Propionate prevents loss of the PDIM virulence lipid in Mycobacterium tuberculosis.

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous PDIM-negative mutants that have attenuated virulence and increased cell wall permeability, thus impacting the relevance of experimental findings. PDIM loss can also reduce the efficacy of the BCG Pasteur vaccine. Here we show that vancomycin susceptibility can rapidly screen for M. tuberculosis PDIM production. We find that metabolic deficiency of methylmalonyl-CoA impedes the growth of PDIM-producing bacilli, selecting for PDIM-negative variants. Supplementation with odd-chain fatty acids, cholesterol or vitamin B12 restores PDIM-positive bacterial growth. Specifically, we show that propionate supplementation enhances PDIM-producing bacterial growth and selects against PDIM-negative mutants, analogous to in vivo conditions. Our study provides a simple approach to screen for and maintain PDIM production, and reveals how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.

The PDIM paradox of Mycobacterium tuberculosis: new solutions to a persistent problem.

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous mutations that cause PDIM loss leading to virulence attenuation and increased cell wall permeability. We discovered that PDIM loss is due to a metabolic deficiency of methylmalonyl-CoA that impedes the growth of PDIM-producing bacilli. This can be remedied by supplementation with odd-chain fatty acids, cholesterol, or vitamin B12. We developed a much-needed facile and scalable routine assay for PDIM production and show that propionate supplementation enhances the growth of PDIM-producing bacilli and selects against PDIM-negative mutants, analogous to in vivo conditions. Our results solve a major issue in tuberculosis research and exemplify how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.

Complete Genome Sequence of a New Zealand Mycobacterium tuberculosis Strain Responsible for Ongoing Transmission over the Past 30 Years.

We report here the complete genome sequence of Mycobacterium tuberculosis strain Colonial S-type 1 (CS1), which has been responsible for ongoing outbreaks of tuberculosis in New Zealand over the past 30 years. CS1 appears to be highly transmissible, with greater rates of progression to active disease, compared to other circulating M. tuberculosis strains; therefore, comparison of its genomic content is of interest.

Are all antibiotic persisters created equal?

Antibiotic persisters are a sub-population of bacteria able to survive in the presence of bactericidal antibiotic despite the lack of heritable drug resistance mechanisms. This phenomenon exists across many bacterial species and is observed for many different antibiotics. Though these bacteria are often described as "multidrug persisters" very few experiments have been carried out to determine the homogeneity of a persister population to different drugs. Further, there is much debate in the field as to the origins of a persister cell. Is it formed spontaneously? Does it form in response to stress? These questions are particularly pressing in the field of Mycobacterium tuberculosis, where persisters may play a crucial role in the required length of treatment and the development of multidrug resistant organisms. Here we aim to interpret the known mechanisms of antibiotic persistence and how they may relate to improving treatments for M. tuberculosis, exposing the gaps in knowledge that prevent us from answering the question: Are all antibiotic persisters created equal?

Capturing Structural Variants of Herpes Simplex Virus Genome in Full Length by Oxford Nanopore Sequencing.

Genome sequencing and assembly of viral genomes within the Herpesviridae family, particularly herpes simplex virus (HSV), have been challenging due to the large size (~154 Kb), high GC content (68%), and nucleotide variations arising during replication. Oxford Nanopore Technology (ONT) has been successful in obtaining read lengths ranging from 100 Kb up to 2.3 Mb. We have optimized DNA extraction and sequencing with ONT to capture the whole genome of HSV-1 as a single read. Although previous studies described the presence of four different genome isomers of HSV, we provided the first report on capturing all four variants' full-length genome as single reads. These isomers were found to be present in almost equal proportion in the sequenced DNA preparation. IMPORTANCE With the advent of next-generation sequencing platforms, genome sequencing of viruses can be performed in a relatively shorter time frame in even the most austere conditions. Ultralong read sequencing platforms, such as Oxford Nanopore Technology (ONT), have made it possible to capture the full-length genome of DNA viruses as a single read. By optimizing ONT for this purpose, we captured the genome (~154 Kb) of a clinical strain of herpes simplex virus 1 (HSV-1). Additionally, we captured full-length sequences of the four isomers of lab-grown HSV-1 virus and were able to determine the frequency of each within the isogenic population. This method will open new directions in studying the significance of these isomers and their clinical relevance to HSV-1 infections. It will also improve basic studies on the recombination and replication of this virus.

Combining CRISPRi and metabolomics for functional annotation of compound libraries.

Molecular profiling of small molecules offers invaluable insights into the function of compounds and allows for hypothesis generation about small-molecule direct targets and secondary effects. However, current profiling methods are limited in either the number of measurable parameters or throughput. Here we developed a multiplexed, unbiased framework that, by linking genetic to drug-induced changes in nearly a thousand metabolites, allows for high-throughput functional annotation of compound libraries in Escherichia coli. First, we generated a reference map of metabolic changes from CRISPR interference (CRISPRi) with 352 genes in all major essential biological processes. Next, on the basis of the comparison of genetic changes with 1,342 drug-induced metabolic changes, we made de novo predictions of compound functionality and revealed antibacterials with unconventional modes of action (MoAs). We show that our framework, combining dynamic gene silencing with metabolomics, can be adapted as a general strategy for comprehensive high-throughput analysis of compound functionality from bacteria to human cell lines.

Nanoluciferase Reporter Mycobacteriophage for Sensitive and Rapid Detection of Mycobacterium tuberculosis Drug Susceptibility.

Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains.IMPORTANCEMycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

Dispersal of Mycobacterium tuberculosis Driven by Historical European Trade in the South Pacific.

Mycobacterium tuberculosis (Mtb) is a globally distributed bacterial pathogen whose population structure has largely been shaped by the activities of its obligate human host. Oceania was the last major global region to be reached by Europeans and is the last region for which the dispersal and evolution of Mtb remains largely unexplored. Here, we investigated the evolutionary history of the Euro-American L4.4 sublineage and its dispersal to the South Pacific. Using a phylodynamics approach and a dataset of 236 global Mtb L4.4 genomes we have traced the origins and dispersal of L4.4 strains to New Zealand. These strains are predominantly found in indigenous Maori and Pacific people and we identify a clade of European, likely French, origin that is prevalent in indigenous populations in both New Zealand and Canada. Molecular dating suggests the expansion of European trade networks in the early 19th century drove the dispersal of this clade to the South Pacific. We also identify historical and social factors within the region that have contributed to the local spread and expansion of these strains, including recent Pacific migrations to New Zealand and the rapid urbanization of Maori in the 20th century. Our results offer new insight into the expansion and dispersal of Mtb in the South Pacific and provide a striking example of the role of historical European migrations in the global dispersal of Mtb.

Rapid molecular diagnosis of the Mycobacterium tuberculosis Rangipo strain responsible for the largest recurring TB cluster in New Zealand.

Despite New Zealand being a low-tuberculosis (TB) burden country, there are disproportionately high rates of TB in particular populations. Here, we report a rapid molecular diagnosis of the Mycobacterium tuberculosis Rangipo strain responsible for the largest recurring TB cluster in New Zealand.