A Ser315Thr Substitution in KatG Is Predominant in Genetically Heterogeneous Multidrug-Resistant Mycobacterium tuberculosis Isolates Originating from the St. Petersburg Area in Russia

Parts of katG and rpoB from 27 Russian Mycobacterium tuberculosis isolates were sequenced to detect mutations causing resistance to isoniazid (INH) and rifampin (RMP), respectively. All 24 INH-resistant isolates had a mutated katG, and 22 of them (91.7%) carried a mutation coding for a Ser315Thr shift. An rpoB mutation was noted for each of the 21 RMP-resistant isolates, with Ser531Leu being the most prevalent change encoded. Only two isolates had identical IS6110 fingerprints.     

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis clinical isolates in Lithuania

Mutations at codon 315 of the katG gene were detected in 312 of 364 (85.7%) isoniazid-resistant Mycobacterium tuberculosis isolates. Seven of 52 (13.5%) isoniazid-resistant isolates with the wild-type Ser315 codon and 10 of 52 (19.2%) isoniazid-resistant isolates with a mutated katG allele had mutation -15C-->T in the promoter of the mabA-inhA operon.     

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.     

Analysis of ahpC gene mutations in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.

The ahpC genes of 57 clinical isolates and one in vitro mutant of Mycobacterium tuberculosis were evaluated by nucleotide sequence analyses. Although compensatory ahpC promoter mutations were identified in 8 catalase-negative, katG-defective strains, the ahpC genes of 25 catalase-positive, isoniazid-resistant isolates and 25 drug-sensitive strains were not altered.     

Molecular characteristics of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates from the Russian Federation

OBJECTIVES: Three Mycobacterium tuberculosis genetic loci--rpoB and katG genes and the fabG1(mabA)-inhA operon promoter region--were studied to reveal the mutations associated with rifampicin and isoniazid resistance. METHODS: Four hundred and twelve isolates of M. tuberculosis from different regions of the Russian Federation were collected during 1997-2005. A matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based minisequencing method was used for the detection of mutations. RESULTS: Thirteen different variants of single mutations in codons 533, 531, 526, 516, 513 and 511 of the rifampicin resistance-determining region of the rpoB gene as well as the TTG insertion in the 514a position were found among the rifampicin-resistant isolates. Single nucleotide substitutions in codons 531, 526 and 516 (64.8%, 10.3% and 7.7%, respectively) were the most prevalent mutations. Codon 526 was shown to be the most variable of all. No mutations were detected in rpoB genes for 29 (10.7%) of the rifampicin-resistant isolates. 76.9% of the isoniazid-resistant isolates carried single mutations in codon 315 of the katG gene. For another 12.9% of them, double mutations in the katG gene and the fabG1(mabA)-inhA promoter region were revealed. No mutations were detected in 8.2% of the isoniazid-resistant isolates. CONCLUSIONS: Molecular analysis of the loci of rpoB and katG genes and the inhA promoter region of 412 M. tuberculosis clinical isolates from various parts of the Russian Federation was carried out. The new MALDI-TOF MS-based method may be used for rapid and accurate monitoring of the spread of drug resistance.     

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.     

Mutations in katG gene sequences in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis are rare.

In this study, a battery of oligonucleotides was directed toward the katG gene and PCR-single-stranded conformation polymorphism (SSCP) analysis was used to search for katG gene deviations in clinical isolates of Mycobacterium tuberculosis from different geographical regions. Since a complete deletion of the katG gene was not found, it is suggested that deletion is not a major mechanism of isoniazid (isonicotinic acid hydrazide; INH) resistance in these isolates. However, 7 of 39 isolates (4 of 25 from South Africa and 3 of 14 from other geographical regions) showed mobility shifts by SSCP analysis, suggesting aberrations in the katG gene. Direct sequence analysis confirmed that the mobility shifts were due to Thr-275-->Ala (Thr275Ala), Arg409Ala, Arg463Leu, and Asp695Ala mutations and a 12-bp deletion in the 5' region of the katG gene. Mutations at codons 275, 463, and 695 created altered restriction sites for HhaI, MspI, and HaeIII, respectively, and sequence results, supported by restriction fragment length polymorphism analysis, suggested that the PCR-SSCP procedure is a good indicator of mutations in PCR-amplified fragments. Identical mutations at codons 463 and 275 were found in isolates from different geographical regions. This may suggest a common evolutionary event, but one of the control isolates (susceptible to INH [3%; n = 30]) also had a mutation at codon 463. The results suggest that variations in the katG coding gene sequences of INH-resistant isolates of M. tuberculosis are infrequent and that defects in other regions of the M. tuberculosis genome are of equal or greater importance in contributing to the acquisition of resistance to INH.     

Molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates from Shanghai, China

China is one of the countries with the highest prevalence of fluoroquinolone-resistant (FQ(r)) Mycobacterium tuberculosis. Nevertheless, knowledge on the molecular characterization of the FQ(r)M. tuberculosis strains of this region remains very limited. This study was performed to investigate the frequencies and types of mutations present in FQ(r)M. tuberculosis clinical isolates collected in Shanghai, China. A total of 206 FQ(r)M. tuberculosis strains and 21 ofloxacin-sensitive (FQ(s)) M. tuberculosis strains were isolated from patients with pulmonary tuberculosis in Shanghai. The phenotypic drug susceptibilities were determined by the proportion method, and the mutations inside quinolone resistance-determining region (QRDR) of gyrA and gyrB genes were identified by DNA sequence analyses. Among 206 FQ(r)M. tuberculosis strains, 44% (90/206) were multidrug-resistant isolates and 39% (81/206) were extensively drug-resistant isolates. Only 9% (19/206) were monoresistant to ofloxacin. In total, 79.1% (163/206) of FQ(r) isolates harboured mutations in either gyrA or gyrB QRDR. Mutations in gyrA QRDR were found in 75.7% (156/206) of FQ(r) clinical isolates. Among those gyrA mutants, a majority (75.6%) harboured mutations at amino acid position 94, with D94G being the most frequent amino acid substitution. Mutations in gyrA QRDR showed 100% positive predictive value for FQ(r)M. tuberculosis in China. Mutations in gyrB were observed in 15.5% (32/206) of FQ(r) clinical isolates. Ten novel mutations were identified in gyrB. However, most of them also harboured mutations in gyrA, limiting their contribution to FQ(r) resistance in M. tuberculosis. Our findings indicated that, similar to other geographic regions, mutations in gyrA were shown to be the major mechanism of FQ(r) resistance in M. tuberculosis isolates. The mutations in gyrA QRDR can be a good molecular surrogate marker for detecting FQ(r)M. tuberculosis in China.     

ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuberculosis isolates

Ethionamide (ETH) is a structural analog of the antituberculosis drug isoniazid (INH). Both of these drugs target InhA, an enzyme involved in mycolic acid biosynthesis. INH requires catalase-peroxidase (KatG) activation, and mutations in katG are a major INH resistance mechanism. Recently an enzyme (EthA) capable of activating ETH has been identified. We sequenced the entire ethA structural gene of 41 ETH-resistant Mycobacterium tuberculosis isolates. We also sequenced two regions of inhA and all or part of katG. The MICs of ETH and INH were determined in order to associate the mutations identified with a resistance phenotype. Fifteen isolates were found to possess ethA mutations, for all of which the ETH MICs were > or =50 microg/ml. The ethA mutations were all different, previously unreported, and distributed throughout the gene. In eight of the isolates, a missense mutation in the inhA structural gene occurred. The ETH MICs for seven of the InhA mutants were > or =100 microg/ml, and these isolates were also resistant to > or =8 microg of INH per ml. Only a single point mutation in the inhA promoter was identified in 14 isolates. A katG mutation occurred in 15 isolates, for which the INH MICs for all but 1 were > or =32 microg/ml. As expected, we found no association between katG mutation and the level of ETH resistance. Mutations within the ethA and inhA structural genes were associated with relatively high levels of ETH resistance. Approximately 76% of isolates resistant to > or =50 microg of ETH per ml had such mutations.     

In vitro susceptibility of Mycobacterium tuberculosis clinical isolates to garenoxacin and DA-7867

The in vitro activities of DA-7867, a novel oxazolidinone, and garenoxacin (BMS-284756) were compared to those of linezolid in 67 susceptible and drug-resistant clinical isolates of Mycobacterium tuberculosis. DA-7867 was the most active drug with an MIC(90) of 0.125 microg/ml, compared to the MIC(90)s of 4 microg/ml of garenoxacin and 2 microg/ml of linezolid.     

Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates from England and Wales reveals the phylogenetic significance of the ahpC -46A polymorphism

The present study investigated the prevalence and diagnostic potential of the most commonly reported mutations associated with isoniazid resistance, katG 315Thr, katG 315Asn, inhA -15T, inhA -8A, and the oxyR-ahpC intergenic region, in a population sample of 202 isoniazid-resistant Mycobacterium tuberculosis isolates and 176 randomly selected fully sensitive isolates from England and Wales identified by using a directed oligonucleotide array and limited DNA sequencing. The strains were recovered from patients originating from 29 countries; 41 isolates were multidrug resistant. Mutations affecting katG 315, the inhA promoter, and the oxyR-ahpC intergenic region were found in 62.7, 21.9, and 30% of 169 genotypically distinct isoniazid-resistant isolates, respectively, whereas they were found in 0, 0, and 8% of susceptible strains, respectively. The frequency of mutation at each locus was unrelated to the resistance profile or previous antituberculous drug therapy. The commonest mutation in the oxyR-ahpC intergenic region, ahpC -46A, was present in 23.7% of isoniazid-resistant isolates and 7.5% of susceptible isolates. This proved to be a phylogenetic marker for a subgroup of M. tuberculosis strains originating on the Indian subcontinent, which shared IS6110-based restriction fragment length polymorphism and spoligotype features with the Delhi strain and Central Asian strain CAS1; and this marker is strongly associated with isoniazid resistance and the katG 315Thr mutation. In total, 82.8% of unrelated isoniazid-resistant isolates could be identified by analysis of just two loci: katG 315 and the inhA promoter. Analysis of the oxyR-ahpC intergenic region, although phylogenetically interesting, does not contribute significantly to further identification of isoniazid-resistant isolates.     

pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil

OBJECTIVES: To investigate the presence of mutations in the pncA gene in 31 pyrazinamide-resistant Mycobacterium tuberculosis and 5 susceptible strains. MICs and pyrazinamidase (PZase) activity were also determined. METHODS: All 36 M. tuberculosis clinical isolates were genotyped by mycobacterial interspersed repetitive units (MIRUs) and most were also typed by spoligotyping. The MIC value necessary to inhibit 99% of the resistant mycobacterial isolates was determined by microplate Alamar Blue assay (MABA) and by L?wenstein-Jensen assay (LJA). The PZase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and the entire pncA sequence including the 410 bp upstream from the start codon was determined by DNA sequencing of purified PCR products. RESULTS: Of the 31 isolates resistant to pyrazinamide, 26 (83.9%) showed at least one mutation in the pncA gene or in its putative regulatory region. Among the 22 different mutations detected in the pncA gene and in its regulatory region, 9 (40.9%) mutations (consisting of six substitutions, two insertions and one deletion) have not been described in previous studies. Three pyrazinamide-resistant isolates, confirmed by MIC varying from 800 to 1600 mg/L, carried the wild-type pncA sequence and retained PZase activity. CONCLUSIONS: These results contribute to the knowledge of the molecular mechanism of pyrazinamide resistance in Brazil and also expand the profile of pncA mutations worldwide. The MABA was successfully used to determine the MICs of pyrazinamide.     

Characterization of novel Mycobacterium tuberculosis pncA gene mutations in clinical isolates from the Ukraine

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis cases in the Ukraine are increasing. Pyrazinamide (PZA) is critically important for first- and second-line tuberculosis (TB) treatment regimes. However, PZA drug susceptibility testing is time consuming and technically challenging. The present study utilized Next-generation sequencing (NGS) to identify mutations in the pncA gene from clinical isolates and to assess the prevalence of pncA gene mutations in MDR/XDR-TB patients. Clinical isolates were inactivated in molecular transport media and shipped from Kharkiv, Ukraine, to San Antonio, TX. Whole-genome and targeted pncA gene sequencing was carried out using Illumina MiSeq instrumentation. Mutations were noted in 67 of 91 (74%) clinical isolates comprising substitutions, insertions, and deletions in the pncA coding and upstream promoter region. Of 45 mutation types, there were 11 novel, i.e., to date unknown, pncA mutations identified of which 3 were confirmed PZA resistant. Seven isolates contained mixed base mutations, whereas 4 harbored doubled mutations. Data reported here further support use of NGS for pncA gene characterization and may contribute in significant fashion to PZA therapy, especially in MDR- and XDR-TB patients.     

Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait and Dubai

Mutations conferring resistance to rifampin in rifampin-resistant clinical Mycobacterium tuberculosis isolates occur mostly in the 81 bp rifampin-resistance-determining region (RRDR) of the rpoB gene. In this study, 29 rifampin-resistant and 12 -susceptible clinical M. tuberculosis isolates were tested for characterization of mutations in the rpoB gene by line probe (INNO-LiPA Rif. TB) assay and the results were confirmed and extended by DNA sequencing of the PCR amplified target DNA. The line probe assay identified all 12 susceptible strains as rifampin-sensitive and the DNA sequence of RRDR in the amplified rpoB gene from two isolates matched perfectly with the wild-type sequence. The line probe assay identified 28 resistant isolates as rifampin-resistant with specific detection of mutation in 22 isolates including one isolate that exhibited hetro-resistance containing both the wild-type pattern as well as a specific mutation within RRDR while one of the rifampin-resistant strain was identified as rifampin-susceptible. DNA sequencing confirmed these results and, in addition, led to the specific detection of mutations in 5 rifampin-resistant isolates in which specific base changes within RRDR could not be determined by the line probe assay. These analyses identified 8 different mutations within RRDR of the rpoB gene including one novel mutation (S522W) that has not been reported so far. The genotyping performed on the isolates carrying similar mutations showed that majority of these isolates were unique as they exhibited varying DNA banding patterns. Correlating the ethnic origin of the infected TB patients with the occurrence of specific mutations at three main codon positions (516, 526 and 531) in the rpoB gene showed that most patients (11 of 15) from South Asian region contained mutations at codon 526 while majority of isolates from patients (6 of 11) of Middle Eastern origin contained mutations at codon 531.     

Limited fluoroquinolone resistance among Mycobacterium tuberculosis isolates from Rwanda: results of a national survey

OBJECTIVES: There is an increasing interest in the possible role of fluoroquinolone antibiotics for the treatment of tuberculosis (TB), but widespread use of these antibiotics for the treatment of other bacterial infections may select for fluoroquinolone-resistant Mycobacterium tuberculosis strains. METHODS: We evaluated fluoroquinolone susceptibility using the proportion method (ofloxacin, critical concentration 2.0 mg/L) in isolates from patients enrolled in a national drug resistance survey in Rwanda from November 2004 to February 2005. RESULTS: Of the 701 M. tuberculosis isolates studied, 617 (88%) were susceptible to all first-line drugs, 32 (4.6%) were multidrug-resistant (MDR) and 52 (7.4%) were resistant to one or more first-line drugs but not MDR. Ofloxacin resistance was found in four (0.6%) of the isolates; three of them being MDR and one susceptible to all first-line drugs. Mutations in the gyrA gene were found in all ofloxacin-resistant strains at codons 80 and 94. CONCLUSIONS: Our finding is not alarming for Rwanda, but highlights the general risk of producing resistance to fluoroquinolones, jeopardizing the potential for these drugs to be used as second-line anti-TB agents in the programmatic management of drug-resistant TB and creating incurable TB strains.     

Susceptibility of clinical Mycobacterium tuberculosis isolates to a potentially less toxic derivate of linezolid, PNU-100480

Susceptibility of clinical Mycobacterium tuberculosis isolates to PNU-100480 and linezolid was evaluated by the MGIT 960 system. The isolates had various susceptibilities to isoniazid (INH), rifampin, ethambutol, and streptomycin. The mean MIC for PNU-100480 was 3.2 times lower than that for linezolid. Therefore, PNU-100480 is a promising candidate to be developed further as an adjunct in the treatment of multidrug- and extensively drug-resistant tuberculosis (MDR/XDR-TB).     

Profiling and identification of novel rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis clinical isolates from Pakistan

IntroductionRifampicin (RIF) is one of the most effective anti-tuberculosis first-line drugs prescribed along with isoniazid. However, the emergence of RIF resistance Mycobacterium tuberculosis (MTB) isolates is a major issue towards tuberculosis (TB) control program in high MDR TB-burdened countries including Pakistan. Molecular data behind phenotypic resistance is essential for better management of RIF resistance which has been linked with mutations in rpoB gene. Since molecular studies on RIF resistance is limited in Pakistan, the current study was aimed to investigate the molecular data of mutations in rpoB gene behind phenotypic RIF resistance isolates in Pakistan.MethodA total of 322 phenotypically RIF-resistant isolates were randomly selected from National TB Reference Laboratory, Pakistan for sequencing while 380 RIF resistance whole-genome sequencing (WGS) of Pakistani isolates (BioProject PRJEB25972), were also analyzed for rpoB mutations.ResultAmong the 702 RIF resistance samples, 675 (96.1%) isolates harbored mutations in rpoB in which 663 (94.4%) were detected within the Rifampicin Resistance Determining Region (RRDR) also known as a mutation hot spot region, including three novel. Among these mutations, 657 (97.3%) were substitutions including 603 (89.3%) single nucleotide polymorphism, 49 (7.25%) double and five (0.8%) triple. About 94.4% of Phenotypic RIF resistance strains, exhibited mutations in RRDR, which were also detectable by GeneXpert.ConclusionMutations in the RRDR region of rpoB is a major mechanism of RIF resistance in MTB circulating isolates in Pakistan. Molecular detection of drug resistance is a faster and better approach than phenotypic drug susceptibility testing to reduce the time for transmission of RIF resistance strains in population. Such insights will inform the deployment of anti-TB drug regimens and disease control tools and strategies in high burden settings, such as Pakistan.     

High prevalence of nalidixic acid resistant,ciprofloxacin susceptible phenotype among clinical isolates of Escherichia coli and other Enterobacteriaceae

Therapeutic failure of infections during their treatment with quinolones has been often described. This may be due to the development of resistance during treatment of an infecting strain which already had diminished susceptibility to quinolones, even though the initial MIC did not exceed the breakpoint. In this study the prevalence of the nalidixic acid resistant, ciprofloxacin susceptible phenotype among Enterobacteriaceae was analyzed. The results showed that 113 out of 151 (74.83%) strains of the Enterobacteriaceae with diminished susceptibility to ciprofloxacin (MICs from 0.06 to 1 microg/ml) were resistant to nalidixic acid (MICs > 32 microg/ml). The Escherichia coli strains presenting this phenotype already have a mutation in the amino acid codon Ser-83 of the gyrA gene, so that the possibility of developing a second mechanism of resistance during treatment is very high.     

Surveillance for antimicrobial resistance among clinical isolates of gram-negative bacteria from intensive care unit patients in China, 1996 to 2002

The objective is to investigate the trend of antimicrobial resistance among nosocomial gram-negative bacteria isolated from intensive care units in China. From 1996 to 2002, the minimum inhibitory concentrations (MICs) of 8 antibiotics for 10,585 isolates of gram-negative bacteria from 19 hospitals in 7 central cities were determined by Etest. From 1996 to 2002, a marked decrease in the susceptibility of Pseudomonas aeruginosa to imipenem was noticed along the years (81-62%). Percentage of multidrug resistance of strains in P. aeruginosa obviously increased (11.5% in 1996, 20.5% in 2002). Imipenem kept active against Escherichia coli (99.2-100% susceptible), Acinetobacter spp. (97.6-93.5%), Klebsiella spp. (94.9-100%), Enterobacter spp. (89-96%). Resistance to cephalosporins, ciprofloxacin, and cefoperazone/sulbactam was observed, particularly among E. coli to ciprofloxacin (42-25%) and cefotaxime (78-54%) and Enterobacter spp. to ceftazidime (51-44%) and cefotaxime (50-37%). Piperacillin/tazobactam kept stable and active against P. aeruginosa, E. coli, and Klebsiella spp. (80%), with an increasing trend, but not good, in Enterobacter spp. (63-58%). Extended-spectrum beta-lactamase-producing strains in E. coli (28.6-45.7%) and Klebsiella spp. (25.5-34.9%) increased during 2001-2002. There was no significant increase resistance in Enterobacteriaceae isolates and Acinetobacter spp. to imipenem, but it has obviously decreased activity in P. aeruginosa throughout the 7-year period in China. Resistance of tested gram-negative bacteria to most comparator antimicrobials increased at different levels from 1996 to 2002 in China.