Clinical Strains Of Mycobacterium Tuberculosis Research Articles

Clinical Strains of Mycobacterium tuberculosis Representing Different Genotype Families Exhibit Distinct Propensities to Adopt the Differentially Culturable State.

Growing evidence points to the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens from individuals with active tuberculosis (TB) disease. These bacteria are unable to grow on solid media but can resuscitate in liquid media. Given the epidemiological success of certain clinical genotype families of Mycobacterium tuberculosis, we hypothesize that different strains may have distinct mechanisms of adaptation and tolerance. We used an in vitro carbon starvation model to determine the propensity of strains from lineages 2 and 4 that included the Beijing and LAM families respectively, to generate DCTB. Beijing strains were associated with a greater propensity to produce DCTB compared to LAM strains. Furthermore, LAM strains required culture filtrate (CF) for resuscitation whilst starved Beijing strains were not dependent on CF. Moreover, Beijing strains showed improved resuscitation with cognate CF, suggesting the presence of unique growth stimulatory molecules in this family. Analysis of starved Beijing and LAM strains showed longer cells, which with resuscitation were restored to a shorter length. Cell wall staining with fluorescent D-amino acids identified strain-specific incorporation patterns, indicating that cell surface remodeling during resuscitation was distinct between clinical strains. Collectively, our data demonstrate that M. tuberculosis clinical strains from different genotype lineages have differential propensities to generate DCTB, which may have implications for TB treatment success.

Bioorthogonal Metabolic Labeling of the Virulence Factor Phenolic Glycolipid in Mycobacteria.

Surface lipids on pathogenic mycobacteria modulate infection outcomes by regulating host immune responses. Phenolic glycolipid (PGL) is a host-modulating surface lipid that varies among clinical Mycobacterium tuberculosis strains. PGL is also found in Mycobacterium marinum, where it promotes infection of zebrafish through effects on the innate immune system. Given the important role this lipid plays in the host-pathogen relationship, tools for profiling its abundance, spatial distribution, and dynamics are needed. Here, we report a strategy for imaging PGL in live mycobacteria using bioorthogonal metabolic labeling. We functionalized the PGL precursor p-hydroxybenzoic acid (pHB) with an azide group (3-azido pHB). When fed to mycobacteria, 3-azido pHB was incorporated into the cell surface, which could then be visualized via the bioorthogonal conjugation of a fluorescent probe. We confirmed that 3-azido pHB incorporates into PGL using mass spectrometry methods and demonstrated selectivity for PGL-producing M. marinum and M. tuberculosis strains. Finally, we applied this metabolic labeling strategy to study the dynamics of PGL within the mycobacterial membrane. This new tool enables visualization of PGL that may facilitate studies of mycobacterial pathogenesis.

Transmission dynamics of drug-resistant tuberculosis in Ningbo, China: an epidemiological and genomic analysis.

Tuberculosis (TB), particularly drug-resistant TB (DR-TB), remains a significant public health concern in Ningbo, China. Understanding its molecular epidemiology and spatial distribution is paramount for effective control. From December 24, 2020, to March 12, 2023, we collected clinical Mycobacterium tuberculosis (MTB) strains in Ningbo, with whole-genome sequencing performed on 130 MTB strains. We analyzed DR-related gene mutations, conducted phylogenetic and phylodynamic analyses, identified recent transmission clusters, and assessed spatial distribution. Among 130 DR-TB cases, 41% were MDR-TB, 36% pre-XDR-TB, 19% RR-TB, and 3% HR-TB. The phylogenetic tree showed that 90% of strains were Lineage 2 (Beijing genotype), while remaining 10% were Lineage 4 (Euro-American genotype). The spatial analysis identified hotspots of DR-TB in Ningbo's northern region, particularly in traditional urban centers. 31 (24%) of the DR-TB cases were grouped into 7 recent transmission clusters with a large outbreak cluster containing 15 pre-XDR-TB patients. Epidemiological analyses suggested a higher risk of recent DR-TB transmission among young adult patients who frequently visited Internet cafes, game rooms, and factories. Our study provides comprehensive insights into the epidemiology and genetics of DR-TB in Ningbo. The presence of genomic clusters highlights recent transmission events, indicating the need for targeted interventions. These findings are vital for informing TB control strategies in Ningbo and similar settings.

Mutations in the promoter region of methionine transporter gene metM (Rv3253c) confer para-aminosalicylic acid (PAS) resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) is a significant global public health threat. Despite the long-standing use of para-aminosalicylic acid (PAS) as a second-line anti-TB drug, its resistance mechanism remains unclear. In this study, we isolated 90 mutants of PAS-resistant Mycobacterium tuberculosis (MTB) H37Ra in 7H11 solid medium and performed whole-genome sequencing, gene overexpression, transcription level comparison and amino acid level determination in MTB, and promoter activity by β-galactosidase assays in Mycobacterium smegmatis to elucidate the mechanism of PAS resistance. Herein, we found that 47 of 90 (52.2%) PAS-resistant mutants had nine different mutations in the intergenic region of metM (Rv3253c) and Rv3254. Beta-galactosidase assays confirmed that mutations increased promoter activity only for metM but not Rv3254. Interestingly, overexpression of MetM or its M. smegmatis homolog (MSMEI_1796) either by its promoter in metM's direction or by exogenous expression in MTB induced PAS resistance in a methionine-dependent manner. Therefore, drug susceptibility results for the metM promoter mutants can be misleading when using standard 7H10 or 7H9 medium, which lacks methionine. At the metabolism level, PAS treatment led to higher intracellular methionine levels in the mutants than the wild type, antagonizing PAS and conferring resistance. Furthermore, 12 different mutations in the metM promoter were identified in clinical MTB strains. In summary, we found a novel mechanism of PAS resistance in MTB. Mutations in the metM (Rv3253c) promoter upregulate metM transcription and elevate intracellular methionine, which antagonize PAS. Our findings shed new light on the mechanism of PAS resistance in MTB and highlight issues with the current PAS susceptibility culture medium.IMPORTANCEAlthough para-aminosalicylic acid (PAS) has been used to treat TB for more than 70 years, the understanding of PAS resistance mechanisms is still vague, living gaps in our ability to predict resistance and apply PAS effectively in clinical practice. This study aimed to address this knowledge gap by inducing in vitro PAS resistance in Mycobacterium tuberculosis (MTB) using 7H11 medium and discovering a new PAS resistance mechanism. Our research revealed that spontaneous mutations occurring in the promoter region of the methionine transporting gene, metM, can upregulate the expression of metM, resulting in increased intracellular transport of methionine and consequently high-level resistance of Mycobacterium tuberculosis to PAS. Notably, this resistance phenotype cannot be observed when using the commonly recommended 7H10 medium, possibly due to the lack of additional methionine supply compared with that when using the 7H11 medium. Mutations on the regulatory region of metM were also found in some clinical MTB strains. These findings may have important implications for the unexplained PAS resistance observed in clinical settings and provide insight into the failures of PAS treatment. Additionally, they underscore the importance of considering the choice of culture media when conducting drug susceptibility testing for MTB.

Temporal Profiling of Host Proteome against Different M. tuberculosis Strains Reveals Delayed Epigenetic Orchestration.

Apart from being preventable and treatable, tuberculosis is the deadliest bacterial disease afflicting humankind owing to its ability to evade host defence responses, many of which are controlled by epigenetic mechanisms. Here, we report the temporal dynamics of the proteome of macrophage-like host cells after infecting them for 6, 18, 30, and 42 h with two laboratory strains (H37Ra and H37Rv) and two clinical strains (BND433 and JAL2287) of Mycobacterium tuberculosis (MTB). Using SWATH-MS, the proteins characterized at the onset of infection broadly represented oxidative stress and cell cytoskeleton processes. Intermediary and later stages of infection are accompanied by a reshaping of the combination of proteins implicated in histone stability, gene expression, and protein trafficking. This study provides strain-specific and time-specific variations in the proteome of the host, which might further the development of host-directed therapeutics and diagnostic tools against the pathogen. Also, our findings accentuate the importance of proteomic tools in delineating the complex recalibration of the host defence enabled as an effect of MTB infection. To the best of our knowledge, this is the first comprehensive proteomic account of the host response to avirulent and virulent strains of MTB at different time periods of the life span of macrophage-like cells. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD022352.

Mycobacterium tuberculosis Rv1916 is an Acetyl-CoA-Binding Protein.

Isocitrate lyase (ICL) isoform 2 is an essential enzyme for some clinical Mycobacterium tuberculosis (Mtb) strains during infection. In the laboratory Mtb strain H37Rv, the icl2 gene encodes two distinct gene products - Rv1915 and Rv1916 - due to a frameshift mutation. This study aims to characterise these two gene products to understand their structure and function. While we were unable to produce Rv1915 recombinantly, soluble Rv1916 was obtained with sufficient yield for characterisation. Kinetic studies using UV-visible spectrophotometry and 1 H-NMR spectroscopy showed that recombinant Rv1916 does not possess isocitrate lyase activity, while waterLOGSY binding experiments demonstrated that it could bind acetyl-CoA. Finally, X-ray crystallography revealed structural similarities between Rv1916 and the C-terminal domain of ICL2. Considering the probable differences between full-length ICL2 and the gene products Rv1915 and Rv1916, care must be taken when using Mtb H37Rv as a model organism to study central carbon metabolism.

The nature of the stimulant differentially impacts the innate memory response of monocytes to Mycobacterium tuberculosis

Abstract Innate immune cells, such as monocytes, can generate a non-specific ‘memory’ response (a.k.a. trained immunity) upon stimulation with an antigen/bacterium (e.g., LPS or BCG), which confers heterologous protection against another infectious agent. However, the association between the nature of stimulants and the corresponding immune correlate of trained immunity is not fully understood. Similarly, the response of macrophages “trained” by various stimulants to subsequent infection by different clinical Mycobacterium tuberculosis(Mtb) strains remains unknown. Here, we report the trained immunity profile of the blood-derived monocytes (BDM) of BCG-vaccinated adults and monocytic THP-1 cells after stimulation with BCG, LPS, Purified Protein Derivative (PPD), heat-killed MtbHN878 or MtbCDC1551 strains. The response of monocytes trained by these agents to subsequent infection with clinical Mtb isolates HN878 and CDC1551, which differ in their virulence potential, was evaluated using standard molecular and cellular immunologic assays. The BCG- and LPS-trained macrophages produced higher proinflammatory, antimicrobial responses, and autophagy induction than unstimulated, PPD, HK-HN878, or CDC1551-stimulated cells. The HN878-infected trained THP-1 cells elicited a more robust inflammatory response than the CDC1551 infection. A significantly reduced intracellular Mtb growth was noted in the BCG and LPS-trained THP-1 cells, which was augmented by adding rapamycin, an autophagy inducer. A similar immunological response was noted in the BDM of BCG-vaccinated adults infected with these Mtb strains. Thus, the nature of the stimulant affects the macrophages’ trained immunity profile and its response to Mtb infection. Supported by grants from NIH (R01 AI161822) and the Bill and Melinda Gates Foundation (INV-009099; formerly OPP1157210).

Emergence of Canonical and Noncanonical Genomic Variants following In Vitro Exposure of Clinical Mycobacterium tuberculosis Strains to Bedaquiline or Clofazimine.

In Mycobacterium tuberculosis, bedaquiline and clofazimine resistance occurs primarily through Rv0678 variants, a gene encoding a repressor protein that regulates mmpS5/mmpL5 efflux pump gene expression. Despite the shared effect of both drugs on efflux, little else is known about other pathways affected. We hypothesized that in vitro generation of bedaquiline- or clofazimine-resistant mutants could provide insight into additional mechanisms of action. We performed whole-genome sequencing and determined phenotypic MICs for both drugs on progenitor and mutant progenies. Mutants were induced through serial passage on increasing concentrations of bedaquiline or clofazimine. Rv0678 variants were identified in both clofazimine- and bedaquiline-resistant mutants, with concurrent atpE SNPs occurring in the latter. Of concern was the acquisition of variants in the F420 biosynthesis pathway in clofazimine-resistant mutants obtained from either a fully susceptible (fbiD: del555GCT) or rifampicin mono-resistant (fbiA: 283delTG and T862C) progenitor. The acquisition of these variants possibly implicates a shared pathway between clofazimine and nitroimidazoles. Pathways associated with drug tolerance and persistence, F420 biosynthesis, glycerol uptake and metabolism, efflux, and NADH homeostasis appear to be affected following exposure to these drugs. Shared genes affected by both drugs include Rv0678, glpK, nuoG, and uvrD1. Genes with variants in the bedaquiline resistant mutants included atpE, fadE28, truA, mmpL5, glnH, and pks8, while clofazimine-resistant mutants displayed ppsD, fbiA, fbiD, mutT3, fadE18, Rv0988, and Rv2082 variants. These results show the importance of epistatic mechanisms as a means of responding to drug pressure and highlight the complexity of resistance acquisition in M. tuberculosis.

Modern Clinical Mycobacterium tuberculosis Strains Leverage Type I IFN Pathway for a Proinflammatory Response in the Host.

Host phagocytes respond to infections by innate defense mechanisms through metabolic shuffling to restrict the invading pathogen. However, this very plasticity of the host provides an ideal platform for pathogen-mediated manipulation. In the human (THP1/THP1 dual/PBMC-derived monocyte-derived macrophages) and mouse (RAW264.7 and C57BL/6 bone marrow-derived) macrophage models of Mycobacterium tuberculosis infection, we have identified an important strategy employed by clinical lineages in regulating the host immune-metabolism axis. We show greater transit via the macrophage phagosomal compartments by Mycobacterium tuberculosis strains of lineage: M. tuberculosis lineage 3 is associated with an ability to elicit a strong and early type I IFN response dependent on DNA (in contrast with the protracted response to lineage: M. tuberculosis lineage 1). This augmented IFN signaling supported a positive regulatory loop for the enhanced expression of IL-6 consequent to an increase in the expression of 25-hydroxycholesterol in macrophages. This amplification of the macrophage innate response-metabolic axis incumbent on a heightened and early type I IFN signaling portrays yet another novel aspect of improved intracellular survival of clinical M. tuberculosis strains.

A five-year review of prevalence and treatment outcomes of pre-extensively drug-resistant plus additional drug-resistant tuberculosis in the Henan Provincial Tuberculosis Clinical Medicine Research Centre

This study investigated the prevalence and significant clinical outcomes of pre-extensively drug-resistant plus additional drug-resistant tuberculosis (pre-XDR-plus) in Henan Provincial Chest Hospital between 2017 and 2021. We analysed and summarized the drug sensitivity test (DST) results of clinical Mycobacterium tuberculosis (MTB) strains in TB patients seeking care in the Tuberculosis Clinical Medical Research Centre of Henan Province between 2017 and 2021. Medical records of pre-extensively drug-resistant plus additional drug-resistant TB patients were statistically analysed, including demographic characteristics, regimens, and outcomes. Of the 3689 Mycobacterium tuberculosis strains, 639 (17.32%), 353 (9.56%), and 109 (2.95%), multidrug-resistant tuberculosis (MDR-TB), pre-extensively drug-resistant tuberculosis (pre-XDR), and pre-XDR-plus, respectively. The proportion of MDR decreased from 19.1% in 2017 to 17.5% in 2021 (χ2=0.686, P = 0.407), the proportion of pre-XDR from 11.4% in 2017 to 9.0% in 2021 (χ2=2.39, P=0.122), and pre-XDR-plus from 4.7% in 2017 to 1.8% in 2020, with the declining trend was significant (χ2=9.348, P=0.002). The most commonly used anti-TB drugs were pyrazinamide (PZA, 37/46, 80.43%) and cycloserine (CS, 32/46, 69.57%), followed by linezolid (LZD, 25/46, 54.35%), protionamide (TH, 25/46, 54.35%), and para-aminosalicylic acid (PAS, 23/46, 50.00%). Patients receiving the LZD regimen were 5 times more likely to have a favourable outcome than those not receiving LZD (OR=6.421, 95% CI 2.101-19.625, P=0.001). Patients receiving a regimen containing CS were 4 times more likely to have a favourable outcome compared to those not taking CS (OR=5.444, 95% CI 1.650-17.926, P=0.005). Our data suggest that the population of pre-XDR-plus had significantly decreased over the past five years in the Henan Provincial Chest Hospital. The COVID-19 and flood disaster affect TB patients' selection of medical services. In addition, the pre-XDR-plus patients whose regimens contain LZD or CS were more likely to have favourable outcomes.

A multi-omics investigation into the mechanisms of hyper-virulence in Mycobacterium tuberculosis

ABSTRACT Clinical manifestations of tuberculosis range from asymptomatic infection to a life-threatening disease such as tuberculous meningitis (TBM). Recent studies showed that the spectrum of disease severity could be related to genetic diversity among clinical strains of Mycobacterium tuberculosis (Mtb). Certain strains are reported to preferentially invade the central nervous system, thus earning the label “hypervirulent strains”.However, specific genetic mutations that accounted for enhanced mycobacterial virulence are still unknown. We previously identified a set of 17 mutations in a hypervirulent Mtb strain that was from TBM patient and exhibited significantly better intracellular survivability. These mutations were also commonly shared by a cluster of globally circulating hyper-virulent strains. Here, we aimed to validate the impact of these hypervirulent-specific mutations on the dysregulation of gene networks associated with virulence in Mtb via multi-omic analysis. We surveyed transcriptomic and proteomic differences between the hyper-virulent and low-virulent strains using RNA-sequencing and label-free quantitative LC-MS/MS approach, respectively. We identified 25 genes consistently differentially expressed between the strains at both transcript and protein level, regardless the strains were growing in a nutrient-rich or a physiologically relevant multi-stress condition (acidic pH, limited nutrients, nitrosative stress, and hypoxia). Based on integrated genomic-transcriptomic and proteomic comparisons, the hypervirulent-specific mutations in FadE5 (g. 295,746 C >T), Rv0178 (p. asp150glu), higB (p. asp30glu), and pip (IS6110-insertion) were linked to deregulated expression of the respective genes and their functionally downstream regulons. The result validated the connections between mutations, gene expression, and mycobacterial pathogenicity, and identified new possible virulence-associated pathways in Mtb.

GENE MUTATION PATTERNS OF RIFAMPICIN IN MULTIDRUG RESISTANT MYCOBACTERIUM TUBERCULOSIS COMPLEX STRAINS

Objective: The aim of this study was to evaluate the GenoType®MTBDRplus test for detection of rifampicin (RIF) resistance in MDR Mycobacterium tuberculosis complex strains. Materials and Methods: Twenty-five multidrug-resistant (MDR) Mycobacterium tuberculosis clinical strains were used, gene mutations causing RIF resistance were investigated by GenoType®MTBDRplus and the results were compared with the results of the BACTEC 460 TB system. The strain was sequenced if there was no mutation and absence Wild Type (WT). Mycobacterium tuberculosis ATCC 35838 was used as a quality control (QC) strain. Results: There was 96% compliance between the GenoType®MTBDRplus and BACTEC 460 TB system for the finding of RIF resistance. The most frequent mutation zone in MDR strains was rpoB S531L promotor zone (13 strains, 52%). The other rpoB gene mutations were H526Y (three strains, 12%) and H526D (three strains, 12%), while five strains (20%) had Δ2-Δ5 mutations in the wild type probes. There was no mutation in only one strain (4%) by GenoType®MTBDRplus but it was found to be as resistant to rifampicin by the BACTEC 460 TB system. This strain was sequenced and detected to have triple mutations. Mutations were found on codons 489, 493, and 503. Conclusion: GenoType®MTBDRplus showed good compatibility with the BACTEC 460 TB system in detecting rifampicin resistance, and it was thought that GenoType®MTBDRplus could be an effective and reliable test for RIF susceptibility testing in MDRTB patients, providing a significant advantage in technical time.

The relationship between IL23R 1142G / A (Arg381Gln) and GM-CSF 3928 C / T (Ile117Thr) gene polymorphism in Iranian patients with tuberculosis disease

The DNA polymorphisms found in clinical strains of Mycobacterium tuberculosis drive altered physiology, virulence, and pathogenesis in them. This study aimed to investigate the association between IL23R 1142 G/A (Arg381Gln) and GM-CSF 3928 C/T (Ile117Thr) gene polymorphisms with the incidence rate of tuberculosis in the population of Sistan. This study was based on the descriptive and applied type. All patients with active pulmonary tuberculosis were referred to the tuberculosis center of Zabol city for one year, with an equal number of healthy people adapted to the patients examined in terms of age. After data collecting to compare the frequency of polymorphisms, the chi-square test and OR index were used using SPSS software version 16. We have found that the IL23R reduced-function allele 1142A and genotypes CC and TC were overrepresented, especially in the Pad subgroup compared with the control group (44% versus 42%, 21% versus 22%, and 44% versus 39%, respectively. Increased risks of TB with minimal/moderate lung involvement, respectively. Our results demonstrate that the reduced-function polymorphism 1142G ¡ A encoded by IL23R influences the outcome of disease severity of active pulmonary TB in ZABOL patients. The genotypic and allelic frequency of IL23R 1142 G/A, and GM-CSF 3928 C/T (Ile117Thr) polymorphism in patients with tuberculosis was significantly different from the control group and this polymorphism was associated with the incidence of tuberculosis in the population of Sistan.

A high-frequency single nucleotide polymorphism in the MtrB sensor kinase in clinical strains of Mycobacterium tuberculosis alters its biochemical and physiological properties.

The DNA polymorphisms found in clinical strains of Mycobacterium tuberculosis drive altered physiology, virulence, and pathogenesis in them. Although the lineages of these clinical strains can be traced back to common ancestor/s, there exists a plethora of difference between them, compared to those that have evolved in the laboratory. We identify a mutation present in ~80% of clinical strains, which maps in the HATPase domain of the sensor kinase MtrB and alters kinase and phosphatase activities, and affects its physiological role. The changes conferred by the mutation were probed by in-vitro biochemical assays which revealed changes in signaling properties of the sensor kinase. These changes also affect bacterial cell division rates, size and membrane properties. The study highlights the impact of DNA polymorphisms on the pathophysiology of clinical strains and provides insights into underlying mechanisms that drive signal transduction in pathogenic bacteria.

Molecular Analysis of Streptomycin Resistance Genes in Clinical Strains of Mycobacterium tuberculosis and Biocomputational Analysis of the MtGidB L101F Variant.

Globally, tuberculosis (TB) remains a prevalent threat to public health. In 2019, TB affected 10 million people and caused 1.4 million deaths. The major challenge for controlling this infectious disease is the emergence and spread of drug-resistant Mycobacterium tuberculosis, the causative agent of TB. The antibiotic streptomycin is not a current first-line anti-TB drug. However, WHO recommends its use in patients infected with a streptomycin-sensitive strain. Several mutations in the M. tuberculosis rpsL, rrs and gidB genes have proved association with streptomycin resistance. In this study, we performed a molecular analysis of these genes in clinical isolates to determine the prevalence of known or novel mutations. Here, we describe the genetic analysis outcome. Furthermore, a biocomputational analysis of the MtGidB L101F variant, the product of a novel mutation detected in gidB during molecular analysis, is also reported as a theoretical approach to study the apparent genotype-phenotype association.

In silico mutational analysis in RNA polymerase β subunit (rpoB) gene of rifampicin-resistant in Mycobacterium tuberculosis from Malaysia

Tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis (MTB) and remains as a key public health problem worldwide. Most of MTB clinical strains are resistant to rifampicin (RIF), the first-line anti-tuberculosis drug. RIF resistance to MTB is due to mutations that mainly found in RIF resistance-determining region (RRDR) in drug target gene, RNA polymerase β subunit (rpoB). Therefore, the aim of the study is to extend the identification of variants in rpoB gene and to elucidate the effect of variants to the RIF resistance. Four of the strains, MTBR1/09, MTBR2/09, MTBR3/09 and MTB221/11 were subjected to drug susceptibility test (DST). All of the strains sequenced and submitted to the National Center for Biotechnology Information Sequence Read Archive were analyzed to identify the variants in the rpoB gene. The identified new variants were then subjected to docking to examine the drug-protein interactions. DST analysis revealed MTBR1/09, MTBR2/09 and MTBR3/09 were resistant to the RIF drug, while MTB221/11 was a presumptive susceptible strain. Two new variants were observed, the deletion (Phe433_Met434delinsLeu in MTBR1/09) and missense (Lys37Arg in MTBR3/09) variants. Meanwhile, the His445Leu, Ser450Leu, Asp103Asp, Ala1075Ala were reported variants. Docking of RIF to MTBR1/09 and MTBR3/09 mutant models revealed the RIF bound to the RIF binding site at different binding affinity and conformation. Concurrently, the new variants caused the RIF to bind to the different active site and neighboring residues. Findings from DST and docking analyses indicate that new variants potentially disturb the RIF inhibition in RpoB mutant proteins, and thus might be responsible to cause the RIF resistance.

Predicted Structural Variability of Mycobacterium tuberculosis PPE18 Protein With Immunological Implications Among Clinical Strains

Recent advancements in vaccinology have led to the development of the M72/AS01E subunit vaccine, of which the major component is the Mycobacterium tuberculosis (MTB) PPE18 protein. Previous studies have demonstrated the genetic variability of the gene encoding PPE18 protein and the resulting peptide changes in diverse clinical strains of MTB; however, none have modeled the structural changes resulting from these peptide changes and their immunological implications. In this study, we investigated the structural predictions of 29 variant PPE18 proteins previously reported. We found evidence that PPE18 is at least a two-domain protein, with a highly conserved first domain and a largely variable second domain that has different coevolutionary clusters. Further, we investigated putative epitope sites in the clinical variants of PPE18 using prediction software. We found a negative relationship between T-cell epitope number and residue variability, while B-cell epitope likelihood was positively correlated with residue variability. Moreover, we found far more residues in the second domain predicted to be B-cell epitopes compared with the first domain. These results suggest an important functional role of the first domain and a role in immune evasion for the second, which extends our knowledge base of the basic biology of the PPE18 protein and indicates the need for further study into non-traditional immunological responses to TB.

The Value of the inhA Mutation Detection in Predicting Ethionamide Resistance Using Melting Curve Technology.

ObjectiveThis study aims to analyze the correlation between gene inhA mutations by melting curve technology and phenotypic drug susceptibility (DST) results of ethionamide (ETH), and evaluate whether gene inhA mutations detection can serve as a molecular marker in predicting ETH resistance.MethodsA retrospective analysis was conducted on 382 strains of Mycobacterium tuberculosis (MTB) with the anti-tuberculosis drugs isoniazid (INH), rifampicin (RIF), ETH, and others. Phenotypic drug susceptibility and the results of inhA and katG genotypes (mutation and no mutation) were obtained using the melting curve technology MeltPro TB assay.ResultsOf the 382 clinical strains of MTB tested, 118 (30.9%) were resistant to INH, and 28 (7.3%) were resistant to ETH. Among the 28 phenotypically ETH-resistant strains, inhA mutations accounted for 42.9% (12/28). These ETH-resistant strains comprise 35.3% (12/34) of the 34 inhA mutant strains. Of 8 single inhA mutation strains (without katG or rpoB mutation), 4(50%) were resistant to INH; however, all of these 8 strains were sensitive to ETH.ConclusionThe inhA mutation test may not be a reliable predictor of ETH resistance. Mutant inhA strains are not necessarily resistant to ETH. The strains with single inhA mutation (without katG or rpoB mutation) may be effective for ETH treatment. The use of ETH in clinical medicine should be guided by gene (other than inhA alone) detection and phenotypic drug susceptibility testing.

Systematic Review of Mutations Associated with Isoniazid Resistance Points to Continuing Evolution and Subsequent Evasion of Molecular Detection, and Potential for Emergence of Multidrug Resistance in Clinical Strains of Mycobacterium tuberculosis.

Molecular testing is rapidly becoming an integral component of global tuberculosis (TB) control. Uncommon mechanisms of resistance escape detection by these platforms and undermine our ability to contain outbreaks. This article is a systematic review of published articles that reported isoniazid (INH) resistance-conferring mutations between September 2013 and December 2019. The genes katG, inhA, and fabG1, and the intergenic region oxyR'-ahpC were considered in this review. Fifty-two articles were included that described 9,306 clinical isolates (5,804 INH resistant [INHr] and 3,502 INH susceptible [INHs]) from 31 countries. The three most frequently mutated loci continue to be locus 315 of katG (katG315; n = 4,271), locus -15 of inhA (inhA-15; n = 787), and locus -8 of inhA (inhA-8; 106). However, the diagnostic value of inhA-8 is far lower than previously thought, as it only appears in 25 (0.4%) of the INHr isolates lacking the first two mutations. I catalogued 45 new loci (29 katG, nine inhA, and seven ahpC) associated with INH resistance and identified 59 loci (common to this and previous reviews) as a reliable basis for molecular diagnostics. Including all observed mutations provides a cumulative sensitivity of 85.6%. In 14.4% of resistant isolates, no mechanism of resistance was detected, making them likely to escape molecular detection, and in the case of INH monoresistance, likely to convert to multidrug-resistant TB (MDR-TB). Integrating the information cataloged in this study into current diagnostic tools is essential for combating the emergence of MDR-TB, and its exclusion can lead to an unintended selection against common mechanisms and to diversifying evolution. Observation of many low-frequency resistance-conferring mutations points to an advantage of whole-genome sequencing (WGS) for diagnostics. Finally, I provide five recommendations for future diagnostic platforms.

Genotypes of Mycobacterium tuberculosis isolates circulating in Shaanxi Province, China.

ObjectivesThe prevalence of drug-resistant TB in Shaanxi Province is higher than other areas. This study was aimed to investigate the genetic diversity and epidemiology of Mycobacterium tuberculosis clinical strains in Shaanxi Province, China.MethodsFrom January to December 2016, a total of 298 Mycobacterium tuberculosis clinical isolates from smear-positive pulmonary tuberculosis patients were genotyped by Mcspoligotyping and 15-locus VNTR.ResultsWe found that the Beijing family strains was the most prominent family(81.54%, 243/298). Other family strains included T family(9.06%, 27/298), U family(0.67%, 2/298), LAM9 family(0.34%, 1/298) and Manu family(0.34%, 1/298). The rates of multidrug-resistant (MDR) M.Tuberculosis, age, type of case and education between Beijing and non-Beijing family strains were not statistically different, while the distribution in the three different regions among these was statistically significant.VNTR results showed that strains were classified into 280 genotypes, and 33 (11.07%) strains could be grouped into 14 clusters. 11 of the 15-VNTR loci were highly or moderately discriminative according to the Hunter-Gaston discriminatory index.ConclusionsWe concluded that the Beijing family genotype was the most prevalent genotype and 15-locus VNTR typing might be suitable for genotyping of M. tuberculosis in Shaanxi Province. There was less association between Beijing family genotypes and drug resistance in our study area.