CARD 2023: expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database.

The Comprehensive Antibiotic Resistance Database (CARD; card.mcmaster.ca) combines the Antibiotic Resistance Ontology (ARO) with curated AMR gene (ARG) sequences and resistance-conferring mutations to provide an informatics framework for annotation and interpretation of resistomes. As of version 3.2.4, CARD encompasses 6627 ontology terms, 5010 reference sequences, 1933 mutations, 3004 publications, and 5057 AMR detection models that can be used by the accompanying Resistance Gene Identifier (RGI) software to annotate genomic or metagenomic sequences. Focused curation enhancements since 2020 include expanded ß-lactamase curation, incorporation of likelihood-based AMR mutations for Mycobacterium tuberculosis, addition of disinfectants and antiseptics plus their associated ARGs, and systematic curation of resistance-modifying agents. This expanded curation includes 180 new AMR gene families, 15 new drug classes, 1 new resistance mechanism, and two new ontological relationships: evolutionary_variant_of and is_small_molecule_inhibitor. In silico prediction of resistomes and prevalence statistics of ARGs has been expanded to 377 pathogens, 21,079 chromosomes, 2,662 genomic islands, 41,828 plasmids and 155,606 whole-genome shotgun assemblies, resulting in collation of 322,710 unique ARG allele sequences. New features include the CARD:Live collection of community submitted isolate resistome data and the introduction of standardized 15 character CARD Short Names for ARGs to support machine learning efforts.

Chronic COVID-19 infection in an immunosuppressed patient shows changes in lineage over time: a case report.

BACKGROUND: The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 virus, emerged in late 2019 and spready globally. Many effects of infection with this pathogen are still unknown, with both chronic and repeated COVID-19 infection producing novel pathologies. CASE PRESENTATION: An immunocompromised patient presented with chronic COVID-19 infection. The patient had history of Hodgkin's lymphoma, treated with chemotherapy and stem cell transplant. During the course of their treatment, eleven respiratory samples from the patient were analyzed by whole-genome sequencing followed by lineage identification. Whole-genome sequencing of the virus present in the patient over time revealed that the patient at various timepoints harboured three different lineages of the virus. The patient was initially infected with the B.1.1.176 lineage before coinfection with BA.1. When the patient was coinfected with both B.1.1.176 and BA.1, the viral populations were found in approximately equal proportions within the patient based on sequencing read abundance. Upon further sampling, the lineage present within the patient during the final two timepoints was found to be BA.2.9. The patient eventually developed respiratory failure and died. CONCLUSIONS: This case study shows an example of the changes that can happen within an immunocompromised patient who is infected with COVID-19 multiple times. Furthermore, this case demonstrates how simultaneous coinfection with two lineages of COVID-19 can lead to unclear lineage assignment by standard methods, which are resolved by further investigation. When analyzing chronic COVID-19 infection and reinfection cases, care must be taken to properly identify the lineages of the virus present.

Sensitivity to Neutralizing Antibodies and Resistance to Type I Interferons in SARS-CoV-2 R.1 Lineage Variants, Canada.

Isolating and characterizing emerging SARS-CoV-2 variants is key to understanding virus pathogenesis. In this study, we isolated samples of the SARS-CoV-2 R.1 lineage, categorized as a variant under monitoring by the World Health Organization, and evaluated their sensitivity to neutralizing antibodies and type I interferons. We used convalescent serum samples from persons in Canada infected either with ancestral virus (wave 1) or the B.1.1.7 (Alpha) variant of concern (wave 3) for testing neutralization sensitivity. The R.1 isolates were potently neutralized by both the wave 1 and wave 3 convalescent serum samples, unlike the B.1.351 (Beta) variant of concern. Of note, the R.1 variant was significantly more resistant to type I interferons (IFN-α/ß) than was the ancestral isolate. Our study demonstrates that the R.1 variant retained sensitivity to neutralizing antibodies but evolved resistance to type I interferons. This critical driving force will influence the trajectory of the pandemic.

ADAMTS1 is regulated by the EP4 receptor in the zebrafish ovary.

Prostaglandins (PGs) are a class of fatty-acid derived hormones that are essential in ovulation of teleosts, but their exact role remains unknown. One putative target of PGs in ovulation is regulation of the expression of members of the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family, which are implicated in follicular rupture. This study investigated the regulation of ADAMTS, other proteases, and their inhibitors in response to treatment with PGE2 or PGF2α. Four members of the ADAMTS family, ADAMTS1, ADAMTS5, ADAMTS9, and ADAMTS16 were shown to be expressed in the ovary of zebrafish, but only adamts1 was upregulated in full-grown follicles following treatment with PGE2. Inhibitors of the PG receptors EP1 and EP2 had no effect on PGE2-stimulated adamts1 expression, while treatment of full-grown follicles with both PGE2 and GW627368x, an inhibitor of EP4 function, prevented the PGE2-induced increase in adamts1 expression. Treatment of full-grown follicles with the maturation-inducing hormone 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) in vitro had no effect on the expression of adamts1 mRNA. These findings suggest that expression of ADAMTS1 in zebrafish ovarian follicles is regulated by the prostaglandin PGE2 via the EP4 series prostaglandin receptor.

Nuclear progesterone receptor regulates ptger4b and PLA2G4A expression in zebrafish (Danio rerio) ovulation.

Previous studies have implicated the nuclear progesterone receptor (Pgr or nPR) as being critical to ovulation in fishes. This study investigated the expression of Pgr in zebrafish ovarian follicles throughout development as well as putative downstream targets of Pgr by searching the promoter regions of selected genes for specific DNA sequences to which Pgr binds and acts as a transcription factor. Expression of Pgr mRNA increases dramatically as follicles grow and mature. In silico analysis of selected genes linked to ovulation showed that the prostaglandin receptors ptger4a and ptger4b contained the progesterone responsive element (PRE) GRCCGGA in their promoter regions. Studies using full-grown follicles incubated in vitro revealed that ptger4b was upregulated in response to 17,20ß-P. Our studies also showed that the expression of phospholipase A2 (PLA2G4A) mRNA and protein, a key enzyme in prostaglandin synthesis, was upregulated in response to 17,20ß-P treatment. pla2g4a was not found to contain a PRE, indicating that it is regulated indirectly by 17,20ß-P or that it may contain an as-of-yet unidentified PRE in its promoter region. Collectively, these studies provide further evidence of the importance of Pgr during the periovulatory periods through its involvement in prostaglandin production and function by controlling expression of PLA2G4A and the receptor EP4b and that these genes appear to be regulated through the actions of 17,20ß-P.

Investigating the role of prostaglandin receptor isoform EP4b in zebrafish ovulation.

Prostaglandins (PGs) are a class of fatty acid-derived hormones that play an essential role in the regulation of ovulation of teleosts. This study investigated the various isoforms of ovarian PG receptors in the zebrafish ovary and their role in ovulation. Using real time qPCR, six PG receptor isoforms (ptger1a, ptger1b, ptger2a, ptger4a, ptger4b, and ptgfr) were shown to be expressed in the ovary. Only the PG receptor isoform ptger4b was upregulated at the time of ovulation in vivo, or following treatment in vivo with Ovaprim, which contains a gonadotropin releasing hormone analogue and a dopamine receptor antagonist and stimulates ovulation. Treatment of full-grown follicles with the maturation-inducing hormone 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ßP) in vitro also induced expression of EP4b mRNA. Females ovulate in vivo after injection with Ovaprim, or injection with Ovaprim and inhibitors of EP1 (ONO-8130) or EP2 (TG4-155) function; they do not ovulate when injected with Ovaprim and an EP4 inhibitor (GW237368x). These findings suggest that the EP4 receptor, in particular the EP4b isoform, is essential for ovulation.

SARS-CoV-2 Outbreak Investigation Using Contact Tracing and Whole-Genome Sequencing in an Ontario Tertiary Care Hospital.

Genomic epidemiology can facilitate an understanding of evolutionary history and transmission dynamics of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak. We used next-generation sequencing techniques to study SARS-CoV-2 genomes isolated from patients and health care workers (HCWs) across five wards of a Canadian hospital with an ongoing SARS-CoV-2 outbreak. Using traditional contact tracing methods, we show transmission events between patients and HCWs, which were also supported by the SARS-CoV-2 lineage assignments. The outbreak predominantly involved SARS-CoV-2 B.1.564.1 across all five wards, but we also show evidence of community introductions of lineages B.1, B.1.1.32, and B.1.231, falsely assumed to be outbreak related. Altogether, our study exemplifies the value of using contact tracing in combination with genomic epidemiology to understand the transmission dynamics and genetic underpinnings of a SARS-CoV-2 outbreak. IMPORTANCE Our manuscript describes a SARS-CoV-2 outbreak investigation in an Ontario tertiary care hospital. We use traditional contract tracing paired with whole-genome sequencing to facilitate an understanding of the evolutionary history and transmission dynamics of this SARS-CoV-2 outbreak in a clinical setting. These advancements have enabled the incorporation of phylogenetics and genomic epidemiology into the understanding of clinical outbreaks. We show that genomic epidemiology can help to explore the genetic evolution of a pathogen in real time, enabling the identification of the index case and helping understand its transmission dynamics to develop better strategies to prevent future spread of SARS-CoV-2 in congregate, clinical settings such as hospitals.