases reflected exclusive mutations at around 15 and 16 regions of the inhA gene and MUT3 accounted to a percentage of 60.3% among the inhA dependant isoniazid resistance.

Conventional and molecular determination of drug resistance in Mycobacterium tuberculosis and Mycobacterium bovis isolates in cattle

This study was aimed out to explore the presence of drug resistance among M. tuberculosis and M. bovis isolates (n = 51) of bovine origin by conventional broth microdilution method and molecular methods. By broth microdilution method, 16 isolates were found to be resistant to isoniazid, 08 isolates were resistant to pyrazinamide, 09 isolates were resistant to rifampicin and 07 isolates were found to be resistant for ethambutol. Two isolates showed resistance to rifampicin and pyrazinamide, one isolate showed resistance to pyrazinamide and ethambutol and 03 isolates showed resistance to isoniazid and ethambutol. None of isolates showed multi drug resistance (MDR). Other than the M. bovis strains, none of the other M. tuberculosis isolates showed any resistance to pyrazinamide. Molecular methods by multiplex PCR targeting katG, pncA, rpoB genes, multiplex allele specific PCR to detect mutation in embB codon 306 and sequencing showed point mutation in katG and rpoB gene. No mutation could be detected in the embB gene by multiplex allele specific PCR. The results indicates that further elaborate studies need to be carried out due to the presence of drug resistant M. tuberculosis in bovines which could be due to spill over from human in tuberculosis endemic areas making TB eradication programme more challenging.

Detection of novel mutations associated with independent resistance and cross-resistance to isoniazid and prothionamide in Mycobacterium tuberculosis clinical isolates

ObjectivesProthionamide, a structural analogue of isoniazid, is used mainly for treating multidrug-resistant tuberculosis (MDR-TB). Both drugs have a common target InhA, so prothionamide can be ineffective against isoniazid-resistant (INHR) Mycobacterium tuberculosis. We aimed to investigate the prevalence of mutations in katG, ethA, ndh, ethR, mshA, inhA and/or its promoter associated with independent resistance and cross-resistance to INHR and/or prothionamide-resistant (PTOR) M. tuberculosis isolates. MethodsWe sequenced the above genes in 206 M. tuberculosis isolates with susceptibility testing against ten drugs. ResultsOf the 173 INHR PTOR isolates, 170 (98.3%) harboured mutations in katG, 111 (64.2%) in ethA, 58 (33.5%) in inhA or its promoter, 5 (2.9%) in ndh, 3 (1.7 %) in ethR and 2 (1.2%) in mshA. Among the 18 INHR PTOS isolates, mutations in katG were found in all of them; one had a mutation in the inhA promoter and another in ndh. Of the five INHS PTOR isolates, four showed mutations in ethA and two in the inhA promoter. Notably, 55 novel non-synonymous mutations were found in them and 20.2% of the PTORM. tuberculosis isolates harboured no known mutations. ConclusionsThis is the first report to investigate cross-resistance between INHR and/or PTOR isolates. Among INHR (94.4% MDR-TB) M. tuberculosis isolates, the high diversity of mutations for independent resistance and cross-resistance with prothionamide highlight the importance of both phenotypic susceptibility and genotypic diagnosis when using it to treat patients with INHR-TB. The high proportion (one-fifth) of PTORM. tuberculosis isolates showed no known mutation related to PTOR genes, so uncovered resistance mechanism(s) of prothionamide exist.

The impact of combined gene mutations in inhA and ahpC genes on high levels of isoniazid resistance amongst katG non-315 in multidrug-resistant tuberculosis isolates from China

Whole-genome sequencing was used to analyze the profiles of isoniazid (INH) resistance-related mutations among 188 multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) and mono-INH-resistant isolates collected in a recent Chinese national survey. Mutations were detected in 18 structural genes and two promoter regions in 96.8% of 188 resistant isolates. There were high mutation frequencies in katG, the inhA promoter, and ahpC-oxyR regulator regions in INH-resistant isolates with frequencies of 86.2%, 19.6%, and 18.6%, respectively. Moreover, a high diversity of mutations was identified as 102 mutants contained various types of single or combined gene mutations in the INH-resistant group of isolates. The cumulative frequencies of katG 315 or inhA-P/inhA mutations was 68.1% (128/188) for the INH-resistant isolates. Of these isolates, 46 isolates (24.5% of 188) exhibited a high level of resistance. A high level of resistance was also observed in 21 isolates (11.2% of 188) with single ahpC-oxyR mutations or a combination of ahpC-oxyR and katG non-315 mutations. The remaining 17 mutations occurred sporadically and emerged in isolates with combined katG mutations. Such development of INH resistance is likely due to an accumulation of mutations under the pressure of drug selection. Thus, these findings provided insights on the levels of INH resistance and its correlation with the combinatorial mutation effect resulting from less frequent genes (inhA and/or ahpC). Such knowledge of other genes (apart from katG) in high-level resistance will aid in developing better strategies for the diagnosis and management of TB.

RpoB, katG and inhA mutations in multi-drug resistant strains of Mycobacterium tuberculosis clinical isolates from southeast Mexico

IntroductionPrevious knowledge of molecular mechanisms related with multi-drug resistances in tuberculosis is important if molecular diagnostic procedures want to be used in specific geographical regions. For that reason, the aim of this study was to investigate the mutations at rpoB, katG and inhA in multi-drug resistant tuberculosis isolates from Southeast Mexico. MethodsIsolates of tuberculosis with a confirmed resistance against rifampicin and isoniazid were collected and sequencing analysis was performed of the rpoB rifampicin resistance-determining region, the katG and the encoding region of inhA. ResultOf 74 isolates with multidrug resistance, 34 (46%) presented six mutations in katG; the most abundant was katG315 in 29 (39%) isolates. At inhA, nine (11%) isolates presented three mutations; the most frequent was inhA21, located in five (6%) strains. Eleven polymorphisms were observed at rpoB in 61 (82%) isolates, prevailing rpoB531 and rpoB 526 in 48 (64%) and ten (12%) isolates, respectively. Eleven double combinations were observed in 39 (52%) isolates, the most common of which was rpoB531+katG315, found in 22 (29%) strains. ConclusionThis study provides valuable information on the diversity of polymorphisms in genes related to multidrug-resistant tuberculosis, as well as the presence of new mutations not previously described; this information should be considered in the implementation of molecular diagnostic tests.

Molecular detection of rifampin, isoniazid, and ofloxacin resistance in Iranian isolates of &lt;em&gt;Mycobacterium tuberculosis&lt;/em&gt; by high-resolution melting analysis

BackgroundThe emergence of drug resistance among Mycobacterium tuberculosis (MTB) strains is a serious health concern worldwide. The development of rapid molecular diagnostic methods in recent years has a significant impact on the early detection of resistance to major anti-TB drugs in MTB isolates, which helps in employing appropriate treatment regimen and prevents the spread of drug-resistant strains. This study was designed to evaluate the efficacy of real-time PCR and high-resolution melting (HRM) curve analysis for the determination of resistance to rifampin (RIF), isoniazid (INH), and ofloxacin (OFX) in MTB isolates and to investigate their resistance-related mutations.MethodsHRM analysis was performed to screen 52 (32 drug-resistant and 20 fully susceptible) MTB clinical isolates for mutations in rpoB, katG, mab-inhA, and gyrA genes. The HRM results were then confirmed by DNA sequencing.ResultsIn total, 32 phenotypically resistant isolates, comprising 18 RIF-, 16 INH-, and five OFX- resistant strains, were investigated. HRM analysis successfully identified 15 out of 18 mutations in rpoB, 14 out of 16 mutations in katG and mab-inhA, and four out of five mutations in gyrA conferring resistance to RIF, INH, and OFX, respectively. The obtained sensitivity and specificity, respectively, for HRM in comparison with phenotypic susceptibility testing were found to be 83.3% and 100% for RIF, 87.5% and 100% for INH, and 80% and 100% for OFX. In five resistant strains (12.8%), no mutation was detected by using HRM and DNA sequencing.ConclusionHRM assay is a rapid, accurate, and cost-effective method possessing high sensitivity and specificity for the determination of antibiotic resistance among MTB clinical isolates and screening of their associated mutations. This method can generate results in a shorter period of time than taken by the phenotypic susceptibility testing and also allows for timely treatment and prevention of the emergence of possible MDR strains.

Correlation between GenoType MTBDRplus Assay and Phenotypic Susceptibility Test for Prothionamide in Patients with Genotypic Isoniazid Resistance.

BackgroundThe purpose of this study was to analyze the relationship between the gene mutation patterns by the GenoType MTBDRplus (MTBDRplus) assay and the phenotypic drug susceptibility test (pDST) results of isoniazid (INH) and prothionamide (Pto).MethodsA total of 206 patients whose MTBDRplus assay results revealed katG or inhA mutations were enrolled in the study. The pDST results were compared to mutation patterns on the MTBDRplus assay.ResultsThe katG and inhA mutations were identified in 68.0% and 35.0% of patients, respectively. Among the 134 isolated katG mutations, three (2.2%), 127 (94.8%) and 11 (8.2%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Among the 66 isolated inhA mutations, 34 (51.5%), 18 (27.3%) and 21 (31.8%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Of the 34 phenotypic Pto resistant isolates, 21 (61.8%), 11 (32.4%), and two (5.9%) had inhA, katG, and both gene mutations.ConclusionIt is noted that Pto may still be selected as one of the appropriate multidrug-resistant tuberculosis regimen, although inhA mutation is detected by the MTBDRplus assay until pDST confirms a Pto resistance. The reporting of detailed mutation patterns of the MTBDRplus assay may be important for clinical practice, rather than simply presenting resistance or susceptibility test results.

Biochemical Characterization of Isoniazid-resistant Mycobacterium tuberculosis: Can the Analysis of Clonal Strains Reveal Novel Targetable Pathways?

Tuberculosis (TB) continues to be an important public health threat worldwide, due in part to drug resistant Mycobacterium tuberculosis (Mtb) strains. The United States recently reported a shortage of isoniazid (INH), which could drive higher INH resistance rates. Changes in the Mtb proteome before and after acquisition of INH resistance in a clean genetic background remain understudied and may elucidate alternate drug targets. Here, we focused on Mtb clonal strains to characterize the consequences of INH resistance on mycobacterial metabolism. Proteomic analysis was conducted by liquid-chromatography tandem mass spectrometry (LC-MS/MS) of cellular and secreted fractions, followed by a normalized spectral counting (NSAF) analysis (data are available via ProteomeXchange with identifier PXD009549). Two different Mtb clonal pairs representing a specific genetic lineage (one clinical and one generated in the laboratory) but sharing a katG mutation associated with INH resistance, were used in our analysis. Overall, we found 26 Mtb proteins with altered abundances after acquisition of INH resistance across both Mtb genetic lineages studied. These proteins were involved in ATP synthesis, lipid metabolism, regulatory events, and virulence, detoxification, and adaptation processes. Proteomic findings were validated by Western blotting analyses whenever possible. Mycolic acid (MA) analysis through LC/MS in the clonal Mtb pairs did not reveal a common trend in the alteration of these fatty acids across both INHr strains but revealed a significant reduction in levels of the two more abundant α-MA features in the clinical INHr strain. Interestingly, the clinical clonal pair demonstrated more variation in the abundance of the proteins involved in the FAS II pathway. Together, the proteomic and lipidomic data highlight the identification of potential drug targets such as alternative lipid biosynthetic pathways that may be exploited to combat clinically relevant Mtb INHr strains.