Foodborne bacterial pathogens: genome-based approaches for enduring and emerging threats in a complex and changing world.

Foodborne illnesses pose a substantial health and economic burden, presenting challenges in prevention due to the diverse microbial hazards that can enter and spread within food systems. Various factors, including natural, political and commercial drivers, influence food production and distribution. The risks of foodborne illness will continue to evolve in step with these drivers and with changes to food systems. For example, climate impacts on water availability for agriculture, changes in food sustainability targets and evolving customer preferences can all have an impact on the ecology of foodborne pathogens and the agrifood niches that can carry microorganisms. Whole-genome and metagenome sequencing, combined with microbial surveillance schemes and insights from the food system, can provide authorities and businesses with transformative information to address risks and implement new food safety interventions across the food chain. In this Review, we describe how genome-based approaches have advanced our understanding of the evolution and spread of enduring bacterial foodborne hazards as well as their role in identifying emerging foodborne hazards. Furthermore, foodborne hazards exist in complex microbial communities across the entire food chain, and consideration of these co-existing organisms is essential to understanding the entire ecology supporting pathogen persistence and transmission in an evolving food system.

Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection.

BACKGROUND: The timely and accurate diagnosis of bloodstream infection (BSI) is critical for patient management. With longstanding challenges for routine blood culture, metagenomics is a promising approach to rapidly provide sequence-based detection and characterisation of bloodborne bacteria. Long-read sequencing technologies have successfully supported the use of clinical metagenomics for syndromes such as respiratory illness, and modified approaches may address two requisite factors for metagenomics to be used as a BSI diagnostic: depletion of the high level of host DNA to then detect the low abundance of microbes in blood. METHODS: Blood samples from healthy donors were spiked with different concentrations of four prevalent causative species of BSI. All samples were then subjected to a modified saponin-based host DNA depletion protocol and optimised DNA extraction, whole genome amplification and debranching steps in preparation for sequencing, followed by bioinformatical analyses. Two related variants of the protocol are presented: 1mL of blood processed without bacterial enrichment, and 5mL of blood processed following a rapid bacterial enrichment protocol-SepsiPURE. RESULTS: After first identifying that a large proportion of host mitochondrial DNA remained, the host depletion process was optimised by increasing saponin concentration to 3% and scaling the reaction to allow more sample volume. Compared to non-depleted controls, the 3% saponin-based depletion protocol reduced the presence of host chromosomal and mitochondrial DNA < 106 and < 103 fold respectively. When the modified depletion method was further combined with a rapid bacterial enrichment method (SepsiPURE; with 5mL blood samples) the depletion of mitochondrial DNA improved by a further > 10X while also increasing detectable bacteria by > 10X. Parameters during DNA extraction, whole genome amplification and long-read sequencing were also adjusted, and subsequently amplicons were detected for each input bacterial species at each of the spiked concentrations, ranging from 50-100 colony forming units (CFU)/mL to 1-5 CFU/mL. CONCLUSION: In this proof-of-concept study, four prevalent BSI causative species were detected in under 12 h to species level (with antimicrobial resistance determinants) at concentrations relevant to clinical blood samples. The use of a rapid and precise metagenomic protocols has the potential to advance the diagnosis of BSI.

Laboratory response checklist for infectious disease outbreaks-preparedness and response considerations for emerging threats.

The purpose of the Laboratory Response Checklist for Infectious Disease Outbreaks (the Checklist) is to provide public health laboratories and laboratory networks operating at multiple jurisdictional levels with a useful, adaptable tool to help rapidly identify important outbreak response considerations, particularly when investigating a previously unknown infectious disease threat. The Checklist was developed by the National Microbiology Laboratory of Canada in collaboration with provincial/territorial, national and international laboratory experts, including the Canadian Public Health Laboratory Network, and the Global Health Security Action Group Laboratory Network. While the Checklist was initially designed to reflect lessons learned through National Microbiology Laboratory participation in extended national and international outbreak responses (e.g. Zika virus epidemic [2015-2016], Ebola virus epidemic, West Africa [2014-2016]), the importance of optimizing laboratory response coordination has only been underscored by the ongoing challenges presented by the coronavirus disease 2019 (COVID-19) pandemic response requirements. The Checklist identifies five highly interdependent laboratory response themes, each of which encompasses multiple considerations that may be critical to a coordinated, strategic outbreak response. As such, the comprehensive review of Checklist considerations by responding laboratory organizations may provide a valuable opportunity to quickly detect key response considerations and interdependencies, and mitigate risks with the potential to impact public health action.

Diagnosis and Management of First Case of COVID-19 in Canada: Lessons Applied From SARS-CoV-1.

We report diagnosis and management of the first laboratory-confirmed case of coronavirus disease 2019 (COVID-19) hospitalized in Toronto, Canada. No healthcare-associated transmission occurred. In the face of a potential pandemic of COVID-19, we suggest sustainable and scalable control measures developed based on lessons learned from severe acute respiratory syndrome.

Outbreak of Escherichia coli O157:H7 Infections Linked to Mechanically Tenderized Beef and the Largest Beef Recall in Canada, 2012.

Contaminated beef is a known vehicle of Escherichia coli O157:H7 infection, although more attention is given to the control of E. coli O157:H7 in ground, rather than whole-cut, beef products. In September 2012, an investigation was initiated at an Alberta, Canada, beef plant after the detection of E. coli O157:H7 in two samples of trim cut from beef originating from this plant. Later in September 2012, Alberta Health Services identified five laboratory-confirmed infections of E. coli O157:H7, and case patients reported eating needle-tenderized beef steaks purchased at a store in Edmonton, Alberta, produced with beef from the Alberta plant. In total, 18 laboratory-confirmed illnesses in Canada in September and October 2012 were linked to beef from the Alberta plant, including the five individuals who ate needle-tenderized steaks purchased at the Edmonton store. A unique strain of E. coli O157:H7, defined by molecular subtyping and whole genome sequencing, was detected in clinical isolates, four samples of leftover beef from case patient homes, and eight samples of Alberta plant beef tested by industry and food safety partners. Investigators identified several deficiencies in the control of E. coli O157:H7 at the plant; in particular, the evaluation of, and response to, the detection of E. coli O157 in beef samples during routine testing were inadequate. To control the outbreak, 4,000 tons of beef products were recalled, making it the largest beef recall in Canadian history. This outbreak, in combination with similar outbreaks in the United States and research demonstrating that mechanical tenderization can transfer foodborne pathogens present on the surface into the interior of beef cuts, prompted amendments to Canada's Food and Drug Regulations requiring mechanically tenderized beef to be labeled as such and to provide safe cooking instructions to consumers. A detailed review of this event also led to recommendations and action to improve the safety of Canada's beef supply.

Interventions to improve emergency department use for mental health reasons: protocol for a mixed-methods systematic review.

BACKGROUND: Healthcare resources are limited and unnecessary, and inappropriate emergency department use is now a highly visible healthcare priority. Individuals visiting the emergency department for mental health-related reasons are often amongst the most frequent presenters. In response, researchers and clinicians have created interventions to streamline emergency department use and several primary studies describe the effects of these interventions. Yet, no consensus exists on the optimal approach, and information on the quality of development, effectiveness, acceptability, and economic considerations is hard to find. The purpose of this study is to systematically review interventions designed to improve appropriate use of the emergency department for mental health reasons. METHOD: A mixed-method systematic review using Joanna Briggs Methodology. Search combining electronic databases (EMBASE, MEDLINE, PsycINFO, CINAHL, HealthSTAR, PROQUEST, Cumulative Index to Nursing and Allied Health) and secondary searches (grey literature and hand search with consultation). Two independent reviewers will screen titles and abstracts using predetermined eligibility criteria and a third reviewer will resolve conflicts. Full texts will also be screened by two independent reviews and conflicts resolved in a consensus meeting with a third reviewer. A pilot-tested data extraction form will be used to retrieve data relevant to the study objectives. We will assess the quality and of all included studies. Data describing interventions will be summarized using logic models and reported narratively. Quality of development will be assessed using the Oxford Implementation Index. For data on intervention effectiveness, we will assess statistical heterogeneity and conduct a meta-analysis using a random effects method, if appropriate. For interventions that cannot be pooled, we will report outcomes narratively and descriptively. Qualitative data on acceptability will be synthesized using meta-aggregation and an economic evaluation of interventions will be done. The reporting of this protocol follows the PRISMA-P statement. DISCUSSION: Using a combined systematic review methodology and integrated knowledge translation plan, the project will provide decision makers with concrete evidence to support the implementation and evaluation of interventions to improve emergency department use for mental health reasons. These interventions reflect widespread priorities in the area of mental health care. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42018087430.

Shared genome analyses of notable listeriosis outbreaks, highlighting the critical importance of epidemiological evidence, input datasets and interpretation criteria.

The persuasiveness of genomic evidence has pressured scientific agencies to supplement or replace well-established methodologies to inform public health and food safety decision-making. This study of 52 epidemiologically defined Listeria monocytogenes isolates, collected between 1981 and 2011, including nine outbreaks, was undertaken (1) to characterize their phylogenetic relationship at finished genome-level resolution, (2) to elucidate the underlying genetic diversity within an endemic subtype, CC8, and (3) to re-evaluate the genetic relationship and epidemiology of a CC8-delimited outbreak in Canada in 2008. Genomes representing Canadian Listeria outbreaks between 1981 and 2010 were closed and manually annotated. Single nucleotide variants (SNVs) and horizontally acquired traits were used to generate phylogenomic models. Phylogenomic relationships were congruent with classical subtyping and epidemiology, except for CC8 outbreaks, wherein the distribution of SNV and prophages revealed multiple co-evolving lineages. Chronophyletic reconstruction of CC8 evolution indicates that prophage-related genetic changes among CC8 strains manifest as PFGE subtype reversions, obscuring the relationship between CC8 isolates, and complicating the public health interpretation of subtyping data, even at maximum genome resolution. The size of the shared genome interrogated did not change the genetic relationship measured between highly related isolates near the tips of the phylogenetic tree, illustrating the robustness of these approaches for routine public health applications where the focus is recent ancestry. The possibility exists for temporally and epidemiologically distinct events to appear related even at maximum genome resolution, highlighting the continued importance of epidemiological evidence.

Antimicrobial susceptibility testing of invasive isolates of Streptococcus pneumoniae from Canadian patients: the SAVE study, 2011-15.

Objectives: To assess antimicrobial susceptibility for 14 agents tested against 6001 invasive isolates of Streptococcus pneumoniae cultured from invasive patient samples from 2011 to 2015 as a part of the annual SAVE study. Methods: Isolates of S. pneumoniae were tested using the standard CLSI broth microdilution method (M07-A10, 2015) with MICs interpreted by CLSI M100 27th Edition (2017) MIC breakpoints. Results: From 2011 to 2015, small but significant increases (P ≤ 0.05) in the percentage susceptibility for penicillin (interpreted by all three CLSI MIC breakpoint criteria) (increase of 1.7%-3.2%), clindamycin (3.1%) and ceftriaxone (interpreted by non-meningitis and meningitis CLSI MIC breakpoint criteria) (1.1%-1.5%) were observed. Susceptibility rates for clarithromycin and other commonly tested antimicrobial agents remained unchanged (P > 0.05) over the 5 year period. Isolates with an MDR phenotype (resistance to three or more antimicrobial agent classes) decreased significantly (P < 0.001) from 8.5% in 2011 to 5.6% in 2015. Antimicrobial susceptibility rates were not generally associated (P > 0.05) with patient gender (exception: clarithromycin) but were associated (P ≤ 0.05) with patient age (chloramphenicol and clindamycin) or specimen source (penicillin, doxycycline, trimethoprim/sulfamethoxazole and clindamycin), as well as geographic location in Canada and concurrent resistance to penicillin or clarithromycin. Conclusions: The in vitro susceptibility of invasive isolates of S. pneumoniae in Canada to penicillin, clindamycin and ceftriaxone increased from 2011 to 2015, coincident with a significant decrease in MDR phenotypes.

Analysis of multidrug resistance in the predominant Streptococcus pneumoniae serotypes in Canada: the SAVE study, 2011-15.

Objectives: This study assessed MDR invasive isolates of Streptococcus pneumoniae, in relation to serotype evolution in Canada between 2011 and 2015 as part of the annual SAVE study. Methods: As part of a collaboration between the Canadian Antimicrobial Resistance Alliance and Public Health Agency of Canada-National Microbiology Laboratory, 6207 invasive isolates of S. pneumoniae were evaluated. All isolates were serotyped and had antimicrobial susceptibility testing performed, in accordance with CLSI guidelines (M07-A10, 2015). Complete susceptibility profiles were available for 6001 isolates. MDR was defined as resistance to three or more classes of antimicrobial agents (with penicillin MIC ≥2 mg/L defined as resistant). Results: The overall rate of MDR S. pneumoniae was 6.2% (372/6001) in SAVE, decreasing significantly from 8.5% in 2011 to 5.6% in 2015 (P = 0.0041). MDR was observed in 32 serotypes, with serotypes 15A and 19A predominating (26.6% and 41.7% of the MDR isolates, respectively). The overall proportion of serotypes 19A, 7F and 33A decreased significantly (P < 0.0001) throughout the study. The annual proportion of serotypes 7C, 8, 9N, 10A, 20, 24F, 29, 31, 33F, 35B and 38 increased throughout the study; however, among these increasing serotypes, MDR was only notable (>5%) for 24F and 33F. Conclusions: In 2015, 56.3% of invasive MDR S. pneumoniae were serotypes included in the PCV-13 vaccine. PCV-13 includes the most commonly identified serotype, 19A; however, other increasingly important MDR serotypes, such as 15A, 24F and 33F, are notably not in the currently used vaccines.

Molecular characterization of predominant Streptococcus pneumoniae serotypes causing invasive infections in Canada: the SAVE study, 2011-15.

Objectives: This study characterized the 11 most predominant serotypes of invasive Streptococcus pneumoniae infections collected by the annual SAVE study in Canada, between 2011 and 2015. Methods: A subset of the 11 most predominant serotypes (7F, 19A, 22F, 3, 12F, 11A, 9N, 8, 33F, 15A and 6C) collected by the SAVE study was analysed using PFGE and MLST, as well as PCR to identify pilus-encoding genes. WGS analyses were performed on a subset of the above isolates plus a random selection of background strains. Results: Of the predominant serotypes analysed, 7F, 33F and 19A were obtained more commonly from children <6 years of age, whereas 15A, 6C, 22F and 11A were more common in adults >65 years of age. Pneumococcal pilus PI-1 was identified in antimicrobial-susceptible serotype 15A (61/212) and <10% of 6C isolates (16/188). PI-2 was found in serotype 7F (683/701) and two-thirds of 11A isolates (162/241). Only serotype 19A-ST320 possessed both pili. Molecular and phylogenetic analyses identified serotypes 19A, 15A, 6C, 9N and 33F as highly diverse, whereas 7F, 22F and 11A demonstrated clonality. Antimicrobial resistance determinants were common within diverse serotypes, and usually similar within a clonal complex. Conclusions: Despite successful use of conjugate vaccines, S. pneumoniae remains a highly diverse organism in Canada. Several predominant serotypes, both antimicrobial susceptible and MDR, have demonstrated rapid clonal expansion or an increase in diversity. As S. pneumoniae continues to evolve in Canada, WGS will be a necessary component in the ongoing surveillance of antimicrobial-resistant and expanding clones.

Zika Virus Causes Persistent Infection in Porcine Conceptuses and may Impair Health in Offspring.

Outcomes of Zika virus (ZIKV) infection in pregnant women vary from the birth of asymptomatic offspring to abnormal development and severe brain lesions in fetuses and infants. There are concerns that offspring affected in utero and born without apparent symptoms may develop mental illnesses. Therefore, animal models are important to test interventions against in utero infection and health sequelae in symptomatic and likely more widespread asymptomatic offspring. To partially reproduce in utero infection in humans, we directly inoculated selected porcine conceptuses with ZIKV. Inoculation resulted in rapid trans-fetal infections, persistent infection in conceptuses, molecular pathology in fetal brains, fetal antibody and type I interferon responses. Offspring infected in utero showed ZIKV in their fetal membranes collected after birth. Some in utero affected piglets were small, depressed, had undersized brains, and showed seizures. Some piglets showed potentially increased activity. Our data suggest that porcine model of persistent in utero ZIKV infection has a strong potential for translational research and can be used to test therapeutic interventions in vivo.

What's in a Name? Species-Wide Whole-Genome Sequencing Resolves Invasive and Noninvasive Lineages of Salmonella enterica Serotype Paratyphi B.

UNLABELLED: For 100 years, it has been obvious that Salmonella enterica strains sharing the serotype with the formula 1,4,[5],12:b:1,2-now known as Paratyphi B-can cause diseases ranging from serious systemic infections to self-limiting gastroenteritis. Despite considerable predicted diversity between strains carrying the common Paratyphi B serotype, there remain few methods that subdivide the group into groups that are congruent with their disease phenotypes. Paratyphi B therefore represents one of the canonical examples in Salmonella where serotyping combined with classical microbiological tests fails to provide clinically informative information. Here, we use genomics to provide the first high-resolution view of this serotype, placing it into a wider genomic context of the Salmonella enterica species. These analyses reveal why it has been impossible to subdivide this serotype based upon phenotypic and limited molecular approaches. By examining the genomic data in detail, we are able to identify common features that correlate with strains of clinical importance. The results presented here provide new diagnostic targets, as well as posing important new questions about the basis for the invasive disease phenotype observed in a subset of strains. IMPORTANCE: Salmonella enterica strains carrying the serotype Paratyphi B have long been known to possess Jekyll and Hyde characteristics; some cause gastroenteritis, while others cause serious invasive disease. Understanding what makes up the population of strains carrying this serotype, as well as the source of their invasive disease, is a 100-year-old puzzle that we address here using genomics. Our analysis provides the first high-resolution view of this serotype, placing strains carrying serotype Paratyphi B into the wider genomic context of the Salmonella enterica species. This work reveals a history of disease dating back to the middle ages, caused by a group of distinct lineages with various abilities to cause invasive disease. By quantifying the key genomic differences between the invasive and noninvasive populations, we are able to identify key virulence-related targets that can form the basis of simple, rapid, point-of-care tests.

Tolerance of Listeria monocytogenes to Quaternary Ammonium Sanitizers Is Mediated by a Novel Efflux Pump Encoded by emrE.

A novel genomic island (LGI1) was discovered in Listeria monocytogenes isolates responsible for the deadliest listeriosis outbreak in Canada, in 2008. To investigate the functional role of LGI1, the outbreak strain 08-5578 was exposed to food chain-relevant stresses, and the expression of 16 LGI1 genes was measured. LGI1 genes with putative efflux (L. monocytogenes emrE [emrELm]), regulatory (lmo1851), and adhesion (sel1) functions were deleted, and the mutants were exposed to acid (HCl), cold (4°C), salt (10 to 20% NaCl), and quaternary ammonium-based sanitizers (QACs). Deletion of lmo1851 had no effect on the L. monocytogenes stress response, and deletion of sel1 did not influence Caco-2 and HeLa cell adherence/invasion, whereas deletion of emrE resulted in increased susceptibility to QACs (P < 0.05) but had no effect on the MICs of gentamicin, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, acriflavine, and triclosan. In the presence of the QAC benzalkonium chloride (BAC; 5 µg/ml), 14/16 LGI1 genes were induced, and lmo1861 (putative repressor gene) was constitutively expressed at 4 °C, 37 °C, and 52 °C and in the presence of UV exposure (0 to 30 min). Following 1 h of exposure to BAC (10 µg/ml), upregulation of emrE (49.6-fold), lmo1851 (2.3-fold), lmo1861 (82.4-fold), and sigB (4.1-fold) occurred. Reserpine visibly suppressed the growth of the ΔemrELm strain, indicating that QAC tolerance is due at least partially to efflux activity. These data suggest that a minimal function of LGI1 is to increase the tolerance of L. monocytogenes to QACs via emrELm. Since QACs are commonly used in the food industry, there is a concern that L. monocytogenes strains possessing emrE will have an increased ability to survive this stress and thus to persist in food processing environments.

Draft Genome Sequence of a Necrotoxigenic Escherichia coli Isolate.

Here, we present the draft genome sequence of a necrotoxigenic Escherichia coli strain isolated from a patient following a very rapidly evolving, lethal necrotizing fasciitis.

Characterization of MDR and XDR Streptococcus pneumoniae in Canada, 2007-13.

OBJECTIVES: The goal of this study was to characterize Streptococcus pneumoniae demonstrating MDR (resistant to three or more antimicrobial classes) or XDR (resistant to five or more classes) phenotypes, collected from Canada during the CANWARD 2007-13 study. METHODS: From 2007 to 2013 inclusive, S. pneumoniae isolates were collected as a part of the CANWARD surveillance study. MDR and XDR isolates were subjected to PFGE, MLST, molecular detection of pneumococcal pili and macrolide resistance determinants mef(A/E) and erm(B), sequencing of PBPs 1A, 2B and 2X and comparison with Pneumococcal Molecular Epidemiology Network (PMEN) clones. RESULTS: Of 2129 S. pneumoniae isolates collected during the CANWARD 2007-13 study, 61 (2.9%) were found to be MDR. Of these MDR isolates, 43 (70.5%) were XDR. The most common serotypes for both MDR and XDR S. pneumoniae were 19A and 19F. Twenty-nine of 61 isolates (48%) demonstrated resistance to clarithromycin, clindamycin, doxycycline, penicillin and trimethoprim/sulfamethoxazole. All isolates possessed at least one macrolide resistance determinant and mutations in PBPs 1A, 2B and 2X. The most common clone was piliated, XDR ST320, an internationally circulating double-locus variant of Taiwan(19F)-14 (ST236). CONCLUSIONS: Though the rate of MDR S. pneumoniae has remained relatively stable since 2007, XDR strains have emerged in Canada. These strains are virulent, possess resistance determinants and are related to international clones.

Tatumella saanichensis sp. nov., isolated from a cystic fibrosis patient.

Polyphasic taxonomic analysis was performed on a clinical isolate (NML 06-3099T) from a cystic fibrosis patient, including whole-genome sequencing, proteomics, phenotypic testing, electron microscopy, chemotaxonomy and a clinical investigation. Comparative whole-genome sequence analysis and multilocus sequence analysis (MLSA) between Tatumella ptyseos ATCC 33301T and clinical isolate NML 06-3099T suggested that the clinical isolate was closely related to, but distinct from, the species T. ptyseos. By 16S rRNA gene sequencing, the clinical isolate shared 98.7 % sequence identity with T. ptyseos ATCC 33301T. A concatenate of six MLSA loci (totalling 4500 bp) revealed < 93.9 % identity between T. ptyseos ATCC 33301T, other members of the genus and the clinical isolate. A whole-genome sequence comparison between NML 06-3099T and ATCC 33301T determined that the average nucleotide identity was 76.24 %. The overall DNA G+C content of NML 06-3099T was 51.27 %, consistent with members of the genus Tatumella. By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis, NML 06-3099T had a genus-level match, but not a species-level match, to T. ptyseos. By shotgun proteomics, T. ptyseos ATCC 33301T and NML 06-3099T were found to have unique proteomes. The two strains had similar morphologies and multiple fimbriae, as observed by transmission electron microscopy, but were distinguishable by phenotypic testing. Cellular fatty acids found were typical for members of the Enterobacteriaceae. NML 06-3099T was susceptible to commonly used antibiotics. Based on these data, NML 06-3099T represents a novel species in the genus Tatumella, for which the name Tatumella saanichensis sp. nov. is proposed (type strain NML 06-3099T = CCUG 55408T = DSM 19846T).

Assessment of multidrug resistance, clonality and virulence in non-PCV-13 Streptococcus pneumoniae serotypes in Canada, 2011-13.

OBJECTIVES: Serotype replacement in Streptococcus pneumoniae following the implementation of a new vaccine has been associated with the emergence of non-vaccine serotypes as prominent causes of invasive pneumococcal disease (IPD). The aim of this study was to characterize specific non-PCV-13 serotypes 15A, 22F, 33F and 35B from IPD, isolated in Canada post-PCV-13 introduction in 2010. METHODS: Of 3802 IPD isolates collected from across Canada in 2011-13, 18.4% were found to be serotypes 15A, 22F, 33F and 35B. These 699 isolates were subjected to antimicrobial susceptibility testing, PFGE, MLST, molecular detection of pneumococcal pili and comparison with Pneumococcal Molecular Epidemiology Network (PMEN) clones. RESULTS: This study demonstrated clonal spread of specific STs, including MDR ST63 and its Sweden(15A)-25-related variants, the increasingly common ST433 and a variant of piliated, penicillin-non-susceptible ST558, related to PMEN clone Utah(35B)-24 (ST377). New STs of serotype 33F were identified. Several potential capsular switching events were identified within these serotypes. CONCLUSIONS: Non-PCV-13 serotype 22F is increasing in Canada through the rapid clonal expansion of ST433. Numerous new STs associated with serotype 33F indicate the potential divergence of the serotype. Serotypes 15A and 35B in Canada are related to international clones of S. pneumoniae.