Bacteria have evolved sophisticated mechanisms to inhibit the growth of competitors. One such mechanism involves type VI secretion systems, which bacteria can use to inject antibacterial toxins directly into neighbouring cells. Many of these toxins target the integrity of the cell envelope, but the full range of growth inhibitory mechanisms remains unknown. Here we identify a type VI secretion effector, Tas1, in the opportunistic pathogen Pseudomonas aeruginosa. The crystal structure of Tas1 shows that it is similar to enzymes that synthesize (p)ppGpp, a broadly conserved signalling molecule in bacteria that modulates cell growth rate, particularly in response to nutritional stress. However, Tas1 does not synthesize (p)ppGpp; instead, it pyrophosphorylates adenosine nucleotides to produce (p)ppApp at rates of nearly 180,000 molecules per minute. Consequently, the delivery of Tas1 into competitor cells drives rapid accumulation of (p)ppApp, depletion of ATP, and widespread dysregulation of essential metabolic pathways, thereby resulting in target cell death. Our findings reveal a previously undescribed mechanism for interbacterial antagonism and demonstrate a physiological role for the metabolite (p)ppApp in bacteria.
See the Commentary at Nature.
Alcock BP, Raphenya AR, Lau TTY, Tsang KK, Bouchard M, Edalatmand A, Huynh W, Nguyen A-LV, Cheng AA, Liu S, Min SY, Miroshnichenko A, Tran H-K, Werfalli RE, Nasir JA, Oloni M, Speicher DJ, Florescu A, Singh B, Faltyn M, Hernandez-Koutoucheva A, Sharma AN, Bordeleau E, Pawlowski AC, Zubyk HL, Dooley D, Griffiths E, Maguire F, Winsor GL, Beiko RG, Brinkman FSL, Hsiao WWL, Van Domselaar G, McArthur AG.
The Comprehensive Antibiotic Resistance Database (CARD; https://card.mcmaster.ca) is a curated resource providing reference DNA and protein sequences, detection models and bioinformatics tools on the molecular basis of bacterial antimicrobial resistance (AMR). CARD focuses on providing high-quality reference data and molecular sequences within a controlled vocabulary, the Antibiotic Resistance Ontology (ARO), designed by the CARD biocuration team to integrate with software development efforts for resistome analysis and prediction, such as CARD’s Resistance Gene Identifier (RGI) software. Since 2017, CARD has expanded through extensive curation of reference sequences, revision of the ontological structure, curation of over 500 new AMR detection models, development of a new classification paradigm and expansion of analytical tools. Most notably, a new Resistomes & Variants module provides analysis and statistical summary of in silico predicted resistance variants from 82 pathogens and over 100 000 genomes. By adding these resistance variants to CARD, we are able to summarize predicted resistance using the information included in CARD, identify trends in AMR mobility and determine previously undescribed and novel resistance variants. Here, we describe updates and recent expansions to CARD and its biocuration process, including new resources for community biocuration of AMR molecular reference data.
Dr. McArthur and PhD student Kara Tsang taught together at the 2019 MacData Institute Summer School, with Dr. McArthur reviewing biocuration and bioinformatics for genomic surviellence of antimicrobial resistance and Kara following up with a lecture on machine learning techniques to predict clinical antimicrobial resistance from raw genomic sequence.
Also congratulations to Kara for being awarded a 2019 Faculty of Health Sciences Graduate Programs Excellence Award!
Updated August 6, 2019: Congratulations to Kara for also winning an Ontario Graduate Scholarship!
Speicher, D.J., K. Luinstra, J. Maciejewski, K.K. Tsang, A.G. McArthur, & M. Smieja. 2019. Clostridioides difficile strain divergence over time. Oral presentation at the Association of Medical Microbiology and Infectious Disease Canada (AMMI Canada) & Canadian Association for Clinical Microbiology and Infectious Diseases (CACMID) Joint Annual Conference, Ottawa, Ontario.
Background: Clostridioides difficileinfection (CDI) is a serious hospital-associated infection with severe outbreaks caused by the hypervirulent NAP1/MLST-1 strain. Whole genome sequencing has shown that most outbreak strains are clonal whereas non-outbreaks display a wide diversity of strains. To examine strain diversity in clinical settings, a subset of C. difficileisolates from symptomatic CDI from an acute care hospital were compared to isolates from C. difficilecolonized (CDC) asymptomatic subjects from the same hospital.
Methods: A subset of PCR-positive stool samples from clinically confirmed CDI isolates from 2016 (13/110), 2017 (8/111), and 2018 (13/65), and CDC from 2017 (17/185) were cultured 3-times consecutively on CHROMagar™ C. difficile, sub-cultured on Columbia colistin-nalidixic acid (CNA) media, had DNA isolated, shotgun sequenced, and genome assembled for both MLST typing and genome-wide SNP phylogenetic analysis.
Results: Based on MLST profiles, the C. difficiletypes detected were diverse. Of the presumed binary toxin positive/NAP1 strains (i.e. PCR tcdA/tcdBpositive) 7/12 (58%) were NAP1/MLST-1 and 3/12 (25%) were NAP7/MLST-11. NAP1/MLST-1 was not detected in any CDC isolate. NAP4/MLST-2,14 were detected in 2016 (n=4), 2017 (n=2), 2018 (n=1), and in CDC isolates (n=3). MLST-42 was dominant in CDC isolates (5/17; 29%) and decreased in prevalence in CDI isolates over time (2016=4; 2017=0; 2018=1).
Conclusion: C. difficilestrains amongst both CDI and CDC individuals are highly divergent. Whilst molecular assays are misclassifying 25% of “NAP1” strains, both NAP1 and NAP7 are hypervirulent. The number of MLST-42 CDC isolates is concerning as it has been reported to be the most common strain causing CDI among U.S. adults. This highlights the need for continued genomic surveillance of both CDI and CDC individuals. Genome-wide SNP phylogenetic analysis is currently being performed.
The Comprehensive Antibiotic Resistance Database has been updated, http://card.mcmaster.ca
CARD Curation: Expanded MCR, OXA & IMP beta-lactamase, and macrolide phosphotransferase (MPH) sequence curation. Updated nomenclature for MPHs and drug resistant dihydrofolate reductases (dfr). Updated classification of ADC beta-lactamases.
Ontologies: Addition of 518 draft virulence ontology (VIRO) terms.
Prevalence, Resistomes, & Variants: Expansion to 82 pathogens (more Brucella species), 81,000+ resistomes, and 173,000+ AMR allele sequences based on sequence data acquired from NCBI on 28-Feb-2019, analyzed using RGI 4.2.2 (DIAMOND homolog detection) and CARD 3.0.1.
During McMaster Spring Mid-Term Recess (February 18-24), the McArthur lab is pleased to present a series of lectures, demonstrations, and training sessions for the Comprehensive Antibiotic Resistance Database (card.mcmaster.ca) and its associated Resistance Gene Identifier (RGI) software, sponsored by the Michael G. DeGroote Institute for Infectious Disease Research (IIDR).
Questions? Email firstname.lastname@example.org
Workshop & Lecture material will be available here: https://github.com/arpcard/state-of-the-card-2019
Dr. David Speicher has joined the McArthur Lab as our new Molecular Epidemiology Postdoctoral Fellow! David joins us via clinical epidemiology research in infectious disease at St. Joseph’s Healthcare Hamilton plus extensive training and experience in Cambodia, India, Sri Lanka, Kenya, and Australia. David has a depth of experience in infectious disease, virology, molecular biology, epidemiology and biostatistics, microbiology, and diagnostic techniques and will be leading infectious disease molecular epidemiology collaborations with McMaster Children’s Hospital, St. Joseph’s Healthcare Hamilton, and Hamilton Health Sciences with an emphasis on antimicrobial resistance, C. difficile, H. pylori, Shigella, Chlamydia trachomatis, and Mycoplasma genitalium. Hear Dr. Speicher talk about his research program on CFMU radio.
Some invitations are more special than others. Dr. Peixoto da Cruz and I went to graduate school together in British Columbia (a long time ago!) and while we have since lived in different hemispheres, the bond remains strong. It was great to visit PUG Goiás and learn about Peixoto’s impressive training program in genetic screening and counselling, plus talk about our AMR surveillance efforts.
Bioinformatics of antimicrobial resistance in the age of molecular epidemiology. Invited Keynote presentation by A.G. McArthur at Reunião de Citogenética do Brasil Central & XII Workshop de Genética da PUC Goiás, Goiânia, Brazil, October 2018.