Molecular analysis of isoniazid resistance in Mycobacterium tuberculosis isolates recovered from South Korea

The katG, inhA and ahpC genes, in 71 isoniazid (INH)-resistant and 26 INH-susceptible Mycobacterium tuberculosis isolates, from South Korea were examined by sequencing and MspI restriction enzyme analysis. Mutations in the katG 315 alone, katG 315 plus inhA, katG 315 plus ahpC, katG 309 alone, katG 309 plus inhA, inhA alone, and ahpC alone, were detected in 54.9, 2.8, 1.4, 1.4, 1.4, 19.7, and 5.6% of the 71 INH-resistant isolates, respectively. There was no statistically significant difference (p > 0.05) in the frequencies of these mutations for the INH-monoresistant compared with the multidrug-resistant isolates. Mutations in the katG codon 315 were associated with the high-level of INH resistance (MIC, >1 microg/ml), whereas the mutation in the inhA promoter region was associated with the low-level of INH resistance (MIC, >0.2 to 1 microg/ml). The previously undescribed GGT-->GAT (Gly-->Asp) mutation in the katG codon 309 was found in two rifampin, including-multidrug-resistant isolates, but we cannot assess if this is predictive of INH resistance. The sensitivity and specificity of molecular analysis of the katG codon 315 and/or the inhA promoter region were 80.3 and 100%, respectively. Therefore, mutations in these regions are highly predictive of INH resistance in South Korea.     

Molecular investigation of resistance to the antituberculous drug ethionamide in multidrug-resistant clinical isolates of Mycobacterium tuberculosis

Ethionamide (ETH) needs to be activated by the mono-oxygenase EthA, which is regulated by EthR, in order to be active against Mycobacterium tuberculosis. The activated drug targets the enzyme InhA, which is involved in cell wall biosynthesis. Resistance to ETH has been reported to result from various mechanisms, including mutations altering EthA/EthR, InhA and its promoter, the NADH dehydrogenase encoded by ndh, and the MshA enzyme, involved in mycothiol biosynthesis. We searched for such mutations in 87 clinical isolates: 47 ETH-resistant (ETH(r)) isolates, 24 ETH-susceptible (ETH(s)) isolates, and 16 isolates susceptible to ETH but displaying an intermediate proportion of resistant cells (ETH(Sip); defined as ≥1% but <10% resistant cells). In 81% (38/47) of the ETH(r) isolates, we found mutations in ethA, ethR, or inhA or its promoter, which mostly corresponded to new alterations in ethA and ethR. The 9 ETH(r) isolates without a mutation in these three genes (9/47, 19%) had no mutation in ndh, and a single isolate had a mutation in mshA. Of the 16 ETH(Sip) isolates, 7 had a mutation in ethA, 8 had no detectable mutation, and 1 had a mutation in mshA. Finally, of the 24 ETH(s) isolates, 23 had no mutation in the studied genes and 1 displayed a yet unknown mutation in the inhA promoter. Globally, the mechanism of resistance to ETH remained unknown for 19% of the ETH(r) isolates, highlighting the complexity of the mechanisms of ETH resistance in M. tuberculosis.     

Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium tuberculosis

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. Previous studies have identified resistance-associated mutations in katG, inhA, kasA, ndh, and the oxyR-ahpC intergenic region. DNA microarray-based experiments have shown that INH induces several genes in Mycobacterium tuberculosis that encode proteins physiologically relevant to the drug's mode of action. To gain further insight into the molecular genetic basis of INH resistance, 20 genes implicated in INH resistance were sequenced for INH resistance-associated mutations. Thirty-eight INH-monoresistant clinical isolates and 86 INH-susceptible isolates of M. tuberculosis were obtained from the Texas Department of Health and the Houston Tuberculosis Initiative. Epidemiologic independence was established for all isolates by IS6110 restriction fragment length polymorphism analysis. Susceptible isolates were matched with resistant isolates by molecular genetic group and IS6110 profiles. Spoligotyping was done with isolates with five or fewer IS6110 copies. A major genetic group was established on the basis of the polymorphisms in katG codon 463 and gyrA codon 95. MICs were determined by the E-test. Semiquantitative catalase assays were performed with isolates with mutations in the katG gene. When the 20 genes were sequenced, it was found that 17 (44.7%) INH-resistant isolates had a single-locus, resistance-associated mutation in the katG, mabA, or Rv1772 gene. Seventeen (44.7%) INH-resistant isolates had resistance-associated mutations in two or more genes, and 76% of all INH-resistant isolates had a mutation in the katG gene. Mutations were also identified in the fadE24, Rv1592c, Rv1772, Rv0340, and iniBAC genes, recently shown by DNA-based microarray experiments to be upregulated in response to INH. In general, the MICs were higher for isolates with mutations in katG and the isolates had reduced catalase activities. The results show that a variety of single nucleotide polymorphisms in multiple genes are found exclusively in INH-resistant clinical isolates. These genes either are involved in mycolic acid biosynthesis or are overexpressed as a response to the buildup or cellular toxicity of INH.     

结核分枝杆菌katG、inhA、ahpC等突变与异烟肼耐药关系的研究

目的：了解结核分枝杆菌katG、inhA、ahpC、fabG1、sodA及sodC基因突变的特征及其与耐异烟肼的关系。方法对127例活动性肺结核患者痰标本进行菌型鉴定及结核分枝杆菌药敏试验,提取结核分枝杆菌菌株DNA,应用PCR扩增katG、inhA及ahpC、fabG1、sodA及sodC基因片段,并进行DNA序列分析。结果结核分枝杆菌药物敏感试验显示127株结核分枝杆菌中,其中47株耐异烟肼,80株对异烟肼敏感,耐异烟肼率为37.01%。47株耐异烟肼中,29株存在katG和（或）inhA基因突变,其中22株（46.81%,22/47）存在katG基因单位点突变,3株（6.38%,3/47）存在inhA基因单位点突变,4株（8.51%,4/47）存在katG及inhA基因联合位点突变。22株katG基因单位点突变中,20株为AGC315ACC、AGC315AAC （42.55%,20/47）突变,2株（2.13%,1/47）分别为CTG378CCG（Leu378Pro）、ACG394ATG（Thr394Met）突变,该突变位点及突变形式尚未见文献报道。18株katG及inhA未突变结核分枝杆菌均未检测到ahpC、fabG1、sodA及sodC基因突变。结论结核分枝杆菌对异烟肼耐药主要与katG和inhA基因突变有关。耐异烟肼结核分枝杆菌临床分离株378和394新突变位点的发现为进一步研究耐药机制以及耐药结核病的快速检测提供了依据。     

Characterization of the katG and inhA genes of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.

Resistance to isoniazid in Mycobacterium tuberculosis has been associated with mutations in genes encoding the mycobacterial catalase-peroxidase (katG) and the InhA protein (inhA). Among the 26 isoniazid-resistant clinical isolates evaluated in this study, mutations in putative inhA regulatory sequences were identified in 2 catalase-positive isolates, katG gene alterations were detected in 20 strains, and 4 isolates had wild-type katG and inhA genes. Mutations in the katG gene were detected in all 11 catalase-negative isolates: one frameshift insertion, two partial gene deletions, and nine different missense mutations were identified. An arginine-to-leucine substitution at position 463 was detected in nine catalase-positive isolates. However, site-directed mutagenesis experiments demonstrated that the presence of a leucine at codon 463 did not alter the activity of the M. tuberculosis catalase-peroxidase and did not affect the capacity of this enzyme to restore isoniazid susceptibility to isoniazid-resistant, KatG-defective Mycobacterium smegmatis BH1 cells. These studies further support the association between katG and inhA gene mutations and isoniazid resistance in M. tuberculosis, while also suggesting that other undefined mechanisms of isoniazid resistance exist.     

Altered NADH/NAD+ ratio mediates coresistance to isoniazid and ethionamide in mycobacteria

The front-line antituberculosis drug isoniazid (INH) and the related drug ethionamide (ETH) are prodrugs that upon activation inhibit the synthesis of mycolic acids, leading to bactericidal activity. Coresistance to INH and ETH can be mediated by dominant mutations in the target gene inhA, encoding an enoyl-ACP reductase, or by recessive mutations in ndh, encoding a type II NADH dehydrogenase (NdhII). To address the mechanism of resistance mediated by the latter, we have isolated novel ndh mutants of Mycobacterium smegmatis and Mycobacterium bovis BCG. The M. smegmatis ndh mutants were highly resistant to INH and ETH, while the M. bovis BCG mutants had low-level resistance to INH and ETH. All mutants had defects in NdhII activity resulting in an increase in intracellular NADH/NAD(+) ratios. Increasing NADH levels were shown to protect InhA against inhibition by the INH-NAD adduct formed upon INH activation. We conclude that ndh mutations mediate a novel mechanism of resistance by increasing the NADH cellular concentration, which competitively inhibits the binding of INH-NAD or ETH-NAD adduct to InhA.     

应用多重PCR-单链构象多态性分析同时检测耐异烟肼和利福平的结核分枝杆菌

目的 探讨应用多重PCR-单链构象多态性分析(multiplexpulymerase chain reaction-single strand conformation polymorphism,multi-PCR-SSCP)方法快速、特异地同时快速检测结核分枝杆菌对异烟肼和利福平耐药性的效能.方法 根据结核分枝杆菌的inhA序列、katG序列、rpoB序列,分别设计出3对特异性寡聚核苷酸引物.采用multi-PCR-SSCP技术,一次性检出耐异烟肼和利福平的结核分枝杆菌.新方法的有效性通过116株临床分离株(70株耐异烟肼,66株耐利福平)的验证.结果 名 Multi-PCR-SSCP方法检测临床分离株基因突变的有效性,以细菌培养和药敏试验结果为金标准.116株临床分离株和H37Rv标准株中除了4株katG缺失突变,其余菌株3个基因katG、inhA和rpoB在单基因PCR中都扩增成功.与H37Rv标准株相比,46株katG基因突变,14株inhA基因突变,58株rpoB基因突变.38株katG和rpoB,4株inhA和rpoB,4株inhA和katG同时突变,还有2株3个基因都有突变.multi-PCR-SSCP对于耐异烟肼和利福平的结核分枝杆菌检出的敏感度分别为80%、82%,特异度分别为100%和92%.结论 multi-PCR-SSCP方法敏感、特异,能同时快速有效地检测耐多药结核分枝杆菌,有望成为临床指导用药的好方法,为深入研究耐药基凶检测奠定了良好的基础.     

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis isolates collected in Australia

Elucidation of the molecular basis of isoniazid (INH) resistance in Mycobacterium tuberculosis has led to the development of different genotypic approaches for the rapid detection of INH resistance in clinical isolates. Mutations in katG, in particular the S315T substitution, are responsible for INH resistance in a large proportion of tuberculosis cases. However, the frequency of the katG S315T substitution varies with population samples. In this study, 52 epidemiologically unrelated clinical INH-resistant M. tuberculosis isolates collected in Australia were screened for mutations at katG codon 315 and the fabG1-inhA regulatory region. Importantly, 52 INH-sensitive isolates, selected to reflect the geographic and genotypic diversity of the isolates, were also included for comparison. The katG S315T substitution and fabG1-inhA -15 C-to-T mutation were identified in 34 and 13 of the 52 INH-resistant isolates, respectively, and none of the INH-sensitive isolates. Three novel katG mutations, D117A, M257I, and G491C, were identified in three INH-resistant strains with a wild-type katG codon 315, fabG1-inhA regulatory region, and inhA structural gene. When analyzed for possible associations between resistance mechanisms, resistance phenotype, and genotypic groups, it was found that neither the katG S315T nor fabG1-inhA -15 C-to-T mutation clustered with any one genotypic group, but that the -15 C-to-T substitution was associated with isolates with intermediate INH resistance and isolates coresistant to ethionamide. In total, 90.4% of unrelated INH-resistant isolates could be identified by analysis of just two loci: katG315 and the fabG1-inhA regulatory region.     

Phenotypic and molecular characterization of Mycobacterium tuberculosis isolates resistant to both isoniazid and ethambutol

In performing radiometric susceptibility testing on over 2,000 patient isolates of Mycobacterium tuberculosis during the past 6 years, we found that resistance to 7.5 microg/ml ethambutol (EMB) occurred only in isolates that are also resistant to 0.4 microg/ml isoniazid (INH). Using 157 selected isolates in the present study, we performed radiometric and agar proportion susceptibility tests and DNA sequencing of genetic regions associated with resistance to these two drugs. The goal was to study the occurrence of the common mutations associated with resistance to each drug and also to determine whether any particular INH-resistance-associated mutation occurred more often in combination with any particular EMB-resistance-associated mutation. In an analysis of 128 isolates resistant to 0.4 microg/ml INH, we found that a mutation at katG Ser315 was more common in isolates also resistant to 7.5 microg/ml EMB (61 of 67=91.0%) than in isolates either susceptible to EMB or resistant to 2.5 microg/ml EMB (39 of 60=65.0%). These observations suggest that INH-resistant strains with a mutation at katG Ser315 are more likely to acquire resistance to 7.5 microg/ml EMB than are isolates with INH-resistance-associated mutations at other sites. In addition, we found that 64 of 67 (95.5%) isolates resistant to 7.5 microg/ml EMB contained a mutation in either codon 306 or codon 406 of embB. Met306Val was the most common embB mutation, present in 52 (77.6%) of the 67 isolates. Most occurrences of this mutation (49 of 52=94.2%) were found in isolates that also contained the katG Ser315Thr mutation. Finally, sequencing this region of embB appears to be sufficiently sensitive for use as a rapid screening tool for detection of high-level resistance to EMB.     

Analysis of the oxyR-ahpC region in isoniazid-resistant and -susceptible Mycobacterium tuberculosis complex organisms recovered from diseased humans and animals in diverse localities.

Automated DNA sequencing was used to analyze the oxyR-ahpC region in 229 Mycobacterium tuberculosis complex isolates recently recovered from diseased humans and animals. The entire 1,221-bp region was studied in 118 isolates, and 111 other isolates were sequenced for oxyR, ahpC, or the 105-bp oxyR-ahpC intergenic region. The sample included isoniazid (INH)-susceptible and -resistant organisms in which the katG gene and inhA locus had previously been sequenced in their entirety to identify polymorphisms. A total of 16 polymorphic sites was identified, including 5 located in oxyR, 2 in ahpC, and 9 in the 105-bp intergenic region. All polymorphic sites located in the intergenic region, and the two missense substitutions identified in ahpC, occurred in INH-resistant organisms. In contrast, there was no preferential association of polymorphisms in oxyR, a pseudogene, with INH-resistant organisms. Surprisingly, most INH-resistant strains with KatG codon 315 substitutions that substantially reduce catalase-peroxidase activity and confer high MICs of INH lacked alterations in the ahpC gene or oxyR-ahpC intervening region. Taken together, the data are consistent with the hypothesis that some polymorphisms located in the ahpC-oxyR intergenic region are selected for after reduction in catalase or peroxidase activity attributable to katG alterations arising with INH therapy. These mutations are uncommon in recently recovered clinically significant organisms, and hence, there is no strict association with INH-resistant patient isolates. The ahpC compensatory mutations are apparently uncommon because strains with a KatG null phenotype are relatively rare among epidemiologically independent INH-resistant organisms.     

Novel Mutations in ndh in Isoniazid-Resistant Mycobacterium tuberculosis Isolates

Novel mutations in NADH dehydrogenase (ndh) were detected in 8 of 84 (9.5%) isoniazid (INH)-resistant isolates (T110A [n = 1], R268H [n = 7]), but not in 22 INH-susceptible isolates of Mycobacterium tuberculosis. Significantly, all eight isolates with mutations at ndh did not have mutations at katG, kasA, or the promoter regions of inhA or ahpC, except for one isolate. Mutations in ndh appear to be an additional molecular mechanism for isoniazid resistance in M. tuberculosis.     

Transfer of embB codon 306 mutations into clinical Mycobacterium tuberculosis strains alters susceptibility to ethambutol, isoniazid, and rifampin

Implicated as a major mechanism of ethambutol (EMB) resistance in clinical studies of Mycobacterium tuberculosis, mutations in codon 306 of the embB gene (embB306) have also been detected in EMB-susceptible clinical isolates. Other studies have found strong associations between embB306 mutations and multidrug resistance, but not EMB resistance. We performed allelic exchange studies in EMB-susceptible and EMB-resistant clinical M. tuberculosis isolates to identify the role of embB306 mutations in any type of drug resistance. Replacing wild-type embB306 ATG from EMB-susceptible clinical M. tuberculosis strain 210 with embB306 ATA, ATC, CTG, or GTG increased the EMB MIC from 2 microg/ml to 7, 7, 8.5, and 14 microg/ml, respectively. Replacing embB306 ATC or GTG from two high-level EMB-resistant clinical strains with wild-type ATG lowered EMB MICs from 20 microg/ml or 28 microg/ml, respectively, to 3 microg/ml. All parental and isogenic mutant strains had identical isoniazid (INH) and rifampin (RIF) MICs. However, embB306 CTG mutants had growth advantages compared to strain 210 at sub-MICs of INH or RIF in monocultures and at sub-MICs of INH in competition assays. CTG mutants were also more resistant to the additive or synergistic activities of INH, RIF, or EMB used in different combinations. These results demonstrate that embB306 mutations cause an increase in the EMB MIC, a variable degree of EMB resistance, and are necessary but not sufficient for high-level EMB resistance. The unusual growth property of embB306 mutants in other antibiotics suggests that they may be amplified during treatment in humans and that a single mutation may affect antibiotic susceptibility against multiple first-line antibiotics.     

Phenotypic characterization of pncA mutants of Mycobacterium tuberculosis

We examined the correlation of mutations in the pyrazinamidase (PZase) gene (pncA) with the pyrazinamide (PZA) resistance phenotype with 60 Mycobacterium tuberculosis isolates. PZase activity was determined by the method of Wayne (L. G. Wayne, Am. Rev. Respir. Dis. 109:147-151, 1974), and the entire pncA nucleotide sequence, including the 74 bp upstream of the start codon, was determined. PZA susceptibility testing was performed by the method of proportions on modified Middlebrook and Cohn 7H10 medium. The PZA MICs were > or =100 microg/ml for 37 isolates, 34 of which had alterations in the pncA gene. These mutations included missense substitutions for 24 isolates, nonsense substitutions for 3 isolates, frameshifts by deletion for 4 isolates, a three-codon insertion for 1 isolate, and putative regulatory mutations for 2 isolates. Among 21 isolates for which PZA MICs were <100 microg/ml, 3 had the same mutation (Thr47-->Ala) and 18 had the wild-type sequence. For the three Thr47-->Ala mutants PZA MICs were 12.5 microg/ml by the method of proportions on 7H10 agar; two of these were resistant to 100 microg of PZA per ml and the third was resistant to 800 microg of PZA per ml by the BACTEC method. In all, 30 different pncA mutations were found among the 37 pncA mutants. No PZase activity was detected in 35 of 37 strains that were resistant to > or =100 microg of PZA per ml or in 34 of 37 pncA mutants. Reduced PZase activity was found in the three mutants with the Thr47-->Ala mutation. This study demonstrates that mutations in the pncA gene may serve as a reliable indicator of resistance to > or =100 microg of PZA per ml.     

New multiplex PCR for rapid detection of isoniazid-resistant Mycobacterium tuberculosis clinical isolates

In this study, we describe a multiplex PCR to detect a AGC-->ACC (serine to threonine) mutation in the katG gene and a -15 C-to-T substitution (inhA(C-15T)) at the 5' end of a presumed ribosome binding site in the promoter of the mabA-inhA operon. These mutations have been reported in the majority of previous studies as the most frequent mutations involved in the resistance to isoniazid (INH) of Mycobacterium tuberculosis clinical strains with high levels of resistance. The method was optimized and validated after an analysis of 30 M. tuberculosis clinical isolates with known sequences of the relevant part of the katG gene and the regulatory region of the mabA-inhA operon. We analyzed 297 INH-resistant M. tuberculosis isolates collected in Spain from 1996 to 2003 by PCR-restriction fragment length polymorphism (using the katG gene), DNA sequencing, and the newly developed multiplex PCR. The results were concordant for all 297 isolates tested. The analysis revealed that 204 (68.7%) of the isolates carried one or both of the mutations. This finding suggests that with further development this multiplex PCR will be able to detect the majority of the INH-resistant M. tuberculosis clinical isolates from Spain and other countries where a high frequency of similar mutations occur.     

耐药结核分枝杆菌基因突变分析

目的 探讨结核分枝杆菌耐药表型与基因突变位点之间的相互关系.方法 采用序列特异性引物分别扩增92株结核分枝杆菌利福平耐药基因rpoB,异烟肼耐药基因katG、inhA、ahpC,链霉素耐药基因rrs、rpsL,乙胺丁醇耐药基因embB及喹诺酮耐药基因gyrA,SSCP筛选出突变序列,DNA测序分析突变性质.结果 59株利福平耐药株rpoB基因突变检出率94.9%(56/59),以Ser450Trp突变最多;90株异烟肼耐药株中,katG基因突变检出率38.9%(35/90),以Ser315Thr最多,3株检出inhA基因突变,ahpC基因无突变检出;34株喹诺酮耐药株中gyrA基因突变检出率82.4%(28/34),主要为Asp94Gly,其次为Ala90Val;31株链霉素耐药株中,15株检出rrs突变,最常见为A514C和A1041G,10株发生rpsL Lys88Arg突变,总的链霉素基因突变检出率为77.4%(24/31);31株乙胺丁醇耐药株中embB 基因突变检出率19.4%(6/31),主要为Met306Val.结论 耐药结核分枝杆菌耐药情况较为严重,以DNA测序为基础的基因突变分析能快速有效地检测结核分枝杆菌的rpoB、katG、gyrA、rrs、rpsL、embB 等耐药分子标识,显示了西安地区耐药性结核分枝杆菌的突变特点,为结核病的临床诊断和合理用药提供了实验依据.     

Mutations in folP associated with elevated sulfonamide MICs for Neisseria meningitidis clinical isolates from five continents

Sulfonamide resistance in meningococci is associated with mutations in the chromosomal gene folP, which encodes dihydropteroate synthase. Several mutations associated with resistance have been previously described, including amino acid substitutions at codons 31 and 194, a glycine-serine insertion at codons 195 and 196, and, recently, an additional mutation at nucleotide 682 (C682A). In this study, sulfisoxazole MICs were determined for 424 geographically diverse clinical isolates of Neisseria meningitidis, including all major subtypes. A subset of 134 isolates with MICs ranging from 0.5 to >64 microg/ml were assayed for the C682A mutation by real-time PCR, and 25 isolates were selected for folP gene sequencing. All isolates for which the sulfisoxazole MIC was >/=8 possessed the C682A mutation by real-time PCR or folP sequencing, and 34 of 35 isolates with a MIC of /=4, 15 possessed previously described mutations, including 10 at codon 31, 1 at codon 194, and 4 with the 2-amino-acid insertion codons 195 and 196; all 16 possessed the C682A mutation. The C682A mutation predicted elevated sulfonamides MICs for a large number of geographically diverse clinical isolates of meningococci. Detection of this mutation by real-time PCR or other methods may allow more wide-scale detection of meningococcal isolates with for which the sulfonamide MICs are elevated without resorting to multiple assays or folP gene sequencing, providing a simple, high-throughput screening method for use in public health and epidemiologic settings.     

Phenylethyl butyrate enhances the potency of second-line drugs against clinical isolates of Mycobacterium tuberculosis

Ethionamide (ETH) is a second-line drug for the treatment of tuberculosis. As a prodrug, ETH has to be activated by EthA. ethA is controlled by its repressor EthR. 2-Phenylethyl-butyrate (2-PEB) inhibits EthR binding, enhances expression of EthA, and thereby enhances the growth-inhibitory effects of ethionamide, isoxyl, and thiacetazone in Mycobacterium tuberculosis strains with resistance to ETH due to inhA promoter mutations but not ethA mutations.     

Isoniazid induces its own resistance in nonreplicating Mycobacterium tuberculosis

Isoniazid (INH) resistance is most frequent among drug-resistant Mycobacterium tuberculosis clinical isolates. This study was conducted to investigate whether INH could induce its own resistance. During INH susceptibility testing in BACTEC 12B and MGIT 960 media, weekly subcultures were made from the drug-containing media into fresh medium without drug and susceptibility testing was performed. Rifampin (RIF) was used as a control drug. M. tuberculosis H37Rv and three clinical isolates were tested in this study. INH-resistant subcultures were analyzed for catalase activity, INH susceptibility, and mutations associated with INH resistance. With inoculum size (10(4) bacilli) smaller than a size that contains spontaneously INH-resistant mutants, INH was found to induce resistance to itself in INH-tolerant persisters but not to other drugs. The minimum time required for induction of INH resistance was 5 to 6 days. In contrast, RIF did not induce RIF resistance. Eight subcultures with INH-induced resistance were analyzed, and two had a MIC of 0.4 microg/ml INH and six had MICs of over 2 microg/ml INH. Four of the eight subcultures with INH-induced resistance had lost catalase activity, with three having katG mutations. Despite being a powerful frontline tuberculosis drug, INH has the potential drawback of inducing its own stable genetic resistance in INH-tolerant persisters. This finding helps to explain the higher frequency and prevalence of INH-resistant isolates than isolates with resistance to other drugs in patients.     

Topoisomerase Sequences of Coagulase-Negative Staphylococcal Isolates Resistant to Ciprofloxacin or Trovafloxacin

Coagulase-negative staphylococcal isolates (n = 188) were screened for susceptibility to oxacillin, ciprofloxacin, and trovafloxacin, a new fluoroquinolone. At an oxacillin concentration of >/=4 microg/ml, 43% were methicillin resistant; of these, 70% were ciprofloxacin resistant (MIC, >/=4 microg/ml). Of the methicillin-resistant, ciprofloxacin-resistant isolates, 46% were susceptible to /=8 microg/ml) and increased trovafloxacin MICs (0.25 to 2 microg/ml) could be conferred by the combined presence of single mutations in each gyrA and grlA gene. Trovafloxacin MICs of >/=8 microg/ml also occurred, but these required an additional mutation in grlA.     

Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis

The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.