Molecular mechanisms of resistance and treatment efficacy of clofazimine and bedaquiline against Mycobacterium tuberculosis.

Clofazimine (CFZ) and bedaquiline (BDQ) are currently used for the treatment of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) strains. In recent years, adding CFZ and BDQ to tuberculosis (TB) drug regimens against MDR Mtb strains has significantly improved treatment results, but these improvements are threatened by the emergence of MDR and extensively drug-resistant (XDR) Mtb strains. Recently, CFZ and BDQ have attracted much attention for their strong clinical efficacy, although very little is known about the mechanisms of action, drug susceptibility test (DST), resistance mechanisms, cross-resistance, and pharmacokinetics of these two drugs. In this current review, we provide recent updates on the mechanisms of action, DST, associated mutations with individual resistance and cross-resistance, clinical efficacy, and pharmacokinetics of CFZ and BDQ against Mtb strains. Presently, known mechanisms of resistance for CFZ and/or BDQ include mutations within the Rv0678, pepQ, Rv1979c, and atpE genes. The cross-resistance between CFZ and BDQ may reduce available MDR-/XDR-TB treatment options. The use of CFZ and BDQ for treatment in the setting of limited DST could allow further spread of drug resistance. The DST and resistance knowledge are urgently needed where CFZ and BDQ resistance do emerge. Therefore, an in-depth understanding of clinical efficacy, DST, cross-resistance, and pharmacokinetics for CFZ and BDQ against Mtb can provide new ideas for improving treatment outcomes, reducing mortality, preventing drug resistance, and TB transmission. Along with this, it will also help to develop rapid molecular diagnostic tools as well as novel therapeutic drugs for TB.

Arabinosyltransferase C Mediates Multiple Drugs Intrinsic Resistance by Altering Cell Envelope Permeability in Mycobacterium abscessus.

Mycobacterium abscessus is an emerging human pathogen leading to significant morbidity and even mortality, intrinsically resistant to almost all the antibiotics available and so can be a nightmare. Mechanisms of its intrinsic resistance remain not fully understood. Here, we selected and confirmed an M. abscessus transposon mutant that is hypersensitive to multiple drugs including rifampin, rifabutin, vancomycin, clofazimine, linezolid, imipenem, levofloxacin, cefoxitin, and clarithromycin. The gene MAB_0189c encoding a putative arabinosyltransferase C was found to be disrupted, using a newly developed highly-efficient strategy combining next-generation sequencing and multiple PCR. Furthermore, selectable marker-free deletion of MAB_0189c recapitulated the hypersensitive phenotype. Disruption of MAB_0189c resulted in an inability to synthesize lipoarabinomannan and markedly enhanced its cell envelope permeability. Complementing MAB_0189c or M. tuberculosis embC restored the resistance phenotype. Importantly, treatment of M. abscessus with ethambutol, a first-line antituberculosis drug targeting arabinosyltransferases of M. tuberculosis, largely sensitized M. abscessus to multiple antibiotics in vitro. We finally tested activities of six selected drugs using a murine model of sustained M. abscessus infection and found that linezolid, rifabutin, and imipenem were active against the MAB_0189c deletion strain. These results identified MAB_0189 as a crucial determinant of intrinsic resistance of M. abscessus, and optimizing inhibitors targeting MAB_0189 might be a strategy to disarm the intrinsic multiple antibiotic resistance of M. abscessus. IMPORTANCE Mycobacterium abscessus is intrinsically resistant to most antibiotics, and treatment of its infections is highly challenging. The mechanisms of its intrinsic resistance remain not fully understood. Here we found a transposon mutant hypersensitive to a variety of drugs and identified the transposon inserted into the MAB_0189c (orthologous embC coding arabinosyltransferase, EmbC) gene by using a newly developed rapid and efficient approach. We further verified that the MAB_0189c gene played a significant role in its intrinsic resistance by decreasing the cell envelope permeability through affecting the production of lipoarabinomannan in its cell envelope. Lastly, we found the arabinosyltransferases inhibitor, ethambutol, increased activities of nine selected drugs in vitro. Knockout of MAB_0189c made M. abscessus become susceptible to 3 drugs in mice. These findings indicated that potential powerful M. abscessus EmbC inhibitor might be used to reverse the intrinsic resistance of M. abscessus to multiple drugs.

Prevalence and molecular characterization of amikacin resistance among Mycobacterium tuberculosis clinical isolates from southern China.

OBJECTIVES: Amikacin is the only second-line injectable antituberculosis (anti-TB) drug still recommended for multidrug-resistant tuberculosis (MDR-TB) treatment when a short MDR-TB regimen is designed. Mutations in rrs and eis are reported to be associated with resistance to amikacin. In this study, we investigated the incidence of rrs, eis, tap and whiB7 mutations in amikacin-resistant Mycobacterium tuberculosis clinical isolates to find the proportion of different mutations related to amikacin resistance. METHODS: A total of 395 clinical isolates of M. tuberculosis were used for phenotypic drug susceptibility testing (DST) to 10 drugs with the Löwenstein-Jensen (L-J) method. We sequenced rrs, eis, tap and whiB7 genes in 178 M. tuberculosis clinical isolates (89 amikacin-resistant isolates and 89 of 306 amikacin-susceptible isolates). RESULTS: Our data showed that 22.53% (89/395) M. tuberculosis clinical isolates were resistant to amikacin. Of the 89 amikacin-resistant isolates, 89.89% (80/89) were MDR-TB, of which 12.36% (11/89) were pre-extensively drug-resistant TB (pre-XDR-TB) and 77.53% (69/89) were XDR-TB. The rrs mutations were found in 82% (73/89) in amikacin-resistant M. tuberculosis clinical isolates. The A1401G alteration in the rrs gene was the most dominant mutation (80.90%; 72/89). Five mutations were detected as new in rrs, tap and whiB7. Notably, 13.48% (12/89) amikacin-resistant isolates had no known mutation in these genes. CONCLUSIONS: Our data reveal that the rrs mutation is a predominant molecular marker of amikacin resistance in southern China. Analysis of the rrs gene mutations will significantly reduce the time and cost to diagnose amikacin resistance in TB patients. Other unknown amikacin resistance mechanism(s) exist.

Detection of Novel Gene Mutations Associated with Pyrazinamide Resistance in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates in Southern China.

OBJECTIVE: Pyrazinamide (PZA) is a cornerstone of modern tuberculosis regimens. This study aimed to investigate the performance of genotypic testing of pncA + upstream region, rpsA, panD, Rv2783c, and clpC1 genes to add insights for more accurate molecular diagnosis of PZA-resistant (R) Mycobacterium tuberculosis. METHODS: Drug susceptibility testing, sequencing analysis of PZA-related genes including the entire operon of pncA (Rv2044c-pncA-Rv2042c) and PZase assay were performed for 448 M. tuberculosis clinical isolates. RESULTS: Our data showed that among 448 M. tuberculosis clinical isolates, 113 were MDR, 195 pre-XDR and 70 XDR TB, while the remaining 70 strains had other combinations of drug-resistance. A total of 60.04% (269/448) M. tuberculosis clinical isolates were resistant to PZA, of which 78/113 were MDR, 119/195 pre-XDR and 29/70 XDR TB strains. PZAR isolates have predominance (83.3%) of Beijing genotype. Genotypic characterization of Rv2044c-pncA-Rv2042c revealed novel nonsynonymous mutations in Rv2044c with negative PZase activity which led to confer PZAR. Compared with phenotypic data, 84.38% (227/269) PZAR strains with mutations in pncA + upstream region exhibited 83.64% sensitivity but the combined evaluation of the mutations in rpsA 2.60% (7/269), panD 1.48% (4/269), Rv2783c 1.11% (3/269) and Rv2044c 0.74% (2/269) increased the sensitivity to 89.59%. Fifty-seven novel mutations were identified in this study. Interestingly, a frameshift deletion (C-114del) in upstream of pncAwt nullified the effect of A-11G mutation and induced positive PZase activity, divergent from five PZase negative A-11G PZAR mutants. Twenty-six PZAR strains having wild-type-sequenced genes with positive or negative PZase suggest the existence of unknown resistance mechanisms. CONCLUSION: Our study revealed that PZAR rate in MDR and pre-XDR TB was markedly higher in southern China. The concomitant evaluation of pncA + UFR, rpsA, panD, Rv2783c, and Rv2044c provides more dependable genotypic results of PZA resistance. Fifty-seven novel mutations/indels in this study may play a vital role as diagnostic markers. The upstream region of pncA and PZase regulation are valuable to explore the unknown mechanism of PZA-resistance.

Comparative Analysis of Whole-Genome and Methylome Profiles of a Smooth and a Rough Mycobacterium abscessus Clinical Strain.

Mycobacterium abscessus is a fast growing Mycobacterium species mainly causing skin and respiratory infections in human. M. abscessus is resistant to numerous drugs, which is a major challenge for the treatment. In this study, we have sequenced the genomes of two clinical M. abscessus strains having rough and smooth morphology, using the single molecule real-time and Illumina HiSeq sequencing technology. In addition, we reported the first comparative methylome profiles of a rough and a smooth M. abscessus clinical strains. The number of N4-methylcytosine (4mC) and N6-methyladenine (6mA) modified bases obtained from smooth phenotype were two-fold and 1.6 fold respectively higher than that of rough phenotype. We have also identified 4 distinct novel motifs in two clinical strains and genes encoding antibiotic-modifying/targeting enzymes and genes associated with intracellular survivability having different methylation patterns. To our knowledge, this is the first report about genome-wide methylation profiles of M. abscessus strains and identification of a natural linear plasmid (15 kb) in this critical pathogen harboring methylated bases. The pan-genome analysis of 25 M. abscessus strains including two clinical strains revealed an open pan genome comprises of 7596 gene clusters. Likewise, structural variation analysis revealed that the genome of rough phenotype strain contains more insertions and deletions than the smooth phenotype and that of the reference strain. A total of 391 single nucleotide variations responsible for the non-synonymous mutations were detected in clinical strains compared to the reference genome. The comparative genomic analysis elucidates the genome plasticity in this emerging pathogen. Furthermore, the detection of genome-wide methylation profiles of M. abscessus clinical strains may provide insight into the significant role of DNA methylation in pathogenicity and drug resistance in this opportunistic pathogen.

Phenotypic and Genotypic Characterization of Streptomycin-Resistant Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates in Southern China.

Streptomycin (STR) is the first antibiotic used in the treatment of tuberculosis (TB) and the earliest antituberculosis drug with acquired resistance developed by Mycobacterium tuberculosis. The high prevalence of such resistance in many parts of the world limits its use for treating multidrug-resistant (MDR) TB. The aims of this study are to characterize of mutations in rpsL, rrs, and gidB genes in MDR M. tuberculosis isolates originating from southern China and to investigate possible relationship between mutations and strain genotypes for precise diagnosis and treatment. Sequences of rpsL, rrs, and gidB genes and the resistance profiles were analyzed for 218 MDR M. tuberculosis isolates. Our study showed that 68.35% of MDR M. tuberculosis isolates were resistant to STR and 89.91% of STR-resistant (STRR) isolates were Beijing lineage strains. Mutations were observed in STRR MDR M. tuberculosis isolates at the following rates: 72.48% in rpsL, 36.91% in rrs, and 15.44% in gidB. Compared with the phenotypic data, the combination of mutations in rpsL, rrs, and gidB has sensitivity and specificity of 96.64% and 100.00%, respectively. The most common mutations in STRR isolates were rpsL128,263 and rrs514,1401, of which rpsL128 showed association with Beijing lineage (p < 0.001). It is noteworthy that a1401g mutation was present in rrs, while MDR M. tuberculosis isolates were resistant to both STR and amikacin. Twenty two novel mutations were found in STRR isolates. These findings could be helpful to develop rapid molecular diagnostic methods and understand STR resistance in China for developing TB precision medicine and disturbance of drug-resistant TB transmission.

Phenotypic and genotypic characterization of levofloxacin- and moxifloxacin-resistant Mycobacterium tuberculosis clinical isolates in southern China.

BACKGROUND: Levofloxacin (LVX) and Moxifloxacin (MXF) are the cornerstones for treatment of multidrug-resistant tuberculosis (MDR-TB). China is one of the highest MDR- and fluoroquinolones (FQ)-resistant TB burdens countries. DNA gyrase encoded by gyr genes is the main target of FQ in Mycobacterium tuberculosis (MTB). The prevalence and molecular characterization of LVX- and MXF-resistant MTB strains from southern China were examined in this study. METHODS: Drug susceptibility testing (DST) of 400 MTB clinical isolates was evaluated by proportion method on Löwenstein-Jensen (LJ) medium against ten drugs. The sequencing of entire gyrA and gyrB genes and multiplex PCR were performed to distinguish the prevalence of mutant types in Beijing and non-Beijing genotypes. RESULTS: Three hundred and twenty-one out of four hundred (80.25%) drug-resistant isolates (resistant > one drug) were categorized as 83/321 (25.80%) MDR, 174/321 (54.20%) pre-XDR and 64/321 (19.93%) XDR-MTB. Overall, 303/400 (75.75%) LVX- and 292/400 (73.00%) MXF-resistant (R) MTB strains were identified. Two hundred seventy-one out of three hundred and three (89.43%) resistant strains carried mutations in gyrA and 91/303 (30.03%) in gyrB. Interestingly, 18 novel mutations were detected in gyrA and gyrB genes. Mutations at (A90, D94) and (T500, G510, G512) frequently existed in QRDR(s) of gyrA and gyrB respectively in 286/400 (71.50%) LVXRMXFR strains. The novel mutations in- and out-side the QRDR of gyrA (L105R, A126E, M127K, D151T, V165A) and gyrB (D461H, N499S, G520A) increased the sensitivity and consistency of genotypic tests. Notably, 25 LVXRMXFR strains were found with unknown resistance mechanisms. CONCLUSIONS: Mutations in QRDR(s) were concomitantly associated with Beijing and non-Beijing genotypes. The prevalence of resistance and cross-resistance between LVX and MXF in MTB isolates from southern China was immensely higher than other countries. Our valuable findings provide the substantial implications to improve the reliability of genotypic diagnostic tests relying on potential resistance conferring mutations in entire gyr genes.

Design, synthesis, and in vitro biological evaluation of novel benzimidazole tethered allylidenehydrazinylmethylthiazole derivatives as potent inhibitors of Mycobacterium tuberculosis.

Tuberculosis (TB) has become one of the most significant public health problems in recent years. Antibiotic therapy remains the mainstay of TB control strategies, but the increasing resistance of mycobacterial species has heightened alarm, requiring the development of novel drugs in order to improve treatment outcomes. Here, as an effort to identify novel and effective antitubercular agents, we designed and synthesized a series of novel substituted benzimidazolallylidenehydrazinylmethylthiazole derivatives via a multi-component molecular hybridization approach with single molecular architecture. Our design strategy involved assembling the antitubercular pharmacophoric fragments benzimidazole, 2-aminothiazole and substituted α,ß-unsaturated ketones via condensation reactions. All the newly synthesized compounds were fully characterized via NMR and mass spectral data and evaluated for in vitro biological activity against the H37Ra strain of Mycobacterium tuberculosis. From the biological evaluation data, we identified some effective compounds, of which 8g and 7e were the most active ones (both having MIC values of 2.5 µg mL-1). In addition, compound 8g exhibited a lower cytotoxicity profile. We conceive that compound 8g may serve as a chemical probe of interest for further lead optimization studies with the general aim of developing novel and effective antitubercular agents.

The compound TB47 is highly bactericidal against Mycobacterium ulcerans in a Buruli ulcer mouse model.

Buruli ulcer (BU) is an emerging infectious disease that causes disfiguring skin ulcers. The causative agent, Mycobacterium ulcerans, secretes toxin called mycolactone that triggers inflammation and immunopathology. Existing treatments are lengthy and consist of drugs developed for tuberculosis. Here, we report that a pyrazolo[1,5-a]pyridine-3-carboxamide, TB47, is highly bactericidal against M. ulcerans both in vitro and in vivo. In the validated mouse model of BU, TB47 alone reduces M. ulcerans burden in mouse footpads by more than 2.5 log10 CFU compared to the standard BU treatment regimen recommended by the WHO. We show that mutations of ubiquinol-cytochrome C reductase cytochrome subunit B confer resistance to TB47 and the dissimilarity of CydABs from different mycobacteria may account for their differences in susceptibility to TB47. TB47 is highly potent against M. ulcerans and possesses desirable pharmacological attributes and low toxicity that warrant further assessment of this agent for treatment of BU.

Advances in the development of molecular genetic tools for Mycobacterium tuberculosis.

Mycobacterium tuberculosis, a clinically relevant Gram-positive bacterium of great clinical relevance, is a lethal pathogen owing to its complex physiological characteristics and development of drug resistance. Several molecular genetic tools have been developed in the past few decades to study this microorganism. These tools have been instrumental in understanding how M. tuberculosis became a successful pathogen. Advanced molecular genetic tools have played a significant role in exploring the complex pathways involved in M. tuberculosis pathogenesis. Here, we review various molecular genetic tools used in the study of M. tuberculosis. Further, we discuss the applications of clustered regularly interspaced short palindromic repeat interference (CRISPRi), a novel technology recently applied in M. tuberculosis research to study target gene functions. Finally, prospective outcomes of the applications of molecular techniques in the field of M. tuberculosis genetic research are also discussed.

Molecular Targets Related Drug Resistance Mechanisms in MDR-, XDR-, and TDR-Mycobacterium tuberculosis Strains.

Tuberculosis (TB) is a formidable infectious disease that remains a major cause of death worldwide today. Escalating application of genomic techniques has expedited the identification of increasing number of mutations associated with drug resistance in Mycobacterium tuberculosis. Unfortunately the prevalence of bacillary resistance becomes alarming in many parts of the world, with the daunting scenarios of multidrug-resistant tuberculosis (MDR-TB), extensively drug-resistant tuberculosis (XDR-TB) and total drug-resistant tuberculosis (TDR-TB), due to number of resistance pathways, alongside some apparently obscure ones. Recent advances in the understanding of the molecular/ genetic basis of drug targets and drug resistance mechanisms have been steadily made. Intriguing findings through whole genome sequencing and other molecular approaches facilitate the further understanding of biology and pathology of M. tuberculosis for the development of new therapeutics to meet the immense challenge of global health.

A Cassette Containing Thiostrepton, Gentamicin Resistance Genes, and dif sequences Is Effective in Construction of Recombinant Mycobacteria.

The genetic manipulation of Mycobacterium tuberculosis genome is limited by the availability of selection markers. Spontaneous resistance mutation rate of M. tuberculosis to the widely used kanamycin is relatively high which often leads to some false positive transformants. Due to the few available markers, we have created a cassette containing thiostrepton resistance gene (tsr) for selection in M. tuberculosis and M. bovis BCG, and gentamicin resistance gene (aacC1) for Escherichia coli and M. smegmatis mc2155, flanked with dif sequences recognized by the Xer system of mycobacteria. This cassette adds to the limited available selection markers for mycobacteria.

Drug resistance mechanisms and novel drug targets for tuberculosis therapy.

Drug-resistant tuberculosis (TB) poses a significant challenge to the successful treatment and control of TB worldwide. Resistance to anti-TB drugs has existed since the beginning of the chemotherapy era. New insights into the resistant mechanisms of anti-TB drugs have been provided. Better understanding of drug resistance mechanisms helps in the development of new tools for the rapid diagnosis of drug-resistant TB. There is also a pressing need in the development of new drugs with novel targets to improve the current treatment of TB and to prevent the emergence of drug resistance in Mycobacterium tuberculosis. This review summarizes the anti-TB drug resistance mechanisms, furnishes some possible novel drug targets in the development of new agents for TB therapy and discusses the usefulness using known targets to develop new anti-TB drugs. Whole genome sequencing is currently an advanced technology to uncover drug resistance mechanisms in M. tuberculosis. However, further research is required to unravel the significance of some newly discovered gene mutations in their contribution to drug resistance.