Drug resistance-conferring mutations in Mycobacterium tuberculosis from Madang, Papua New Guinea

ABSTRACT: BACKGROUND: Monitoring drug resistance in Mycobacterium tuberculosis is essential to curb the spread of tuberculosis (TB). Unfortunately, drug susceptibility testing is currently not available in Papua New Guinea (PNG) and that impairs TB control in this country. We report for the first time M. tuberculosis mutations associated with resistance to first and second-line anti-TB drugs in Madang, PNG. A molecular cluster analysis was performed to identify M. tuberculosis transmission in that region. RESULTS: Phenotypic drug susceptibility tests showed 15.7% resistance to at least one drug and 5.2% multidrug resistant (MDR) TB. Rifampicin resistant strains had the rpoB mutations D516F, D516Y or S531L; isoniazid resistant strains had the mutations katG S315T or inhA promoter C15T; streptomycin resistant strains had the mutations rpsL K43R, K88Q, K88R), rrs A514C or gidB V77G. The molecular cluster analysis indicated evidence for transmission of resistant strain. CONCLUSIONS: We observed a substantial rate of MDR-TB in the Madang area of PNG associated with mutations in specific genes. A close monitoring of drug resistance is therefore urgently required, particularly in the presence of drug-resistant M. tuberculosis transmission. In the absence of phenotypic drug susceptibility testing in PNG, molecular assays for drug resistance monitoring would be of advantage.     

新疆南疆MTB临床分离株异烟肼耐药情况及耐药相关基因的突变研究

目的:探索新疆南疆部分地区维吾尔族结核病人群中分离到的结核分枝杆菌基因组中的katG、inhA、kasA、ahpC基因突变与耐异烟肼的关联,揭示新疆肺结核病高患病率、高死亡率的可能原因.方法:收集新疆南疆部分地区维吾尔族结核病患者痰液标本,对MTB进行分离培养后,应用比例法检测其对异烟肼耐药性,运用PCR技术对所分离菌株的上述基因相关片段扩增并进行测序及序列分析.结果:筛选出实验菌株99株,其中敏感株63株,耐异烟肼菌株36株,耐药率为36.4%.耐药株中katG基因突变率为63.9%,315位点突变,突变类型AGC→ACC(Ser→Thr)、AGC→AAC(Ser→Asn);inhA基因突变率为47.2%,有缺失、同义突变和错义突变;kasA基因突变率为41.7%,突变类型为缺失和错义突变;ahpC基因突变率为8.3%,属于错义突变.结论:新疆南疆结核病高患病率、高死亡率的可能原因之一是结核分枝杆菌对INH产生了耐药,结核分枝杆菌对INH耐药机制可能与耐药菌株基因组内katG、inhA、kasA和ahpC基因发生了突变有关.     

Detection and characterization of mutations in genes related to isoniazid resistance in Mycobacterium tuberculosis clinical isolates from Iran

Molecular Biology Reports - The global rise in drug-resistant Mycobacterium tuberculosis (M.tb), and especially the significant prevalence of isoniazid (INH)-resistance constitute a significant...     

Characterization of mutations causing rifampicin and isoniazid resistance of Mycobacterium tuberculosis in Syria

In order to characterize mutations causing rifampicin and isoniazid resistance of M. tuberculosis in Syria, 69 rifampicin resistant (Rif(r)) and 72 isoniazid resistant (Inh(r)) isolates were screened for point mutations in hot spots of the rpoB, katG and inhA genes by DNA sequencing and real time PCR. Of 69 Rif(r) isolates, 62 (90%) had mutations in the rifampin resistance determining region (RRDR) of the rpoB gene, with codons 531 (61%), 526 (13%), and 516 (8.7%) being the most commonly mutated. We found two new mutations (Asp516Thr and Ser531Gly) described for the first time in the rpoB-RRDR in association with rifampicin resistance. Only one mutation (Ile572Phe) was found outside the rpoB-RRDR. Of 72 Inh(r) strains, 30 (41.6%) had a mutation in katGcodon315 (with Ser315Thr being the predominant alteration), and 23 (32%) harbored the inhA(-15C-->T) mutation. While the general pattern of rpoB-RRDR and katG mutations reflected those found worldwide, the prevalence of the inhA(-15C-->T mutation was above the value found in most other countries, emphasizing the great importance of testing the inhA(-15C-->T) mutation for prediction of isoniazid resistance in Syria. Sensitivity of a rapid test using real time PCR and 3'-Minor groove binder (MGB) probes in detecting Rif(r) and Inh(r) isolates was 90% and 69.4%, respectively. This demonstrates that a small set of MGB-probes can be used in real time PCR in order to detect most mutations causing resistance to rifampicin and isoniazid.     

Structures of Neisseria gonorrhoeae MtrR-operator complexes reveal molecular mechanisms of DNA recognition and antibiotic resistance-conferring clinical mutations

Mutations within the mtrR gene are commonly found amongst multidrug resistant clinical isolates of Neisseria gonorrhoeae, which has been labelled a superbug by the Centers for Disease Control and Prevention. These mutations appear to contribute to antibiotic resistance by interfering with the ability of MtrR to bind to and repress expression of its target genes, which include the mtrCDE multidrug efflux transporter genes and the rpoH oxidative stress response sigma factor gene. However, the DNA-recognition mechanism of MtrR and the consensus sequence within these operators to which MtrR binds has remained unknown. In this work, we report the crystal structures of MtrR bound to the mtrCDE and rpoH operators, which reveal a conserved, but degenerate, DNA consensus binding site 5′-MCRTRCRN₄YGYAYGK-3′. We complement our structural data with a comprehensive mutational analysis of key MtrR-DNA contacts to reveal their importance for MtrR-DNA binding both in vitro and in vivo. Furthermore, we model and generate common clinical mutations of MtrR to provide plausible biochemical explanations for the contribution of these mutations to multidrug resistance in N. gonorrhoeae. Collectively, our findings unveil key biological mechanisms underlying the global stress responses of N. gonorrhoeae.     

Missense mutations in the catalase-peroxidase gene, katG, are associated with isoniazid resistance in Mycobacterium tuberculosis

The toxicity of the powerful anti-tuberculosis drug isoniazid (INH) is believed to be mediated by the haem-containing enzyme catalase-peroxidase, encoded by the katG gene of Mycobacterium tuberculosis. Compelling evidence for this was obtained by studying a panel of INH-resistant clinical isolates using a novel strategy based on the polymerase chain reaction and single-strand-conformation polymorphism analysis (PCR-SSCP) to detect mutations in katG. In most cases INH resistance was associated with missense mutations while in a small number of strains the gene had been completely, or partially, deleted. The missense mutations fell into two groups, the larger of which contained several independent mutations that affected the N-terminal peroxidase domain of the protein, resulting in the production of a catalase peroxidase with strongly reduced enzyme activity and increased heat lability. The effects of these substitutions could be interpreted by means of molecular modelling using the crystal structure of the related enzyme cytochrome c peroxidase from yeast as a template. The second group comprises a frequently occurring amino acid substitution and a single mutation that are both located in the C-terminal domain but do not noticeably alter either enzyme activity or heat stability.     

The effects of isoniazid on the carbohydrates of Mycobacterium tuberculosis BCG

1. Mycobacterium tuberculosis BCG was usually grown in glycerol-asparagine-casein hydrolysate medium. A soluble fraction was obtained from the cells with aq. 50% ethanol; unbound lipids were then removed and the cells were treated with dilute alkali to give, after acidification, an alkali-extractable fraction and an insoluble fraction. On occasion, lipopolysaccharides were obtained by extracting with phenol or dimethyl sulphoxide instead of alkali. The soluble fraction contained, particularly after long extraction, polysaccharide containing mainly glucose, in addition to trehalose and monosaccharides and their derivatives. The alkali-extractable fraction contained polysaccharides containing mannose, glucose, arabinose, galactose and 6-O-methylglucose. These could be resolved into three fractions of markedly different molecular size. It is argued that the high-molecular-weight materials originated from the outside of the cell envelope and the medium-molecular-weight materials from a middle layer of the envelope. 2. Exposure of the growing cells to isoniazid, usually at 1 or 10mug/ml for 6-12h, increased the total cell carbohydrate, mainly due to an increase in trehalose and in insoluble glucan. It also facilitated the extraction of polysaccharide into the medium and the soluble fraction. This produced about a 25% decrease in the amount of carbohydrate in the alkaline-extractable fraction, mainly due to a fall in glucose, arabinose and 6-O-methylglucose. The decrease was confined to polysaccharides of large and medium molecular weight. When intact lipopolysaccharides were extracted, their amount was also decreased by isoniazid. 3. Substitution of ammonium sulphate for asparagine and casein hydrolysate in the medium, so that glycerol was the sole carbon source, decreased the carbohydrate accumulation brought about by isoniazid but did not alter its effect on polysaccharide extraction. 4. Growth with (14)C-labelled substrates showed that glycerol provided two to four times as much of the cell carbon as did asparagine, when both were present. Under these conditions isoniazid inhibited the incorporation of carbon atoms from asparagine into the cells, but had little effect on the total incorporation from glycerol. These experiments also showed that the effect of isoniazid on alkali-extractable polysaccharides was due to their loss to the soluble fraction and external medium. 5. It is suggested that isoniazid inhibits a pathway (probably the synthesis of mycolic acid) involved in the formation of the cell envelope, and that this inhibition results in some re-channelling of intermediates into carbohydrate synthesis and in some loss of polysaccharides through damage to the envelope.     

A real-time PCR assay for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates

A real-time PCR genotypic assay was developed for the detection of isoniazid (INH) resistance in Mycobacterium tuberculosis. The assay detects mutations C(-15)T and, possibly, G(-24)T in the regulatory region of the inhA gene and proved as sensitive and specific as nucleotide sequencing in all the clinical isolates tested. Our assays mapped the mutations efficiently in 10 out of 35 resistant isolates, thereby covering 29% of all resistant strains.     

Detection of mutations associated with multidrug-resistant Mycobacterium tuberculosis clinical isolates

Antimicrobial resistance was studied in 100 Mycobacterium tuberculosis strains selected randomly from sputum cultures of newly diagnosed tuberculosis patients. Resistance of the isolates to rifampicin, isoniazid, and ethambutol was tested by both drug susceptibility testing (DST) and allele-specific PCR (AS-PCR). A total of 19 (19%) isolates were found resistant to at least one of the antituberculosis drugs investigated by PCR compared with 14 (14%) resistant isolates detected by DST. Eleven mutations were detected by AS-PCR in the rpoB gene (codons 516, 526, and 531), associated with rifampicin resistance, a marker of multidrug-resistant tuberculosis (MDR-TB), 14 mutations in the katG gene codon 315 that confers resistance to isoniazid, and nine mutations in the embB gene codon 306 that confers resistance to ethambutol. Mutations in the six multidrug-resistant isolates were confirmed by DNA sequencing. Results were compared with phenotypic DST data. Nineteen different mutation types to at least one of the drugs were found; six isolates (6%) were classified as MDR-TB, defined as resistance to at least rifampicin and isoniazid. The rates of concordance of the PCR with the phenotypic susceptibility test were 71.4, 54.5, and 44.4 for isoniazid, rifampicin, and ethambutol, respectively. These results highlight the importance of molecular epidemiology studies of tuberculosis in understudied regions with a tuberculosis burden to uncover the true prevalence of the MDR-TB.     

The overexpression of acetylcholinesterase compensates for the reduced catalytic activity caused by resistance-conferring mutations in Tetranychus urticae

The mutations (G228S, A391T and F439W) and duplication of the acetylcholinesterase (AChE) gene (Tuace) are involved in monocrotophos resistance in the two-spotted spider mites, Tetranychus urticae (Kwon et al., 2010a, Kwon et al., 2010b). The overexpression of T. urticae AChE (TuAChE) as a result of Tuace duplication was confirmed in several field-collected populations by Western blotting using an AChE-specific antibody. To investigate the effects of each mutation on the insensitivity and fitness cost of AChE, eight variants of TuAChE were expressed in vitro using the baculovirus expression system. Kinetic analysis revealed that the G228S and F439W mutations confer approximately 26-fold and 99-fold increases in the insensitivity to monocrotophos, respectively, whereas the insensitivity increased over 1165-fold in the AChE with double mutations. Nevertheless, the presence of these mutations reduced the catalytic efficiency of AChE significantly. In particular, the TuAChE having both mutations together exhibited a 17.8～27.1-fold reduced catalytic efficiency, suggesting an apparent fitness cost in the monocrotophos-resistant mites. The A391T mutation did not change the kinetic properties of either the substrate or inhibitor when present alone but mitigated the negative impacts of the F439 mutation. To simulate the catalytic activity of the overexpressed TuAChE in two T. urticae strains (approximately 6 copies for AD strain vs. 2 copies for PyriF strain), appropriate TuAChE variants were combined to make up the desired AChE copies and mutation frequencies, and their enzyme kinetics were determined. The reconstituted 6-copy and 2-copy TuAChEs exhibited catalytic efficiency levels comparable to those of a single-copy wildtype TuAChE, suggesting that, if mutations are present, multiple copies of AChE are required to restore a normal level of catalytic activity in the monocrotophos-resistant mites. In summary, the present study provides clear evidence that Tuace duplication resulted in the proportional overexpression of AChE, which was necessary to compensate for the reduced catalytic activity of AChE caused by mutations.     

Decreased resistance of multiresistant mycobacteria to isoniazid during the treatment of experimental tuberculosis with ozone and isoniazid

Mycobacteria (MB) of the clinical strain resistant to streptomycin, isoniazid (IN), rifampicin and kanamycin were injected intravenously into 68 BALB/c mice. The animals were divided into 5 groups: two control groups 0 and 1 (intact and infected without subsequent treatment), group 2 (treated with IN), group 3 (treated with IN and injected intraperitoneally with dissolved ozone, or dO3), group 4 (injected with dO3). The animals started to die by month 4 after the infection. By month 5 all mice died with the exception of intact mice and those treated with dO3). By month 4 the study of MB cultures isolated from the lungs revealed a decrease in their resistance to IN in the groups undergoing treatment with dO3. Hepatic and splenic lesions were observed after treatment with IN only were greater than in the absence of treatment. After the use of IN + dO3 such lesions were the least. The mechanism of a decrease in the medicinal resistance of MB under the action of dO3 and the expediency of the simultaneous use IN and dO3 in cases of the unknown medicinal resistance of MB are discussed.     

Detection of mutations associated with resistance to rifampicin and isoniazid in Mycobacterium tuberculosis by polymerase chain reaction-ligase detection reaction

Mycobacterium tuberculosis （MTB） infection remains a serious infectious disease worldwide, causing 8.8 million new infections and 1.45 million deaths in 2010 [1]. The emergence of drug-resistant strains of MTB poses a significant threat to the control of the disease globally. Multidrugresistant MTB （MDR-TB）, defined as being resistant to at least rifampicin （RMP） and isoniazid （1NH）,     

Proteome-wide profiling of isoniazid targets in Mycobacterium tuberculosis

Isoniazid (INH) is an essential drug used to treat tuberculosis. The mycobactericidal agents are INH adducts [INH-NAD(P)] of the pyridine nucleotide coenzymes, which are generated in vivo after INH activation and which bind to, and inhibit, essential enzymes. The NADH-dependent enoyl-ACP reductase (InhA) and the NADPH-dependent dihydrofolate reductase (DfrA) have both been shown to be inhibited by INH-NAD(P) adducts with nanomolar affinity. In this paper, we profiled the Mycobacterium tuberculosis proteome using both the INH-NAD and INH-NADP adducts coupled to solid supports and identified, in addition to InhA and DfrA, 16 other proteins that bind these adducts with high affinity. The majority of these are predicted to be pyridine nucleotide-dependent dehydrogenases/reductases. They are involved in many cellular processes, including S-adenosylmethionine-dependent methyl transfer reactions, pyrimidine and valine catabolism, the arginine degradative pathway, proton and potassium transport, stress response, lipid metabolism, and riboflavin biosynthesis. The targeting of multiple enzymes could, thus, account for the pleiotropic effects of, and powerful mycobactericidal properties of, INH.     

Detection of resistance to isoniazid by denaturing gradient-gel electrophoresis DNA sequencing in Mycobacterium tuberculosis clinical isolates

Isoniazid (INH) resistance was genotypically assessed in 104 (37 INH-susceptible, 67 INH-resistant) genetically unrelated Mycobacterium tuberculosis strains cultured in North Italy. The PCR products of selected regions of the katG gene, the oxyR-ahpC intergenic region, and the inhA regulatory region were analyzed utilizing the double gradient-denaturing gradient gel electrophoresis (DG-DGGE) technique and confirmed by DNA sequencing. Mutations were detected in 61 (91%) of the INH-resistant strains, the relative frequency of the mutations being 65.7% in katG, 23.9% in oxyR-ahpC, and 13.4% in inhA. Previously described alterations, invariably associated with drug resistance, accounted for 95.1% of the mutations. No alterations were found in the INH-susceptible strains. DG-DGGE analysis and DNA sequencing were equally sensitive, but the former is cheaper, easier and more robust. Rapid genotypic assessment of INH resistance by means of the methodology described here could reasonably be used in clinical mycobacteriology laboratories.