Mutations in theembC-embAIntergenic Region Contribute to Mycobacterium tuberculosis Resistance to Ethambutol

ABSTRACTThe rapid increase inMycobacterium tuberculosisresistance to ethambutol (EMB) threatens the diagnosis and treatment of tuberculosis (TB). We investigated the role of mutations in theembC-embAintergenic region (IGR) in EMB-resistant clinical strains from east China. A total of 767M. tuberculosisclinical strains were collected and analyzed for their drug susceptibility to EMB using the MGIT 960 system and MIC assay, and theembC-embAIGRs of these strains were sequenced. The transcriptional activity of theembC-embAIGR mutations was examined by reporter gene assays in recombinantMycobacterium smegmatisstrains, and the effect of IGR mutations on its binding to EmbR, a transcription regulator ofembAB, was analyzed by gel mobility shift assays. Correlation coefficient analysis showed that theembC-embAIGR mutation is associated with EMB resistance. The clinical strains carrying IGR mutations had a much higher level ofembAandembBmRNA as well as higher MICs to EMB. IGR mutations had higher transcriptional activity when transformed intoM. smegmatisstrains. Mutated IGRs bound to EmbR with much higher affinity than wild-type fragments. The sensitivity of molecular drug susceptibility testing (DST) with IGR mutations as an additional marker increased from 65.5% to 73.5%. Mutations of theembC-embAIGR enhance the binding of EmbR to the promoter region ofembABand increase the expression ofembAB, thus contributing to EMB resistance. Therefore, identification of IGR mutations as markers of EMB resistance could increase the sensitivity of molecular DST.

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Evaluation of the Abbott RealTi m e MTB and RealTi m e MTB INH/RIF Assays for Direct Detection of Mycobacterium tuberculosis Complex and Resistance Markers in Respiratory and Extrapulmonary Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.01144-16 ◽

2016 ◽

Vol 54 (12) ◽

pp. 3022-3027 ◽

Cited By ~ 27

Author(s):

Sabine Hofmann-Thiel ◽

Nikolay Molodtsov ◽

Uladzimir Antonenka ◽

Harald Hoffmann

Keyword(s):

Mycobacterium Tuberculosis ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Nucleic Acid Amplification ◽

Content Type ◽

Clinical Specimens ◽

Different Types ◽

Resistance Markers ◽

Smear Negative

The Abbott RealTi m e MTB (RT MTB) assay is a new automated nucleic acid amplification test for the detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. In combination with the RealTi m e MTB INH/RIF (RT MTB INH/RIF) resistance assay, which can be applied to RT MTB-positive specimens as an add-on assay, the tests also indicate the genetic markers of resistance to isoniazid (INH) and rifampin (RIF). We aimed to evaluate the diagnostic sensitivity and specificity of RT MTB using different types of respiratory and extrapulmonary specimens and to compare performance characteristics directly with those of the FluoroType MTB assay. The resistance results obtained by RT MTB INH/RIF were compared to those from the GenoType MTBDR plus and from phenotypic drug susceptibility testing. A total of 715 clinical specimens were analyzed. Compared to culture, the overall sensitivity of RT MTB was 92.1%; the sensitivity rates for smear-positive and smear-negative samples were 100% and 76.2%, respectively. The sensitivities of smear-negative specimens were almost identical for respiratory (76.3%) and extrapulmonary (76%) specimens. Specificity rates were 100% and 95.8% for culture-negative specimens and those that grew nontuberculous mycobacteria, respectively. RT MTB INH/RIF was applied to 233 RT MTB-positive samples and identified resistance markers in 7.7% of samples. Agreement with phenotypic and genotypic drug susceptibility testing was 99.5%. In conclusion, RT MTB and RT MTB INH/RIF allow for the rapid and accurate diagnosis of tuberculosis (TB) in different types of specimens and reliably indicate resistance markers. The strengths of this system are the comparably high sensitivity with paucibacillary specimens, its ability to detect INH and RIF resistance, and its high-throughput capacities.

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Susceptibility Testing of Extensively Drug-Resistant and Pre-Extensively Drug-Resistant Mycobacterium tuberculosis against Levofloxacin, Linezolid, and Amoxicillin-Clavulanate

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02020-12 ◽

2013 ◽

Vol 57 (6) ◽

pp. 2522-2525 ◽

Cited By ~ 20

Author(s):

Imran Ahmed ◽

Kauser Jabeen ◽

Raunaq Inayat ◽

Rumina Hasan

Keyword(s):

Mycobacterium Tuberculosis ◽

Susceptibility Testing ◽

Critical Concentration ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Fluoroquinolone Resistance ◽

Drug Resistant ◽

Content Type ◽

Extensively Drug Resistant ◽

Amoxicillin Clavulanate

ABSTRACTPakistan is a high-burden country for tuberculosis (TB). The emergence and increasing incidence of extensively drug-resistant (XDR) TB has been reported in Pakistan. Similarly, the prevalence of multidrug-resistant TB infections with fluoroquinolone resistance (pre-XDR) is also increasing. To treat these infections, local drug susceptibility patterns of alternate antituberculosis agents, including levofloxacin (LVX), linezolid (LZD), and amoxicillin-clavulanate (AMC), is urgently needed. The aim of this study was to determine the susceptibility frequencies of drug-resistant (DR)Mycobacterium tuberculosisagainst LVX, LZD, and AMC. All susceptibilities were determined on Middlebrook 7H10 agar. A critical concentration was used for LVX (1 μg/ml), whereas MICs were determined for LZD and AMC.M. tuberculosisH37Rv was used as a control strain. A total of 102M. tuberculosisisolates (XDR,n= 59; pre-XDR,n= 43) were tested. Resistance to LVX was observed in 91.2% (93/102). Using an MIC value of 0.5 μg/ml as a cutoff, resistance to LZD (MIC ≥ 1 μg/ml) was noted in 5.9% (6/102). Although the sensitivity breakpoints are not established for AMC, the MIC values were high (>16 μg/ml) in 97.1% (99/102). Our results demonstrate that LZD may be effective for the treatment of XDR and pre-XDR cases from Pakistan. High resistance rates against LVX in our study suggest the use of this drug with caution for DR-TB cases from this area. Drug susceptibility testing against LVX and AMC may be helpful in complicated and difficult-to-manage cases.

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Disparities in Capreomycin Resistance Levels Associated with therrsA1401G Mutation in Clinical Isolates of Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04438-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 444-449 ◽

Cited By ~ 19

Author(s):

Analise Z. Reeves ◽

Patricia J. Campbell ◽

Melisa J. Willby ◽

James E. Posey

Keyword(s):

Mycobacterium Tuberculosis ◽

Strong Predictor ◽

Critical Concentration ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Drug Susceptibility Testing ◽

Clinical Isolates ◽

Cross Resistance ◽

Second Line ◽

Content Type

ABSTRACTAs the prevalence of multidrug-resistant and extensively drug-resistant tuberculosis strains continues to rise, so does the need to develop accurate and rapid molecular tests to complement time-consuming growth-based drug susceptibility testing. Performance of molecular methods relies on the association of specific mutations with phenotypic drug resistance and while considerable progress has been made for resistance detection of first-line antituberculosis drugs, rapid detection of resistance for second-line drugs lags behind. TherrsA1401G allele is considered a strong predictor of cross-resistance between the three second-line injectable drugs, capreomycin (CAP), kanamycin, and amikacin. However, discordance is often observed between therrsA1401G mutation and CAP resistance, with up to 40% ofrrsA1401G mutants being classified as CAP susceptible. We measured the MICs to CAP in 53 clinical isolates harboring therrsA1401G mutation and found that the CAP MICs ranged from 8 μg/ml to 40 μg/ml. These results were drastically different from engineered A1401G mutants generated in isogenicMycobacterium tuberculosis, which exclusively exhibited high-level CAP MICs of 40 μg/ml. These data support the results of prior studies, which suggest that the critical concentration of CAP (10 μg/ml) used to determine resistance by indirect agar proportion may be too high to detect all CAP-resistant strains and suggest that a larger percentage of resistant isolates could be identified by lowering the critical concentration. These data also suggest that differences in resistance levels among clinical isolates are possibly due to second site or compensatory mutations located elsewhere in the genome.

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Pyrazinamide Is Active against Mycobacterium tuberculosis Cultures at Neutral pH and Low Temperature

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00654-16 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4956-4960 ◽

Cited By ~ 15

Author(s):

Alice L. den Hertog ◽

Sandra Menting ◽

Richard Pfeltz ◽

Matthew Warns ◽

Salman H. Siddiqi ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Low Temperature ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Acidic Ph ◽

Content Type ◽

The Past ◽

Neutral Ph

ABSTRACTFor the past decades, an acidic pH has been used to renderMycobacterium tuberculosissusceptible to pyrazinamide forin vitrotesting. Here, we show that at the standard breakpoint concentration and reduced culture temperatures, pyrazinamide (PZA) is active against tuberculosis (TB) at neutral pH. This finding should help unravel the mechanism of action of PZA and allow drug susceptibility testing (DST) methods to be optimized.

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A Diagnostic Algorithm To Investigate Pyrazinamide and Ethambutol Resistance in Rifampin-Resistant Mycobacterium tuberculosis Isolates in a Low-Incidence Setting

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01798-18 ◽

2018 ◽

Vol 63 (2) ◽

pp. e01798-18 ◽

Cited By ~ 5

Author(s):

Söenke Andres ◽

Matthias I. Gröschel ◽

Doris Hillemann ◽

Matthias Merker ◽

Stefan Niemann ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Sanger Sequencing ◽

Rational Design ◽

Clinical Decision Making ◽

Susceptibility Testing ◽

Diagnostic Algorithm ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Content Type ◽

Phenotypic Resistance

ABSTRACT Phenotypic drug susceptibility testing (DST) for the two first-line tuberculosis drugs ethambutol and pyrazinamide is known to yield unreliable and inaccurate results. In this prospective study, we propose a diagnostic algorithm combining phenotypic DST with Sanger sequencing to inform clinical decision-making for drug-resistant Mycobacterium tuberculosis complex isolates. Sequencing results were validated using whole-genome sequencing (WGS) of the isolates. Resistance-conferring mutations obtained by pncA sequencing correlated well with phenotypic DST results for pyrazinamide. Phenotypic resistance to ethambutol was only partly explained by mutations in the embB 306 codon. Additional resistance-conferring mutations were found in the embB gene at codons 354, 406, and 497. In several isolates that tested ethambutol susceptibility by phenotypic DST, well-known resistance-conferring embB mutations were determined. Thus, targeted Sanger sequencing beyond the embB 306 codon or WGS together with phenotypic DST should be employed to ensure reliable ethambutol drug susceptibility testing, as a basis for the rational design of multidrug-resistant tuberculosis regimens with or without ethambutol.

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A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02712-15 ◽

2016 ◽

Vol 60 (7) ◽

pp. 3869-3883 ◽

Cited By ~ 17

Author(s):

Charlotte Louise Hendon-Dunn ◽

Kathryn Sarah Doris ◽

Stephen Richard Thomas ◽

Jonathan Charles Allnutt ◽

Alice Ann Neville Marriott ◽

...

Keyword(s):

Flow Cytometry ◽

Mycobacterium Tuberculosis ◽

Mode Of Action ◽

Ester Group ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Additional Advantage ◽

Content Type ◽

Fluorescent Marker ◽

Sytox Green

ABSTRACTCurrent methods for assessing the drug susceptibility ofMycobacterium tuberculosisare lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations ofM. tuberculosisgrowing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria.M. tuberculosiswas exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic.

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DNA Gyrase Inhibition Assays Are Necessary To Demonstrate Fluoroquinolone Resistance Secondary togyrBMutations in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00707-11 ◽

2011 ◽

Vol 55 (10) ◽

pp. 4524-4529 ◽

Cited By ~ 27

Author(s):

Alix Pantel ◽

Stéphanie Petrella ◽

Stéphanie Matrat ◽

Florence Brossier ◽

Sylvaine Bastian ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Susceptibility Testing ◽

Dna Gyrase ◽

Fluoroquinolone Resistance ◽

Wild Type ◽

Content Type ◽

Clinical Strains ◽

Highly Active ◽

The Impact

ABSTRACTThe main mechanism of fluoroquinolone (FQ) resistance inMycobacterium tuberculosisis mutation in DNA gyrase (GyrA2GyrB2), especially ingyrA. However, the discovery of unknown mutations ingyrBwhose implication in FQ resistance is unclear has become more frequent. We investigated the impact on FQ susceptibility of eightgyrBmutations inM. tuberculosisclinical strains, three of which were previously identified in an FQ-resistant strain. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineeredgyrBalleles by mutagenesis were overexpressed inEscherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. We demonstrated that the eight substitutions in GyrB (D473N, P478A, R485H, S486F, A506G, A547V, G551R, and G559A), recently identified in FQ-resistant clinical strains or encountered inM. tuberculosisstrains isolated in France, are not implicated in FQ resistance. These results underline that, as opposed to phenotypic FQ susceptibility testing, the DNA gyrase inhibition assay is the only way to prove the role of a DNA gyrase mutation in FQ resistance. Therefore, the use of FQ in the treatment of tuberculosis (TB) patients should not be ruled out only on the basis of the presence of mutations ingyrB.

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Validation of the FluoroType MTBDR Assay for Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Complex Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.00072-18 ◽

2018 ◽

Vol 56 (6) ◽

pp. e00072-18 ◽

Cited By ~ 13

Author(s):

Doris Hillemann ◽

Carsten Haasis ◽

Sönke Andres ◽

Tobias Behn ◽

Katharina Kranzer

Keyword(s):

Mycobacterium Tuberculosis ◽

Sanger Sequencing ◽

High Sensitivity ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Molecular Diagnostic ◽

Isoniazid Resistance ◽

Content Type ◽

Specificity And Sensitivity ◽

Genotype Mtbdrplus

ABSTRACT For Mycobacterium tuberculosis complex (MTBC), the rapid and accurate diagnosis of drug resistance is crucial to ensure early initiation of appropriate therapy. Recently, a new molecular diagnostic test, the FluoroType MTBDR, aimed at detecting rifampin and isoniazid resistance has become available. This study aimed to evaluate the FluoroType MTBDR in comparison to phenotypic drug susceptibility testing (DST) using M. tuberculosis complex isolates. MTBC isolates underwent phenotypic DST and were tested using the FluoroType MTBDR and Genotype MTBDRplus. Sanger sequencing of the key regions of rpoB, katG, inhA, and aphC was performed for isolates with discordant phenotypic and molecular results. Furthermore, isolates with specific wild-type bands missing in the Genotype MTBDRplus, indicating the presence of a mutation, were investigated by Sanger sequencing. Specificity and sensitivity, defined as the proportions of isolates correctly determined as susceptible and resistant by the FluoroType MTBDR compared to phenotypic DST, were calculated. A total of 180 culture isolates were included; phenotypic DST showed 85 isolates susceptible to isoniazid and rifampin, 7 with isoniazid monoresistance, 7 with rifampin monoresistance, and 81 with multidrug resistance. The specificity of the FluoroType MTBDR was 100% (95% confidence interval [CI], 96.0 to 100%) for both rifampin and isoniazid. The sensitivity was 91.7% (95% CI, 83.6 to 96.6%) for isoniazid and 98.9% (95% CI, 93.8 to 100.0%) for rifampin. The FluoroType MTBDR has a high sensitivity and specificity for the detection of rifampin and isoniazid resistance when using culture isolates.

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Sensititre MYCOTB MIC Plate for Testing Mycobacterium tuberculosis Susceptibility to First- and Second-Line Drugs

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01209-13 ◽

2013 ◽

Vol 58 (1) ◽

pp. 11-18 ◽

Cited By ~ 52

Author(s):

Jongseok Lee ◽

Derek T. Armstrong ◽

Willy Ssengooba ◽

Jeong-ae Park ◽

Yeuni Yu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Susceptibility Testing ◽

Critical Concentration ◽

Microtiter Plate ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Second Line ◽

Content Type ◽

The Republic ◽

Phenotypic Methods

ABSTRACTForMycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first- and second-line antituberculosis drugs. To evaluate the performance of MYCOTB forM. tuberculosisdrug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archivedM. tuberculosisisolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was ≥92% for 7 of 12 drugs when a strict definition was used and ≥96% for 10 of 12 drugs when agreement was defined by allowing a ± one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was ≥95% for all drugs. MYCOTB provided reliable results forM. tuberculosissusceptibility testing of first- and second-line drugs except ethambutol, and results were available sooner than those determined by APM.

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Non-pncAGene-Mutated but Pyrazinamide-Resistant Mycobacterium tuberculosis: Why Is That?

Journal of Clinical Microbiology ◽

10.1128/jcm.02532-16 ◽

2017 ◽

Vol 55 (6) ◽

pp. 1920-1927 ◽

Cited By ~ 20

Author(s):

Jim Werngren ◽

Erik Alm ◽

Mikael Mansjö

Keyword(s):

Susceptibility Testing ◽

Gene Mutations ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Drug Susceptibility Testing ◽

Molecular Techniques ◽

Content Type ◽

Resistant Strains ◽

Clear Majority

ABSTRACTPyrazinamide (PZA) is a key component for the effective treatment of drug-susceptible and PZA-susceptible multidrug-resistant (MDRPZA-S) tuberculosis (TB).pncAgene mutations are usually detected in a clear majority (>90%) of PZA-resistant strains but obviously not in all. Rapid and reliable PZA drug susceptibility testing (DST) is critical whenever PZA is to be used in a treatment regimen, not least for the treatment of MDRPZA-STB. In this study, we selected 26 PZA-resistant isolates reported to carry a wild-typepncAgene. To confirm resistance, susceptibility testing was repeated using 100 mg/liter and 200 mg/liter PZA for all the 26 isolates and Sanger sequencing was repeated on the 18 isolates that remained PZA resistant. Apart from the eight isolates initially misclassified as PZA resistant, the retests identified three factors responsible for the phenotype-genotype discrepancy:panDorrpsAmutations identified by whole-genome sequencing (WGS) (n= 7), heteroresistance (n= 8), and mixed populations withMycobacterium avium(n= 3). Additionally, we performed WGS on 400 PZA-susceptible isolates and 15 consecutive MDRPZA-Rclinical isolates. Of the 400 PZA-susceptible isolates, only 1 harbored a nonsynonymouspncAmutation (Thr87Met), whereas a nonsynonymousrpsAmutation was found in 17 isolates. None of these isolates carried a nonsynonymouspanDmutation, while all 15 of the MDRPZA-Risolates harbored a nonsynonymouspncAmutation. Our findings indicate that it is necessary to consider the occurrence ofpanDmutations in PZA-resistant isolates, as well as heteroresistance, for the development and evaluation of new molecular techniques to ensure high-quality DST performance. The identification of nonsynonymousrpsAmutations in both PZA-susceptible and PZA-resistant isolates also implies that further studies are needed in order to determine the role ofrpsAin PZA resistance.

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