Research ArticleInvestigation of Ser315 Substitutions withinkatG Gene in Isoniazid-Resistant Clinical Isolates ofMycobacterium tuberculosis from South India

A Nusrath Unissa N Selvakumar Sujatha Narayanan C Suganthi and L E Hanna

Division of Biomedical Informatics Department of Clinical Research National Institute for Research in Tuberculosis (NIRT)Indian Council of Medical Research (ICMR) No 1 Mayor Sathyamoorthy Road Chetput Chennai Tamil Nadu 600 031 India

Correspondence should be addressed to L E Hanna hannatrcchennaiin

Received 2 July 2014 Accepted 20 October 2014

Academic Editor Filippo Canducci

Copyright copy 2015 A Nusrath Unissa et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Mutation at codon 315 of katG gene is themajor cause for isoniazid (INH) resistance inMycobacterium tuberculosis (M tuberculosis)Substitution at codon 315 of katG gene was analyzed in 85 phenotypically resistant isolates collected from various parts of southernIndia by direct sequencing method The obtained results were interpreted in the context of minimum inhibitory concentration(MIC) of INH Of the 85 phenotypically resistant isolates 56 (66) were also correlated by the presence of resistance mutations inthe katG gene 47 of these isolates had ACC 6 had AAC 2 had ATC and one had CGC codonThe frequency of Ser315 substitutionin katG gene was found to be higher (70) amongstmultidrug-resistant (MDR) strains than among non-MDR (61) INH-resistantisolates Further the frequency of mutations was found to be greater (74) in isolates with higher MIC values in contrast to thoseisolates with lowMIC values (58)Therefore the study identified high prevalence of Ser315Thr substitution in katG gene of INH-resistant isolates from south India Also isolates harboring this substitution were found to be associated with multidrug and highlevel INH resistance

1 Introduction

Although tuberculosis (TB) is a preventable treatable andcurable disease it still remains as a major public healthproblem The period of 6ndash9 months needed to treat thedisease is too long and the treatment is often associated withsignificant toxicity These factors make patient complianceto therapy very difficult leading to the emergence andselection of drug-resistant TB bacteria [1] The emergenceof multidrug-resistant (MDR) TB defined as strains whichare resistant to two most potent anti-TB drugs namelyisoniazid (INH) and rifampicin (RIF) and XDR-TB definedasMDR-TB strains that are resistant to second-line TB drugsthat is fluoroquinolones and at least one of the injectableaminoglycosides (capreomycin kanamycin) or amikacinhas worsened the situation further [2] Globally 450000people developed MDR-TB in 2012 more than half of thesecases were from India China and the Russian Federation

It is estimated that about 96 of MDR-TB cases had XDR-TB About 170000 MDR-TB deaths are estimated to haveoccurred in 2012 [3]

Resistance against all known anti-TB drugs has beenreportedHowever isolates ofM tuberculosis resistant to INHare seen with increasing frequency (1 in 106) as comparedto isolates resistant to other drugs [4] Also resistance toINH alone or in combination with other drugs is now thesecond most common cause for resistance Globally it hasbeen estimated that INH resistancewas found in 103of newcases and 277 of treated cases [5] An earlier study basedon susceptibility testing from south India also reported highresistance to INH in 154 of cases compared to 44 casesof RIF resistance [6]

INH has been used extensively as the frontline anti-TBdrug and a drug of choice for chemoprophylaxis acting asa principle component in the current six-month short coursechemotherapy regimen It has long been recognized that INH

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 257983 5 pageshttpdxdoiorg1011552015257983

2 BioMed Research International

resistance inM tuberculosis correlateswith the loss of catalaseand peroxidase (CP) activity in resistant strains [7ndash9] Inother words INH resistance is often accompanied by lossandor reduction of CP or KatG activity coded by katG geneIn fact INH is a prodrug that requires cellular activationby KatG protein to its active form before it can exert itstoxic effect on the bacillus [7] There has been considerableinterest to understand the molecular basis of INH resistanceMutations in several genes (katG inhA ahpC kasA andothers) have been associated with INH resistance [10ndash13] Ofthese inhA and ahpC promoter mutations lead to low-levelINH resistance whereas mutation in katG is responsible forhigh-level resistance Zhang et al (1992) demonstrated thatmutation in the katG gene coding forKatGprotein is amajorcontributor to INH resistance inM tuberculosis [10]

The most prevalent mutation found to occur in katGgene is AGC to ACC at codon 315 [14] or Ser315Thr (S315T)substitution The appearance of this mutation was mostfrequent amongst MDR strains [15] However this mutationwas also reported to be associated with intermediate orhigh levels of resistance to INH (1 to 10120583gmL) [16] Whencompared to other resistance-conferring mutations in katGS315T substitution was found to result in near-normal CPactivities Also it maintains the levels of virulence and confersresistance to INH simultaneously [17] Therefore it may beconsidered as a reliable biomarker for the detection of INHresistance

There are very few studies [18ndash20] on molecular charac-terization of INH resistance from India and there is paucityof data for south Indian isolates Previous studies have clearlyshown that the south Indian INH-resistant (INHR) isolateswere quite distinct and had lower virulence and highersusceptibility to H

2O2[21ndash23]

Hence it was of interest to

analyze mutations in katG gene encompassing codon 315 inINHR clinical isolates ofM tuberculosis from south India byDNA sequencing method in the present study

2 Methods

21 INH-Resistant Clinical Isolates of M tuberculosis A totalof 85 INHR clinical isolates ofM tuberculosis were randomlyselected from south Indian TB patients (20ndash60 years old)belonging to both the sexes from 2006 to 2009 at the NationalInstitute for Research in Tuberculosis (NIRT) ChennaiIndia Of the 85 isolates collected 67 were from Tamil Nadu14 were from Andhra Pradesh 3 were from Karnataka andone was from Kerala Drug susceptibility testing (DST) wasperformed on the cultures they were coded and subjected toDNA sequencing for identification of mutations in the katGgene

22 DNA Extraction from INH-Resistant Strains GenomicDNA was prepared from the resistant strains using sodiumchloride and cetyltrimethylammonium bromide method asdescribed previously [18]

23 Amplification of katG Gene Amplification was per-formed in the isolated genomicDNAusing a reactionmixture

containing 1 120583L of forward and reverse primers (10 pmol)each 6120583L of deoxyribonucleoside triphosphates (dNTPs)mix (25mM) 25 120583L of 10X PCR buffer 10ndash50 ng of templategenomic DNA and 1U of Taq DNA polymerase (AmershamBiosciences UK) The amplification was performed in athermal controller (MJ Research USA) with 30 cycles (1minute (min) at 95∘C 30 seconds at 63∘C and 1min at 72∘Cfollowed by a final extension step at 72∘C for 10min) Theprimers of katG gene forward sequence (51015840-A A A C A GC G G C G C T G G A T C G T 31015840) and reverse sequence(51015840-G T T G T C C C A T T T C G T C G G G G 31015840)were used to generate a 209 bp fragment encompassing theS315T codon [24] The amplicons were purified using GFXCOLUMN (Amersham Biosciences UK) according to themanufacturerrsquos instructions

24 DNA Sequencing Sequencing of the amplicon wascarried out using an automated DNA sequencer (ABIPrism 310Genetic Analyzer-Applied BiosystemsUSA) usingthe above-mentioned primers and the BigDye terminatorsequencing kit (Applied Biosystems) To 4120583L of the termi-nator ready reaction mix l 120583L of the amplified fragment(2-3 ng) and 1 120583L of the primer (1 pmol120583L) were addedand the volume was made up to 20 120583L using deionisedwater The reaction mixture was subjected to cycle sequenc-ing The samples were vortexed and spun then heated at95∘C for 2min and immediately chilled on ice They werevortexed and spun again and placed on ice and loadedonto the DNA sequencer We refer to supporting docu-ments for the figures (see Supplementary Material avail-able online at httpdxdoiorg1011552014257983) Thesequence obtained was compared with sequences available inEMBOSS database via httpwwwebiacukembossalignusing the alignment toolTheGenBank accession number forkatG is X68081

3 Results and Discussion

On the basis of DST results 46 strains were found to have aminimum inhibitory concentration (MIC) of 1mgliter (L) 17had an MIC of 5mgL and the remaining strains had MICsof gt5mgL Amongst the 85 isolates 51 were MDR casesand 48 were found to be INH monoresistant cases withor without resistance to other TB drugs (data not shown)A laboratory reference strain of M tuberculosis H

37Rv was

used as the control Of the 85 phenotypically INHR isolatesincluded in the present study 56 isolates (66) were foundto possess resistance conferring mutations to INH in thekatG gene upon genotypingThe remaining 29 phenotypicallyresistant isolates had no known mutations in the katG geneOf the 56 isolates with mutations 47 had ACC (Thr) 6 hadAAC (Asn) two had ATC (Ile) and one had CGC (Arg)substitution at codon 315 (Table 1) This observation suggestsa high frequency distribution of S315 substitutions in southIndian isolates which is consistent with the global pattern[14] therefore this region may be regarded as a hot spotregion The predominance of 315 mutations in the katG genein INHR clinical isolates has been documented in several

BioMed Research International 3

Table 1 Mutational profile of INH-resistant clinical isolates ofM tuberculosis from south India

Serial number DST katG States Serial number DST katG States1 MDR ACC TN 44 MDR AAC TN2 MDR ACC TN 45 MDR ACC TN3 MDR ACC TN 46 H AAC TN4 H TN 47 H AAC TN5 H TN 48 H TN6 H TN 49 H TN7 H ACC TN 50 H ACC TN8 MDR TN 51 H ACC TN9 MDR ACC TN 52 H ACC TN10 MDR ATC TN 53 MDR ATC TN11 MDR ACC TN 54 MDR ACC TN12 MDR ACC TN 55 H CGC TN13 H TN 56 MDR TN14 H TN 57 MDR ACC TN15 MDR ACC TN 58 H TN16 MDR TN 59 MDR TN17 MDR AAC TN 60 MDR ACC TN18 H TN 61 H ACC TN19 H AAC TN 62 H ACC TN20 MDR ACC TN 63 H TN21 MDR ACC TN 64 MDR ACC TN22 H TN 65 MDR ACC TN23 MDR ACC TN 66 MDR ACC TN24 MDR TN 67 MDR ACC TN25 H ACC TN 68 MDR ACC AP26 H ACC TN 69 H AP27 H ACC TN 70 H ACC AP28 MDR TN 71 MDR AP29 H TN 72 MDR AP30 MDR TN 73 MDR ACC AP31 H TN 74 MDR AP32 MDR AAC TN 75 MDR AP33 H ACC TN 76 MDR ACC AP34 H ACC TN 77 MDR AP35 H ACC TN 78 MDR ACC AP36 H ACC TN 79 MDR ACC AP37 MDR ACC TN 80 H ACC AP38 MDR ACC TN 81 H ACC AP39 H ACC TN 82 H KA40 H ACC TN 83 H KA41 MDR TN 84 H ACC KA42 H ACC TN 85 H ACC K43 MDR ACC TNStrains with MIC of 1mgliter (number 1ndash28 68ndash85) 5mgliter (number 29ndash45) and gt5mgliter (number 46ndash67) Tamil Nadu cultures (number 1ndash67) andother states cultures (number 68ndash85) AP Andhra Pradesh KA Karnataka K Kerala H isoniazid and MDR multidrug resistant

studies with observed frequencies ranging between 30and gt90 In countries such as Scotland Spain Italy andUruguay [25ndash28] the prevalence of INHR strains with S315Tsubstitution was found to be relatively low (20ndash50) Studies

from countries like Netherlands India Africa China andDubai have reported moderate levels of prevalence of the 315substitutions ranging between 50 and 70 [16 18 19 29ndash31] The S315T substitutions have been reported to be highly

4 BioMed Research International

prevalent (60 to gt90) among INHR strains circulating incounties like South Africa Russia Brazil Lithuania and Peru[24 32ndash35]

In this study the frequency of S315T substitution inkatG gene was slightly higher (70) in MDR strains thanin non-MDR (61) INHR isolates It has also been previ-ously observed that S315T substitution is associated withMDR strains of M tuberculosis Further the mutation hasimportant implications for the transmission and control ofMDR-TB [17]We also observed a strong correlation betweenthe MICs of INH and occurrence of S315T substitutions inour isolates with 74 of them showing high MIC values(ge5mgL) and were associated with S315T substitution whileonly 58 of isolates with low MIC value of 1mgL hadthis mutation These findings are in agreement with theobservations made earlier by van Soolingen et al [16]

In one our previous study we compared virulence usingfive clinical mutants of KatG and found that the virulence ofS315mutants from south India was comparable to that of wildtype H

37Rv [36]This is in contrast to the classical notion that

INHR M tuberculosis isolates from south India are inherentlyless virulent than fully susceptible organisms [22 23] Ourpresent findings on the high prevalence of S315 mutantsin south Indian isolates suggest their wider transmissionMoreover Cohen et al (2004) provided ecological evidenceto support the theory that mutations at position 315 of katGconfer INH resistance inM tuberculosiswithout diminishingvirulence or transmissibility [37] Also in general the con-cept of virulence ismultifactorial and comprises several genesand factors The functional loss of a single gene (katG) dueto point mutation that is AGC to ACC may lead resistanceto INH but need not necessarily compromise virulenceTherefore the classical concept of less virulent nature ofINHR M tuberculosis isolates from south India should berevisited

Further the present finding goes in agreement with astudy from eastern rural areas of China which demon-strated that INHR isolates were widely transmitted Also thecorrelation of prevalence and transmission between INHR

isolates especially with the katG S315T substitution andMDR-TB was confirmed The study also recognized thekatG S315T mutants among INHR strains which could beseen as an unsafe factor for subsequent development ofMDR-TB Early detection of the patients with INHR strainswould facilitate the modification of treatment regimens andappropriate infection control measures can be taken in timeto reduce the risk of further development and transmissionof MDR-TB [38] Additionally the impact of INH resistanceupon treatment outcomes is a matter of great concernPatients with katG S315T substitution are hypothesizedto have worse outcomes than those with inhA promotermutations especially when INH is used in the treatment[39]

Thus the present study shows a high prevalence ofS315T substitution in katG gene of INHR clinical isolates ofM tuberculosis from south India Also this substitution isassociated with high level resistance to INH and correlateswith multidrug resistance

Conflict of Interests

There is no conflict of interests amongst the authors

Acknowledgment

Dr Ameeruddin Nusrath Unissa receives financial supportfor postdoctoral fellowship from the Indian Council ofMedical Research

References

[1] R Prasad ldquoMDRTB current statusrdquo Indian Journal of Tubercu-losis vol 52 pp 121ndash131 2005

[2] Centers for Disease Control and Prevention ldquoRevised defini-tion of extensively drug-resistant tuberculosisrdquo Morbidity andMortality Weekly Report vol 55 no 43 p 1176 2006

[3] ldquoWorld Health Organization Media centrerdquo Fact sheet No 1042013

[4] J B Nachega and R E Chaisson ldquoTuberculosis drug resistancea global threatrdquo Clinical Infectious Diseases vol 36 no 1 ppS24ndashS30 2003

[5] World Health Organization International Union against Tuber-culosis and Lung Disease Global Project on Anti-TuberculosisDrug Resistance Surveillance 2002ndash2007 FourthGlobal ReportWorld Health Organization 2008

[6] C N Paramasivan K Bhaskaran and V Venkataraman ldquoSur-veillance of drug resistance in tuberculosis in the state of TamilNadurdquo Indian Journal of Tuberculosis vol 47 pp 27ndash33 2000

[7] F G Winder P Brennan and C Ratledge ldquoSynthesis of fattyacids by extracts of mycobacteria and the absence of inhibitionby isoniazidrdquo Biochemical Journal vol 93 no 3 pp 635ndash6401964

[8] G Middlebrook ldquoIsoniazid-resistance and catalase activity oftubercle bacilli a preliminary reportrdquo American Review ofTuberculosis vol 69 pp 471ndash472 1954

[9] G Middlebrook and M L Cohn ldquoSome observations on thepathogenicity of isoniazid-resistant variants of tubercle bacillirdquoScience vol 118 no 3063 pp 297ndash299 1953

[10] Y Zhang B Heym B Allen D Young and S Cole ldquoThecatalase-peroxidase gene and isoniazid resistance of Mycobac-terium tuberculosisrdquoNature vol 358 no 6387 pp 591ndash593 1992

[11] A Banerjee E Dubnau AQuemard et al ldquoinhA a gene encod-ing a target for isoniazid and ethionamide in Mycobacteriumtuberculosisrdquo Science vol 263 no 5144 pp 227ndash230 1994

[12] K Mdluli R A Slayden Y Zhu et al ldquoInhibition of aMycobac-terium tuberculosis 120573-Ketoacyl ACP synthase by isoniazidrdquoScience vol 280 no 5369 pp 1607ndash1610 1998

[13] S Sreevatsan X Pan Y Zhang V Deretic and J M MusserldquoAnalysis of the oxyR-ahpC region in isoniazid-resistant and- susceptible Mycobacterium tuberculosis complex organismsrecovered from diseased humans and animals in diverse locali-tiesrdquo Antimicrobial Agents and Chemotherapy vol 41 no 3 pp600ndash606 1997

[14] S Ramaswamy and J M Musser ldquoMolecular genetic basis ofantimicrobial agent resistance in Mycobacterium tuberculosis1998 updaterdquo Tubercle and Lung Disease vol 79 no 1 pp 3ndash291998

[15] H J Marttila H Soini E Eerola et al ldquoA Ser315Thr substi-tution in KatG is predominant in genetically heterogeneous

BioMed Research International 5

multidrug-resistant Mycobacterium tuberculosis isolates origi-nating from the St Petersburg area in Russiardquo AntimicrobialAgents and Chemotherapy vol 42 no 9 pp 2443ndash2445 1998

[16] D van Soolingen P E W de Haas H R van Doorn E KuijperH Rinder and M W Borgdorff ldquoMutations at amino acidposition 315 of the katG gene are associated with high-levelresistance to isoniazid other drug resistance and successfultransmission of Mycobacterium tuberculosis in The Nether-landsrdquo The Journal of Infectious Diseases vol 182 no 6 pp1788ndash1790 2000

[17] A S Pym B Saint-Joanis and S T Cole ldquoEffect of katGmutations on the virulence of Mycobacterium tuberculosis andthe implication for transmission in humansrdquo Infection andImmunity vol 70 no 9 pp 4955ndash4960 2002

[18] N Siddiqi M Shamim N K Jain et al ldquoMolecular geneticanalysis of multi-drug resistance in Indian isolates ofMycobac-terium tuberculosisrdquo Memorias do Instituto Oswaldo Cruz vol93 no 5 pp 589ndash594 1998

[19] ANUnissaN Selvakumar SNarayanan andP RNarayananldquoMolecular analysis of isoniazid-resistant clinical isolates ofMycobacterium tuberculosis from Indiardquo International Journalof Antimicrobial Agents vol 31 no 1 pp 71ndash75 2008

[20] A N Unissa S Narayanan C Suganthi and N SelvakumarldquoDetection of isoniazid-resistant clinical isolates of Mycobac-terium tuberculosis from India using Ser315Thr marker bycomparison of molecular methodsrdquo International Journal ofMolecular and Clinical Microbiology vol 1 pp 52ndash59 2011

[21] D A Mitchison J B Selkon and J Lloyd ldquoVirulence in theguinea-pig susceptibility to hydrogen peroxide and catalaseactivity of isoniazid-sensitive tubercle bacilli from South Indianand British PatientsrdquoThe Journal of Pathology and Bacteriologyvol 86 pp 377ndash386 1963

[22] T V Subbaiah D A Mitchison and J B Selkon ldquoThe suscep-tibility to hydrogen peroxide of Indian and British isoniazid-sensitive and isoniazid-resistant tubercle bacillirdquo Tubercle vol41 no 5 pp 323ndash333 1960

[23] Tuberculosis Chemotherapy Centre Madras ldquoA concurrentcomparison of isoniazid plus PAS with three regimens ofisoniazid alone in the domiciliary treatment of pulmonarytuberculosis in South Indiardquo Bulletin of the World HealthOrganization vol 23 no 4-5 pp 535ndash585 1963

[24] P Kiepiela K S Bishop A N Smith L Roux and DF York ldquoGenomic mutations in the katG inhA and aphCgenes are useful far the prediction of isoniazid resistance inMycobacterium tuberculosis isolates from Kwazulu Natal SouthAfricardquo Tubercle and Lung Disease vol 80 no 1 pp 47ndash562000

[25] Z Fang C Doig A Rayner D T Kenna B Watt andK J Forbes ldquoMolecular evidence for heterogeneity of themultiple-drug-resistantMycobacterium tuberculosis populationin Scotland (1990 to 1997)rdquo Journal of Clinical Microbiology vol37 no 4 pp 998ndash1003 1999

[26] S Samper M J Iglesias M J Rabanaque et al ldquoSystematicmolecular characterization of multidrug-resistant Mycobac-terium tuberculosis complex isolates from Spainrdquo Journal ofClinical Microbiology vol 43 no 3 pp 1220ndash1227 2005

[27] L Rindi L Bianchi E Tortoli N Lari D Bonanni and CGarzelli ldquoMutations responsible forMycobacterium tuberculosisisoniazid resistance in Italyrdquo International Journal of Tuberculo-sis and Lung Disease vol 9 no 1 pp 94ndash97 2005

[28] G Varela S Gonzalez P Gadea et al ldquoPrevalence and dissem-ination of the Ser315Thr substitution within the KatG enzyme

in isoniazid-resistant strains of Mycobacterium tuberculosisisolated in Uruguayrdquo Journal of Medical Microbiology vol 57no 12 pp 1518ndash1522 2008

[29] W H Haas K Schilke J Brand et al ldquoMolecular analysis ofkatG gene mutations in strains of Mycobacterium tuberculosiscomplex fromAfricardquoAntimicrobial Agents and Chemotherapyvol 41 no 7 pp 1601ndash1603 1997

[30] M Zhang J Yue Y-P Yang et al ldquoDetection of mutations asso-ciated with isoniazid resistance in Mycobacterium tuberculosisisolates from Chinardquo Journal of Clinical Microbiology vol 43no 11 pp 5477ndash5482 2005

[31] S Ahmad E Fares G F Araj T D Chugh and A SMustafa ldquoPrevalence of S315Tmutation within the katG gene inisoniazid-resistant clinical Mycobacterium tuberculosis isolatesfrom Dubai and Beirutrdquo International Journal of Tuberculosisand Lung Disease vol 6 no 10 pp 920ndash926 2002

[32] I Mokrousov O Narvskaya T Otten E Limeschenko LSteklova and B Vyshnevskiy ldquoHigh prevalence of KatGSer315Thr substitution among isoniazid-resistant Mycobac-terium tuberculosis clinical isolates from northwestern Russia1996 to 2001rdquo Antimicrobial Agents and Chemotherapy vol 46no 5 pp 1417ndash1424 2002

[33] M S N Silva S G Senna M O Ribeiro et al ldquoMutationsin katG inhA and ahpC genes of Brazilian isoniazid-resistantisolates of Mycobacterium tuberculosisrdquo Journal of ClinicalMicrobiology vol 41 no 9 pp 4471ndash4474 2003

[34] D Bakonyte A Baranauskaite J Cicenaite A Sosnovskajaand P Stakenas ldquoMolecular characterization of isoniazid-resistantMycobacterium tuberculosis clinical isolates in Lithua-niardquo Antimicrobial Agents and Chemotherapy vol 47 no 6 pp2009ndash2011 2003

[35] P Escalante S Ramaswamy H Sanabria et al ldquoGenotypiccharacterization of drug-resistant Mycobacterium tuberculosisisolates from Perurdquo Tubercle and Lung Disease vol 79 no 2pp 111ndash118 1998

[36] A N Unissa S Narayanan and N Selvakumar ldquoVirulence inisoniazid-resistant clinical isolates of Mycobacterium tubercu-losis from south Indiardquo International Journal of Molecular andClinical Microbiology vol 1 pp 87ndash96 2011

[37] T Cohen M C Becerra and M B Murray ldquoIsoniazid resis-tance and the future of drug-resistant tuberculosisrdquo MicrobialDrug Resistance vol 10 no 4 pp 280ndash285 2004

[38] Y Hu S Hoffner W Jiang W Wang and B Xu ldquoExtensivetransmission of isoniazid resistant M tuberculosis and itsassociation with increased multidrug-resistant TB in two ruralcounties of Eastern China a molecular epidemiological studyrdquoBMC Infectious Diseases vol 10 article 43 2010

[39] K R Jacobson D Theron T C Victor E M Streicher R MWarren and M B Murray ldquoTreatment outcomes of isoniazid-resistant tuberculosis patients Western Cape Province SouthAfricardquo Clinical Infectious Diseases vol 53 no 4 pp 369ndash3722011

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2 BioMed Research International

resistance inM tuberculosis correlateswith the loss of catalaseand peroxidase (CP) activity in resistant strains [7ndash9] Inother words INH resistance is often accompanied by lossandor reduction of CP or KatG activity coded by katG geneIn fact INH is a prodrug that requires cellular activationby KatG protein to its active form before it can exert itstoxic effect on the bacillus [7] There has been considerableinterest to understand the molecular basis of INH resistanceMutations in several genes (katG inhA ahpC kasA andothers) have been associated with INH resistance [10ndash13] Ofthese inhA and ahpC promoter mutations lead to low-levelINH resistance whereas mutation in katG is responsible forhigh-level resistance Zhang et al (1992) demonstrated thatmutation in the katG gene coding forKatGprotein is amajorcontributor to INH resistance inM tuberculosis [10]

The most prevalent mutation found to occur in katGgene is AGC to ACC at codon 315 [14] or Ser315Thr (S315T)substitution The appearance of this mutation was mostfrequent amongst MDR strains [15] However this mutationwas also reported to be associated with intermediate orhigh levels of resistance to INH (1 to 10120583gmL) [16] Whencompared to other resistance-conferring mutations in katGS315T substitution was found to result in near-normal CPactivities Also it maintains the levels of virulence and confersresistance to INH simultaneously [17] Therefore it may beconsidered as a reliable biomarker for the detection of INHresistance

There are very few studies [18ndash20] on molecular charac-terization of INH resistance from India and there is paucityof data for south Indian isolates Previous studies have clearlyshown that the south Indian INH-resistant (INHR) isolateswere quite distinct and had lower virulence and highersusceptibility to H

2O2[21ndash23]

Hence it was of interest to

analyze mutations in katG gene encompassing codon 315 inINHR clinical isolates ofM tuberculosis from south India byDNA sequencing method in the present study

2 Methods

21 INH-Resistant Clinical Isolates of M tuberculosis A totalof 85 INHR clinical isolates ofM tuberculosis were randomlyselected from south Indian TB patients (20ndash60 years old)belonging to both the sexes from 2006 to 2009 at the NationalInstitute for Research in Tuberculosis (NIRT) ChennaiIndia Of the 85 isolates collected 67 were from Tamil Nadu14 were from Andhra Pradesh 3 were from Karnataka andone was from Kerala Drug susceptibility testing (DST) wasperformed on the cultures they were coded and subjected toDNA sequencing for identification of mutations in the katGgene

22 DNA Extraction from INH-Resistant Strains GenomicDNA was prepared from the resistant strains using sodiumchloride and cetyltrimethylammonium bromide method asdescribed previously [18]

23 Amplification of katG Gene Amplification was per-formed in the isolated genomicDNAusing a reactionmixture

containing 1 120583L of forward and reverse primers (10 pmol)each 6120583L of deoxyribonucleoside triphosphates (dNTPs)mix (25mM) 25 120583L of 10X PCR buffer 10ndash50 ng of templategenomic DNA and 1U of Taq DNA polymerase (AmershamBiosciences UK) The amplification was performed in athermal controller (MJ Research USA) with 30 cycles (1minute (min) at 95∘C 30 seconds at 63∘C and 1min at 72∘Cfollowed by a final extension step at 72∘C for 10min) Theprimers of katG gene forward sequence (51015840-A A A C A GC G G C G C T G G A T C G T 31015840) and reverse sequence(51015840-G T T G T C C C A T T T C G T C G G G G 31015840)were used to generate a 209 bp fragment encompassing theS315T codon [24] The amplicons were purified using GFXCOLUMN (Amersham Biosciences UK) according to themanufacturerrsquos instructions

24 DNA Sequencing Sequencing of the amplicon wascarried out using an automated DNA sequencer (ABIPrism 310Genetic Analyzer-Applied BiosystemsUSA) usingthe above-mentioned primers and the BigDye terminatorsequencing kit (Applied Biosystems) To 4120583L of the termi-nator ready reaction mix l 120583L of the amplified fragment(2-3 ng) and 1 120583L of the primer (1 pmol120583L) were addedand the volume was made up to 20 120583L using deionisedwater The reaction mixture was subjected to cycle sequenc-ing The samples were vortexed and spun then heated at95∘C for 2min and immediately chilled on ice They werevortexed and spun again and placed on ice and loadedonto the DNA sequencer We refer to supporting docu-ments for the figures (see Supplementary Material avail-able online at httpdxdoiorg1011552014257983) Thesequence obtained was compared with sequences available inEMBOSS database via httpwwwebiacukembossalignusing the alignment toolTheGenBank accession number forkatG is X68081

3 Results and Discussion

On the basis of DST results 46 strains were found to have aminimum inhibitory concentration (MIC) of 1mgliter (L) 17had an MIC of 5mgL and the remaining strains had MICsof gt5mgL Amongst the 85 isolates 51 were MDR casesand 48 were found to be INH monoresistant cases withor without resistance to other TB drugs (data not shown)A laboratory reference strain of M tuberculosis H

37Rv was

used as the control Of the 85 phenotypically INHR isolatesincluded in the present study 56 isolates (66) were foundto possess resistance conferring mutations to INH in thekatG gene upon genotypingThe remaining 29 phenotypicallyresistant isolates had no known mutations in the katG geneOf the 56 isolates with mutations 47 had ACC (Thr) 6 hadAAC (Asn) two had ATC (Ile) and one had CGC (Arg)substitution at codon 315 (Table 1) This observation suggestsa high frequency distribution of S315 substitutions in southIndian isolates which is consistent with the global pattern[14] therefore this region may be regarded as a hot spotregion The predominance of 315 mutations in the katG genein INHR clinical isolates has been documented in several

BioMed Research International 3

Table 1 Mutational profile of INH-resistant clinical isolates ofM tuberculosis from south India

Serial number DST katG States Serial number DST katG States1 MDR ACC TN 44 MDR AAC TN2 MDR ACC TN 45 MDR ACC TN3 MDR ACC TN 46 H AAC TN4 H TN 47 H AAC TN5 H TN 48 H TN6 H TN 49 H TN7 H ACC TN 50 H ACC TN8 MDR TN 51 H ACC TN9 MDR ACC TN 52 H ACC TN10 MDR ATC TN 53 MDR ATC TN11 MDR ACC TN 54 MDR ACC TN12 MDR ACC TN 55 H CGC TN13 H TN 56 MDR TN14 H TN 57 MDR ACC TN15 MDR ACC TN 58 H TN16 MDR TN 59 MDR TN17 MDR AAC TN 60 MDR ACC TN18 H TN 61 H ACC TN19 H AAC TN 62 H ACC TN20 MDR ACC TN 63 H TN21 MDR ACC TN 64 MDR ACC TN22 H TN 65 MDR ACC TN23 MDR ACC TN 66 MDR ACC TN24 MDR TN 67 MDR ACC TN25 H ACC TN 68 MDR ACC AP26 H ACC TN 69 H AP27 H ACC TN 70 H ACC AP28 MDR TN 71 MDR AP29 H TN 72 MDR AP30 MDR TN 73 MDR ACC AP31 H TN 74 MDR AP32 MDR AAC TN 75 MDR AP33 H ACC TN 76 MDR ACC AP34 H ACC TN 77 MDR AP35 H ACC TN 78 MDR ACC AP36 H ACC TN 79 MDR ACC AP37 MDR ACC TN 80 H ACC AP38 MDR ACC TN 81 H ACC AP39 H ACC TN 82 H KA40 H ACC TN 83 H KA41 MDR TN 84 H ACC KA42 H ACC TN 85 H ACC K43 MDR ACC TNStrains with MIC of 1mgliter (number 1ndash28 68ndash85) 5mgliter (number 29ndash45) and gt5mgliter (number 46ndash67) Tamil Nadu cultures (number 1ndash67) andother states cultures (number 68ndash85) AP Andhra Pradesh KA Karnataka K Kerala H isoniazid and MDR multidrug resistant

studies with observed frequencies ranging between 30and gt90 In countries such as Scotland Spain Italy andUruguay [25ndash28] the prevalence of INHR strains with S315Tsubstitution was found to be relatively low (20ndash50) Studies

from countries like Netherlands India Africa China andDubai have reported moderate levels of prevalence of the 315substitutions ranging between 50 and 70 [16 18 19 29ndash31] The S315T substitutions have been reported to be highly

4 BioMed Research International

prevalent (60 to gt90) among INHR strains circulating incounties like South Africa Russia Brazil Lithuania and Peru[24 32ndash35]

In this study the frequency of S315T substitution inkatG gene was slightly higher (70) in MDR strains thanin non-MDR (61) INHR isolates It has also been previ-ously observed that S315T substitution is associated withMDR strains of M tuberculosis Further the mutation hasimportant implications for the transmission and control ofMDR-TB [17]We also observed a strong correlation betweenthe MICs of INH and occurrence of S315T substitutions inour isolates with 74 of them showing high MIC values(ge5mgL) and were associated with S315T substitution whileonly 58 of isolates with low MIC value of 1mgL hadthis mutation These findings are in agreement with theobservations made earlier by van Soolingen et al [16]

In one our previous study we compared virulence usingfive clinical mutants of KatG and found that the virulence ofS315mutants from south India was comparable to that of wildtype H

37Rv [36]This is in contrast to the classical notion that

INHR M tuberculosis isolates from south India are inherentlyless virulent than fully susceptible organisms [22 23] Ourpresent findings on the high prevalence of S315 mutantsin south Indian isolates suggest their wider transmissionMoreover Cohen et al (2004) provided ecological evidenceto support the theory that mutations at position 315 of katGconfer INH resistance inM tuberculosiswithout diminishingvirulence or transmissibility [37] Also in general the con-cept of virulence ismultifactorial and comprises several genesand factors The functional loss of a single gene (katG) dueto point mutation that is AGC to ACC may lead resistanceto INH but need not necessarily compromise virulenceTherefore the classical concept of less virulent nature ofINHR M tuberculosis isolates from south India should berevisited

Further the present finding goes in agreement with astudy from eastern rural areas of China which demon-strated that INHR isolates were widely transmitted Also thecorrelation of prevalence and transmission between INHR

isolates especially with the katG S315T substitution andMDR-TB was confirmed The study also recognized thekatG S315T mutants among INHR strains which could beseen as an unsafe factor for subsequent development ofMDR-TB Early detection of the patients with INHR strainswould facilitate the modification of treatment regimens andappropriate infection control measures can be taken in timeto reduce the risk of further development and transmissionof MDR-TB [38] Additionally the impact of INH resistanceupon treatment outcomes is a matter of great concernPatients with katG S315T substitution are hypothesizedto have worse outcomes than those with inhA promotermutations especially when INH is used in the treatment[39]

Thus the present study shows a high prevalence ofS315T substitution in katG gene of INHR clinical isolates ofM tuberculosis from south India Also this substitution isassociated with high level resistance to INH and correlateswith multidrug resistance

Conflict of Interests

There is no conflict of interests amongst the authors

Acknowledgment

Dr Ameeruddin Nusrath Unissa receives financial supportfor postdoctoral fellowship from the Indian Council ofMedical Research

References

[1] R Prasad ldquoMDRTB current statusrdquo Indian Journal of Tubercu-losis vol 52 pp 121ndash131 2005

[2] Centers for Disease Control and Prevention ldquoRevised defini-tion of extensively drug-resistant tuberculosisrdquo Morbidity andMortality Weekly Report vol 55 no 43 p 1176 2006

[3] ldquoWorld Health Organization Media centrerdquo Fact sheet No 1042013

[4] J B Nachega and R E Chaisson ldquoTuberculosis drug resistancea global threatrdquo Clinical Infectious Diseases vol 36 no 1 ppS24ndashS30 2003

[5] World Health Organization International Union against Tuber-culosis and Lung Disease Global Project on Anti-TuberculosisDrug Resistance Surveillance 2002ndash2007 FourthGlobal ReportWorld Health Organization 2008

[6] C N Paramasivan K Bhaskaran and V Venkataraman ldquoSur-veillance of drug resistance in tuberculosis in the state of TamilNadurdquo Indian Journal of Tuberculosis vol 47 pp 27ndash33 2000

[7] F G Winder P Brennan and C Ratledge ldquoSynthesis of fattyacids by extracts of mycobacteria and the absence of inhibitionby isoniazidrdquo Biochemical Journal vol 93 no 3 pp 635ndash6401964

[8] G Middlebrook ldquoIsoniazid-resistance and catalase activity oftubercle bacilli a preliminary reportrdquo American Review ofTuberculosis vol 69 pp 471ndash472 1954

[9] G Middlebrook and M L Cohn ldquoSome observations on thepathogenicity of isoniazid-resistant variants of tubercle bacillirdquoScience vol 118 no 3063 pp 297ndash299 1953

[10] Y Zhang B Heym B Allen D Young and S Cole ldquoThecatalase-peroxidase gene and isoniazid resistance of Mycobac-terium tuberculosisrdquoNature vol 358 no 6387 pp 591ndash593 1992

[11] A Banerjee E Dubnau AQuemard et al ldquoinhA a gene encod-ing a target for isoniazid and ethionamide in Mycobacteriumtuberculosisrdquo Science vol 263 no 5144 pp 227ndash230 1994

[12] K Mdluli R A Slayden Y Zhu et al ldquoInhibition of aMycobac-terium tuberculosis 120573-Ketoacyl ACP synthase by isoniazidrdquoScience vol 280 no 5369 pp 1607ndash1610 1998

[13] S Sreevatsan X Pan Y Zhang V Deretic and J M MusserldquoAnalysis of the oxyR-ahpC region in isoniazid-resistant and- susceptible Mycobacterium tuberculosis complex organismsrecovered from diseased humans and animals in diverse locali-tiesrdquo Antimicrobial Agents and Chemotherapy vol 41 no 3 pp600ndash606 1997

[14] S Ramaswamy and J M Musser ldquoMolecular genetic basis ofantimicrobial agent resistance in Mycobacterium tuberculosis1998 updaterdquo Tubercle and Lung Disease vol 79 no 1 pp 3ndash291998

[15] H J Marttila H Soini E Eerola et al ldquoA Ser315Thr substi-tution in KatG is predominant in genetically heterogeneous

BioMed Research International 5

multidrug-resistant Mycobacterium tuberculosis isolates origi-nating from the St Petersburg area in Russiardquo AntimicrobialAgents and Chemotherapy vol 42 no 9 pp 2443ndash2445 1998

[16] D van Soolingen P E W de Haas H R van Doorn E KuijperH Rinder and M W Borgdorff ldquoMutations at amino acidposition 315 of the katG gene are associated with high-levelresistance to isoniazid other drug resistance and successfultransmission of Mycobacterium tuberculosis in The Nether-landsrdquo The Journal of Infectious Diseases vol 182 no 6 pp1788ndash1790 2000

[17] A S Pym B Saint-Joanis and S T Cole ldquoEffect of katGmutations on the virulence of Mycobacterium tuberculosis andthe implication for transmission in humansrdquo Infection andImmunity vol 70 no 9 pp 4955ndash4960 2002

[18] N Siddiqi M Shamim N K Jain et al ldquoMolecular geneticanalysis of multi-drug resistance in Indian isolates ofMycobac-terium tuberculosisrdquo Memorias do Instituto Oswaldo Cruz vol93 no 5 pp 589ndash594 1998

[19] ANUnissaN Selvakumar SNarayanan andP RNarayananldquoMolecular analysis of isoniazid-resistant clinical isolates ofMycobacterium tuberculosis from Indiardquo International Journalof Antimicrobial Agents vol 31 no 1 pp 71ndash75 2008

[20] A N Unissa S Narayanan C Suganthi and N SelvakumarldquoDetection of isoniazid-resistant clinical isolates of Mycobac-terium tuberculosis from India using Ser315Thr marker bycomparison of molecular methodsrdquo International Journal ofMolecular and Clinical Microbiology vol 1 pp 52ndash59 2011

[21] D A Mitchison J B Selkon and J Lloyd ldquoVirulence in theguinea-pig susceptibility to hydrogen peroxide and catalaseactivity of isoniazid-sensitive tubercle bacilli from South Indianand British PatientsrdquoThe Journal of Pathology and Bacteriologyvol 86 pp 377ndash386 1963

[22] T V Subbaiah D A Mitchison and J B Selkon ldquoThe suscep-tibility to hydrogen peroxide of Indian and British isoniazid-sensitive and isoniazid-resistant tubercle bacillirdquo Tubercle vol41 no 5 pp 323ndash333 1960

[23] Tuberculosis Chemotherapy Centre Madras ldquoA concurrentcomparison of isoniazid plus PAS with three regimens ofisoniazid alone in the domiciliary treatment of pulmonarytuberculosis in South Indiardquo Bulletin of the World HealthOrganization vol 23 no 4-5 pp 535ndash585 1963

[24] P Kiepiela K S Bishop A N Smith L Roux and DF York ldquoGenomic mutations in the katG inhA and aphCgenes are useful far the prediction of isoniazid resistance inMycobacterium tuberculosis isolates from Kwazulu Natal SouthAfricardquo Tubercle and Lung Disease vol 80 no 1 pp 47ndash562000

[25] Z Fang C Doig A Rayner D T Kenna B Watt andK J Forbes ldquoMolecular evidence for heterogeneity of themultiple-drug-resistantMycobacterium tuberculosis populationin Scotland (1990 to 1997)rdquo Journal of Clinical Microbiology vol37 no 4 pp 998ndash1003 1999

[26] S Samper M J Iglesias M J Rabanaque et al ldquoSystematicmolecular characterization of multidrug-resistant Mycobac-terium tuberculosis complex isolates from Spainrdquo Journal ofClinical Microbiology vol 43 no 3 pp 1220ndash1227 2005

[27] L Rindi L Bianchi E Tortoli N Lari D Bonanni and CGarzelli ldquoMutations responsible forMycobacterium tuberculosisisoniazid resistance in Italyrdquo International Journal of Tuberculo-sis and Lung Disease vol 9 no 1 pp 94ndash97 2005

[28] G Varela S Gonzalez P Gadea et al ldquoPrevalence and dissem-ination of the Ser315Thr substitution within the KatG enzyme

in isoniazid-resistant strains of Mycobacterium tuberculosisisolated in Uruguayrdquo Journal of Medical Microbiology vol 57no 12 pp 1518ndash1522 2008

[29] W H Haas K Schilke J Brand et al ldquoMolecular analysis ofkatG gene mutations in strains of Mycobacterium tuberculosiscomplex fromAfricardquoAntimicrobial Agents and Chemotherapyvol 41 no 7 pp 1601ndash1603 1997

[30] M Zhang J Yue Y-P Yang et al ldquoDetection of mutations asso-ciated with isoniazid resistance in Mycobacterium tuberculosisisolates from Chinardquo Journal of Clinical Microbiology vol 43no 11 pp 5477ndash5482 2005

[31] S Ahmad E Fares G F Araj T D Chugh and A SMustafa ldquoPrevalence of S315Tmutation within the katG gene inisoniazid-resistant clinical Mycobacterium tuberculosis isolatesfrom Dubai and Beirutrdquo International Journal of Tuberculosisand Lung Disease vol 6 no 10 pp 920ndash926 2002

[32] I Mokrousov O Narvskaya T Otten E Limeschenko LSteklova and B Vyshnevskiy ldquoHigh prevalence of KatGSer315Thr substitution among isoniazid-resistant Mycobac-terium tuberculosis clinical isolates from northwestern Russia1996 to 2001rdquo Antimicrobial Agents and Chemotherapy vol 46no 5 pp 1417ndash1424 2002

[33] M S N Silva S G Senna M O Ribeiro et al ldquoMutationsin katG inhA and ahpC genes of Brazilian isoniazid-resistantisolates of Mycobacterium tuberculosisrdquo Journal of ClinicalMicrobiology vol 41 no 9 pp 4471ndash4474 2003

[34] D Bakonyte A Baranauskaite J Cicenaite A Sosnovskajaand P Stakenas ldquoMolecular characterization of isoniazid-resistantMycobacterium tuberculosis clinical isolates in Lithua-niardquo Antimicrobial Agents and Chemotherapy vol 47 no 6 pp2009ndash2011 2003

[35] P Escalante S Ramaswamy H Sanabria et al ldquoGenotypiccharacterization of drug-resistant Mycobacterium tuberculosisisolates from Perurdquo Tubercle and Lung Disease vol 79 no 2pp 111ndash118 1998

[36] A N Unissa S Narayanan and N Selvakumar ldquoVirulence inisoniazid-resistant clinical isolates of Mycobacterium tubercu-losis from south Indiardquo International Journal of Molecular andClinical Microbiology vol 1 pp 87ndash96 2011

[37] T Cohen M C Becerra and M B Murray ldquoIsoniazid resis-tance and the future of drug-resistant tuberculosisrdquo MicrobialDrug Resistance vol 10 no 4 pp 280ndash285 2004

[38] Y Hu S Hoffner W Jiang W Wang and B Xu ldquoExtensivetransmission of isoniazid resistant M tuberculosis and itsassociation with increased multidrug-resistant TB in two ruralcounties of Eastern China a molecular epidemiological studyrdquoBMC Infectious Diseases vol 10 article 43 2010

[39] K R Jacobson D Theron T C Victor E M Streicher R MWarren and M B Murray ldquoTreatment outcomes of isoniazid-resistant tuberculosis patients Western Cape Province SouthAfricardquo Clinical Infectious Diseases vol 53 no 4 pp 369ndash3722011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 3

Table 1 Mutational profile of INH-resistant clinical isolates ofM tuberculosis from south India

Serial number DST katG States Serial number DST katG States1 MDR ACC TN 44 MDR AAC TN2 MDR ACC TN 45 MDR ACC TN3 MDR ACC TN 46 H AAC TN4 H TN 47 H AAC TN5 H TN 48 H TN6 H TN 49 H TN7 H ACC TN 50 H ACC TN8 MDR TN 51 H ACC TN9 MDR ACC TN 52 H ACC TN10 MDR ATC TN 53 MDR ATC TN11 MDR ACC TN 54 MDR ACC TN12 MDR ACC TN 55 H CGC TN13 H TN 56 MDR TN14 H TN 57 MDR ACC TN15 MDR ACC TN 58 H TN16 MDR TN 59 MDR TN17 MDR AAC TN 60 MDR ACC TN18 H TN 61 H ACC TN19 H AAC TN 62 H ACC TN20 MDR ACC TN 63 H TN21 MDR ACC TN 64 MDR ACC TN22 H TN 65 MDR ACC TN23 MDR ACC TN 66 MDR ACC TN24 MDR TN 67 MDR ACC TN25 H ACC TN 68 MDR ACC AP26 H ACC TN 69 H AP27 H ACC TN 70 H ACC AP28 MDR TN 71 MDR AP29 H TN 72 MDR AP30 MDR TN 73 MDR ACC AP31 H TN 74 MDR AP32 MDR AAC TN 75 MDR AP33 H ACC TN 76 MDR ACC AP34 H ACC TN 77 MDR AP35 H ACC TN 78 MDR ACC AP36 H ACC TN 79 MDR ACC AP37 MDR ACC TN 80 H ACC AP38 MDR ACC TN 81 H ACC AP39 H ACC TN 82 H KA40 H ACC TN 83 H KA41 MDR TN 84 H ACC KA42 H ACC TN 85 H ACC K43 MDR ACC TNStrains with MIC of 1mgliter (number 1ndash28 68ndash85) 5mgliter (number 29ndash45) and gt5mgliter (number 46ndash67) Tamil Nadu cultures (number 1ndash67) andother states cultures (number 68ndash85) AP Andhra Pradesh KA Karnataka K Kerala H isoniazid and MDR multidrug resistant

studies with observed frequencies ranging between 30and gt90 In countries such as Scotland Spain Italy andUruguay [25ndash28] the prevalence of INHR strains with S315Tsubstitution was found to be relatively low (20ndash50) Studies

from countries like Netherlands India Africa China andDubai have reported moderate levels of prevalence of the 315substitutions ranging between 50 and 70 [16 18 19 29ndash31] The S315T substitutions have been reported to be highly

4 BioMed Research International

prevalent (60 to gt90) among INHR strains circulating incounties like South Africa Russia Brazil Lithuania and Peru[24 32ndash35]

In this study the frequency of S315T substitution inkatG gene was slightly higher (70) in MDR strains thanin non-MDR (61) INHR isolates It has also been previ-ously observed that S315T substitution is associated withMDR strains of M tuberculosis Further the mutation hasimportant implications for the transmission and control ofMDR-TB [17]We also observed a strong correlation betweenthe MICs of INH and occurrence of S315T substitutions inour isolates with 74 of them showing high MIC values(ge5mgL) and were associated with S315T substitution whileonly 58 of isolates with low MIC value of 1mgL hadthis mutation These findings are in agreement with theobservations made earlier by van Soolingen et al [16]

In one our previous study we compared virulence usingfive clinical mutants of KatG and found that the virulence ofS315mutants from south India was comparable to that of wildtype H

37Rv [36]This is in contrast to the classical notion that

INHR M tuberculosis isolates from south India are inherentlyless virulent than fully susceptible organisms [22 23] Ourpresent findings on the high prevalence of S315 mutantsin south Indian isolates suggest their wider transmissionMoreover Cohen et al (2004) provided ecological evidenceto support the theory that mutations at position 315 of katGconfer INH resistance inM tuberculosiswithout diminishingvirulence or transmissibility [37] Also in general the con-cept of virulence ismultifactorial and comprises several genesand factors The functional loss of a single gene (katG) dueto point mutation that is AGC to ACC may lead resistanceto INH but need not necessarily compromise virulenceTherefore the classical concept of less virulent nature ofINHR M tuberculosis isolates from south India should berevisited

Further the present finding goes in agreement with astudy from eastern rural areas of China which demon-strated that INHR isolates were widely transmitted Also thecorrelation of prevalence and transmission between INHR

isolates especially with the katG S315T substitution andMDR-TB was confirmed The study also recognized thekatG S315T mutants among INHR strains which could beseen as an unsafe factor for subsequent development ofMDR-TB Early detection of the patients with INHR strainswould facilitate the modification of treatment regimens andappropriate infection control measures can be taken in timeto reduce the risk of further development and transmissionof MDR-TB [38] Additionally the impact of INH resistanceupon treatment outcomes is a matter of great concernPatients with katG S315T substitution are hypothesizedto have worse outcomes than those with inhA promotermutations especially when INH is used in the treatment[39]

Thus the present study shows a high prevalence ofS315T substitution in katG gene of INHR clinical isolates ofM tuberculosis from south India Also this substitution isassociated with high level resistance to INH and correlateswith multidrug resistance

Conflict of Interests

There is no conflict of interests amongst the authors

Acknowledgment

Dr Ameeruddin Nusrath Unissa receives financial supportfor postdoctoral fellowship from the Indian Council ofMedical Research

References

[1] R Prasad ldquoMDRTB current statusrdquo Indian Journal of Tubercu-losis vol 52 pp 121ndash131 2005

[2] Centers for Disease Control and Prevention ldquoRevised defini-tion of extensively drug-resistant tuberculosisrdquo Morbidity andMortality Weekly Report vol 55 no 43 p 1176 2006

[3] ldquoWorld Health Organization Media centrerdquo Fact sheet No 1042013

[4] J B Nachega and R E Chaisson ldquoTuberculosis drug resistancea global threatrdquo Clinical Infectious Diseases vol 36 no 1 ppS24ndashS30 2003

[5] World Health Organization International Union against Tuber-culosis and Lung Disease Global Project on Anti-TuberculosisDrug Resistance Surveillance 2002ndash2007 FourthGlobal ReportWorld Health Organization 2008

[6] C N Paramasivan K Bhaskaran and V Venkataraman ldquoSur-veillance of drug resistance in tuberculosis in the state of TamilNadurdquo Indian Journal of Tuberculosis vol 47 pp 27ndash33 2000

[7] F G Winder P Brennan and C Ratledge ldquoSynthesis of fattyacids by extracts of mycobacteria and the absence of inhibitionby isoniazidrdquo Biochemical Journal vol 93 no 3 pp 635ndash6401964

[8] G Middlebrook ldquoIsoniazid-resistance and catalase activity oftubercle bacilli a preliminary reportrdquo American Review ofTuberculosis vol 69 pp 471ndash472 1954

[9] G Middlebrook and M L Cohn ldquoSome observations on thepathogenicity of isoniazid-resistant variants of tubercle bacillirdquoScience vol 118 no 3063 pp 297ndash299 1953

[10] Y Zhang B Heym B Allen D Young and S Cole ldquoThecatalase-peroxidase gene and isoniazid resistance of Mycobac-terium tuberculosisrdquoNature vol 358 no 6387 pp 591ndash593 1992

[11] A Banerjee E Dubnau AQuemard et al ldquoinhA a gene encod-ing a target for isoniazid and ethionamide in Mycobacteriumtuberculosisrdquo Science vol 263 no 5144 pp 227ndash230 1994

[12] K Mdluli R A Slayden Y Zhu et al ldquoInhibition of aMycobac-terium tuberculosis 120573-Ketoacyl ACP synthase by isoniazidrdquoScience vol 280 no 5369 pp 1607ndash1610 1998

[13] S Sreevatsan X Pan Y Zhang V Deretic and J M MusserldquoAnalysis of the oxyR-ahpC region in isoniazid-resistant and- susceptible Mycobacterium tuberculosis complex organismsrecovered from diseased humans and animals in diverse locali-tiesrdquo Antimicrobial Agents and Chemotherapy vol 41 no 3 pp600ndash606 1997

[14] S Ramaswamy and J M Musser ldquoMolecular genetic basis ofantimicrobial agent resistance in Mycobacterium tuberculosis1998 updaterdquo Tubercle and Lung Disease vol 79 no 1 pp 3ndash291998

[15] H J Marttila H Soini E Eerola et al ldquoA Ser315Thr substi-tution in KatG is predominant in genetically heterogeneous

BioMed Research International 5

multidrug-resistant Mycobacterium tuberculosis isolates origi-nating from the St Petersburg area in Russiardquo AntimicrobialAgents and Chemotherapy vol 42 no 9 pp 2443ndash2445 1998

[16] D van Soolingen P E W de Haas H R van Doorn E KuijperH Rinder and M W Borgdorff ldquoMutations at amino acidposition 315 of the katG gene are associated with high-levelresistance to isoniazid other drug resistance and successfultransmission of Mycobacterium tuberculosis in The Nether-landsrdquo The Journal of Infectious Diseases vol 182 no 6 pp1788ndash1790 2000

[17] A S Pym B Saint-Joanis and S T Cole ldquoEffect of katGmutations on the virulence of Mycobacterium tuberculosis andthe implication for transmission in humansrdquo Infection andImmunity vol 70 no 9 pp 4955ndash4960 2002

[18] N Siddiqi M Shamim N K Jain et al ldquoMolecular geneticanalysis of multi-drug resistance in Indian isolates ofMycobac-terium tuberculosisrdquo Memorias do Instituto Oswaldo Cruz vol93 no 5 pp 589ndash594 1998

[19] ANUnissaN Selvakumar SNarayanan andP RNarayananldquoMolecular analysis of isoniazid-resistant clinical isolates ofMycobacterium tuberculosis from Indiardquo International Journalof Antimicrobial Agents vol 31 no 1 pp 71ndash75 2008

[20] A N Unissa S Narayanan C Suganthi and N SelvakumarldquoDetection of isoniazid-resistant clinical isolates of Mycobac-terium tuberculosis from India using Ser315Thr marker bycomparison of molecular methodsrdquo International Journal ofMolecular and Clinical Microbiology vol 1 pp 52ndash59 2011

[21] D A Mitchison J B Selkon and J Lloyd ldquoVirulence in theguinea-pig susceptibility to hydrogen peroxide and catalaseactivity of isoniazid-sensitive tubercle bacilli from South Indianand British PatientsrdquoThe Journal of Pathology and Bacteriologyvol 86 pp 377ndash386 1963

[22] T V Subbaiah D A Mitchison and J B Selkon ldquoThe suscep-tibility to hydrogen peroxide of Indian and British isoniazid-sensitive and isoniazid-resistant tubercle bacillirdquo Tubercle vol41 no 5 pp 323ndash333 1960

[23] Tuberculosis Chemotherapy Centre Madras ldquoA concurrentcomparison of isoniazid plus PAS with three regimens ofisoniazid alone in the domiciliary treatment of pulmonarytuberculosis in South Indiardquo Bulletin of the World HealthOrganization vol 23 no 4-5 pp 535ndash585 1963

[24] P Kiepiela K S Bishop A N Smith L Roux and DF York ldquoGenomic mutations in the katG inhA and aphCgenes are useful far the prediction of isoniazid resistance inMycobacterium tuberculosis isolates from Kwazulu Natal SouthAfricardquo Tubercle and Lung Disease vol 80 no 1 pp 47ndash562000

[25] Z Fang C Doig A Rayner D T Kenna B Watt andK J Forbes ldquoMolecular evidence for heterogeneity of themultiple-drug-resistantMycobacterium tuberculosis populationin Scotland (1990 to 1997)rdquo Journal of Clinical Microbiology vol37 no 4 pp 998ndash1003 1999

[26] S Samper M J Iglesias M J Rabanaque et al ldquoSystematicmolecular characterization of multidrug-resistant Mycobac-terium tuberculosis complex isolates from Spainrdquo Journal ofClinical Microbiology vol 43 no 3 pp 1220ndash1227 2005

[27] L Rindi L Bianchi E Tortoli N Lari D Bonanni and CGarzelli ldquoMutations responsible forMycobacterium tuberculosisisoniazid resistance in Italyrdquo International Journal of Tuberculo-sis and Lung Disease vol 9 no 1 pp 94ndash97 2005

[28] G Varela S Gonzalez P Gadea et al ldquoPrevalence and dissem-ination of the Ser315Thr substitution within the KatG enzyme

in isoniazid-resistant strains of Mycobacterium tuberculosisisolated in Uruguayrdquo Journal of Medical Microbiology vol 57no 12 pp 1518ndash1522 2008

[29] W H Haas K Schilke J Brand et al ldquoMolecular analysis ofkatG gene mutations in strains of Mycobacterium tuberculosiscomplex fromAfricardquoAntimicrobial Agents and Chemotherapyvol 41 no 7 pp 1601ndash1603 1997

[30] M Zhang J Yue Y-P Yang et al ldquoDetection of mutations asso-ciated with isoniazid resistance in Mycobacterium tuberculosisisolates from Chinardquo Journal of Clinical Microbiology vol 43no 11 pp 5477ndash5482 2005

[31] S Ahmad E Fares G F Araj T D Chugh and A SMustafa ldquoPrevalence of S315Tmutation within the katG gene inisoniazid-resistant clinical Mycobacterium tuberculosis isolatesfrom Dubai and Beirutrdquo International Journal of Tuberculosisand Lung Disease vol 6 no 10 pp 920ndash926 2002

[32] I Mokrousov O Narvskaya T Otten E Limeschenko LSteklova and B Vyshnevskiy ldquoHigh prevalence of KatGSer315Thr substitution among isoniazid-resistant Mycobac-terium tuberculosis clinical isolates from northwestern Russia1996 to 2001rdquo Antimicrobial Agents and Chemotherapy vol 46no 5 pp 1417ndash1424 2002

[33] M S N Silva S G Senna M O Ribeiro et al ldquoMutationsin katG inhA and ahpC genes of Brazilian isoniazid-resistantisolates of Mycobacterium tuberculosisrdquo Journal of ClinicalMicrobiology vol 41 no 9 pp 4471ndash4474 2003

[34] D Bakonyte A Baranauskaite J Cicenaite A Sosnovskajaand P Stakenas ldquoMolecular characterization of isoniazid-resistantMycobacterium tuberculosis clinical isolates in Lithua-niardquo Antimicrobial Agents and Chemotherapy vol 47 no 6 pp2009ndash2011 2003

[35] P Escalante S Ramaswamy H Sanabria et al ldquoGenotypiccharacterization of drug-resistant Mycobacterium tuberculosisisolates from Perurdquo Tubercle and Lung Disease vol 79 no 2pp 111ndash118 1998

[36] A N Unissa S Narayanan and N Selvakumar ldquoVirulence inisoniazid-resistant clinical isolates of Mycobacterium tubercu-losis from south Indiardquo International Journal of Molecular andClinical Microbiology vol 1 pp 87ndash96 2011

[37] T Cohen M C Becerra and M B Murray ldquoIsoniazid resis-tance and the future of drug-resistant tuberculosisrdquo MicrobialDrug Resistance vol 10 no 4 pp 280ndash285 2004

[38] Y Hu S Hoffner W Jiang W Wang and B Xu ldquoExtensivetransmission of isoniazid resistant M tuberculosis and itsassociation with increased multidrug-resistant TB in two ruralcounties of Eastern China a molecular epidemiological studyrdquoBMC Infectious Diseases vol 10 article 43 2010

[39] K R Jacobson D Theron T C Victor E M Streicher R MWarren and M B Murray ldquoTreatment outcomes of isoniazid-resistant tuberculosis patients Western Cape Province SouthAfricardquo Clinical Infectious Diseases vol 53 no 4 pp 369ndash3722011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 BioMed Research International

prevalent (60 to gt90) among INHR strains circulating incounties like South Africa Russia Brazil Lithuania and Peru[24 32ndash35]

In this study the frequency of S315T substitution inkatG gene was slightly higher (70) in MDR strains thanin non-MDR (61) INHR isolates It has also been previ-ously observed that S315T substitution is associated withMDR strains of M tuberculosis Further the mutation hasimportant implications for the transmission and control ofMDR-TB [17]We also observed a strong correlation betweenthe MICs of INH and occurrence of S315T substitutions inour isolates with 74 of them showing high MIC values(ge5mgL) and were associated with S315T substitution whileonly 58 of isolates with low MIC value of 1mgL hadthis mutation These findings are in agreement with theobservations made earlier by van Soolingen et al [16]

In one our previous study we compared virulence usingfive clinical mutants of KatG and found that the virulence ofS315mutants from south India was comparable to that of wildtype H

37Rv [36]This is in contrast to the classical notion that

INHR M tuberculosis isolates from south India are inherentlyless virulent than fully susceptible organisms [22 23] Ourpresent findings on the high prevalence of S315 mutantsin south Indian isolates suggest their wider transmissionMoreover Cohen et al (2004) provided ecological evidenceto support the theory that mutations at position 315 of katGconfer INH resistance inM tuberculosiswithout diminishingvirulence or transmissibility [37] Also in general the con-cept of virulence ismultifactorial and comprises several genesand factors The functional loss of a single gene (katG) dueto point mutation that is AGC to ACC may lead resistanceto INH but need not necessarily compromise virulenceTherefore the classical concept of less virulent nature ofINHR M tuberculosis isolates from south India should berevisited

Further the present finding goes in agreement with astudy from eastern rural areas of China which demon-strated that INHR isolates were widely transmitted Also thecorrelation of prevalence and transmission between INHR

isolates especially with the katG S315T substitution andMDR-TB was confirmed The study also recognized thekatG S315T mutants among INHR strains which could beseen as an unsafe factor for subsequent development ofMDR-TB Early detection of the patients with INHR strainswould facilitate the modification of treatment regimens andappropriate infection control measures can be taken in timeto reduce the risk of further development and transmissionof MDR-TB [38] Additionally the impact of INH resistanceupon treatment outcomes is a matter of great concernPatients with katG S315T substitution are hypothesizedto have worse outcomes than those with inhA promotermutations especially when INH is used in the treatment[39]

Thus the present study shows a high prevalence ofS315T substitution in katG gene of INHR clinical isolates ofM tuberculosis from south India Also this substitution isassociated with high level resistance to INH and correlateswith multidrug resistance

Conflict of Interests

There is no conflict of interests amongst the authors

Acknowledgment

Dr Ameeruddin Nusrath Unissa receives financial supportfor postdoctoral fellowship from the Indian Council ofMedical Research

References

[1] R Prasad ldquoMDRTB current statusrdquo Indian Journal of Tubercu-losis vol 52 pp 121ndash131 2005

[2] Centers for Disease Control and Prevention ldquoRevised defini-tion of extensively drug-resistant tuberculosisrdquo Morbidity andMortality Weekly Report vol 55 no 43 p 1176 2006

[3] ldquoWorld Health Organization Media centrerdquo Fact sheet No 1042013

[4] J B Nachega and R E Chaisson ldquoTuberculosis drug resistancea global threatrdquo Clinical Infectious Diseases vol 36 no 1 ppS24ndashS30 2003

[5] World Health Organization International Union against Tuber-culosis and Lung Disease Global Project on Anti-TuberculosisDrug Resistance Surveillance 2002ndash2007 FourthGlobal ReportWorld Health Organization 2008

[6] C N Paramasivan K Bhaskaran and V Venkataraman ldquoSur-veillance of drug resistance in tuberculosis in the state of TamilNadurdquo Indian Journal of Tuberculosis vol 47 pp 27ndash33 2000

[7] F G Winder P Brennan and C Ratledge ldquoSynthesis of fattyacids by extracts of mycobacteria and the absence of inhibitionby isoniazidrdquo Biochemical Journal vol 93 no 3 pp 635ndash6401964

[8] G Middlebrook ldquoIsoniazid-resistance and catalase activity oftubercle bacilli a preliminary reportrdquo American Review ofTuberculosis vol 69 pp 471ndash472 1954

[9] G Middlebrook and M L Cohn ldquoSome observations on thepathogenicity of isoniazid-resistant variants of tubercle bacillirdquoScience vol 118 no 3063 pp 297ndash299 1953

[10] Y Zhang B Heym B Allen D Young and S Cole ldquoThecatalase-peroxidase gene and isoniazid resistance of Mycobac-terium tuberculosisrdquoNature vol 358 no 6387 pp 591ndash593 1992

[11] A Banerjee E Dubnau AQuemard et al ldquoinhA a gene encod-ing a target for isoniazid and ethionamide in Mycobacteriumtuberculosisrdquo Science vol 263 no 5144 pp 227ndash230 1994

[12] K Mdluli R A Slayden Y Zhu et al ldquoInhibition of aMycobac-terium tuberculosis 120573-Ketoacyl ACP synthase by isoniazidrdquoScience vol 280 no 5369 pp 1607ndash1610 1998

[13] S Sreevatsan X Pan Y Zhang V Deretic and J M MusserldquoAnalysis of the oxyR-ahpC region in isoniazid-resistant and- susceptible Mycobacterium tuberculosis complex organismsrecovered from diseased humans and animals in diverse locali-tiesrdquo Antimicrobial Agents and Chemotherapy vol 41 no 3 pp600ndash606 1997

[14] S Ramaswamy and J M Musser ldquoMolecular genetic basis ofantimicrobial agent resistance in Mycobacterium tuberculosis1998 updaterdquo Tubercle and Lung Disease vol 79 no 1 pp 3ndash291998

[15] H J Marttila H Soini E Eerola et al ldquoA Ser315Thr substi-tution in KatG is predominant in genetically heterogeneous

BioMed Research International 5

multidrug-resistant Mycobacterium tuberculosis isolates origi-nating from the St Petersburg area in Russiardquo AntimicrobialAgents and Chemotherapy vol 42 no 9 pp 2443ndash2445 1998

[16] D van Soolingen P E W de Haas H R van Doorn E KuijperH Rinder and M W Borgdorff ldquoMutations at amino acidposition 315 of the katG gene are associated with high-levelresistance to isoniazid other drug resistance and successfultransmission of Mycobacterium tuberculosis in The Nether-landsrdquo The Journal of Infectious Diseases vol 182 no 6 pp1788ndash1790 2000

[17] A S Pym B Saint-Joanis and S T Cole ldquoEffect of katGmutations on the virulence of Mycobacterium tuberculosis andthe implication for transmission in humansrdquo Infection andImmunity vol 70 no 9 pp 4955ndash4960 2002

[18] N Siddiqi M Shamim N K Jain et al ldquoMolecular geneticanalysis of multi-drug resistance in Indian isolates ofMycobac-terium tuberculosisrdquo Memorias do Instituto Oswaldo Cruz vol93 no 5 pp 589ndash594 1998

[19] ANUnissaN Selvakumar SNarayanan andP RNarayananldquoMolecular analysis of isoniazid-resistant clinical isolates ofMycobacterium tuberculosis from Indiardquo International Journalof Antimicrobial Agents vol 31 no 1 pp 71ndash75 2008

[20] A N Unissa S Narayanan C Suganthi and N SelvakumarldquoDetection of isoniazid-resistant clinical isolates of Mycobac-terium tuberculosis from India using Ser315Thr marker bycomparison of molecular methodsrdquo International Journal ofMolecular and Clinical Microbiology vol 1 pp 52ndash59 2011

[21] D A Mitchison J B Selkon and J Lloyd ldquoVirulence in theguinea-pig susceptibility to hydrogen peroxide and catalaseactivity of isoniazid-sensitive tubercle bacilli from South Indianand British PatientsrdquoThe Journal of Pathology and Bacteriologyvol 86 pp 377ndash386 1963

[22] T V Subbaiah D A Mitchison and J B Selkon ldquoThe suscep-tibility to hydrogen peroxide of Indian and British isoniazid-sensitive and isoniazid-resistant tubercle bacillirdquo Tubercle vol41 no 5 pp 323ndash333 1960

[23] Tuberculosis Chemotherapy Centre Madras ldquoA concurrentcomparison of isoniazid plus PAS with three regimens ofisoniazid alone in the domiciliary treatment of pulmonarytuberculosis in South Indiardquo Bulletin of the World HealthOrganization vol 23 no 4-5 pp 535ndash585 1963

[24] P Kiepiela K S Bishop A N Smith L Roux and DF York ldquoGenomic mutations in the katG inhA and aphCgenes are useful far the prediction of isoniazid resistance inMycobacterium tuberculosis isolates from Kwazulu Natal SouthAfricardquo Tubercle and Lung Disease vol 80 no 1 pp 47ndash562000

[25] Z Fang C Doig A Rayner D T Kenna B Watt andK J Forbes ldquoMolecular evidence for heterogeneity of themultiple-drug-resistantMycobacterium tuberculosis populationin Scotland (1990 to 1997)rdquo Journal of Clinical Microbiology vol37 no 4 pp 998ndash1003 1999

[26] S Samper M J Iglesias M J Rabanaque et al ldquoSystematicmolecular characterization of multidrug-resistant Mycobac-terium tuberculosis complex isolates from Spainrdquo Journal ofClinical Microbiology vol 43 no 3 pp 1220ndash1227 2005

[27] L Rindi L Bianchi E Tortoli N Lari D Bonanni and CGarzelli ldquoMutations responsible forMycobacterium tuberculosisisoniazid resistance in Italyrdquo International Journal of Tuberculo-sis and Lung Disease vol 9 no 1 pp 94ndash97 2005

[28] G Varela S Gonzalez P Gadea et al ldquoPrevalence and dissem-ination of the Ser315Thr substitution within the KatG enzyme

in isoniazid-resistant strains of Mycobacterium tuberculosisisolated in Uruguayrdquo Journal of Medical Microbiology vol 57no 12 pp 1518ndash1522 2008

[29] W H Haas K Schilke J Brand et al ldquoMolecular analysis ofkatG gene mutations in strains of Mycobacterium tuberculosiscomplex fromAfricardquoAntimicrobial Agents and Chemotherapyvol 41 no 7 pp 1601ndash1603 1997

[30] M Zhang J Yue Y-P Yang et al ldquoDetection of mutations asso-ciated with isoniazid resistance in Mycobacterium tuberculosisisolates from Chinardquo Journal of Clinical Microbiology vol 43no 11 pp 5477ndash5482 2005

[31] S Ahmad E Fares G F Araj T D Chugh and A SMustafa ldquoPrevalence of S315Tmutation within the katG gene inisoniazid-resistant clinical Mycobacterium tuberculosis isolatesfrom Dubai and Beirutrdquo International Journal of Tuberculosisand Lung Disease vol 6 no 10 pp 920ndash926 2002

[32] I Mokrousov O Narvskaya T Otten E Limeschenko LSteklova and B Vyshnevskiy ldquoHigh prevalence of KatGSer315Thr substitution among isoniazid-resistant Mycobac-terium tuberculosis clinical isolates from northwestern Russia1996 to 2001rdquo Antimicrobial Agents and Chemotherapy vol 46no 5 pp 1417ndash1424 2002

[33] M S N Silva S G Senna M O Ribeiro et al ldquoMutationsin katG inhA and ahpC genes of Brazilian isoniazid-resistantisolates of Mycobacterium tuberculosisrdquo Journal of ClinicalMicrobiology vol 41 no 9 pp 4471ndash4474 2003

[34] D Bakonyte A Baranauskaite J Cicenaite A Sosnovskajaand P Stakenas ldquoMolecular characterization of isoniazid-resistantMycobacterium tuberculosis clinical isolates in Lithua-niardquo Antimicrobial Agents and Chemotherapy vol 47 no 6 pp2009ndash2011 2003

[35] P Escalante S Ramaswamy H Sanabria et al ldquoGenotypiccharacterization of drug-resistant Mycobacterium tuberculosisisolates from Perurdquo Tubercle and Lung Disease vol 79 no 2pp 111ndash118 1998

[36] A N Unissa S Narayanan and N Selvakumar ldquoVirulence inisoniazid-resistant clinical isolates of Mycobacterium tubercu-losis from south Indiardquo International Journal of Molecular andClinical Microbiology vol 1 pp 87ndash96 2011

[37] T Cohen M C Becerra and M B Murray ldquoIsoniazid resis-tance and the future of drug-resistant tuberculosisrdquo MicrobialDrug Resistance vol 10 no 4 pp 280ndash285 2004

[38] Y Hu S Hoffner W Jiang W Wang and B Xu ldquoExtensivetransmission of isoniazid resistant M tuberculosis and itsassociation with increased multidrug-resistant TB in two ruralcounties of Eastern China a molecular epidemiological studyrdquoBMC Infectious Diseases vol 10 article 43 2010

[39] K R Jacobson D Theron T C Victor E M Streicher R MWarren and M B Murray ldquoTreatment outcomes of isoniazid-resistant tuberculosis patients Western Cape Province SouthAfricardquo Clinical Infectious Diseases vol 53 no 4 pp 369ndash3722011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 5

multidrug-resistant Mycobacterium tuberculosis isolates origi-nating from the St Petersburg area in Russiardquo AntimicrobialAgents and Chemotherapy vol 42 no 9 pp 2443ndash2445 1998

[16] D van Soolingen P E W de Haas H R van Doorn E KuijperH Rinder and M W Borgdorff ldquoMutations at amino acidposition 315 of the katG gene are associated with high-levelresistance to isoniazid other drug resistance and successfultransmission of Mycobacterium tuberculosis in The Nether-landsrdquo The Journal of Infectious Diseases vol 182 no 6 pp1788ndash1790 2000

[17] A S Pym B Saint-Joanis and S T Cole ldquoEffect of katGmutations on the virulence of Mycobacterium tuberculosis andthe implication for transmission in humansrdquo Infection andImmunity vol 70 no 9 pp 4955ndash4960 2002

[18] N Siddiqi M Shamim N K Jain et al ldquoMolecular geneticanalysis of multi-drug resistance in Indian isolates ofMycobac-terium tuberculosisrdquo Memorias do Instituto Oswaldo Cruz vol93 no 5 pp 589ndash594 1998

[19] ANUnissaN Selvakumar SNarayanan andP RNarayananldquoMolecular analysis of isoniazid-resistant clinical isolates ofMycobacterium tuberculosis from Indiardquo International Journalof Antimicrobial Agents vol 31 no 1 pp 71ndash75 2008

[20] A N Unissa S Narayanan C Suganthi and N SelvakumarldquoDetection of isoniazid-resistant clinical isolates of Mycobac-terium tuberculosis from India using Ser315Thr marker bycomparison of molecular methodsrdquo International Journal ofMolecular and Clinical Microbiology vol 1 pp 52ndash59 2011

[21] D A Mitchison J B Selkon and J Lloyd ldquoVirulence in theguinea-pig susceptibility to hydrogen peroxide and catalaseactivity of isoniazid-sensitive tubercle bacilli from South Indianand British PatientsrdquoThe Journal of Pathology and Bacteriologyvol 86 pp 377ndash386 1963

[22] T V Subbaiah D A Mitchison and J B Selkon ldquoThe suscep-tibility to hydrogen peroxide of Indian and British isoniazid-sensitive and isoniazid-resistant tubercle bacillirdquo Tubercle vol41 no 5 pp 323ndash333 1960

[23] Tuberculosis Chemotherapy Centre Madras ldquoA concurrentcomparison of isoniazid plus PAS with three regimens ofisoniazid alone in the domiciliary treatment of pulmonarytuberculosis in South Indiardquo Bulletin of the World HealthOrganization vol 23 no 4-5 pp 535ndash585 1963

[24] P Kiepiela K S Bishop A N Smith L Roux and DF York ldquoGenomic mutations in the katG inhA and aphCgenes are useful far the prediction of isoniazid resistance inMycobacterium tuberculosis isolates from Kwazulu Natal SouthAfricardquo Tubercle and Lung Disease vol 80 no 1 pp 47ndash562000

[25] Z Fang C Doig A Rayner D T Kenna B Watt andK J Forbes ldquoMolecular evidence for heterogeneity of themultiple-drug-resistantMycobacterium tuberculosis populationin Scotland (1990 to 1997)rdquo Journal of Clinical Microbiology vol37 no 4 pp 998ndash1003 1999

[26] S Samper M J Iglesias M J Rabanaque et al ldquoSystematicmolecular characterization of multidrug-resistant Mycobac-terium tuberculosis complex isolates from Spainrdquo Journal ofClinical Microbiology vol 43 no 3 pp 1220ndash1227 2005

[27] L Rindi L Bianchi E Tortoli N Lari D Bonanni and CGarzelli ldquoMutations responsible forMycobacterium tuberculosisisoniazid resistance in Italyrdquo International Journal of Tuberculo-sis and Lung Disease vol 9 no 1 pp 94ndash97 2005

[28] G Varela S Gonzalez P Gadea et al ldquoPrevalence and dissem-ination of the Ser315Thr substitution within the KatG enzyme

in isoniazid-resistant strains of Mycobacterium tuberculosisisolated in Uruguayrdquo Journal of Medical Microbiology vol 57no 12 pp 1518ndash1522 2008

[29] W H Haas K Schilke J Brand et al ldquoMolecular analysis ofkatG gene mutations in strains of Mycobacterium tuberculosiscomplex fromAfricardquoAntimicrobial Agents and Chemotherapyvol 41 no 7 pp 1601ndash1603 1997

[30] M Zhang J Yue Y-P Yang et al ldquoDetection of mutations asso-ciated with isoniazid resistance in Mycobacterium tuberculosisisolates from Chinardquo Journal of Clinical Microbiology vol 43no 11 pp 5477ndash5482 2005

[31] S Ahmad E Fares G F Araj T D Chugh and A SMustafa ldquoPrevalence of S315Tmutation within the katG gene inisoniazid-resistant clinical Mycobacterium tuberculosis isolatesfrom Dubai and Beirutrdquo International Journal of Tuberculosisand Lung Disease vol 6 no 10 pp 920ndash926 2002

[32] I Mokrousov O Narvskaya T Otten E Limeschenko LSteklova and B Vyshnevskiy ldquoHigh prevalence of KatGSer315Thr substitution among isoniazid-resistant Mycobac-terium tuberculosis clinical isolates from northwestern Russia1996 to 2001rdquo Antimicrobial Agents and Chemotherapy vol 46no 5 pp 1417ndash1424 2002

[33] M S N Silva S G Senna M O Ribeiro et al ldquoMutationsin katG inhA and ahpC genes of Brazilian isoniazid-resistantisolates of Mycobacterium tuberculosisrdquo Journal of ClinicalMicrobiology vol 41 no 9 pp 4471ndash4474 2003

[34] D Bakonyte A Baranauskaite J Cicenaite A Sosnovskajaand P Stakenas ldquoMolecular characterization of isoniazid-resistantMycobacterium tuberculosis clinical isolates in Lithua-niardquo Antimicrobial Agents and Chemotherapy vol 47 no 6 pp2009ndash2011 2003

[35] P Escalante S Ramaswamy H Sanabria et al ldquoGenotypiccharacterization of drug-resistant Mycobacterium tuberculosisisolates from Perurdquo Tubercle and Lung Disease vol 79 no 2pp 111ndash118 1998

[36] A N Unissa S Narayanan and N Selvakumar ldquoVirulence inisoniazid-resistant clinical isolates of Mycobacterium tubercu-losis from south Indiardquo International Journal of Molecular andClinical Microbiology vol 1 pp 87ndash96 2011

[37] T Cohen M C Becerra and M B Murray ldquoIsoniazid resis-tance and the future of drug-resistant tuberculosisrdquo MicrobialDrug Resistance vol 10 no 4 pp 280ndash285 2004

[38] Y Hu S Hoffner W Jiang W Wang and B Xu ldquoExtensivetransmission of isoniazid resistant M tuberculosis and itsassociation with increased multidrug-resistant TB in two ruralcounties of Eastern China a molecular epidemiological studyrdquoBMC Infectious Diseases vol 10 article 43 2010

[39] K R Jacobson D Theron T C Victor E M Streicher R MWarren and M B Murray ldquoTreatment outcomes of isoniazid-resistant tuberculosis patients Western Cape Province SouthAfricardquo Clinical Infectious Diseases vol 53 no 4 pp 369ndash3722011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom