INH-resistant Strains Research Articles

Genetic mechanisms of co-emergence of INH-resistant Mycobacterium tuberculosis strains during the standard course of antituberculosis therapy.

The incidence of isoniazid (INH) resistant Mycobacterium tuberculosis is increasing globally. This study aimed to identify the molecular mechanisms behind the development of INH resistance in M. tuberculosis strains collected from the same patients during the standard course of treatment. Three M. tuberculosis strains were collected from a patient before and during antituberculosis (anti-TB) therapy. The strains were characterized using phenotypic drug susceptibility tests, Mycobacterial Interspersed Repeated Unit-Variable-Number Tandem Repeats (MIRU-VNTR), and whole-genome sequencing (WGS) to identify mutations associated with INH resistance. To validate the role of the novel mutations in INH resistance, the mutated katG genes were electroporated into a KatG-deleted M. tuberculosis strain (GA03). Three-dimensional structures of mutated KatG were modeled to predict their impact on INH binding. The pre-treatment strain was susceptible to INH. However, two INH-resistant strains were isolated from the patient after anti-TB therapy. MIRU-VNTR and WGS revealed that the three strains were clonally identical. A missense mutation (P232L) and a nonsense mutation (Q461Stop) were identified in the katG of the two post-treatment strains, respectively. Transformation experiments showed that katG of the pre-treatment strain restored INH susceptibility in GA03, whereas the mutated katG genes from the post-treatment strains rendered negative catalase activity and INH resistance. The protein model indicated that P232L reduced INH-KatG binding affinity while Q461Stop truncated gene transcription. Our results showed that the two katG mutations, P232L and Q461Stop, accounted for the co-emergence of INH-resistant clones during anti-TB therapy. The inclusion of these mutations in the design of molecular assays could increase the diagnostic performance.IMPORTANCEThe evolution of drug-resistant strains of Mycobacterium tuberculosis within the lung lesions of a patient has a detrimental impact on treatment outcomes. This is particularly concerning for isoniazid (INH), which is the most potent first-line antimycobacterial drug. However, the precise genetic factors responsible for drug resistance in patients have not been fully elucidated, with approximately 15% of INH-resistant strains harboring unknown genetic factors. This raises concerns about the emergence of drug-resistant clones within patients, further contributing to the global epidemic of resistance. In this study, we revealed the presence of two novel katG mutations, which emerged independently due to the stress exerted by antituberculosis (anti-TB) treatment on a parental strain. Importantly, we experimentally demonstrated the functional significance of both mutations in conferring resistance to INH. Overall, this research sheds light on the genetic mechanisms underlying the evolution of INH resistance within patients and provides valuable insights for improving diagnostic performance by targeting specific mutations.

Identification of potential inhibitors of Mtb InhA: a pharmacoinformatics approach

The emergence of superbugs of multi-drug resistant (MDR/RR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (Mtb) strains at a faster rate is posing a serious threat to Tuberculosis (TB) control worldwide. Mtb enoyl-acyl carrier protein reductase (InhA) is a well-established target of the front-line anti-TB prodrug Isoniazid (INH), which requires activation by Catalase-peroxidase enzyme (KatG) in order to inhibit InhA enzyme, that is crucial for the biosynthesis of the mycobacterial cell wall. Currently, due to widespread resistance to this drug, it is necessary to identify new clinical candidates that directly inhibit InhA enzyme and do not require activation by KatG, thereby circumventing most of the resistance mechanisms. In the present study, high-throughput virtual screening of ASINEX database was carried out to identify potential direct inhibitors of Mtb InhA. Best twenty compounds with good binding energies ranging between −12.36 and −9.27 kcal/mol were selected as promising virtual screening hits. These molecules were subjected to ADME study followed by toxicity prediction. Finally, four top-ranked molecules which are structurally diverse and possess best binding affinity than the co-crystalized ligand have been chosen for MD simulation studies followed by MM-GBSA analysis to validate and ensure the stability of hits in the active site of the enzyme. Based on the 100 ns MD simulation studies and binding free energy estimates, three hit molecules B244, B369, and B310 could be considered as potential inhibitors for Mtb InhA, which are likely to be potent against INH-resistant Mtb strains after successful experimental validation. Communicated by Ramaswamy H. Sarma

The Detection of Mutations and Genotyping of Drug-Resistant Mycobacterium tuberculosis Strains Isolated from Patients in the Rural Eastern Cape Province.

Drug-resistant tuberculosis (DR-TB) is still a major public health concern in South Africa. Mutations in M. tuberculosis can cause varying levels of phenotypic resistance to anti-TB medications. There have been no prior studies on gene mutations and the genotyping of DR-TB in the rural Eastern Cape Province; hence, we aimed to identify DR-TB mutations, genetic diversity, and allocated lineages among patients in this area. Using Xpert® MTB/RIF, we assessed the rifampin resistance of sputum samples collected from 1157 patients suspected of having tuberculosis. GenoType MTBDR plus VER 2.0 was used for the detection of mutations causing resistance to anti-TB medications. The next step was to spoligotype 441 isolates. The most prevalent rifampin resistance-conferring mutations were in rpoB codon S531L in INH-resistant strains; the katG gene at codon S315TB and the inhA gene at codon C-15TB had the most mutations; 54.5% and 24.7%, respectively. In addition, 24.6% of strains showed mutations in both the rpoB and inhA genes, while 69.9% of strains showed mutations in both the katG and rpoB genes. Heteroresistance was seen in 17.9% of all cases in the study. According to spoligotyping analysis, Beijing families predominated. Investigation of the evolutionary lineages of M. tuberculosis isolates can be carried out using the information provided by the study's diversity of mutations. In locations wherein these mutations have been discovered, decision-making regarding the standardization of treatment regimens or individualized treatment may be aided by the detection frequency of rpoB, katG, and inhA mutations in various study areas.

Using Microfluidic Chip and Allele-Specific PCR to Rapidly Identify Drug Resistance-Associated Mutations of Mycobacterium tuberculosis.

The currently used conventional susceptibility testing for drug-resistant Mycobacterium tuberculosis (M.TB) is limited due to being time-consuming and having low efficiency. Herein, we propose the use of a microfluidic-based method to rapidly detect drug-resistant gene mutations using Kompetitive Allele-Specific PCR (KASP). A total of 300 clinical samples were collected, and DNA extraction was performed using the "isoChip®" Mycobacterium detection kit. Phenotypic susceptibility testing and Sanger sequencing were performed to sequence the PCR products. Allele-specific primers targeting 37 gene mutation sites were designed, and a microfluidic chip (KASP) was constructed using 112 reaction chambers to simultaneously detect multiple mutations. Chip validation was performed using clinical samples. Phenotypic susceptibility of clinical isolates revealed 38 rifampicin (RIF)-resistant, 64 isoniazid (INH)-resistant, 48 streptomycin (SM)-resistant and 23 ethambutol (EMB)-resistant strains, as well as 33 multi-drug-resistant TB (MDR-TB) strains and 20 strains fully resistant to all four drugs. Optimization of the chip-based detection system for drug resistance detection showed satisfactory specificity and maximum fluorescence at a DNA concentration of 1×101 copies/µL. Further analysis revealed that 76.32% of the RIF-resistant strains harbored rpoB gene mutations (sensitivity, 76.32%; specificity 100%), 60.93% of the INH-resistant strains had katG gene mutations (sensitivity, 60.93%; specificity, 100%), 66.66% of the SM-resistant strains carried drug resistance gene mutations (sensitivity, 66.66%; specificity, 99.2%), and 69.56% of the EMB-resistant strains had embB gene mutations (sensitivity, 69.56%; specificity, 100%). Further, the overall agreement between the microfluidic chip and Sanger sequencing was satisfactory, with a turnaround time of the microfluidic chip was approximately 2 hours, much shorter than the conventional DST method. The proposed microfluidic-based KASP assay provides a cost-effective and convenient method for detecting mutations associated with drug resistance in M. tuberculosis. It represents a promising alternative to the traditional DST method, with satisfactory sensitivity and specificity and a much shorter turnaround time.

Discovery of potent triterpenoid and limonoid compounds targeting Mycobacterium tuberculosis enoyl acyl carrier protein reductase (InhA): An in silico investigation

Drug resistance remains one of the primary concerns in treating tuberculosis (TB), making it imperative to find new and effective drug candidates for improved TB treatment. Enoyl acyl carrier protein reductase (InhA), an enzyme involved in mycolic acid biosynthesis, is crucial for anti-TB drug activity such as isoniazid (INH). Triterpenoids and limonoids are widespread in the plant kingdom and have gained much attention in their development as anti-TB agents. This study aims to examine the potential of triterpenoid and limonoid compounds as InhA inhibitors based on binding energy, interaction mode, and binding stability using docking and molecular dynamics (MD) investigation. Chisopatens A (−10.93 kcal/mol), 3-epi-cabraleahydroxy lactone (−9.85 kcal/mol), and 3-oxo-olean-11(12)en-28-oic acid (−9.73 kcal/mol), as well as 6α-(acetoxy)-14β,15β-epoxyazadirone (−9.77 kcal/mol), an azadirone-type limonoid derivative, have the best affinity for Leu63, Gly96, and Lys165 amino acid residues from InhA compared to native ligand (NAD) analyzed by docking studies. Chisopatens A, 3-epi-cabraleahydroxy lactone, and 3-oxo-olean-11(12)en-28-oic also had the best affinity for InhA based on MM-PBSA analysis, with values of −140.676 kJ/mol, −108.888 kJ/mol, and −162.605 kJ/mol, respectively. During dynamics studies, these compounds also showed good stability, protein compactness, and high-intensity interaction with InhA. These results strongly support future in-vitro studies to evaluate the efficacy of the hit compounds for treating INH-resistant MTB strains.

Isoniazid Linked to Sulfonate Esters via Hydrazone Functionality: Design, Synthesis, and Evaluation of Antitubercular Activity.

Isoniazid (INH) is one of the key molecules employed in the treatment of tuberculosis (TB), the most deadly infectious disease worldwide. However, the efficacy of this cornerstone drug has seriously decreased due to emerging INH-resistant strains of Mycobacterium tuberculosis (Mtb). In the present study, we aimed to chemically tailor INH to overcome this resistance. We obtained thirteen novel compounds by linking INH to in-house synthesized sulfonate esters via a hydrazone bridge (SIH1–SIH13). Following structural characterization by FTIR, 1H NMR, 13C NMR, and HRMS, all compounds were screened for their antitubercular activity against Mtb H37Rv strain and INH-resistant clinical isolates carrying katG and inhA mutations. Additionally, the cytotoxic effects of SIH1–SIH13 were assessed on three different healthy host cell lines; HEK293, IMR-90, and BEAS-2B. Based on the obtained data, the synthesized compounds appeared as attractive antimycobacterial drug candidates with low cytotoxicity. Moreover, the stability of the hydrazone moiety in the chemical structure of the final compounds was confirmed by using UV/Vis spectroscopy in both aqueous medium and DMSO. Subsequently, the compounds were tested for their inhibitory activities against enoyl acyl carrier protein reductase (InhA), the primary target enzyme of INH. Although most of the synthesized compounds are hosted by the InhA binding pocket, SIH1–SIH13 do not primarily show their antitubercular activities by direct InhA inhibition. Finally, in silico determination of important physicochemical parameters of the molecules showed that SIH1–SIH13 adhered to Lipinski’s rule of five. Overall, our study revealed a new strategy for modifying INH to cope with the emerging drug-resistant strains of Mtb.

Significance of the coexistence of non-codon 315 katG, inhA, and oxyR-ahpC intergenic gene mutations among isoniazid-resistant and multidrug-resistant isolates of Mycobacterium tuberculosis: a report of novel mutations

ABSTRACT Tuberculosis (TB) is a global threat due to the emergence and spread of drug-resistant Mycobacterium tuberculosis (MTB). Isoniazid (INH) is the main antibiotic used for prevention and treatment of TB. Evidence shows that accumulated mutations can produce INH resistant (INHR) strains, resulting in the progression of multidrug-resistant (MDR) TB. Since point mutations in katG gene, inhA gene, and oxyR-ahpC region correlated with the INH resistance, in this study, we aimed to identify mutations in these three genes in INHR and MDR clinical isolates of MTB by Sanger DNA sequencing analysis. Thirty-three out of 438 isolates were resistant, including 66.7% INHR and 30.3% MDR isolates. In the katG gene, 68.2% INHR isolates had non-synonymous point mutations, mainly R463L (63.6%), and non-synonymous point mutation KatG L587P was seen in one of the MDR isolate. A novel silent substitution L649L was identified in the inhA gene of the MDR isolates. The oxyR-ahpC intergenic region g-88a common mutations (63.6%) in INHR and two distinct novel mutations were found at positions −76 and −77 of the oxyR-ahpC intergenic region. The coexistence of katG non-codon 315 with oxyR-ahpC intergenic region mutations was highly frequent in INHR 59.1% and MDR isolates 70%. Since mutations of all three genes 95.5% lead to the detection of INHR, they might be useful for molecular detection. Our results indicated the continuous evolution and region-specific prevalence of INH resistance. Overall, identification of new mutations in INH resistance can improve the available strategies for diagnosis and control of TB.

Molecular Characterization of Drug-Resistant Mycobacterium tuberculosis Isolated from HIV-Positive Patients in Rivers State, Nigeria

Aim: Drug resistant tuberculosis is a major challenge in the global bid to control the disease burden and improve treatment outcomes of DRTB infected individuals.
 Methods: The Line Probe Assay (LPA) was used to assess the drug resistance pattern and gene mutations in 260 sputum specimens collected consecutively from 260 adult, HIV sero-positive subjects presenting with symptoms suggestive of TB in clinical settings across Rivers State, Nigeria.
 Results: The results showed a 61.2% (n = 159) prevalence of TB among all study subjects. LPA analysis showed that 16 (10.1%) were multidrug resistant strains, 17 (10.7%) Rifampicin (RIF) monoresistant strains, 24 (15.1%) Isonaizid (INH) monoresistant strains and 102 (64.2%) drug susceptible strains. Among all 32 RIF-resistant strains, 24 (75%) had a mutation in rpoB S531L at the MUT3 band. Another mutation was observed at rpoB H526D (1/17) in RIF-monoresistant strains but not in MDR-TB strains. In INH resistant strains, mutations were observed at the katG gene [32/39 (82.1%)] which constituted 13/15 (86.7%) in MDR-TB strains and 19/24 (79.2%) in INH-monoresistant strains (p =0.002). Overall frequency of inhA mutation was 6/39 (15.4%); MDR-TB strains [2/15 (13.3%)] and INH-monoresistant strains [4/24 (16.7%), p = 0.2]. Combined KatG and inhA mutation was found in 1/39 (2.6%) of INH-monoresistant strains and in none of MDR-TB strains. Of the single inhA mutation bands, only 1/39 (4.2%) MUT 3B (T8A mutation) was observed in INH-monoresistant strains.
 Conclusion: The results showed that drug-resistant TB is prevalent in Rivers State with various gene mutations observed in the different TB strains isolated, making LPA analysis a necessity in the bid to improving prevention and control efforts in the region.

Genomic epidemiological analysis identifies high relapse among individuals with recurring tuberculosis and provides evidence of recent household-related transmission of tuberculosis in Ghana

ObjectiveTo retrospectively investigate the cause of recurring tuberculosis (rcTB) among participants with pulmonary TB recruited from a prospective population-based study conducted between July 2012 and December 2015. MethodsMycobacterium tuberculosis complex isolates obtained from rcTB cases were characterized by standard mycobacterial genotyping tools, whole-genome sequencing, and phylogenetic analysis carried out to assess strain relatedness. ResultsThe majority (58.3%, 21/36) of study participants with rcTB episodes had TB recurrence within 12 months post treatment. TB strains with isoniazid (INH) resistance were found in 19.4% (7/36) of participants at the primary episode, of which 29% (2/7) were also rifampicin-resistant. On TB recurrence, an INH-resistant strain was found in a larger proportion of participants, 27.8% (10/36), of which 40% (4/10) were MDR-TB strains. rcTB was attributed to relapse (same strain) in 75.0% (27/36) of participants and 25.0% (9/36) to re-infection. ConclusionOur findings indicate that previous unresolved infectiondue to inadequate treatment, may be the major cause of rcTB.

Drug Resistance Conferring Mutation and Genetic Diversity of Mycobacterium tuberculosis Isolates in Tuberculosis Lymphadenitis Patients; Ethiopia.

BackgroundTuberculosis lymphadenitis (TBLN) is a growing public health concern in Ethiopia. However, there is limited information available on gene mutations conferring drug resistance and genetic diversity of M. tuberculosis isolates from TBLN patients.MethodsDrug resistance and genetic diversity analysis were done on 91 M. tuberculosis isolates from culture positive TBLN patients collected between 2016 and 2017. Detection of mutations conferring resistance was carried out using GenoType MTBDRplus VER 2.0. Thereafter, isolates were typed using spoligotyping.ResultsOut of the 91 strains, mutations conferring resistance to rifampicin (RIF) and isoniazid (INH) were observed in two (2.2%) and six (6.6%) isolates, respectively. The two RIF resistant isolates displayed a mutation at codon 531 in the rpoB gene with amino acid change of S531L. Among the six INH resistant strains, four isolates had shown mutation at the KatG gene at codon 315 with amino acid change of S315T, one isolate had a mutation at the inhA gene at codon 15 with amino acid change of C15T and one isolate had a mutation at the inhA gene with unknown amino acid change. All drug resistant isolates were from treatment naive TBLN patients. The dominantly identified Spoligo International Types (SITs) were SIT25, SIT149, and SIT53, respectively; these accounted for 43% of the total number of strains. The isolates were grouped into four main lineages; Lineage 1 (2, 2.2%), Lineage 3 (38, 41.7%), Lineage 4 (49, 53.8%) and Lineage 7 (2, 2.2%). Four out of six (66.7%) isolates with drug resistance conferring mutations belonged to clustered strains (strains with shared SIT).ConclusionThe detection of drug resistant conferring mutation in treatment naïve TBLN patients together with detection of drug resistant isolates among clustered strains might suggest resistant strains' transmission in the community. This needs to be carefully considered to prevent the spread of drug resistant clones in the country.

Acetylation of Isoniazid Is a Novel Mechanism of Isoniazid Resistance in Mycobacterium tuberculosis.

Isoniazid (INH), one of the first-line drugs used for the treatment of tuberculosis, is a prodrug which is activated by the intracellular KatG enzyme of Mycobacterium tuberculosis The activated drug hinders cell wall biosynthesis by inhibiting the InhA protein. INH-resistant strains of M. tuberculosis usually have mutations in katG, inhA, ahpC, kasA, and ndh genes. However, INH-resistant strains which do not have mutations in any of these genes are reported, suggesting that these strains may adopt some other mechanism to become resistant to INH. In the present study, we characterized Rv2170, a putative acetyltransferase in M. tuberculosis, to elucidate its role in inactivating isoniazid. The purified recombinant protein was able to catalyze the transfer of the acetyl group to INH from acetyl coenzyme A (acetyl-CoA). High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses showed that following acetylation by Rv2170, INH is broken down into isonicotinic acid and acetylhydrazine. A drug susceptibility assay and confocal analysis showed that Mycobacterium smegmatis, which is susceptible to INH, is not inhibited by INH acetylated with Rv2170. Mutant proteins of Rv2170 failed to acetylate INH. Recombinant M. smegmatis and M. tuberculosis H37Ra overexpressing Rv2170 were found to be resistant to INH at MICs that inhibited wild-type strains. Besides, intracellular M. tuberculosis H37Ra overexpressing Rv2170 survived better in macrophages when treated with INH. Our results strongly indicate that Rv2170 acetylates INH, and this could be one of the strategies adopted by at least some M. tuberculosis strains to overcome INH toxicity, although this needs to be tested in INH-resistant clinical strains.

Benzimidazoquinazolines as new potent anti-TB chemotypes: Design, synthesis, and biological evaluation.

In search for new molecular entities as anti-TB agents, the benzimidazoquinazoline polyheterocyclic scaffold has been designed adopting the scaffold hopping strategy. Thirty-two compounds have been synthesized through an improved tandem decarboxylative nucleophilic addition cyclocondensation reaction of o-phenylenediamine with isatoic anhydride followed by further cyclocondensation of the intermediately formed 2-(o-aminoaryl)benzimidazole with trialkyl orthoformate/acetate. The resultant benzimidazoquinazolines were evaluated in vitro for anti-TB activity against M. tuberculosis H37Rv (ATCC27294 strain). Fourteen compounds exhibiting MIC values in the range of 0.4-6.25µg/mL were subjected to cell viability test against RAW 264.7 cell lines and were found to be non-toxic (<30% inhibition at 50µg/mL). The active compounds were further evaluated against INH resistant Mtb strains. The most active compound 6x [MIC (H37Rv) of 0.4µg/mL] and the compound 6d [MIC (H37Rv) of 0.78µg/mL] were also found to be active against INH resistant Mtb strain with MIC values of 12.5 and 0.78µg/mL, respectively.

The Introduction of Hydrazone, Hydrazide, or Azepane Moieties to the Triterpenoid Core Enhances an Activity Against M. tuberculosis.

Triterpenoids exhibit a wide spectrum of antimicrobial activity. The objective of this study was to synthesize a series of nitrogen derivatives based on lupane, oleanane, and ursane triterpenoids with high antitubercular activity. Isonicotinoylhydrazones were prepared via the reaction of 3-oxotriterpenic acids or betulonic aldehyde with isoniazid (INH) in yields of 54-72%. N-Acylation of betulonic or azepanobetulinic acids led to lupane C28 hydrazides and dihydrazides. The derivatives were evaluated for their in vitro antimycobacterial activities against Mycobacterium tuberculosis (MTB) H37RV and single-drug resistance (SDR)-TB in the National Institute of Allergy and Infectious Diseases, USA. Molecular docking was performed to evaluate the possible binding modes of investigated compounds in the active site of Diterpene synthase (Rv3378c). The obtained compounds are represented by C3 or C28 conjugates with hydrazine hydrate or INH. Some compounds demonstrated from high minimum inhibitory concentration (MIC ≤ 10 μg/mL) to excellent (MICs from 0.19 to 1.25 μg/mL) activity against MTB H37RV. Two lupane conjugates with INH were the leading compounds against MTB H37RV and some SDR-strains with MICs ranged from 0.19 to 1.70 μg/mL. Molecular docking of active compounds to diterpene synthase showed that these moieties accommodate the active site of the enzyme. It was revealed that the conjugation of lupanes with INH at C3 is more effective than at C28 and the lupane skeleton is preferable among oleanane and ursane types. The replacement of native hexacarbocyclic A ring to seven-member azepane ring is favorably for inhibition of both MTB H37RV and SDR-strains. These data could possibly mean that the antitubercular activity against INH-resistant strains (INH-R) came from both triterpenoid and isoniazid parts of the hybrid molecules. Azepanobetulin showed the highest activity against both INH-R strains in comparison with other triterpenoids and INH. Thus, the introduction of hydrazone, hydrazide (dihydrazide), or azepane moieties into the triterpenoid core is a promising way for the development of new anti-tubercular agents.

Molecular Dynamics Assisted Mechanistic Insight of Val430-Ala Mutation of Rv1592c Protein in Isoniazid Resistant Mycobacterium Tuberculosis.

Multi drug-resistant tuberculosis is a major health threat to humans. Whole genome sequencing of several isoniazid (INH) resistant strains of M. tuberculosis revealed mutations in several genes. Rv1592c was demonstrated as lipolytic enzyme and its expression was up-regulated during isoniazid (INH) treatment. The valine at position 430 of Rv1592c was mutated to alanine frequently in the INH resistant strain of M. tuberculosis. In this report, an array of computational approaches was used to understand the role of Val430-Ala mutation in Rv1592c in INH resistance. The impact of mutations on structural stability and degree of INH modification was demonstrated using the molecular dynamics method. The mutation in the Rv1592c gene at V430 position was created by the PCR primer walking method. Mutant and wild type gene was cloned into E. coli-mycobacteria shuttle vector (pVV-16) and expressed in Mycobacterium smegmatis system. The isoniazid susceptibility assay was performed by agar plate culture spot and CFUs count assay. This study demonstrated that the Val430 in Rv1592c makes the part of flap covering the substrate binding cavity. Mutation at Val430-Ala in Rv1592c caused the displacement of the flap region, resulting in uncovering a cavity, which allows accessibility of substrate to the active site cleft. The Val430-Ala mutation in Rv1592c created its structure energetically more stable. RMSD, RMSF and Rg simulation of mutant maintained overall stability throughout the simulation period while the native protein displayed comparatively more fluctuations. Moreover, docking studies showed that INH was bound into the active pocket of the mutant with considerable binding energy (-6.3 kcal/mol). In order to observe constant binding for INH, complexes were simulated for 50 ns. It was observed that after simulation, INH remained bound in the pocket with an increased molecular bonding network with the neighbor amino acid residues. In vitro studies clearly suggested that M. smegmatis expressing mutant has a better survival rate in isoniazid treatment as compared to wild type. Overall, this study at the outset suggested that the mutation observed in drug resistant strain provides stability to the Rv1592c protein and increased affinity towards the INH due to flap displacement, leading to the possibility for its modification. In vitro results supported our in silico findings.

Genotypic Characterization of katG, inhA, and ahpC in Isoniazid-Resistant Mycobacterium tuberculosis Clinical Isolates in Shanghai, China

BackgroundMycobacterium tuberculosis is a pathogen that causes Tuberculosis and can invade various organs in infected patients. Its high morbidity and high mortality seriously threaten human health. In recent years, the continuous emergence of drug-resistant Tuberculosis bacteria has brought severe challenges to the prevention and control of Tuberculosis.ObjectivesThis study aimed to characterize the most frequent mutations of the katG, inhA, and ahpC genes in isoniazid (INH)-resistant M. tuberculosis clinical isolates in Shanghai Pulmonary Hospital, China, and investigate the relationship between gene mutations and the minimum inhibitory concentrations (MICs) of INH against M. tuberculosis.MethodsWe collected 92 INH-resistant and 30 INH-susceptible clinical isolates of M. tuberculosis. The drug resistance profiles of M. tuberculosis clinical isolates against common anti-tuberculosis drugs were determined and sequencing analysis was performed.ResultsOf 92 INH-resistant strains, mutations in the katG and inhA genes were observed in 64 (69.6%) isolates and five (5.4%) isolates, respectively, and only had one (1.1%) strain both katG and inhA mutations. Among them, 62 (67.4%) strains carried a single mutation at codon 315 of the katG gene and a new mutation site was found in the katG gene of two strains. We detected a single mutation site at codon 271 and three simultaneous mutation sites at codons 315, 431, and 439. Only one (3.3%) of the 30 isoniazid-sensitive strains had the katG mutation. The AhpC mutation was detected in no experimental strains. The KatG Ser 315 Thr (AGC315ACC) mutation occurred in 53 (68.8%) out of 77 strains with high MICs (≥ 1 g/mL) of isoniazid-resistant M. tuberculosis while five (33.3%) out of 15 strains with low MICs (less than 1 g/mL) had katG Ser 315 Thr (AGC315ACC) mutation.ConclusionsIsoniazid-resistant strains were dominated by Ser315 → Thr (AGC → ACC) substitution, which seems to be associated with multidrug resistance and high-level resistance to INH. Multisite mutations are related to multidrug-resistant M. tuberculosis and the discovery of new mutation sites provides a new basis for the detection of drug-resistant M. tuberculosis.

Pattern of InhA and KatG mutations in isoniazid monoresistant Mycobacterium tuberculosis isolates.

Aims and Objectives:The aim of the study is to detect the pattern of genetic mutation, i.e., InhA or KatG or both (InhA and katG) in isoniazid (INH) monoresistant mycobacteria and to correlate with the pattern in multidrug-resistant (MDR) isolates.Materials and Methods:In this study, a quantitative research approach was used. The research design was descriptive observational study. The study was conducted at the Department of Respiratory Medicine, JLN Medical College, Ajmer, Rajasthan, and Intermediate Referral Laboratory, State TB Demonstration Centre, Ajmer. A total of 298 samples found to have resistant strains of Mycobacterium tuberculosis were enrolled with purposive sampling.Results:The mean age of patients was 40.27 ± 13.82 years. There were 250 (83.9%) males, while 48 (16.1%) were females. One hundred ninety-two (64.4%) were resistant for INH only, while the rest were resistant to both INH as well as rifampicin (MDR-tuberculosis). The most common mutation in INH monoresistance was katG (125; 65.1%) as compared to inhA (54; 28.1%) and both inhA and katG (13; 6.7%). Among katG mutations, the most common gene pattern was the absence of WT (S315T) and the presence of MUT1 (S315T1).Conclusion:Knowledge about mutation patterns of different INH resistant strains is important in the present era where there is a provision of separate regimens for INH monoresistant TB. Since these mutations are very closely related to high- or low-degree resistance to INH, the therapeutic regimens cannot be generalized.

Whole genome sequencing, analyses of drug resistance-conferring mutations, and correlation with transmission of Mycobacterium tuberculosis carrying katG-S315T in Hanoi, Vietnam

Drug-resistant tuberculosis (TB) is a serious global problem, and pathogen factors involved in the transmission of isoniazid (INH)-resistant TB have not been fully investigated. We performed whole genome sequencing of 332 clinical Mycobacterium tuberculosis (Mtb) isolates collected from patients newly diagnosed with smear-positive pulmonary TB in Hanoi, Vietnam. Using a bacterial genome-wide approach based on linear mixed models, we investigated the associations between 31-bp k-mers and clustered strains harboring katG-S315T, a major INH-resistance mutation in the present cohort and in the second panel previously published in South Africa. Five statistically significant genes, namely, PPE18/19, gid, emrB, Rv1588c, and pncA, were shared by the two panels. We further identified variants of the genes responsible for these k-mers, which are relevant to the spread of INH-resistant strains. Phylogenetic convergence test showed that variants relevant to PPE46/47-like chimeric genes were significantly associated with the same phenotype in Hanoi. The associations were further confirmed after adjustment for the confounders. These findings suggest that genomic variations of the pathogen facilitate the expansion of INH-resistance TB, at least in part, and our study provides a new insight into the mechanisms by which drug-resistant Mtb maintains fitness and spreads in Asia and Africa.

1347. Tuberculosis Caused by Mycobacterium bovis in Children: A Retrospective Review of Cases From 2010 to 2019 in a Pediatric Tertiary-care Center in Mexico

BackgroundTuberculosis (Tb) is a leading cause of death worldwide. The most common cause of Tb is due to M. tuberculosis, however, M. bovis is considered a public health issue and an often-undetected cause of Tb. Information regarding Tb by M. bovis in children is scarce.MethodsRetrospective analysis of culture-proven cases of Tb by M. bovis from 2010 to 2019 in a pediatric tertiary-care center in Mexico. Clinical and paraclinical characteristics were compared.ResultsWe included 22 cases of Tb by M. bovis in children younger than 18 years of age. 64 percent were men, mean age was 3.8 years. All subjects had a positive history of BCG immunization. Eight subjects (36.4%) consumed unpasteurized dairy products, 3 (13.6%) referred contact with Tb-infected people, and none had contact with cattle. Twelve patients (54.5%) had an immunodeficiency (ID). The most common ID was defects in the IL-12/IFN-γ axis (36.3%), followed by severe-combined ID (9%). All of the subjects presented any form of extrapulmonary Tb, and 8 (36.4%) had disseminated Tb. The most common extrapulmonary presentations were lymph-node (40.9%), abdominal (18.2%), and skeletal Tb (13.6%). Fever was present in 21.3% of the cases, weight-loss in 12%, and diaphoresis in 9.3%. Hepatomegaly was present in 83% of patients with abdominal Tb (P = 0.001). We found a negative association between lymph-node enlargement and skeletal Tb by M. bovis (P = 0.01). Five cases (22.7%) were identified as M. bovis BCG strain. Infection by BCG strain was associated with a shorter time of presentation (< 4 months) (P = 0.005). Polydrug resistance (resistance to ≥2 drugs, excluding those classified as MDR) was observed in five strains (22.7%). We detected 3 RIF-resistant strains (19%), 2 INH-resistant strains (9.1%), and 1 EMB-resistant strain (4.5%). No MDR strains were detected. We report an 86% cure rate, and 5% mortality. Nine percent are still in treatment.ConclusionWe report 22 cases of culture-proven cases of Tb by M. bovis, from 2010 to 2019 in a pediatric tertiary-care center in Mexico. Disseminated presentation was common, as well as extrapulmonary involvement. Infection by M. bovis BCG strain was associated with a shorter time of presentation. Further studies are required in order to expand our knowledge on M. bovis infection in children.Disclosures All authors: No reported disclosures.

Change in prevalence and molecular characteristics of isoniazid-resistant tuberculosis over a 10-year period in China

BackgroundIsoniazid (INH) represents the cornerstone for the treatment of cases infected with Mycobacterium tuberculosis (MTB) strains. Several molecular mechanisms have been shown to be the major causes for INH resistance, while the dynamic change of mutations conferring INH resistance among MTB strains during the past decade is still unknown in China.MethodsIn this study, we carried out a comparative analysis of the INH minimal inhibitory concentration (MIC) distribution, and investigate the dynamic change of molecular characteristics among INH-resistant MTB strains between 2005 and 2015.ResultsThe proportion of INH resistance (39.0%, 105/269) in 2015 was significantly higher than in 2005 (30.0%, 82/273; P = 0.03). Among 269 isolates collected in 2015, 76 (28.3%, 76/269) exhibited high-level INH-resistance (MIC≥32 mg/L), which was significantly higher than that in 2005 (20.5%, 56/273, P = 0.04). In addition, a significantly higher percentage of INH-resistant isolates carried inhA promoter mutations in 2015 (26.7%) versus that in 2005 (14.6%, P = 0.04), while no significant difference was observed in the rates of isolates containing katG mutations between 2005 (76.8%) and 2015 (70.5%, P = 0.33). Notably, the proportion of MTB isolates with inhA mutations (26.7%, 28/105) for patients who had previous exposure to protionamide (PTH) was higher than that for patients who had no previous exposure to PTH (21.4%, 6/28).ConclusionsIn conclusion, our results demonstrated that the proportion of INH-resistant MTB isolates significantly increased during the last decade, which was mainly attributed to an increase of high-level INH-resistant MTB. In addition, prior exposure to PTH may be associated with the increased frequency of INH-resistant tuberculosis strains with inhA mutations in China.

Design of hybrid molecules as antimycobacterial compounds: Synthesis of isoniazid-naphthoquinone derivatives and their activity against susceptible and resistant strains of Mycobacterium tuberculosis

Isoniazid-naphthoquinone hybrids were synthesized and evaluated against a susceptible (H37Rv) strain and two isoniazid-resistant strains (INHR1 and INHR2) of Mycobacterium tuberculosis. The antimycobacterial activity of the derivatives was determined based on the resazurin microtiter assay and their cytotoxicity in adhered mouse monocyte macrophage J774.A1 cells (ATCC TIB-67). Of the twenty-two compounds evaluated against the three strains of M. tuberculosis, twenty-one presented some activity against the H37Rv and INHR1 (katG S315T) or INHR2 (inhA C(−5)T) strains. Compounds 1a, 2a, and 8a were effective against the INHR1 strain, and compounds 1a, 1b, 2a, 3a, 5a, 5b and 8a were effective against the INHR2 strain, with MICs in the range of 3.12–6.25 µg/mL. Compounds 1b and 5b were the most active against H37Rv, with MIC of 0.78 µg/mL. Based on the selectivity index, 1b and 5b can be considered safe as a drug candidate compounds. These results demonstrate that quinoidal compounds can be used as promising scaffolds for the development of new anti-TB drugs and hybrids with activity against M. tuberculosis-susceptible and INH-resistant strains.