Genomic investigation of the most common Streptococcus pneumoniae serotypes causing invasive infections in Canada: the SAVE study, 2011-2020.

OBJECTIVES: To investigate the lineages and genomic antimicrobial resistance (AMR) determinants of the 10 most common pneumococcal serotypes identified in Canada during the five most recent years of the SAVE study, in the context of the 10-year post-PCV13 period in Canada. METHODS: The 10 most common invasive Streptococcus pneumoniae serotypes collected by the SAVE study from 2016 to 2020 were 3, 22F, 9N, 8, 4, 12F, 19A, 33F, 23A and 15A. A random sample comprising ∼5% of each of these serotypes collected during each year of the full SAVE study (2011-2020) were selected for whole-genome sequencing (WGS) using the Illumina NextSeq platform. Phylogenomic analysis was performed using the SNVPhyl pipeline. WGS data were used to identify virulence genes of interest, sequence types, global pneumococcal sequence clusters (GPSC) and AMR determinants. RESULTS: Of the 10 serotypes analysed in this study, six increased significantly in prevalence from 2011 to 2020: 3, 4, 8, 9N, 23A and 33F (P ≤ 0.0201). Serotypes 12F and 15A remained stable in prevalence over time, while serotype 19A decreased in prevalence (P < 0.0001). The investigated serotypes represented four of the most prevalent international lineages causing non-vaccine serotype pneumococcal disease in the PCV13 era: GPSC3 (serotypes 8/33F), GPSC19 (22F), GPSC5 (23A) and GPSC26 (12F). Of these lineages, GPSC5 isolates were found to consistently possess the most AMR determinants. Commonly collected vaccine serotypes 3 and 4 were associated with GPSC12 and GPSC27, respectively. However, a more recently collected lineage of serotype 4 (GPSC192) was highly clonal and possessed AMR determinants. CONCLUSIONS: Continued genomic surveillance of S. pneumoniae in Canada is essential to monitor for the appearance of new and evolving lineages, including antimicrobial-resistant GPSC5 and GPSC162.

Linear Regression Equations To Predict ß-Lactam, Macrolide, Lincosamide, and Fluoroquinolone MICs from Molecular Antimicrobial Resistance Determinants in Streptococcus pneumoniae.

Antimicrobial resistance in Streptococcus pneumoniae represents a threat to public health, and monitoring the dissemination of resistant strains is essential to guiding health policy. Multiple-variable linear regression modeling was used to determine the contributions of molecular antimicrobial resistance determinants to antimicrobial MICs for penicillin, ceftriaxone, erythromycin, clarithromycin, clindamycin, levofloxacin, and trimethoprim-sulfamethoxazole. Training data sets consisting of Canadian S. pneumoniae isolates obtained from 1995 to 2019 were used to generate multiple-variable linear regression equations for each antimicrobial. The regression equations were then applied to validation data sets of Canadian (n = 439) and U.S. (n = 607 and n = 747) isolates. The MICs for ß-lactam antimicrobials were fully explained by amino acid substitutions in motif regions of the penicillin binding proteins PBP1a, PPB2b, and PBP2x. Accuracies of predicted MICs within 1 doubling dilution to phenotypically determined MICs were 97.4% for penicillin, 98.2% for ceftriaxone, 94.8% for erythromycin, 96.6% for clarithromycin, 98.2% for clindamycin, 100% for levofloxacin, and 98.8% for trimethoprim-sulfamethoxazole, with an overall sensitivity of 95.8% and specificity of 98.0%. Accuracies of predicted MICs to the phenotypically determined MICs were similar to those of phenotype-only MIC comparison studies. The ability to acquire detailed antimicrobial resistance information directly from molecular determinants will facilitate the transition from routine phenotypic testing to whole-genome sequencing analysis and can fill the surveillance gap in an era of increased reliance on nucleic acid assay diagnostics to better monitor the dynamics of S. pneumoniae.

Characterization of invasive meningococcal disease case isolates in Atlantic Canada, 2014 to 2020: spatial-temporal variations of clones and predicted meningococcal B vaccine coverage.

Introduction. Invasive meningococcal disease (IMD) caused by Neisseria meningitidis may show temporal and geographical changes in both the epidemiology and the characteristics of the strains involved.Gap statement. A study that examined invasive N. meningitidis causing IMD in Atlantic Canada from 2009 to 2013 was published in 2014. Data from subsequent years have not been described.Aim. This study examined the molecular epidemiology of IMD in four Atlantic Provinces of Canada as well as potential serogroup B (MenB) vaccine coverage.Methods. Individual IMD case isolates recovered from 2014 to 2020 were analysed for serotype and serosubtype antigens as well as by whole-genome sequencing (WGS) for prediction of potential MenB vaccine coverage.Results. Of the 56 IMD isolates, 42, 8, 5 and 1 were MenB, serogroup Y, serogroup W (MenW) and serogroup C, respectively. Geographical differences in the distribution of MenB clones revealed concentration of sequence type (ST)-269 clonal complex (cc) and ST-60 cc in Newfoundland and Labrador, while ST-41/44 cc (particularly ST-154) was predominantly found in New Brunswick and Nova Scotia. Core genome multi-locus sequence typing (cgMLST) also separated the New Brunswick and Nova Scotia ST-154 isolates into two clusters, with differences in their nhba and penA alleles. Furthermore, cgMLST also separated the ST-269 cc isolates in Atlantic Canada into the ST-1611 and the ST-269/ST-8924 clusters, with the latter showing high similarity to the ST-269 that first emerged in the Province of Quebec. Genetic Meningococcal Antigen Typing System showed that 54.8 % of MenB were predicted to be covered by the MenB vaccine Bexsero, with a further 38.1 % potentially covered by virtue of the presence of genes that encoded factor H-binding protein variant 1 proteins. Meningococcal deduced vaccine antigen reactivity predicted from WGS data showed that 95.3 % of MenB were covered by Trumenba. Four cases of IMD due to MenW ST-11 cc were also identified, with the first case found in 2018.Conclusions. This study provided evidence concerning the dynamics of N. meningitidis strains causing IMD in Atlantic Canada, with both geographical and temporal differences found. MenB vaccine appeared to provide good coverage of MenB IMD, especially towards the predominant strain of ST-154.

Invasive pneumococcal disease surveillance in Canada, 2020.

Background: Invasive pneumococcal disease (IPD), which is caused by Streptococcus pneumoniae, has been a nationally notifiable disease in Canada since 2000. The use of conjugate vaccines has markedly decreased the incidence of IPD in Canada; however, the distribution of serotypes has shifted in favour of non-vaccine types. This report summarizes the demographics, serotypes and antimicrobial resistance of IPD infections in Canada in 2020. Methods: The Public Health Agency of Canada's National Microbiology Laboratory (Winnipeg, Manitoba) collaborates with provincial and territorial public health laboratories to conduct national surveillance of IPD. A total of 2,108 IPD isolates were reported in 2020. Serotyping was performed by Quellung reaction and antimicrobial susceptibilities were determined in collaboration with the University of Manitoba/Canadian Antimicrobial Resistance Alliance. Population-based IPD incidence rates were obtained through the Canadian Notifiable Disease Surveillance System. Results: Overall incidence of IPD in Canada decreased significantly from 11.5 (95% confidence interval [CI]: 10.1-13.1) to 6.0 (95% CI: 5.0-7.2), and from 10.0 (95% CI: 9.7-10.3) to 5.9 (95% CI: 5.7-6.2) cases per 100,000 from 2019 to 2020; in those younger than five years and those five years and older, respectively. The most common serotypes overall were 4 (11.2%, n=237), 3 (10.9%, n=229) and 8 (7.2%, n=151). From 2016 to 2020, serotypes with increasing trends (p<0.05) included 4 (6.4%-11.2%), 3 (9.5%-10.9%), 8 (5.2%-7.2%) and 12F (3.6%-5.7%). The overall prevalence of PCV13 serotypes increased over the same period (30.3%-34.9%, p<0.05). Antimicrobial resistance rates in 2020 included 23.0% clarithromycin and 9.9% penicillin (IV meningitis breakpoints). Multidrug-resistant IPD has significantly increased since 2016 (4.2%-9.5%, p<0.05). Conclusion: Though the incidence of IPD decreased in 2020 in comparison to previous years across all age groups, disease due to PCV13 serotypes 3 and 4, as well as non-PCV13 serotypes such as 8 and 12F, increased in prevalence. Continued surveillance of IPD is imperative to monitor shifts in serotype distribution and antimicrobial resistance.

Invasive group A streptococcal disease surveillance in Canada, 2020.

Background: Invasive group A streptococcal (iGAS) disease (caused by Streptococcus pyogenes) has been a nationally notifiable disease in Canada since 2000. This report summarizes the demographics, emm types and antimicrobial resistance of iGAS infections in Canada in 2020. Methods: The Public Health Agency of Canada's National Microbiology Laboratory (Winnipeg, Manitoba) collaborates with provincial and territorial public health laboratories to conduct national surveillance of invasive S. pyogenes. Emm typing was performed on all isolates using the Centers for Disease Control and Prevention emm sequencing protocol. Antimicrobial susceptibilities were determined using Kirby-Bauer disk diffusion according to Clinical and Laboratory Standards Institute guidelines. Population-based iGAS disease incidence rates up to 2019 were obtained through the Canadian Notifiable Disease Surveillance System. Results: Overall, the incidence of iGAS disease in Canada has increased from 4.0 to 8.1 cases per 100,000 population from 2009 to 2019. The 2019 incidence represents a slight decrease from the 2018 rate of 8.6 cases per 100,000 population. A total of 2,867 invasive S. pyogenes isolates that were collected during 2020 are included in this report, representing a decrease from 2019 (n=3,194). The most common emm types in 2020 were emm49 (16.8%, n=483) and emm76 (15.0%, n=429), both increasing significantly in prevalence since 2016 (p<0.001). The former most prevalent type, emm1, decreased to 7.6% (n=217) in 2020 from 15.4% (n=325) in 2016. Antimicrobial resistance rates in 2020 included 11.5% resistance to erythromycin, 3.2% resistance to clindamycin and 1.6% nonsusceptibility to chloramphenicol. Conclusion: Though the number of collected invasive S. pyogenes isolates decreased slightly in 2020 in comparison to previous years, iGAS disease remains an important public health concern. The emm distribution in Canada has been subtly shifting over the past five years, away from common and well-known emm1 and towards emm49 and emm76. It is important to continue surveillance of S. pyogenes in Canada to monitor expanding replacement emm types, as well as outbreak clones and antimicrobial resistance.

lfnA from Pseudomonas aeruginosa O12 and wbuX from Escherichia coli O145 encode membrane-associated proteins and are required for expression of 2,6-dideoxy-2-acetamidino-L-galactose in lipopolysaccharide O antigen.

The rare sugar 2,6-dideoxy-2-acetamidino-L-galactose (L-FucNAm) is found only in bacteria and is a component of cell surface glycans in a number of pathogenic species, including the O antigens of Pseudomonas aeruginosa serotype O12 and Escherichia coli O145. P. aeruginosa is an important opportunistic pathogen, and the O12 serotype is associated with multidrug-resistant epidemic outbreaks. O145 is one of the classic non-O157 serotypes associated with Shiga toxin-producing, enterohemorrhagic E. coli. The acetamidino (NAm) moiety of L-FucNAm is of interest, because at neutral pH it contributes a positive charge to the cell surface, and we aimed to characterize the biosynthesis of this functional group. The pathway is not known, but expression of NAm-modified sugars coincides with the presence of a pseA homologue in the relevant biosynthetic locus. PseA is a putative amidotransferase required for synthesis of a NAm-modified sugar in Campylobacter jejuni. In P. aeruginosa O12 and E. coli O145, the pseA homologues are lfnA and wbuX, respectively, and we hypothesized that these genes function in L-FucNAm biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and nuclear magnetic resonance analysis of the lfnA mutant O-antigen structure indicated that the mutant expresses 2,6-dideoxy-2-acetamido-L-galactose (L-FucNAc) in place of L-FucNAm. The mutation could be complemented by expression of either His(6)-tagged lfnA or wbuX in trans, confirming that these genes are functional homologues and that they are required for NAm moiety synthesis. Both proteins retained their activity when fused to a His(6) tag and localized to the membrane fraction. These data will assist future biochemical investigation of this pathway.