McArthur, A.G., Waglechner, N., Nizam, F., Pereira, S.K., Jia, B., Sardar, D., Westman, E.L., Pawlowski, A.C., Johnson, T., Lo, R., Courtot, M., Brinkman, F.S., Williams, L.E., Frye, J.G., & Wright, G.D. 2015. The Comprehensive Antibiotic Resistance Database. Presentation at the 4th ASM Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens, Washington, District of Columbia.
Antimicrobial resistance (AMR) is among the most pressing public health crises of the 21st Century. Despite the importance of resistance to health, this field has been slow to take advantage of genome scale tools. Rather, phenotype based criteria dominate the epidemiology of antibiotic action and effectiveness. As a result, there is a poor understanding of which antibiotic resistance genes are in circulation, which ones are a threat, and how clinicians and public health workers can manage the crisis of resistance. However, DNA sequencing is rapidly decreasing in cost and as such we are on the cusp of an age of high-throughput molecular epidemiology. What are needed are tools for rapid, accurate analysis of DNA sequence data for the genetic underpinnings of antibiotic resistance. In an effort to address this problem, we have created the Comprehensive Antibiotic Resistance Database (arpcard.mcmaster.ca). This database is a rigorously curated collection of known antibiotics, targets, and resistance determinants. It integrates disparate molecular and sequence data, provides a unique organizing principle in the form of the Antibiotic Resistance Ontology (ARO), and can quickly identify putative antibiotic resistance genes in raw genome sequences using the novel Resistance Gene Identifier (RGI). Here we review the current state of the CARD, particularly recent advances in the curation of resistance determinants and the structure of the ARO. We will also present our plans for development of semi- and fully-automated text mining algorithms for curation of broader AMR data, construction of Probabilistic Graphic Models for improved AMR phenotype prediction, and development of portable command-line genome analysis tools.
Dr. Nancy Hopkins’ article is a worthy read. Hopkins, Nancy. Reflecting on Fifty Years of Progress for Women in Science. DNA and Cell Biology. March 2015, 34(3): 159-161.
The McArthur Lab welcomes Tariq Elsayegh from the Royal College of Surgeons in Ireland, where he is in the six year medical program. Tariq is undertaking a 6 week project to build bioinformatics models for colistin resistance in the Comprehensive Antibiotic Resistance Database.
The major histocompatibility complex (Mhc) is subject to pathogen-mediated balancing selection and can link natural selection with mate choice. We characterized two Mhc class II B loci in Leach’s storm-petrel, Oceanodroma leucorhoa, focusing on exon 2 which encodes the portion of the protein that binds pathogen peptides. We amplified and sequenced exon 2 with locus-specific nested PCR and Illumina MiSeq using individually barcoded primers. Repeat genotyping of 78 single-locus genotypes produced identical results in 77 cases (98.7 %). Sequencing of messenger RNA (mRNA) from three birds confirmed expression of both loci, consistent with the observed absence of stop codons or frameshifts in all alleles. In 48 birds, we found 9 and 12 alleles at the two loci, respectively, and all 21 alleles translated to unique amino acid sequences. Unlike many studies of duplicated Mhc genes, alleles of the two loci clustered into monophyletic groups. Consistent with this phylogenetic result, interlocus gene conversion appears to have affected only two short fragments of the exon. As predicted under a paradigm of pathogen-mediated selection, comparison of synonymous and non-synonymous substitution rates found evidence of a history of positive selection at putative peptide binding sites. Overall, the results suggest that the gene duplication event leading to these two loci is not recent and that point mutations and positive selection on the peptide binding sites may be the predominant forces acting on these genes. Characterization of these loci sets the stage for population-level work on the evolutionary ecology of Mhc in this species.
Justin Jia (left) and Daim Sardar (right) have joined the McArthur Lab. Justin has joined as a 8 month McMaster Honours Biochemistry – Biomedical Research Specialization Co-Op placement and will be working as a Comprehensive Antibiotic Resistance Database biocurator. Daim will be in the lab 8 weeks performing an Independent Project on 16S rRNA mutations conferring antibiotic resistance as part of his Honours Integrated Science Program (iSci) 3A12 coursework.
Serial Analysis of Gene Expression (SAGE) was used to quantify transcriptional changes in Giardia intestinalis during its interaction with human intestinal epithelial cells (IECs, HT-29) in serum free M199 medium. Transcriptional changes were compared to those in trophozoites alone in M199 and in TYI-S-33 Giardia growth medium. In total, 90 genes were differentially expressed, mainly those involved in cellular redox homeostasis, metabolism and small molecule transport but also cysteine proteases and structural proteins of the giardin family. Only 29 genes changed their expression due to IEC interaction and the rest were due to M199 medium. Although our findings generated a small dataset, it was consistent with our earlier microarray studies performed under different interaction conditions. This study has confined the number of genes in Giardia to a small subset that specifically change their expression due to interaction with IECs.
Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis and to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. The developmental expression and inducibility of anti-oxidant defenses through activation of NF-E2-related factor 2 (NRF2) affect susceptibility to oxidants, but the embryonic response to oxidants is not well understood. To assess the response to chemically mediated oxidative stress and how it may vary during development, zebrafish embryos, eleutheroembryos, or larvae at 1, 2, 3, 4, 5, and 6 days post fertilization (dpf) were exposed to DMSO (0.1%), tert-butylhydroquinone (tBHQ; 10 µM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 2 nM) for 6 hr. Transcript abundance was assessed by real-time qRT-PCR and microarray. qRT-PCR showed strong (4- to 5-fold) induction of gstp1 by tBHQ as early as 1 dpf. tBHQ also induced gclc (2 dpf), but not sod1, nqo1, or cyp1a. TCDD induced cyp1a but none of the other genes. Microarray analysis showed that 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated eleutheroembryos at 4 dpf. There was substantial overlap between genes induced in developing zebrafish and a set of marker genes induced by oxidative stress in mammals. Genes induced by tBHQ in 4-dpf zebrafish included those involved in glutathione synthesis and utilization, signal transduction, and DNA damage/stress response. The strong induction of hsp70 determined by microarray was confirmed by qRT-PCR and by use of transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under control of the hsp70 promoter. Genes strongly down-regulated by tBHQ included mitfa, providing a molecular explanation for the loss of pigmentation in tBHQ-exposed embryos. These data show that zebrafish embryos are responsive to oxidative stress as early as 1 dpf, that responsiveness varies with development in a gene-specific manner, and that the oxidative stress response is substantially conserved in vertebrate animals.