Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin

ABSTRACTAntofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying itsin vitroactivity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinicalMycobacterium tuberculosisstrains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions ofgyrA(Rv0006) andgyrB(Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective.

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Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosis against Pyrazinamide Using Wild-Type MIC Distributions andpncAGene Sequencing

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05894-11 ◽

2011 ◽

Vol 56 (3) ◽

pp. 1253-1257 ◽

Cited By ~ 35

Author(s):

J. Werngren ◽

E. Sturegård ◽

P. Juréen ◽

K. Ängeby ◽

S. Hoffner ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Critical Concentration ◽

Gene Mutations ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Clinical Isolates ◽

Wild Type ◽

First Line ◽

Content Type ◽

Resistant Strains

ABSTRACTPyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistantMycobacterium tuberculosisstrains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates ofMycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinicalM. tuberculosisisolates and compared the results topncAsequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n= 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤8 to 64 mg/liter), and nopncAgene mutations were detected. In strains resistant in both Bactec systems (n= 18), the PZA MICs ranged from 256 to ≥1,024 mg/liter. The discordances betweenpncAsequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated topncAmutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.

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How To Optimally Combine Genotypic and Phenotypic Drug Susceptibility Testing Methods for Pyrazinamide

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01003-20 ◽

2020 ◽

Vol 64 (9) ◽

Cited By ~ 1

Author(s):

Claudio U. Köser ◽

Daniela M. Cirillo ◽

Paolo Miotto

Keyword(s):

Mycobacterium Tuberculosis ◽

Current Practice ◽

Susceptibility Testing ◽

Critical Concentration ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Resistance Mutations ◽

Testing Methods ◽

Content Type

ABSTRACT False-susceptible phenotypic drug-susceptibility testing (DST) results for pyrazinamide due to mutations with MICs close to the critical concentration (CC) confound the classification of pncA resistance mutations, leading to an underestimate of the specificity of genotypic DST. This could be minimized by basing treatment decisions on well-understood mutations and by adopting an area of technical uncertainty for phenotypic DST rather than only testing the CC, as is current practice for the Mycobacterium tuberculosis complex.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Characterization of Guided Entry of Tail-Anchored Proteins 3 Homologues in Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.00159-19 ◽

2019 ◽

Vol 201 (14) ◽

Author(s):

Kuan Hu ◽

Ashley T. Jordan ◽

Susan Zhang ◽

Avantika Dhabaria ◽

Amanda Kovach ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Structure Prediction ◽

Bacterial Species ◽

Normal Growth ◽

Anion Transport ◽

Content Type ◽

Reading Frame ◽

Anion Transporters ◽

Anion Transporter

ABSTRACT We characterized an operon in Mycobacterium tuberculosis, Rv3679-Rv3680, in which each open reading frame is annotated to encode “anion transporter ATPase” homologues. Using structure prediction modeling, we found that Rv3679 and Rv3680 more closely resemble the guided entry of tail-anchored proteins 3 (Get3) chaperone in eukaryotes. Get3 delivers proteins into the membranes of the endoplasmic reticulum and is essential for the normal growth and physiology of some eukaryotes. We sought to characterize the structures of Rv3679 and Rv3680 and test if they have a role in M. tuberculosis pathogenesis. We solved crystal structures of the nucleotide-bound Rv3679-Rv3680 complex at 2.5 to 3.2 Å and show that while it has some similarities to Get3 and ArsA, there are notable differences, including that these proteins are unlikely to be involved in anion transport. Deletion of both genes did not reveal any conspicuous growth defects in vitro or in mice. Collectively, we identified a new class of proteins in bacteria with similarity to Get3 complexes, the functions of which remain to be determined. IMPORTANCE Numerous bacterial species encode proteins predicted to have similarity with Get3- and ArsA-type anion transporters. Our studies provide evidence that these proteins, which we named BagA and BagB, are unlikely to be involved in anion transport. In addition, BagA and BagB are conserved in all mycobacterial species, including the causative agent of leprosy, which has a highly decayed genome. This conservation suggests that BagAB constitutes a part of the core mycobacterial genome and is needed for some yet-to-be-determined part of the life cycle of these organisms.

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Whole-Genome Sequencing for Drug Resistance Profile Prediction inMycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02175-18 ◽

2019 ◽

Vol 63 (4) ◽

Cited By ~ 11

Author(s):

Sebastian M. Gygli ◽

Peter M. Keller ◽

Marie Ballif ◽

Nicolas Blöchliger ◽

Rico Hömke ◽

...

Keyword(s):

Drug Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Critical Concentration ◽

Drug Susceptibility Testing ◽

Quality Data ◽

Whole Genome ◽

Resistance Mutations ◽

Content Type ◽

Agar Dilution

ABSTRACTWhole-genome sequencing allows rapid detection of drug-resistantMycobacterium tuberculosisisolates. However, the availability of high-quality data linking quantitative phenotypic drug susceptibility testing (DST) and genomic data have thus far been limited. We determined drug resistance profiles of 176 genetically diverse clinicalM. tuberculosisisolates from the Democratic Republic of the Congo, Ivory Coast, Peru, Thailand, and Switzerland by quantitative phenotypic DST for 11 antituberculous drugs using the BD Bactec MGIT 960 system and 7H10 agar dilution to generate a cross-validated phenotypic DST readout. We compared DST results with predicted drug resistance profiles inferred by whole-genome sequencing. Classification of strains by the two phenotypic DST methods into resistotype/wild-type populations was concordant in 73 to 99% of cases, depending on the drug. Our data suggest that the established critical concentration (5 mg/liter) for ethambutol resistance (MGIT 960 system) is too high and misclassifies strains as susceptible, unlike 7H10 agar dilution. Increased minimal inhibitory concentrations were explained by mutations identified by whole-genome sequencing. Using whole-genome sequences, we were able to predict quantitative drug resistance levels for the majority of drug resistance mutations. Predicting quantitative levels of drug resistance by whole-genome sequencing was partially limited due to incompletely understood drug resistance mechanisms. The overall sensitivity and specificity of whole-genome-based DST were 86.8% and 94.5%, respectively. Despite some limitations, whole-genome sequencing has the potential to infer resistance profiles without the need for time-consuming phenotypic methods.

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In Vitro Activity and MIC of Sitafloxacin against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis Isolated in Thailand

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00825-17 ◽

2017 ◽

Vol 62 (1) ◽

Cited By ~ 1

Author(s):

Manoon Leechawengwongs ◽

Therdsak Prammananan ◽

Sarinya Jaitrong ◽

Pamaree Billamas ◽

Nampueng Makhao ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Critical Concentration ◽

Multidrug Resistant ◽

Quinolone Resistance ◽

Drug Resistant ◽

Content Type ◽

Extensively Drug Resistant ◽

Resistant Strains ◽

Drug Resistant Strains

ABSTRACT New fluoroquinolones (FQs) have been shown to be more active against drug-resistant Mycobacterium tuberculosis strains than early FQs, such as ofloxacin. Sitafloxacin (STFX) is a new fluoroquinolone with in vitro activity against a broad range of bacteria, including M. tuberculosis. This study aimed to determine the in vitro activity of STFX against all groups of drug-resistant strains, including multidrug-resistant M. tuberculosis (MDR M. tuberculosis), MDR M. tuberculosis with quinolone resistance (pre-XDR), and extensively drug-resistant (XDR) strains. A total of 374 drug-resistant M. tuberculosis strains were tested for drug susceptibility by the conventional proportion method, and 95 strains were randomly submitted for MIC determination using the microplate alamarBlue assay (MABA). The results revealed that all the drug-resistant strains were susceptible to STFX at a critical concentration of 2 μg/ml. Determination of the MIC90s of the strains showed different MIC levels; MDR M. tuberculosis strains had a MIC90 of 0.0625 μg/ml, whereas pre-XDR and XDR M. tuberculosis strains had identical MIC90s of 0.5 μg/ml. Common mutations within the quinolone resistance-determining region (QRDR) of gyrA and/or gyrB did not confer resistance to STFX, except that double mutations of GyrA at Ala90Val and Asp94Ala were found in strains with a MIC of 1.0 μg/ml. The results indicated that STFX had potent in vitro activity against all the groups of drug-resistant M. tuberculosis strains and should be considered a new repurposed drug for treatment of multidrug-resistant and extensively drug-resistant TB.

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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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On the Consequences of Poorly Defined Breakpoints for Rifampin Susceptibility Testing of Mycobacterium tuberculosis Complex

Journal of Clinical Microbiology ◽

10.1128/jcm.02328-20 ◽

2021 ◽

Vol 59 (4) ◽

Author(s):

Claudio U. Köser ◽

Sophia B. Georghiou ◽

Thomas Schön ◽

Max Salfinger

Keyword(s):

Mycobacterium Tuberculosis ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Antimicrobial Susceptibility Testing ◽

World Health ◽

The European Union ◽

Reference Standard ◽

Resistance Mutations ◽

Content Type ◽

Indicator Tube

ABSTRACT In a recent report of a systematic review of critical concentrations (CCs), the World Health Organization (WHO) lowered the rifampin (RIF) CC for antimicrobial susceptibility testing (AST) of the Mycobacterium tuberculosis complex using Middlebrook 7H10 medium and the Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 system from 1 to 0.5 μg/ml. The previous RIF CC for 7H10 had been in use for over half a century. Because it had served as the de facto reference standard, it contributed to the endorsement of inappropriately high CCs for other AST methods, including the U.S. Food and Drug Administration (FDA)-approved MGIT system. Moreover, this resulted in confusion about the interpretation of seven borderline resistance mutations in rpoB (i.e., L430P, D435Y, H445L, H445N, H445S, L452P, and I491F). In this issue of the Journal of Clinical Microbiology, Shea et al. (J Clin Microbiol 59:e01885-20, 2021, https://doi.org/10.1128/JCM.01885-20) provide evidence that the CC endorsed by the Clinical and Laboratory Standards Institute for the Sensititre MYCOTB system, which is not FDA approved but is CE-IVD marked in the European Union, is likely also too high. These findings underscore the importance of calibrating AST methods against a rigorously defined reference standard, as recently proposed by the European Committee on Antimicrobial Susceptibility Testing, as well as the value of routine next-generation sequencing for investigating discordant AST results.

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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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