The identification and association of the nucleotide sequences encoding antibiotic resistance elements is critical to improve surveillance and monitor trends in antibiotic resistance. Current methods to study antibiotic resistance in various environments rely on extensive deep sequencing or laborious culturing of fastidious organisms, which are both heavily time-consuming operations. An accurate and sensitive method to identify both rare and common resistance elements in complex metagenomic samples is needed. Referencing the Comprehensive Antibiotic Resistance Database, we designed a set of 37,826 probes to specifically target over 2000 nucleotide sequences associated with antibiotic resistance in clinically relevant bacteria. Testing of this probeset on DNA libraries generated from multi-drug resistant bacteria to selectively capture resistance genes reproducibly produced higher reads on-target at greater length of coverage when compared to shotgun sequencing. We also identified additional resistance gene sequences from human gut microbiome samples that sequencing alone was not able to detect. Our method to capture the resistome enables sensitive gene detection in diverse environments where antibiotic resistance represents less than 0.1% of the metagenome.
Glycopeptide antibiotics are produced by Actinobacteria through biosynthetic gene clusters that include genes supporting their regulation, synthesis, export and resistance. The chemical and biosynthetic diversities of glycopeptides are the product of an intricate evolutionary history. Extracting this history from genome sequences is difficult as conservation of the individual components of these gene clusters is variable and each component can have a different trajectory. We show that glycopeptide biosynthesis and resistance in Actinobacteria maps to approximately 150-400 million years ago. Phylogenetic reconciliation reveals that the precursors of glycopeptide biosynthesis are far older than other components, implying that these clusters arose from a pre-existing pool of genes. We find that resistance appeared contemporaneously with biosynthetic genes, raising the possibility that the mechanism of action of glycopeptides was a driver of diversification in these gene clusters. Our results put antibiotic biosynthesis and resistance into an evolutionary context and can guide the future discovery of compounds possessing new mechanisms of action, which are especially needed as the usefulness of the antibiotics available at present is imperilled by human activity.
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Novel gene targets are needed in accurate diagnosis of malaria. Previous studies show that the dynein light chains (dlc) in Plasmodium are uniquely conserved within the species, possibly due to their role as the cargo adptor moiety. This study aimed at the development of PCR assay for the detection of Plasmodium based on the (dlc-Tctex) as a genus and species-specific tool in malaria diagnosis. Multiple primers were designed based on Plasmodium spp dlc(Tctex) genes. The primers were applied on PCR to detect malaria on clinical samples and on laboratory maintained isolates of P. falciparum and P. vivax for human infecting species and P. knowlesi and P. cynomolgi for zoonoses infection involving primates. The amplified PCR fragments were gene cleaned and sequenced. BLASTn e-values output from the raw nucleotide queries supports that the genes are uniquely conserved. Species-specific primers amplified P. falciparum infections with no cross-reactivity to P. vivax, P. knowlesi or P. cynomolgi species. In this assay only 11 out of the 30 microscope positive malaria positive clinical blood samples were positive for PCR detection of P. falciparum infection. Primers designed for Plasmodium genus amplified the target band in all clinical malaria samples but also had another specific band amplification. This preliminary data demonstrate that a species-specific dlc(Tctex) PCR assay can be used for detection of P. falciparum and optimized genus primers can be applied to differentiate mixed malaria infections.
Faltyn, M., B. Alcock, & A.G. McArthur. 2019. Evolution and nomenclature of the trimethoprim resistant dihydrofolate (dfr) reductases. Preprints 2019, 2019050137. [Preprint]
Tsang, K.K., D.J. Speicher, & A.G. McArthur. 2019. Pathogen taxonomy updates at the Comprehensive Antibiotic Resistance Database: Implications for molecular epidemiology. Preprints 2019, 2019070222. [Preprint]
Srinivasan, K.A., S.K. Virdee, & A.G. McArthur. 2019. Strandedness during cDNA synthesis, the stranded parameter in htseq-count, and analysis of RNA-Seq data. Preprints 2019, 2019030124. [Preprint]
Matthews, T., F. Bristow, E. Griffiths, A. Petkau, J. Adam, D. Dooley, P. Kruczkiewicz, J. Curatcha, J. Cabral, D. Fornika, G. Winsor, M. Courtot, C. Bertelli, A. Roudgar, P. Fejaio, P. Mabon, E. Enns, J. Thiessen, A.W.S. Keddy, J. Isaac-Renton, J. Gardy, P. Tang, The IRIDA consortium, J. Carriço, L. Chindelevitch, C. Chauve, M. Graham, A.G. McArthur, E. Taboada, R. Beiko, F.S.L. Brinkman, W.W.L. Hsiao, & G. Van Domselaar. 2018. The Integrated Rapid Infectious Disease Analysis (IRIDA) Platform. BioRxiv 381830 [Preprint].
BACKGROUND: Physical inactivity impairs insulin sensitivity, which is exacerbated with aging. We examined the impact of 2 wk of acute inactivity and recovery on glycemic control, and integrated rates of muscle protein synthesis in older men and women.
METHODS: Twenty-two overweight, prediabetic older adults (12 men, 10 women, 69 ± 4 y) undertook 7 d of habitual activity (baseline; BL), step reduction (SR; <1,000 steps.d-1 for 14 d), followed by 14 d of recovery (RC). An oral glucose tolerance test was used to assess glycemic control and deuterated water ingestion to measure integrated rates of muscle protein synthesis.
RESULTS: Daily step count was reduced (all p < .05) from BL at SR (7362 ± 3294 to 991 ± 97) and returned to BL levels at RC (7117 ± 3819). Homeostasis model assessment-insulin resistance increased from BL to SR and Matsuda insulin sensitivity index decreased and did not return to BL in RC. Glucose and insulin area under the curve were elevated from BL to SR and did not recover in RC. Integrated muscle protein synthesis was reduced during SR and did not return to BL in RC.
CONCLUSIONS: Our findings demonstrate that 2 wk of SR leads to lowered rates of muscle protein synthesis and a worsening of glycemic control that unlike younger adults is not recovered during return to normal activity in overweight, prediabetic elderly humans.
Round whitefish (Prosopium cylindraceum) have a broad, disjunct range across northern North America and Eurasia, and little is known about their genetic population structure. We performed genetic analyses of round whitefish from 17 sites across its range using nine microsatellites, two mitochondrial DNA (mtDNA) loci, and 4918 to 8835 single-nucleotide polymorphism (SNP) loci. Our analyses identified deep phylogenetic division between eastern and western portions of the range, likely indicative of origins from at least two separate Pleistocene glacial refugia. Regionally, microsatellites and SNPs identified congruent patterns in subdivision, and population structure was consistent with expectations based on hydrologic connectivity. Within the Laurentian Great Lakes, Lake Huron and Lake Ontario were identified as key areas of interest. Lake Huron appears to be a contemporary source population for several other Great Lakes, and Lake Ontario contains a genetically discrete group of round whitefish. In all cases, multiple genetic markers yielded similar patterns, but SNPs offered substantially enhanced resolution. We conclude that round whitefish have population subdivision on several scales important for understanding their evolutionary history and conservation planning.
Type III Secretion Systems (T3SSs) are structurally conserved nanomachines that span the inner and outer bacterial membranes, and via a protruding needle complex contact host cell membranes and deliver type III effector proteins. T3SS are phylogenetically divided into several families based on structural basal body components. Here we have studied the evolutionary and functional conservation of four T3SS proteins from the Inv/Mxi-Spa family: a cytosolic chaperone, two hydrophobic translocators that form a plasma membrane-integral pore, and the hydrophilic ‘tip complex’ translocator that connects the T3SS needle to the translocon pore. Salmonella enterica serovar Typhimurium (S. Typhimurium), a common cause of food-borne gastroenteritis, possesses two T3SSs, one belonging to the Inv/Mxi-Spa family. We used invasion-deficient S. Typhimurium mutants as surrogates for expression of translocator orthologs identified from an extensive phylogenetic analysis, and type III effector translocation and host cell invasion as a readout for complementation efficiency, and identified several Inv/Mxi-Spa orthologs that can functionally substitute for the S. Typhimurium chaperone and translocator proteins. Functional complementation correlates with amino acid sequence identity between orthologs, but varies considerably between the four proteins. This is the first in-depth survey of the functional interchangeability of Inv/Mxi-Spa T3SS proteins acting directly at the host-pathogen interface.
The loss of effective antimicrobials is reducing our ability to protect the global population from infectious disease. However, the field of antibiotic drug discovery and the public health monitoring of antimicrobial resistance (AMR) is beginning to exploit the power of genome and metagenome sequencing. The creation of novel AMR bioinformatics tools and databases and their continued development will advance our understanding of the molecular mechanisms and threat severity of antibiotic resistance, while simultaneously improving our ability to accurately predict and screen for antibiotic resistance genes within environmental, agricultural, and clinical settings. To do so, efforts must be focused toward exploiting the advancements of genome sequencing and information technology. Currently, AMR bioinformatics software and databases reflect different scopes and functions, each with its own strengths and weaknesses. A review of the available tools reveals common approaches and reference data but also reveals gaps in our curated data, models, algorithms, and data-sharing tools that must be addressed to conquer the limitations and areas of unmet need within the AMR research field before DNA sequencing can be fully exploited for AMR surveillance and improved clinical outcomes.
Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, Lago BA, Dave BM, Pereira S, Sharma AN, Doshi S, Courtot M, Lo R, Williams LE, Frye JG, Elsayegh T, Sardar D, Westman EL, Pawlowski AC, Johnson TA, Brinkman FS, Wright GD, & McArthur AG.
The Comprehensive Antibiotic Resistance Database (CARD; http://arpcard.mcmaster.ca) is a manually curated resource containing high quality reference data on the molecular basis of antimicrobial resistance (AMR), with an emphasis on the genes, proteins and mutations involved in AMR. CARD is ontologically structured, model centric, and spans the breadth of AMR drug classes and resistance mechanisms, including intrinsic, mutation-driven and acquired resistance. It is built upon the Antibiotic Resistance Ontology (ARO), a custom built, interconnected and hierarchical controlled vocabulary allowing advanced data sharing and organization. Its design allows the development of novel genome analysis tools, such as the Resistance Gene Identifier (RGI) for resistome prediction from raw genome sequence. Recent improvements include extensive curation of additional reference sequences and mutations, development of a unique Model Ontology and accompanying AMR detection models to power sequence analysis, new visualization tools, and expansion of the RGI for detection of emergent AMR threats. CARD curation is updated monthly based on an interplay of manual literature curation, computational text mining, and genome analysis.