The genome of antibiotic-producing colonies of the Pelagophyte alga Chrysophaeum taylorii reveals a diverse and non-canonical capacity for secondary metabolism.

Chrysophaeum taylorii is a member of an understudied clade of marine algae that can be responsible for harmful coastal blooms and is known to accumulate bioactive natural products including antibiotics of the chrysophaentin class. Whole genome sequencing of laboratory-cultivated samples revealed an extensive and diverse complement of secondary metabolite biosynthetic genes in C. taylorii, alongside a small microbiome with a more limited biosynthetic potential. 16S microbiome analysis of laboratory cultured alongside wild-collected samples revealed several common taxa; however, analysis of biosynthetic genes suggested an algal origin for the chrysophaentins, possibly via one of several non-canonical polyketide synthase genes encoded within the genome.

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.

Screening Repurposed Antiviral Small Molecules as Antimycobacterial Compounds by a Lux-Based phoP Promoter-Reporter Platform.

The emergence of multidrug-resistant strains and hyper-virulent strains of Mycobacterium tuberculosis are big therapeutic challenges for tuberculosis (TB) control. Repurposing bioactive small-molecule compounds has recently become a new therapeutic approach against TB. This study aimed to identify novel anti-TB agents from a library of small-molecule compounds via a rapid screening system. A total of 320 small-molecule compounds were used to screen for their ability to suppress the expression of a key virulence gene, phop, of the M. tuberculosis complex using luminescence (lux)-based promoter-reporter platforms. The minimum inhibitory and bactericidal concentrations on drug-resistant M. tuberculosis and cytotoxicity to human macrophages were determined. RNA sequencing (RNA-seq) was conducted to determine the drug mechanisms of the selected compounds as novel antibiotics or anti-virulent agents against the M. tuberculosis complex. The results showed that six compounds displayed bactericidal activity against M. bovis BCG, of which Ebselen demonstrated the lowest cytotoxicity to macrophages and was considered as a potential antibiotic for TB. Another ten compounds did not inhibit the in vitro growth of the M. tuberculosis complex and six of them downregulated the expression of phoP/R significantly. Of these, ST-193 and ST-193 (hydrochloride) showed low cytotoxicity and were suggested to be potential anti-virulence agents for M. tuberculosis.

Lentzeacins A-E, New Bacterial-Derived 2,5- and 2,6-Disubstituted Pyrazines from a BGC-Rich Soil Bacterium Lentzea sp. GA3-008.

Pyrazines (1,4-diazirines) are an important group of natural products that have tremendous monetary value in the food and fragrance industries and can exhibit a wide range of biological effects including antineoplastic, antidiabetic and antibiotic activities. As part of a project investigating the secondary metabolites present in understudied and chemically rich Actinomycetes, we isolated a series of six pyrazines from a soil-derived Lentzea sp. GA3-008, four of which are new. Here we describe the structures of lentzeacins A-E (1, 3, 5 and 6) along with two known analogues (2 and 4) and the porphyrin zincphyrin. The structures were determined by NMR spectroscopy and HR-ESI-MS. The suite of compounds present in Lentzea sp. includes 2,5-disubstituted pyrazines (compounds 2, 4, and 6) together with the new 2,6-disubstituted isomers (compounds 1, 3 and 5), a chemical class that is uncommon. We used long-read Nanopore sequencing to assemble a draft genome sequence of Lentzea sp. which revealed the presence of 40 biosynthetic gene clusters. Analysis of classical di-modular and single module non-ribosomal peptide synthase genes, and cyclic dipeptide synthases narrows down the possibilities for the biosynthesis of the pyrazines present in this strain.

Genome-Guided Discovery of Natural Products through Multiplexed Low-Coverage Whole-Genome Sequencing of Soil Actinomycetes on Oxford Nanopore Flongle.

Genome mining is an important tool for discovery of new natural products; however, the number of publicly available genomes for natural product-rich microbes such as actinomycetes, relative to human pathogens with smaller genomes, is small. To obtain contiguous DNA assemblies and identify large (ca. 10 to greater than 100 kb) biosynthetic gene clusters (BGCs) with high GC (>70%) and high-repeat content, it is necessary to use long-read sequencing methods when sequencing actinomycete genomes. One of the hurdles to long-read sequencing is the higher cost. In the current study, we assessed Flongle, a recently launched platform by Oxford Nanopore Technologies, as a low-cost DNA sequencing option to obtain contiguous DNA assemblies and analyze BGCs. To make the workflow more cost-effective, we multiplexed up to four samples in a single Flongle sequencing experiment while expecting low-sequencing coverage per sample. We hypothesized that contiguous DNA assemblies might enable analysis of BGCs even at low sequencing depth. To assess the value of these assemblies, we collected high-resolution mass spectrometry data and conducted a multi-omics analysis to connect BGCs to secondary metabolites. In total, we assembled genomes for 20 distinct strains across seven sequencing experiments. In each experiment, 50% of the bases were in reads longer than 10 kb, which facilitated the assembly of reads into contigs with an average N50 value of 3.5 Mb. The programs antiSMASH and PRISM predicted 629 and 295 BGCs, respectively. We connected BGCs to metabolites for N,N-dimethyl cyclic-di-tryptophan, two novel lasso peptides, and three known actinomycete-associated siderophores, namely, mirubactin, heterobactin, and salinichelin. IMPORTANCE Short-read sequencing of GC-rich genomes such as those from actinomycetes results in a fragmented genome assembly and truncated biosynthetic gene clusters (often 10 to >100 kb long), which hinders our ability to understand the biosynthetic potential of a given strain and predict the molecules that can be produced. The current study demonstrates that contiguous DNA assemblies, suitable for analysis of BGCs, can be obtained through low-coverage, multiplexed sequencing on Flongle, which provides a new low-cost workflow ($30 to 40 per strain) for sequencing actinomycete strain libraries.

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.

MIRU-profiler: a rapid tool for determination of 24-loci MIRU-VNTR profiles from assembled genomes of Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) resulted in an estimated 1.7 million deaths in the year 2016. The disease is caused by the members of Mycobacterium tuberculosis complex, which includes Mycobacterium tuberculosis, Mycobacterium bovis and other closely related TB causing organisms. In order to understand the epidemiological dynamics of TB, national TB control programs often conduct standardized genotyping at 24 Mycobacterial-Interspersed-Repetitive-Units (MIRU)-Variable-Number-of-Tandem-Repeats (VNTR) loci. With the advent of next generation sequencing technology, whole-genome sequencing (WGS) has been widely used for studying TB transmission. However, an open-source software that can connect WGS and MIRU-VNTR typing is currently unavailable, which hinders interlaboratory communication. In this manuscript, we introduce the MIRU-profiler program which could be used for prediction of MIRU-VNTR profile from WGS of M. tuberculosis. IMPLEMENTATION: The MIRU-profiler is implemented in shell scripting language and depends on EMBOSS software. The in-silico workflow of MIRU-profiler is similar to those described in the laboratory manuals for genotyping M. tuberculosis. Given an input genome sequence, the MIRU-profiler computes alleles at the standard 24-loci based on in-silico PCR amplicon lengths. The final output is a tab-delimited text file detailing the 24-loci MIRU-VNTR pattern of the input sequence. VALIDATION: The MIRU-profiler was validated on four datasets: complete genomes from NCBI-GenBank (n = 11), complete genomes for locally isolated strains sequenced using PacBio (n = 4), complete genomes for BCG vaccine strains (n = 2) and draft genomes based on 250 bp paired-end Illumina reads (n = 106). RESULTS: The digital MIRU-VNTR results were identical to the experimental genotyping results for complete genomes of locally isolated strains, BCG vaccine strains and five out of 11 genomes from the NCBI-GenBank. For draft genomes based on short Illumina reads, 21 out of 24 loci were inferred with a high accuracy, while a number of inaccuracies were recorded for three specific loci (ETRA, QUB11b and QUB26). One of the unique features of the MIRU-profiler was its ability to process multiple genomes in a batch. This feature was tested on all complete M. tuberculosis genome (n = 157), for which results were successfully obtained in approximately 14 min. CONCLUSION: The MIRU-profiler is a rapid tool for inference of digital MIRU-VNTR profile from the assembled genome sequences. The tool can accurately infer repeat numbers at the standard 24 or 21/24 MIRU-VNTR loci from the complete or draft genomes respectively. Thus, the tool is expected to bridge the communication gap between the laboratories using WGS and those using the conventional MIRU-VNTR typing.

Comprehensive Evaluation of the MBT STAR-BL Module for Simultaneous Bacterial Identification and ß-Lactamase-Mediated Resistance Detection in Gram-Negative Rods from Cultured Isolates and Positive Blood Cultures.

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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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.

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.

The Use of Omic Technologies Applied to Traditional Chinese Medicine Research.

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.

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.