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.

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.

Natural products represent one of the most important reservoirs of structural and chemical diversity for the generation of leads in the drug development process. A growing number of researchers have shown interest in the development of drugs based on Chinese herbs. In this review, the use and potential of omic technologies as powerful tools in the modernization of traditional Chinese medicine are discussed. The analytical combination from each omic approach is crucial for understanding the working mechanisms of cells, tissues, organs, and organisms as well as the mechanisms of disease. Gradually, omic approaches have been introduced in every stage of the drug development process to generate high-quality Chinese medicine-based drugs. Finally, the future picture of the use of omic technologies is a promising tool and arena for further improvement in the modernization of traditional Chinese medicine.

Objective: This study evaluated the capability of a MALDI Biotyper system equipped with the newly introduced MBT STAR-BL module to simultaneously perform species identification and β-lactamase-mediated resistance detection in bacteremia -causing bacteria isolated from cultured isolates and patient-derived blood cultures (BCs).Methods: Two hundred retrospective cultured isolates and 153 prospective BCs containing Gram-negative rods (GNR) were collected and subjected to direct bacterial identification, followed by the measurement of β-lactamase activities against ampicillin, piperacillin, cefotaxime, ceftazidime, and meropenem using the MBT STAR-BL module. The results and turnaround times were compared with those of routine microbiological processing. All strains were also characterized by beta-lactamase PCR and sequencing.Results: Using the saponin-based extraction method, MALDI-TOF MS correctly identified bacteria in 116/134 (86.6%) monomicrobial BCs. The detection sensitivities for β-lactamase activities against ampicillin, piperacillin, third-generation cephalosporin and meropenem were 91.3, 100, 97.9, and 100% for cultured isolates, and 80.4, 100, 68.8, and 40% for monomicrobial BCs (n = 134) respectively. The overall specificities ranged from 91.5 to 100%. Furthermore, the MBT STAR-BL and conventional drug susceptibility test results were concordant in 14/19 (73.7%) polymicrobial cultures. Reducing the logRQ cut-off value from 0.4 to 0.2 increased the direct detection sensitivities for β-lactamase activities against ampicillin, cefotaxime and meropenem in BCs to 85.7, 87.5, and 100% respectively. The MBT STAR-BL test enabled the reporting of β-lactamase-producing GNR at 14.16 and 47.64 h before the interim and final reports of routine BCs processing, respectively, were available.Conclusion: The MALDI Biotyper system equipped with the MBT STAR-BL module enables the simultaneous rapid identification of bacterial species and β-lactamase-mediated resistance from BCs and cultured isolates. Adjustment of the logRQ cut-off value to 0.2 significantly increased the detection sensitivities for clinically important drug-resistant pathogens.

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.