Lack of Clinical Significance for the Common Arginine‐to‐Leucine Substitution at Codon 463 of the katG Gene in Isoniazid‐Resistant Mycobacterium tuberculosis in Singapore

Lee, Ann Siew‐Gek; Tang, Lynn L. H.; Lim, Irene H. K.; Ml, Ling; Tay, L.; Wong, Serre-Yu

doi:10.1086/517320

Cited by 15 publications

(12 citation statements)

References 3 publications

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“…However, in previous studies, the mutation at codon 463 was found in 3 to 61% of INH-susceptible M. tuberculosis isolates (5,6,13,17,18,23). Also, it was found that the activity of catalase, a katG-encoded enzyme, did not differ among isolates having either Arg or Leu at codon 463 (21,22).…”

Section: Tuberculosis Isolates and Assessment Of Inh Resistancementioning

confidence: 88%

The Susceptibility of Mycobacterium tuberculosis to Isoniazid and the Arg→Leu Mutation at Codon 463 of katG Are Not Associated

et al. 2001

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Tuberculosis is the leading cause of death due to infectious diseases worldwide (4), although various drugs against Mycobacterium tuberculosis are available. One of the mainstay drugs for the treatment of tuberculosis is isoniazid (INH). Its effectiveness against M. tuberculosis was initially reported in 1952 (3, 15). Today, INH-resistant M. tuberculosis organisms are not rare anymore; prevalence was reported to be 7% in a recent study performed in The Netherlands (25). The emergence of multidrug-resistant strains (resistant to at least INH and rifampin) (7,11,12,20) has further complicated the treatment of tuberculosis. Therefore, and because of the organism's slow growth rate, rapid methods for detecting drug resistance in clinical isolates of M. tuberculosis are required. The primary mechanism of resistance in M. tuberculosis is the accumulation of mutations in genes coding for drug targets or drug-converting enzymes (16).In the last decade, mutations in katG (24, 26) and inhA (2) have been found to account for 60 to 70% and 10 to 15% of INH resistance cases among M. tuberculosis isolates, respectively (11). The two predominant mutations of katG, and those most referred to, are found within codons 315 and 463 (17).The mutation at codon 315 has been found to be an important indicator for INH resistance as well as for multidrug resistance among isolates of M. tuberculosis organisms recovered from patients in The Netherlands (25).The aims of this study were to assess whether the Arg463Leu mutation is also predictive of INH resistance and, if so, to develop a diagnostic PCR-based screening method for this type of INH resistance.(This work was presented in part at the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 17 to 20 September 2000.) M. tuberculosis isolates and assessment of INH resistance.M. tuberculosis isolates from 395 patients who were diagnosed with tuberculosis in The Netherlands in the period of 1993 to 1997 were used in this study. The isolates were sent by medical microbiology laboratories in The Netherlands to the National Institute of Public Health and the Environment (RIVM, Bilthoven, The Netherlands) for routine typing and susceptibility tests. Susceptibility to INH was measured with the MIC method using 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 g of INH/ml in Middlebrook 7H10 medium (8). Isolates were considered resistant if more than 1% of the bacteria in the inoculum grew in the presence of INH concentrations of Ն1 g/ml. If growth of more than 1% of the inoculum in the presence of 0.5 g of INH/ml occurred, then the isolates were classified as having intermediate susceptibility.DNA isolation. M. tuberculosis isolates were grown on Lö-wenstein-Jensen solid medium or Middlebrook 7H9 liquid medium for 7 days to an optical density corresponding to 10 8 bacteria/ml and were harvested by centrifugation (4,500 ϫ g for 15 min). Chromosomal DNA was isolated as described by Ausubel et al. (1). Briefly, the bacteria were killed by heating at 80°C for 20 min and the...

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Section: Tuberculosis Isolates and Assessment Of Inh Resistancementioning

confidence: 88%

The Susceptibility of Mycobacterium tuberculosis to Isoniazid and the Arg→Leu Mutation at Codon 463 of katG Are Not Associated

et al. 2001

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“…Though R463 is almost exclusively present in the katG gene in isoniazid-susceptible clinical isolates of M. tuberculosis from Europe and North America, the substitution R463L has been found in some clinical isolates that are weakly resistant to isoniazid (MIC !1 Ìg/ml) [9,12,15]. However, the significance of substitution R463L in conferring resistance to isoniazid is not clear.…”

Section: Discussionmentioning

confidence: 99%

“…Introduction of this mutation in wild-type M. tuberculosis by in vitro mutagenesis did not significantly affect the sensitivity to isoniazid [14]. Characterization of clinical isolates of M. tuberculosis from Singapore and China showed that the majority of isoniazid-susceptible strains contained R463L substitution suggesting that this mutation does not contribute to the resistance of M. tuberculosis to isoniazid [15].…”

Section: Introductionmentioning

confidence: 88%

Genetic Polymorphism at Codon 463 in the <i>katG</i> Gene in Isoniazid-Sensitive and -Resistant Isolates of <i>Mycobacterium tuberculosis</i> from the Middle East

Ahmad¹,

Mokaddas²,

Abal

et al. 2001

Med Princ Pract

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Objective: To determine whether substitution R463L within the katG gene is associated with resistance to isoniazid in clinical isolates of Mycobacterium tuberculosis recovered from tuberculosis (TB) patients from the Middle East. Methods: A total of 113 clinical isolates of M. tuberculosis obtained from TB patients residing in Kuwait, UAE and Lebanon were used. The isolates were characterized as isoniazid-susceptible (n = 25) or -resistant (n = 88) based on drug susceptibility testing by BACTEC 460 TB system. The presence of R463L mutation within the katG gene of each isolate was detected by amplification of the DNA region around codon 463 by polymerase chain reaction (PCR) followed by digestion with restriction endonuclease NciI to generate PCR-restriction fragment length polymorphism. The results with selected isolates were further confirmed by DNA sequencing. Results: The mutation R463L was detected in 46 of 59 isoniazid-resistant clinical M. tuberculosis isolates from Kuwait, 9 of 12 from UAE and 3 of 17 from Lebanon. The prevalence of this substitution was much higher in isolates recovered from patients of South Asian/Southeast Asian origin (41/49, 84%) than from patients of Middle Eastern origin (17/39, 44%). Although all the 7 isoniazid-susceptible isolates of M. tuberculosis from Lebanon (recovered exclusively from Middle Eastern patients) contained arginine at codon 463, 3/7 and 10/11 isoniazid-susceptible strains from Kuwait, recovered from Middle Eastern and South Asian patients, respectively, contained leucine at codon 463. Conclusion: The results show that genetic polymorphism exists at codon 463 in the katG gene of M. tuberculosis recovered from patients of different ethnic backgrounds. The data also show that substitution R463L within the katG gene does not contribute to isoniazid resistance.

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“…A similar scenario was observed in studies defining the role of the katG gene in isoniazid resistance in M. tuberculosis. Members of our group and others have shown that the predominant alteration in katG is R463L, which is detected in both isoniazid-resistant and -susceptible isolates and hence is considered a polymorphism and an unreliable indicator of isoniazid resistance (11,13,27). Thus, it is imperative for all studies elucidating the molecular mechanisms of drug resistance in M. tuberculosis to include drug-susceptible isolates as controls.…”

Section: Ethambutol (Emb) [(Ssј)-22јmentioning

confidence: 99%

Novel Mutations within the embB Gene in Ethambutol-Susceptible Clinical Isolates of Mycobacterium tuberculosis

Lee

Othman

et al. 2004

Antimicrob Agents Chemother

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Genetic analysis of the embB gene revealed mutations in 17 (68%) of 25 ethambutol (EMB) resistant isolates (M306I, M306V, M306L, Q497R) but also in 4 (20%) of 20 EMB-susceptible isolates of Mycobacterium tuberculosis, namely, an ATG3ATM substitution resulting in M306I, G406N, and the novel alterations M423I and A659T. Ethambutol (EMB) [(S,SЈ)-2,2Ј-(ethylenediimino)di-1-butanol] is a first-line drug used for antituberculosis therapy. It is often used in combination with isoniazid, rifampin, pyrazinamide, and streptomycin. Membrane-associated arabinosyl transferases have been implicated as the targets for EMB (2,3,14,15). The Mycobacterium tuberculosis emb operon is a gene cluster of three contiguous genes, namely, embC, embA, and embB, which encode mycobacterial arabinosyl transferases (26). These enzymes are involved in the polymerization of the cell wall arabinan (4,6,9,24,25,32). Inhibition of arabinan synthesis by EMB results in the accumulation of mycolic acids, leading to cell death.Alterations at codon 306 of embB have been identified as being the most common alteration in EMB-resistant M. tuberculosis clinical isolates (8, 12, 17-20, 23, 29). Initial work on 51 EMB-resistant isolates had shown that 89% of these isolates had alterations at residue 306 of embB, but these alterations were not detected in 30 EMB-susceptible isolates (23). A subsequent study confirmed this high frequency of embB306 alterations, with 67% of 75 EMB-resistant isolates having mutations not found in EMB-susceptible strains (19). This led to several groups developing targeted strategies for the detection of embB306 alterations (7,16,21,30). Amino acids within the EMB resistance-determining region of EmbB proteins are well conserved among mycobacterial species, including those from M. tuberculosis, M. leprae, and M. smegmatis (2), and mutations within this region have been detected in EMB-resistant isolates of M. tuberculosis.The aim of this present work was to screen all regions of the embB gene with previously reported mutations in order to assess the contribution of mutations within this gene to EMB resistance in M. tuberculosis clinical isolates from Singapore.Drug susceptibility testing was done using the BACTEC 460 radiometric method (Becton Dickinson, Towson, Md.) (2.5 g/ml). Twenty-five consecutive M. tuberculosis isolates resistant to EMB and 20 EMB-susceptible isolates from Singapore were collected as previously described (5, 10).DNA extracted from the isolates was analyzed by amplifying four fragments, using the PCR primers shown in Table 1. The PCR products were purified (QIAquick PCR purification kit or QIAquick gel extraction kit; QIAGEN) and directly sequenced using the BigDye Terminator sequencing kit and the ABI PRISM 377 automated sequencer (PE Biosystems, Branchburg, N.J.). Confirmation of mutations was done by reamplification and resequencing.IS6110 profiling was done according to standard procedures to determine if the isolates were epidemiologically independent (28). All isolates with the same nucleotide substit...

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Lack of Clinical Significance for the Common Arginine‐to‐Leucine Substitution at Codon 463 of the katG Gene in Isoniazid‐Resistant Mycobacterium tuberculosis in Singapore

Cited by 15 publications

References 3 publications

The Susceptibility of Mycobacterium tuberculosis to Isoniazid and the Arg→Leu Mutation at Codon 463 of katG Are Not Associated

The Susceptibility of Mycobacterium tuberculosis to Isoniazid and the Arg→Leu Mutation at Codon 463 of katG Are Not Associated

Genetic Polymorphism at Codon 463 in the <i>katG</i> Gene in Isoniazid-Sensitive and -Resistant Isolates of <i>Mycobacterium tuberculosis</i> from the Middle East

Novel Mutations within the embB Gene in Ethambutol-Susceptible Clinical Isolates of Mycobacterium tuberculosis

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