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.

Long-Read Sequencing with Hierarchical Clustering for Antiretroviral Resistance Profiling of Mixed Human Immunodeficiency Virus Quasispecies.

BACKGROUND: HIV infections often develop drug resistance mutations (DRMs), which can increase the risk of virological failure. However, it has been difficult to determine if minor mutations occur in the same genome or in different virions using Sanger sequencing and short-read sequencing methods. Oxford Nanopore Technologies (ONT) sequencing may improve antiretroviral resistance profiling by allowing for long-read clustering. METHODS: A new ONT sequencing-based method for profiling DRMs in HIV quasispecies was developed and validated. The method used hierarchical clustering of long amplicons that cover regions associated with different types of antiretroviral drugs. A gradient series of an HIV plasmid and 2 plasma samples was prepared to validate the clustering performance. The ONT results were compared to those obtained with Sanger sequencing and Illumina sequencing in 77 HIV-positive plasma samples to evaluate the diagnostic performance. RESULTS: In the validation study, the abundance of detected quasispecies was concordant with the predicted result with the R2 of > 0.99. During the diagnostic evaluation, 59/77 samples were successfully sequenced for DRMs. Among 18 failed samples, 17 were below the limit of detection of 303.9 copies/µL. Based on the receiver operating characteristic analysis, the ONT workflow achieved an F1 score of 0.96 with a cutoff of 0.4 variant allele frequency. Four cases were found to have quasispecies with DRMs, in which 2 harbored quasispecies with more than one class of DRMs. Treatment modifications were recommended for these cases. CONCLUSIONS: Long-read sequencing coupled with hierarchical clustering could differentiate the quasispecies resistance profiles in HIV-infected samples, providing a clearer picture for medical care.

Evaluation of Mycobacterium tuberculosis enrichment in metagenomic samples using ONT adaptive sequencing and amplicon sequencing for identification and variant calling.

Sensitive detection of Mycobacterium tuberculosis (TB) in small percentages in metagenomic samples is essential for microbial classification and drug resistance prediction. However, traditional methods, such as bacterial culture and microscopy, are time-consuming and sometimes have limited TB detection sensitivity. Oxford nanopore technologies (ONT) MinION sequencing allows rapid and simple sample preparation for sequencing. Its recently developed adaptive sequencing selects reads from targets while allowing real-time base-calling to achieve sequence enrichment or depletion during sequencing. Another common enrichment method is PCR amplification of the target TB genes. In this study, we compared both methods using ONT MinION sequencing for TB detection and variant calling in metagenomic samples using both simulation runs and those with synthetic and patient samples. We found that both methods effectively enrich TB reads from a high percentage of human (95%) and other microbial DNA. Adaptive sequencing with readfish and UNCALLDE achieved a 3.9-fold and 2.2-fold enrichment compared to the control run. We provide a simple automatic analysis framework to support the detection of TB for clinical use, openly available at https://github.com/HKU-BAL/ONT-TB-NF . Depending on the patient's medical condition and sample type, we recommend users evaluate and optimize their workflow for different clinical specimens to improve the detection limit.

Clinical utility of target amplicon sequencing test for rapid diagnosis of drug-resistant Mycobacterium tuberculosis from respiratory specimens.

An in-house-developed target amplicon sequencing by next-generation sequencing technology (TB-NGS) enables simultaneous detection of resistance-related mutations in Mycobacterium tuberculosis (MTB) against 8 anti-tuberculosis drug classes. In this multi-center study, we investigated the clinical utility of incorporating TB-NGS for rapid drug-resistant MTB detection in high endemic regions in southeast China. From January 2018 to November 2019, 4,047 respiratory specimens were available from patients suffering lower respiratory tract infections in Hong Kong and Guangzhou, among which 501 were TB-positive as detected by in-house IS6110-qPCR assay with diagnostic sensitivity and specificity of 97.9 and 99.2%, respectively. Preliminary resistance screening by GenoType MTBDRplus and MTBDRsl identified 25 drug-resistant specimens including 10 multidrug-resistant TB. TB-NGS was performed using MiSeq on all drug-resistant specimens alongside 67 pan-susceptible specimens, and demonstrated 100% concordance to phenotypic drug susceptibility test. All phenotypically resistant specimens with dominating resistance-related mutations exhibited a mutation frequency of over 60%. Three quasispecies were identified with mutation frequency of less than 35% among phenotypically susceptible specimens. They were well distinguished from phenotypically resistant cases and thus would not complicate TB-NGS results interpretations. This is the first large-scale study that explored the use of laboratory-developed NGS platforms for rapid TB diagnosis. By incorporating TB-NGS with our proposed diagnostic algorithm, the workflow would provide a user-friendly, cost-effective routine diagnostic solution for complicated TB cases with an average turnaround time of 6 working days. This is critical for timely management of drug resistant TB patients and expediting public health control on the emergence of drug-resistant TB.

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

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.

Nosocomial Outbreak of Coronavirus Disease 2019 by Possible Airborne Transmission Leading to a Superspreading Event.

BACKGROUND: Nosocomial outbreaks with superspreading of coronavirus disease 2019 due to a possible airborne transmission have not been reported. METHODS: Epidemiological analysis, environmental samplings, and whole-genome sequencing (WGS) were performed for a hospital outbreak. RESULTS: A superspreading event that involved 12 patients and 9 healthcare workers (HCWs) occurred within 9 days in 3 of 6 cubicles at an old-fashioned general ward with no air exhaust built within the cubicles. The environmental contamination by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was significantly higher in air grilles (>2 m from patients' heads and not within reach) than on high-touch clinical surfaces (36.4%, 8 of 22 vs 3.4%, 1 of 29, P = .003). Six (66.7%) of 9 contaminated air exhaust grilles were located outside patient cubicles. The clinical attack rate of patients was significantly higher than of HCWs (15.4%, 12 of 78 exposed patients vs 4.6%, 9 of 195 exposed HCWs, P = .005). Moreover, the clinical attack rate of ward-based HCWs was significantly higher than of nonward-based HCWs (8.1%, 7 of 68 vs 1.8%, 2 of 109, P = .045). The episodes (mean ±â€…standard deviation) of patient-care duty assignment in the cubicles was significantly higher among infected ward-based HCWs than among noninfected ward-based HCWs (6.0 ±â€…2.4 vs 3.0 ±â€…2.9, P = .012) during the outbreak period. The outbreak strains belong to SARS-CoV-2 lineage B.1.36.27 (GISAID clade GH) with the unique S-T470N mutation on WGS. CONCLUSIONS: This nosocomial point source superspreading event due to possible airborne transmission demonstrates the need for stringent SARS-CoV-2 screening at admission to healthcare facilities and better architectural design of ventilation systems to prevent such outbreaks. Portable high-efficiency particulate filters were installed in each cubicle to improve ventilation before resumption of clinical service.

Territorywide Study of Early Coronavirus Disease Outbreak, Hong Kong, China.

Initial cases of coronavirus disease in Hong Kong were imported from mainland China. A dramatic increase in case numbers was seen in February 2020. Most case-patients had no recent travel history, suggesting the presence of transmission chains in the local community. We collected demographic, clinical, and epidemiologic data from 50 patients, who accounted for 53.8% of total reported case-patients as of February 28, 2020. We performed whole-genome sequencing to determine phylogenetic relationship and transmission dynamics of severe acute respiratory syndrome coronavirus 2 infections. By using phylogenetic analysis, we attributed the community outbreak to 2 lineages; 1 harbored a common mutation, Orf3a-G251V, and accounted for 88.0% of the cases in our study. The estimated time to the most recent common ancestor of local coronavirus disease outbreak was December 24, 2019, with an evolutionary rate of 3.04 × 10-3 substitutions/site/year. The reproduction number was 1.84, indicating ongoing community spread.

Targeted-Sequencing Workflows for Comprehensive Drug Resistance Profiling of Mycobacterium tuberculosis Cultures Using Two Commercial Sequencing Platforms: Comparison of Analytical and Diagnostic Performance, Turnaround Time, and Cost.

BACKGROUND: The emergence of Mycobacterium tuberculosis with complex drug resistance profiles necessitates a rapid and comprehensive drug susceptibility test for guidance of patient treatment. We developed two targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the prediction of drug resistance in M. tuberculosis toward 12 antibiotics. METHODS: A total of 163 M. tuberculosis isolates collected from Hong Kong and Ethiopia were subjected to a multiplex PCR for simultaneous amplification of 19 drug resistance-associated genetic regions. The amplicons were then barcoded and sequenced in parallel on MiSeq and MinION in respective batch sizes of 24 and 12 samples. A web-based bioinformatics pipeline, BacterioChek-TB, was developed to translate the raw datasets into clinician-friendly reports. RESULTS: Both platforms successfully sequenced all samples with mean read depths of 1,127× and 1,649×, respectively. The variant calling by MiSeq and MinION could achieve 100% agreement if variants with an allele frequency of <40% reported by MinION were excluded. Both workflows achieved a mean clinical sensitivity of 94.8% and clinical specificity of 98.0% when compared with phenotypic drug susceptibility test (pDST). Turnaround times for the MiSeq and MinION workflows were 38 and 15 h, facilitating the delivery of treatment guidance at least 17-18 days earlier than pDST, respectively. The higher cost per sample on the MinION platform ($71.56) versus the MiSeq platform ($67.83) was attributed to differences in batching capabilities. CONCLUSION: Our study demonstrates the interchangeability of MiSeq and MinION platforms for generation of accurate and actionable results for the treatment of tuberculosis.

Direct Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis by Use of pncA PCR Sequencing.

An in-house-developed pncA sequencing assay for analysis of pyrazinamide (PZA) resistance was evaluated using 162 archived Mycobacterium tuberculosis complex (MTBC) isolates with phenotypic PZA susceptibility profiles that were well defined by analysis of Bactec MGIT 960 PZA kit and PZase activity data. Preliminary results showed 100% concordance between pncA sequencing and phenotypic PZA drug susceptibility test (DST) results among archived isolates. Also, 637 respiratory specimens were prospectively collected, and 158 were reported as MTBC positive by the Abbott Realtime MTB assay (96.3% sensitivity [95% confidence interval {CI}: 92.2% to 98.7%]; 100% specificity [95% CI: 99.2% to 100.0%]). Genotypic and phenotypic PZA resistance profiles of these 158 MTBC-positive specimens were analyzed by pncA sequencing and Bactec MGIT 960 PZA kit, respectively. For analysis of PZA resistance, pncA sequencing detected pncA mutations in 5/5 (100%) phenotypic PZA-resistant respiratory specimens within 4 working days. No pncA mutations were detected among PZA-susceptible specimens. Combining archived isolates with prospective specimens, 27 were identified as phenotypic PZA resistant with pncA mutation. Among these 27 samples, 6/27 (22.2%) phenotypic PZA-resistant strains carried novel pncA mutations without rpsA and panD mutations. These included 5 with mutations (a deletion [Del] at 383T [Del383T], Del 380 to 390, insertion of A [A Ins] at position 127, A Ins at position 407, and G Ins at position 508) in pncA structural genes and 1 with a mutation (T-12C) at the pncA promoter region. All six of these strains had no or reduced PZase activities, indicating that the novel mutations might confer PZA resistance. Additionally, 25/27 phenotypic PZA-resistant strains were confirmed multidrug-resistant tuberculosis (MDR-TB) strains. As PZA is commonly used in MDR-TB treatment regimens, direct pncA sequencing will rapidly detect PZA resistance and facilitate judicious use of PZA in treating PZA-susceptible MDR-TB.

Molecular Characterization of HIV-1 Minority Subtypes in Hong Kong: A Recent Epidemic of CRF07_BC among the Men who have Sex with Men Population.

BACKGROUND: Over the past years, an increasing trend was noticed for non-B and non- CRF01_AE HIV-1 strains prevalence in Hong Kong. OBJECTIVE: In this study, we aimed at using the available HIV-1 pol sequences collected from 1994 to 2013 through our local antiretroviral resistance surveillance program to investigate the molecular epidemiology and evolution of HIV-1 minority subtypes in Hong Kong. We also aimed at investigating their potential association and impact of those transmission risk groups. METHODS: A total of 2,315 HIV-1 partial pol sequences were included. HIV-1 genotypes were determined by REGA Genotyping Tool and phylogenetic analysis with reference sequences. The viral evolutionary rates and time of the most common ancestor (tMRCA) were estimated by Bayesian Markov Chain Monte Carlo (MCMC) interference. RESULTS: Apart from the two prevalent HIV-1 genotypes in Hong Kong (subtype B,41.6%, CRF01_AE,40.5%), phylogenetic analysis revealed a broad viral diversity including CRF07_BC(5.1%), subtype C(4.5%), CRF02_AG(1.1%), CRF08_BC(0.8%), subtype A1(0.8%), subtype G(0.4%), subtype D(0.4%), CRF06_cpx(0.4%), subtype F(0.1%), CRF12_BF(0·04%) and other recombinants(4.5%). The top five minority subtypes were further analyzed which demonstrated distinct epidemiological and phylogenetic patterns. Over 70% of subtypes A1, C and CRF02_AG infections were circulated among non-Chinese Asians or African community in Hong Kong and were mainly transmitted between heterosexual regular partners. Instead, over 90% of CRF07_BC and CRF08_BC patients were Chinese. An epidemic cluster was identified in CRF07_BC and estimated to expand from 2002 onwards based on skyline plot and molecular clock analysis. CONCLUSION: Our results highlighted the emergence of CRF07_BC epidemic in local MSM community, public health interventions targeting the community should be further enhanced to tackle the epidemic.

Diagnostic evaluation of an in-house developed single-tube, duplex, nested IS6110 real-time PCR assay for rapid pulmonary tuberculosis diagnosis.

OBJECTIVE: To perform a prospective evaluation on the diagnostic performance of an in-house developed, duplex nested IS6110 real-time Polymerase-Chain-Reaction (PCR) assay (IS6110-qPCR assay) for rapid pulmonary TB diagnosis. METHODS: A total of 503 sputum specimens were prospectively collected from July 2016 to November 2016. Diagnostic accuracy and optimal cut-off Cycle-threshold (Ct) value for IS6110-qPCR assay was determined by Receiver Operating Characteristic (ROC) curve. Using the optimal cut-off Ct, diagnostic performance of IS6110-qPCR assay was assessed with reference to both bacteriological and clinical information. Meanwhile, limit of detection (LOD) was calculated using Mycobacterium tuberculosis H37Rv as reference strain. RESULT: ROC curve analysis of IS6110-qPCR assay showed a high Area Under the Curve (AUC) value (0.948) with optimal Ct value at 24.140. Prospective analysis of IS6110-qPCR assay with cut-off Ct = 24.140 showed a high overall sensitivity and specificity of 97.2% and 99.7%, respectively. No cross reactivity was observed among all non-tuberculous mycobacteria specimens in this study. LOD analysis on MTB-spiked sputum showed an average detection limit of 5.0 CFU/mL at Ct = 23.18 (±SD, 0.57). CONCLUSION: IS6110-qPCR assay is a highly accurate and cost-effective assay developed for primary screening of suspected TB cases, which is particularly suitable for regions with limited resources but high TB burden.

Whole genome sequencing data of 1110 Mycobacterium tuberculosis isolates identifies insertions and deletions associated with drug resistance.

BACKGROUND: Drug resistance in Mycobacterium tuberculosis (MTB) is one of the major challenges in tuberculosis (TB) treatment. However, known mutations cannot explain all of the cases of resistance and little research has focused on the relationship between insertions / deletions (indels) and drug resistance. RESULTS: Here, we retrieved whole genome sequencing data of 743 drug-resistant MTB strains and 367 pan-susceptible strains from TB patients from the public domain to identify novel genomic markers of drug resistance. A total of 20 region markers containing genes and intergenic regions (IGRs) with significant statistical correlation with antibiotic resistance were revealed, four of which have been previously reported to be associated with drug resistance. In addition, 83 point markers containing frameshift (FS) mutations and IGR indels were also identified independently based on differences in their incidence rates between drug-sensitive and -resistant strains. Among the 83 point markers, eight indels were detected in known drug-associated genes or IGRs. Furthermore, the overlap between 20 region markers and 83 point markers further indicated their associations with drug resistance. The markers identified were involved in essential bacterial metabolic functions, including cell wall and transmembrane transporter functions. A strong correlation between FS mutations and mutations in DNA repair genes including I21V in alkA, R48G in mutT4 and P2R in nth was also found. CONCLUSIONS: This study identified a set of novel genetic markers with FS mutations and IGR indels associated with MTB drug resistance, which greatly broadens the pool of mutations related to MTB drug resistance. This insight may be important in identifying novel mechanisms of drug resistance in MTB.

Comparative Genomic Analysis of Two Clonally Related Multidrug Resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing.

Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB under in vivo challenge of anti-TB drugs. Result: Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations at rpoB S531L, inhA C-15T, and embB M306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 of rv0888 in pre-treatment strain, and a missense mutation at rv3303c (lpdA) V44I and a 6-bp inframe deletion at codon 67-68 in rv2071c (cobM) in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function. Conclusion: This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations at rv0888, lpdA, and cobM that might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.

Direct detection of Mycobacterium tuberculosis and drug resistance in respiratory specimen using Abbott Realtime MTB detection and RIF/INH resistance assay.

Abbott RealTime MTB (Abbott-RT) in conjunction with Abbott RealTime MTB RIF/INH Resistance (Abbott-RIF/INH) is a new, high-throughput automated nucleic acid amplification platform (Abbott-MDR) for detection of Mycobacterium tuberculosis complex (MTBC) and the genotypic markers for rifampicin (RIF) and isoniazid (INH) resistance directly from respiratory specimens. This prospective study evaluated the diagnostic performance of this new platform for MTBC and multidrug-resistant tuberculosis (MDR-TB) using 610 sputum specimens in a tuberculosis high-burden setting. Using conventional culture results and clinical background as reference standards, Abbott-RT exhibited an overall sensitivity and specificity of 95.2% and 99.8%, respectively. Genotypic RIF/INH resistance of 178 "MTB detected" specimens was subsequently analyzed by Abbott-RIF/INH. Compared to phenotypic drug susceptibility test results, Abbott-RIF/INH detected resistance genotypic markers in 84.6% MDR-TB, 80% mono-RIF-resistant and 66.7% mono-INH-resistant specimens. Two of the RIF-resistant specimens carried a novel single, nonsense mutation at rpoB Q513 and in silico simulation demonstrated that the truncated RpoB protein failed to bind with other subunits for transcription. Overall, Abbott-MDR platform provided high throughput and reliable diagnosis of MDR-TB within a TB high-burden region.

Comparative Whole-Genomic Analysis of an Ancient L2 Lineage Mycobacterium tuberculosis Reveals a Novel Phylogenetic Clade and Common Genetic Determinants of Hypervirulent Strains.

Background: Development of improved therapeutics against tuberculosis (TB) is hindered by an inadequate understanding of the relationship between disease severity and genetic diversity of its causative agent, Mycobacterium tuberculosis. We previously isolated a hypervirulent M. tuberculosis strain H112 from an HIV-negative patient with an aggressive disease progression from pulmonary TB to tuberculous meningitis-the most severe manifestation of tuberculosis. Human macrophage challenge experiment demonstrated that the strain H112 exhibited significantly better intracellular survivability and induced lower level of TNF-α than the reference virulent strain H37Rv and other 123 clinical isolates. Aim: The present study aimed to identify the potential genetic determinants of mycobacterial virulence that were common to strain H112 and hypervirulent M. tuberculosis strains of the same phylogenetic clade isolated in other global regions. Methods: A low-virulent M. tuberculosis strain H54 which belonged to the same phylogenetic lineage (L2) as strain H112 was selected from a collection of 115 clinical isolates. Both H112 and H54 were whole-genome-sequenced using PacBio sequencing technology. A comparative genomics approach was adopted to identify mutations present in strain H112 but absent in strain H54. Subsequently, an extensive phylogenetic analysis was conducted by including all publically available M. tuberculosis genomes. Single-nucleotide-polymorphisms (SNPs) and structural variations (SVs) common to hypervirulent strains in the global collection of genomes were considered as potential genetic determinants of hypervirulence. Results:Sequencing data revealed that both H112 and H54 were identified as members of the same sub-lineage L2.2.1. After excluding the lineage-related mutations shared between H112 and H54, we analyzed the phylogenetic relatedness of H112 with global collection of M. tuberculosis genomes (n = 4,338), and identified a novel phylogenetic clade in which four hypervirulent strains isolated from geographically diverse regions were clustered together. All hypervirulent strains in the clade shared 12 SNPs and 5 SVs with H112, including those affecting key virulence-associated loci, notably, a deleterious SNP (rv0178 p. D150E) within mce1 operon and an intergenic deletion (854259_ 854261delCC) in close-proximity to phoP. Conclusion: The present study identified common genetic factors in a novel phylogenetic clade of hypervirulent M. tuberculosis. The causative role of these mutations in mycobacterial virulence should be validated in future study.

Phylogenetic and Structural Significance of Dihydrofolate Synthase (folC) Mutations in Drug-Resistant Mycobacterium tuberculosis.

The aim of this study is to investigate the mutation pattern of the folC gene in drug-resistant Mycobacterium tuberculosis (MTB) clinical isolates of global and Hong Kong cohorts. The public sequence read archives of 1,124 MTB genomes from three independent studies were retrieved and folC mutations existing solely in drug-resistant MTB strains were identified. A phylogenetic tree was constructed to analyze the segregation of mutation-related amino acid residues in the FolC structure. These mutation sites were further supported by direct Sanger sequencing of the folC gene among 254 clinical MTB isolates in a Hong Kong cohort. Homology modeling of wild-type and mutated FolC was performed, and the predicted structures were docked with hydroxydihydropteroate, the metabolic derivative of para-aminosalicylic acid (PAS), to evaluate the resultant binding affinity changes. Combining the results of three previous cohorts and our cohort, E40, I43, S150, and E153 are the most frequently affected amino acid residues in resistant isolates. Based on the distribution of mutations in the genome-based phylogenetic tree, lineage-specific mutation patterns were observed. Regarding the segregation of affected amino acid residues, the four most frequently affected residues are all in close proximity of the binding pocket for the PAS derivative. Molecular modeling results showed that mutations at E40, I43, and S150 can alter the structure of FolC putative binding pocket, causing the PAS derivative to bind outside of the now deformed pocket. This might ablate the interaction between the protein and the PAS derivative. To conclude, this study is the first comprehensive mutation pattern and bioinformatics analysis of the folC gene in MTB drug-resistant isolates. The distribution of mutations in phylogenetic lineages and protein structure is reported, analyzed, and discussed.

The involvement of DNA methylation and histone modification on the epigenetic regulation of embryonic stem cells and induced pluripotent stem cells.

Stem cell research has been developing rapidly in diverse areas such as the fields of genetics and molecular biology over the past decades. Genomic studies on both embryonic stem cells (ESCs) and terminally-differentiated cells illustrated that factors apart from their hereditary information disparity are associated with gene expression patterns of ESCs. Therefore, current research is trying to explore the effects of epigenetic processes in stem cell physiology and phenotypic changes. In-depth analyses of the molecular mechanisms underpinning such epigenetic-mediated functions have also been conducted. These findings suggest the importance of understanding the epigenetic influences in stem cell activities. Accordingly this review will describe the regulatory machineries of stem cells development targeting the two epigenetic processes: (1) DNA methylation and (2) histones modification. In addition, up-to-date findings concerning the functional roles of these processes in stem cells homeostasis will be covered.