| Abstract|| |
Background: Isoniazid (INH) is one of the main line drugs used in the treatment of tuberculosis (TB) and the development of resistance against this compound can result in serious problems in the treatment procedures. Resistance to INH is associated with a variety of mutations. Objectives: The aim of the present study is to determine the frequency of major drug resistance mutations across katG and inhA loci of multidrug-resistant (MDR)-MTB isolates using a molecular test. Materials and Methods: 125 Mycobacterium Tuberculosis (MTB) clinical isolates were studied. The Centers for Disease Control and Prevention (CDC) standard conventional proportional method was implied to test drug susceptibility. DNA extraction, katG and InhA amplification, and microarray were performed. Results: From 125 MTB clinical isolates, 34 strains were INH-resistant and 91 strains were INH-sensitive. Of 34 INH-resistant strains, the mutation was identified in 32% KatG 315 (Ser→Thr), in 14% KatG315 (Ser→ Asn), in 52% InhA 15 (C→T), and in 2.9% InhA 17 (G→T). Conclusions: Rapid diagnosis of MDR-TB is essential for the prompt initiation of effective second-line therapy to improve the treatment outcome and limit transmission of this obstinate disease. A range of molecular methods that aid in rapid detection of mutations implicated in MDR-TB have been developed. The sensitivity of the methods is dependent, in principle, on the repertoire of mutations being detected, which is typically limited to mutations in the genes katG and the promoter region of inhA.
Keywords: inhA gene, isoniazid (INH) resistance; katG gene, Mycobacterium tuberculosis (MTB)
|How to cite this article:|
Sadri H, Farahani A, Mohajeri P. Frequency of mutations associated with isoniazid-resistant in clinical Mycobacterium tuberculosis strains by low-cost and density (LCD) DNA microarrays. Ann Trop Med Public Health 2016;9:307-11
|How to cite this URL:|
Sadri H, Farahani A, Mohajeri P. Frequency of mutations associated with isoniazid-resistant in clinical Mycobacterium tuberculosis strains by low-cost and density (LCD) DNA microarrays. Ann Trop Med Public Health [serial online] 2016 [cited 2020 Apr 2];9:307-11. Available from: http://www.atmph.org/text.asp?2016/9/5/307/190166
| Background|| |
Tuberculosis (TB) is still one of the greatest threats to human health in the contemporary world especially in the developing countries, where the highest burdens of TB are found. It is estimated that 1/3 of the human population (more than 2 billion people) are infected with Mycobacterium tuberculosis (MTB). About 9 million new TB cases and 2 million deaths from the disease are noted each year. , The latest appearance of drug-resistant TB strains is the most severe threat correlated to the control of the TB. This appearance has been tempted by widespread consumption of the standard regimen short-course drug. In this regard, the special concern is the emergence of multidrug-resistant TB (MDR-TB) that is defined as TB resistant to at least isoniazid (INH) and rifampin (RIF). ,
Resistance to INH is associated with a variety of mutations that affect several genes including those encoding catalase-peroxidase (katG) that converts INH to an active form and enoyl-acyl carrier protein reductase (enoyl-ACP-reductase) (inhA) involved in mycolic acid biosynthesis in mycobacterial cell wall formation.  Enoyl-ACP-reductase binds nicotinamide adenine dinucleotide to which catalase peroxidase activated INH forms a complex and lets biosynthesis of mycolic acid to be inhibited. KatG is the most commonly altered, with the majority of mutations occurring in codon 315. Mutations in the katG codon 315 and the promoter region of inhA have been identified in INH resistant, but not in INH-susceptible MTB isolates. ,
MDR-TB was characterized by late diagnosis, insufficient treatment regimens, and mortality. It must be ensured that all patients are diagnosed and treated effectively to mitigate the creation and transmission of the resistant strains in the community.
It often lasts 6-10 weeks to determine the drug resistance in MTB by the use of conventional culture methods. In order to shorten this period, targeted molecular approaches have been developed. Genomic mutations frequently associated with resistance to each of the primary anti-TB drugs have been identified. 
Due to the initiation of molecular detection technique, it can be visualized that the understanding of the genetic basis of drug resistance in a more systematic could simplify the development of rapid methods for assessing the antibiotic susceptibility phenotypes of clinical MTB isolates, thus allowing more operative drug usage and treatment of the disease, and later reduction in resistance development. 
The aim of the present study is to determine the frequency of major drug resistance mutations across katG and inhA loci of MDR-MTB isolates using a molecular test.
| Materials and Methods|| |
A total of 125 clinical MTB isolates were collected from TB reference laboratory of Kermanshah, Iran, in 2011-2013. MTB strains isolated from sputum samples. They were tested by conventional biochemical methods. Bacterial culture was accomplished in Lowenstein-Jensen (LJ) medium and the isolates were identified by standard microbiological methods such as Ziehl-Neelsen staining, morphology of colony, pigment and niacin production, and nitrate and catalase tests.Briefly, sputum specimens were decontaminated with 4% NaOH and cultured on Lowenstein solid medium by incubation at 37°C for 3-10 weeks, until MTB colonies appeared on the medium surface. Resistance INH was tested by culturing bacterial suspensions (0.5 McFarland) on Lowenstein solid medium containing INH (0.2 g/mL). MTB isolate was considered as drug resistant only when the number of colonies emerging on drug containing medium was at least 1% of the number of colonies emerging on drug free medium. For all experiments, MTB HRV37 was used as standard. Chromosomal DNA was prepared from MTB extracted using the automation QIAamp system by QIAamp genomic DNA Kits followed by the low-cost, the low-cost and density (LCD) DNA microarrays array that has been developed for rapid, easy, and reliable identification of point mutations in the genes rpoB, katG, and inhA of MTB, associated with resistance to rifampin and INH.
Each LCD chip contains identical arrays in rectangular reaction chambers that can be addressed individually. LCD arrays were manufactured by Myco Resist 3.5 Kit (Chipron, Germany). The LCD array protocol for the analysis of MTB resistance is based on a polymerase chain reaction (PCR) amplification of the two regions of interest KatG and InhA.
The labeled PCR fragments are joined with the hybridization buffer (provided) and hybridized to the individual array fields of one chip. While hybridization, the labeled PCR fragments will be fixed to specific capture probes immobilized as spots on the bottom of each field. After a short procedure of washing, each field is incubated with a secondary label solution (enzyme conjugate). Next to second washing step, those positions (spots) in which PCR fragments and secondary label are bounded can be envisioned by a blue precipitate formed by the enzyme substrate provided as "STAIN."
Though these signals are obviously noticeable to the human eyes, high-resolution grayscale images (~10 mm resolution) were taken to use a transmission-light film scanning device (Chipron, Germany). These images allow using commercially available software to analyze image and store data. Two software packages (Slide Reader 1.1, Chipron, Germany and GenePix Pro 5.0, Axon Instruments, Inc., USA) and the online analysis module (Chipron, Germany) are available at www.chipron.com were implied for the analyze the hybridization signals gained from "STAIN." 
| Results|| |
A total of 125 MTB strains were isolated from TB patients, including 85 (68%) males and 40 (32%) females. All isolates were from newly diagnosed and previously untreated cases in our study. The mean age of the patients was 44.2 ± 17.4 (SD). Of the 34 INH-resistant specimens, 18 specimens (52%) had a mutation in the inhA gene, C15T, which is the most common mutation found in INH-resistant strains, 16 specimens (53.3%) had a mutation in katG (codon 315). One of the specimens (2%) had a mutation in the inhA gene, G17T. The most frequent mutation in the region of katG was AGC< ACC (32%), changing serine to threonine (Ser315thr). We found also one isolates bearing two mutations in katG (codon 315) [Table 1].
|Table 1: Frequency of amino acid and nucleotide changes in different codons of the KatG and InhA Gene in INH-resistant strains|
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Analysis of INH MIC among 34 INH-resistant strains demonstrated that the MIC was 0.2 μg/mL in 5 strains, 0.4 μg/mL in 12 strains, 0.8 μg/mL in 15 strains, and 1.6 μg/mL in 2 strains.
| Discussion|| |
The appearance and development of drug- resistant strains from MTB makes a severe threat to global understanding of the connotation of antibiotic resistance and transmissibility and virulence of MTB is vital for predicting future burden of drug resistant disease.  INH resistance is a surrogate marker for MDR MTB (RIF and INH resistance) and is the result of mutation within certain region of the katG gene, which encodes the catalase peroxidase. It is significant to be aware of the correlation of clinical states of patients with TB to mutations and high resistance levels to INH to decide if the patients are at the beginning infected by MDR MTB strain or if the emergence of MDR MTB outbreaks , due to inadequate antibiotic treatment that led to the acquisition of mutations and antibiotic resistant. DNA microarray is a simple and exact tool to identify MTB and to diagnose RMP and INH resistance. It seems that this system is likely to be effective to spinal TB specimens. The automated readout and short turnaround time make this assay suitable to test and decrease delays in diagnosis, without the necessity of building large numbers of advanced biosafety facilities. 
The known genes related to INH-resistant are katG, inhA, ahpC, and kasA. Several investigators have reported that MTB resistance to INH corresponds to amino acid changes in codon 315. In our study, we have observed 47% (N = 16) of all INH-resistant isolates that showed mutations in codon 315.
Prevalence of mutations in the katG gene among INH-resistant MTB isolates found in this study (47%) lies within the international range (40-95%) and is comparable to most previous reports. A large number of different mutations in the katG gene have been described so far. However, the Ser315thr mutation is found most often, occurring in approximately 32% of all INH-resistant strains. , We found almost exclusively the Ser315thr substitution among this set of diverse INH-resistant isolates. This highlights the selective advantage conferred by the Ser315thr mutation, which provides enough catalase-peroxidase activity to protect the cell inform oxidative stress while reducing the conversion of the INH producing to its active form by KatG. , The active form of INH is thought to inactivate the fatty-acid enoyl-acyl carrier protein reductase (InhA) involved in synthesis of mycolic acids. It is proposed that the −15C< T mutation in the ribosome binding site of the promoter of the mabA-inhA operon causes overexpression of InhA, thus providing excess active enzyme and leading to INH-resistance in mutant strains.  Of 34 INH-resistant isolates included in our study, 18 (52%) harbored the inhA −15C< T mutation. This rate is higher than values found in most other countries (15% in eastern China, 19.2% in Lithuania, 23.6% in Spain, and 27.2% in England). ,,,
The finding that the G17T mutation in inhA gene occurs less frequently in this study in this region has a direct impact on the application of molecular techniques for rapid detection of MDR-TB. Among the 34 INH-resistant isolates in this study, the inhA mutations associated with resistance were inhA G 17T (2%) and other loci (52%). The sensitivity of this chip for detection of the inhA G17T appears to be low for the isolates in this study. Therefore, it is reasonable to develop new probes of the katG genes for the detection of new mutation in different geographic areas.
It is recognized that rifampin and INH-resistance-associated genes are related with cross-resistance to other anti-TB drugs, TB transmission, and treatment outcome.  For example, MTB isolates that are unaffected by a low level (0.2 mg/L) of INH are susceptible to a high level (1 mg/L) of INH are usually resistant to ethionamide. Specific mutations in the katG gene basis INH resistance at a very high level, while mutations in the inhA regulatory region, mainly C-15T, and inhA structural region, principally S94A and I194T, have been stated to be related with high-level resistance to both INH and ethionamide in Latin American-Mediterranean family MDR MTB strains in Lisbon. , The katG 315 mutation is more likely to make extra drug resistance and has less fitness cost. The influence of the katG 315 mutation on TB transmission was discovered to be associated with the main lineage of MTB. Thus, the current array can also be used for the initial examination and identification of epidemiologically unlinked cases. 
| Conclusion|| |
To conclude, the search to identify additional genes related with INH-resistance as defined in this current study will be vital for developing of comprehensiveness of molecular detection strategies, more efficient than current susceptibility testing methods based on the culture. Such methods of molecular screening could help to have more proper treatment provided earlier to the patients and have the potential to mitigate transmission of the resistant strains. Moreover, the detection of the new loci stated here may reveal novel targets appropriate for the development of alternative therapeutic options.  In contrast to the high frequencies of the inhA C15T gene mutations, the frequency (1 of 34 INH-resistant isolates) of the inhA G17T gene mutation was low in this study.
The limitation of this study is that small number of INH-resistant isolates and an insufficient amount of INH-susceptible isolates would result in bias in the proportion of resistant isolates among the collection. Therefore, we focused on the frequency of each point mutation in the resistant isolates rather than the proportion in all of the isolates.
The study shows the necessity of further investigations concerning MTB isolate genotypes and their association with the drug resistance in our region. Molecular genotyping methods plays significant role in spotting the domination of transmission or reinfection in population. More studies in relation to the determination of genotype of Georgian MTB isolates are needed. Moreover, the analysis of phylogenetically wrapped series of motifs among members of the MTB complex joining with geographical and epidemiological data will provide important information to track the phylogenetic spread of these pathogens. In general, microarray is capable of detecting most of rifampicin- resistant and INH-resistant isolates. The method is rapid and requires simple equipment and the interpretation of the results is easy. Being not expensive of the test represents a benefit for TB patients and can be used as first line test for rapid detection of MDR isolates in the developing countries, such as Iran, as a complement to the phenotypic tests.
We gratefully acknowledge Vice-Chancellor for Research and Technology, Kermanshah University of Medical Sciences for financial support, this article resulted from the Masters Degrees in medical Microbiology thesis of Hadis Sadri, Kermanshah University of Medical Sciences, Kermanshah, Iran (grant No. 93281).
Financial support and sponsorship
Kermanshah University of Medical Sciences.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Jagielski T, Grzeszczuk M, Kamiñski M, Roeske K, Napiórkowska A, Stachowiak R, et al
. Identification and analysis of mutations in the katG
gene in multidrug-resistant Mycobacterium tuberculosis clinical isolates. Pneumonol Alergol Pol 2013;81:298-307.
Jagielski T, Augustynowicz-Kopeæ E, Zwolska Z. Epidemiology of tuberculosis: A global, European and Polish perspective. Wiad Lek 2010;63:230-46.
Li J, Xin J, Zhang L, Jiang L, Cao H, Li L. Rapid detection of rpoB mutations in rifampin resistant M. tuberculosis from sputum samples by denaturing gradient gel electrophoresis. Int J Med Sci 2012;9: 148-56.
Zhang SL, Shen JG, Xu PH, Li DX, Sun ZQ, Li L, et al
. A novel genotypic test for rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates by a multiplex probe array. J Appl Microbiol 2007;103:1262-71.
Haas WH, Schilke K, Brand J, Amthor B, Weyer K, Fourie PB, et al
. Molecular analysis of katG
gene mutations in strains of Mycobacterium tuberculosis complex from Africa. Antimicrob Agents Chemother 1997;41:1601-3.
Kiepiela P, Bishop KS, Smith AN, Roux L, York DF. Genomic mutations in the katG, inhA
genes are useful for the prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from Kwazulu Natal, South Africa. Tuber Lung Dis 2000;80:47-56.
Dobner P, Rüsch-Gerdes S, Bretzel G, Feldmann K, Rifai M, Löscher T, et al
. Usefulness of Mycobacterium tuberculosis genomic mutations in the genes katG
for the prediction of isoniazid resistance. Int J Tuberc Lung Dis 1997;1:365-9.
Khadka DK, Eampokalap B, Panitchakorn J, Ramasoota P, Khusmith S. Multiple mutations in katG
identified in Thai isoniazid-resistant Mycobacterium tuberculosis isolates. Southeast Asian J Trop Med Public Health 2007;38:376-82.
Chan RC, Hui M, Chan EW, Au TK, Chin ML, Yip CK, et al
. Genetic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates in Hong Kong. J Antimicrob Chemother 2007;59:866-73.
Mohajeri P, Sadri H, Farahani A, Norozi B, Atashi S. Frequency of mutations associated with rifampicin resistance in mycobacterium tuberculosis
strains isolated from patients in west of Iran. Microb Drug Resist 2015;21:315-9.
Shubladze N, Tadumadze N, Bablishvili N. Molecular patterns of multidrug resistance of mycobacterium tuberculosis
in Georgia. Int J Mycobacteriol 2013;2:73-8.
Zhang Z, Li L, Luo F, Cheng P, Wu F, Wu Z, et al
. Rapid and accurate detection of RMP- and INH- resistant mycobacterium tuberculosis
in spinal tuberculosis specimens by CapitalBio™ DNA microarray: A prospective validation study. BMC Infect Dis 2012;12:303.
Vilchèze C, Wang F, Arai M, Hazbón MH, Colangeli R, Kremer L, et al
. Transfer of a point mutation in mycobacterium tuberculosis inhA
resolves the target of isoniazid. Nat Med 2006;12:1027-9.
Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agent resistance in mycobacterium tuberculosis
: 1998 update. Tuber Lung Dis 1998;79:3-29.
Rouse DA, DeVito JA, Li Z, Byer H, Morris SL. Site-directed mutagenesis of the katG gene of Mycobacterium tuberculosis: Effects on catalase-peroxidase activities and isoniazid resistance. Mol Microbiol 1996;22:583-92.
van Soolingen D, de Haas PE, van Doorn HR, Kuijper E, Rinder H, Borgdorff MW. Mutations at amino acid position 315 of the katG gene are associated with high-level resistance to isoniazid, other drug resistance, and successful transmission of mycobacterium tuberculosis
in the Netherlands. J Infect Dis 2000;182:1788-90.
Slayden RA, Lee RE, Barry CE 3 rd
. Isoniazid affects multiple components of the type II fatty acid synthase system of mycobacterium tuberculosis
. Mol Microbiol 2000;38:514-25.
Bakonyte D, Baranauskaite A, Cicenaite J, Sosnovskaja A, Stakenas P. Molecular characterization of isoniazid-resistant mycobacterium tuberculosis
clinical isolates in Lithuania. Antimicrob Agents Chemother 2003;47:2009-11.
Herrera-León L, Molina T, Saíz P, Sáez-Nieto JA, Jiménez MS. New multiplex PCR for rapid detection of isoniazid-resistant mycobacterium tuberculosis
clinical isolates. Antimicrob Agents Chemother 2005;49:144-7.
Baker LV, Brown TJ, Maxwell O, Gibson AL, Fang Z, Yates MD, et al
. Molecular analysis of isoniazid-resistant mycobacterium tuberculosis
isolates from England and Wales reveals the phylogenetic significance of the ahpC -46A polymorphism. Antimicrob Agents Chemother 2005;49:1455-64.
Li WM, Wang SM, Li CY, Liu YH, Shen GM, Zhang XX, et al
. Molecular epidemiology of mycobacterium tuberculosis
in China: A nationwide random survey in 2000. Int J Tuberc Lung Dis 2005;9:1314-9.
Köser CU, Feuerriegel S, Summers DK, Archer JA, Niemann S. Importance of the genetic diversity within the mycobacterium tuberculosis
complex for the development of novel antibiotics and diagnostic tests of drug resistance. Antimicrob Agents Chemother 2012;56:6080-7.
Machado D, Perdigão J, Ramos J, Couto I, Portugal I, Ritter C, et al
. High-level resistance to isoniazid and ethionamide in multidrug-resistant mycobacterium tuberculosis
of the Lisboa family is associated with inhA
double mutations. J Antimicrob Chemother 2013;68:1728-32.
Huang WL, Hsu ZJ, Chang TC, Jou R. Rapid and accurate detection of rifampin and isoniazid-resistant mycobacterium tuberculosis
using an oligonucleotide array. Clin Microbiol Infect 2014;20:O542-9.
Shekar S, Yeo ZX, Wong JC, Chan MK, Ong DC, Tongyoo P, et al
. Detecting novel genetic variants associated with isoniazid-resistant mycobacterium tuberculosis
. PLoS One 2014;9:e102383.
Department of Microbiology, Faculty of Medicine, Kermanshah University of Medical Sciences, Shirudi Shahid Boulevard, Daneshgah Street, Kermanshah
Source of Support: None, Conflict of Interest: None