Antimicrobial susceptibility testing of invasive isolates of Streptococcus pneumoniae from Canadian patients: the SAVE study, 2011-2020.

OBJECTIVES: To assess the antimicrobial susceptibility of 14 138 invasive Streptococcus pneumoniae isolates collected in Canada from 2011 to 2020. METHODS: Antimicrobial susceptibility testing was performed using the CLSI M07 broth microdilution reference method. MICs were interpreted using 2022 CLSI M100 breakpoints. RESULTS: In 2020, 90.1% and 98.6% of invasive pneumococci were penicillin-susceptible when MICs were interpreted using CLSI meningitis or oral and non-meningitis breakpoints, respectively; 96.9% (meningitis breakpoint) and 99.5% (non-meningitis breakpoint) of isolates were ceftriaxone-susceptible, and 99.9% were levofloxacin-susceptible. Numerically small, non-temporal, but statistically significant differences (P < 0.05) in the annual percentage of isolates susceptible to four of the 13 agents tested was observed across the 10-year study: chloramphenicol (4.4% difference), trimethoprim-sulfamethoxazole (3.9%), penicillin (non-meningitis breakpoint, 2.7%) and ceftriaxone (meningitis breakpoint, 2.7%; non-meningitis breakpoint, 1.2%). During the same period, annual differences in percent susceptible values for penicillin (meningitis and oral breakpoints) and all other agents did not achieve statistical significance. The percentage of isolates with an MDR phenotype (resistance to ≥3 antimicrobial classes) in 2011 and 2020 (8.5% and 9.4%) was not significantly different (P = 0.109), although there was a significant interim decrease observed between 2011 and 2015 (P < 0.001) followed by a significant increase between 2016 and 2020 (P < 0.001). Statistically significant associations were observed between resistance rates to most antimicrobial agents included in the MDR analysis (penicillin, clarithromycin, clindamycin, doxycycline, trimethoprim/sulfamethoxazole and chloramphenicol) and patient age, specimen source, geographic location in Canada or concurrent resistance to penicillin or clarithromycin, but not biological sex of patients. Given the large isolate collection studied, statistical significance did not necessarily imply clinical or public health significance in some analyses. CONCLUSIONS: Invasive pneumococcal isolates collected in Canada from 2011 to 2020 generally exhibited consistent in vitro susceptibility to commonly tested antimicrobial agents.

Streptococcus pneumoniae serotyping and antimicrobial susceptibility: assessment for vaccine efficacy in Canada after the introduction of PCV13.

BACKGROUND: Streptococcus pneumoniae continues to be an important bacterial pathogen associated with invasive (e.g. bacteraemia, meningitis) and non-invasive (e.g. community-acquired respiratory tract) infections worldwide. Surveillance studies conducted nationally and globally assist in determining trends over geographical areas and allow comparisons between countries. OBJECTIVES: To characterize invasive isolates of S. pneumoniae in terms of their serotype, antimicrobial resistance, genotype and virulence and to use the serotype data to determine the level of coverage by different generations of pneumococcal vaccines. METHODS: SAVE (Streptococcus pneumoniae Serotyping and Antimicrobial Susceptibility: Assessment for Vaccine Efficacy in Canada) is an ongoing, annual, national collaborative study between the Canadian Antimicrobial Resistance Alliance (CARE) and the National Microbiology Laboratory, focused on characterizing invasive isolates of S. pneumoniae obtained across Canada. Clinical isolates from normally sterile sites were forwarded by participating hospital public health laboratories to the Public Health Agency of Canada-National Microbiology Laboratory and CARE for centralized phenotypic and genotypic investigation. RESULTS: The four articles in this Supplement provide a comprehensive examination of the changing patterns of antimicrobial resistance and MDR, serotype distribution, genotypic relatedness and virulence of invasive S. pneumoniae obtained across Canada over a 10 year period (2011-2020). CONCLUSIONS: The data highlight the evolution of S. pneumoniae under pressure by vaccination and antimicrobial usage, as well as vaccine coverage, allowing both clinicians and researchers nationally and globally to view the current status of invasive pneumococcal infections in Canada.

Analysis of MDR in the predominant Streptococcus pneumoniae serotypes in Canada: the SAVE study, 2011-2020.

OBJECTIVES: To investigate the levels of MDR in the predominant serotypes of invasive Streptococcus pneumoniae isolated in Canada over a 10 year period. METHODS: All isolates were serotyped and had antimicrobial susceptibility testing performed, in accordance with CLSI guidelines (M07-11 Ed., 2018). Complete susceptibility profiles were available for 13 712 isolates. MDR was defined as resistance to three or more classes of antimicrobial agents (penicillin MIC ≥2 mg/L defined as resistant). Serotypes were determined by Quellung reaction. RESULTS: In total, 14 138 invasive isolates of S. pneumoniae were tested in the SAVE study (S. pneumoniae Serotyping and Antimicrobial Susceptibility: Assessment for Vaccine Efficacy in Canada), a collaboration between the Canadian Antimicrobial Resistance Alliance and Public Health Agency of Canada-National Microbiology Laboratory. The rate of MDR S. pneumoniae in SAVE was 6.6% (902/13 712). Annual rates of MDR S. pneumoniae decreased between 2011 and 2015 (8.5% to 5.7%) and increased between 2016 and 2020 (3.9% to 9.4%). Serotypes 19A and 15A were the most common serotypes demonstrating MDR (25.4% and 23.5% of the MDR isolates, respectively); however, the serotype diversity index increased from 0.7 in 2011 to 0.9 in 2020 with a statistically significant linear increasing trend (P < 0.001). In 2020, MDR isolates were frequently serotypes 4 and 12F in addition to serotypes 15A and 19A. In 2020, 27.3%, 45.5%, 50.5%, 65.7% and 68.7% of invasive MDR S. pneumoniae were serotypes included in the PCV10, PCV13, PCV15, PCV20 and PPSV23 vaccines, respectively. CONCLUSIONS: Although current vaccine coverage of MDR S. pneumoniae in Canada is high, the increasing diversity of serotypes observed among the MDR isolates highlights the ability of S. pneumoniae to rapidly evolve.

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.

Comparison of PCV10, PCV13, PCV15, PCV20 and PPSV23 vaccine coverage of invasive Streptococcus pneumoniae isolate serotypes in Canada: the SAVE study, 2011-20.

BACKGROUND: As pneumococci evolve under vaccine, antimicrobial and other selective pressures, it is important to track isolates covered by established (PCV10, PCV13 and PPSV23) and new (PCV15 and PCV20) vaccine formulations. OBJECTIVES: To compare invasive pneumococcal disease (IPD) isolates from serotypes covered by PCV10, PCV13, PCV15, PCV20 and PPSV23, collected in Canada from 2011 to 2020, by demographic category and antimicrobial resistance phenotype. METHODS: IPD isolates from the SAVE study were initially collected by members of the Canadian Public Health Laboratory Network (CPHLN) as part of a collaboration between the Canadian Antimicrobial Resistance Alliance (CARA) and the Public Health Agency of Canada (PHAC). Serotypes were determined by quellung reaction, and antimicrobial susceptibility testing was performed using the CLSI broth microdilution method. RESULTS: A total of 14 138 invasive isolates were collected from 2011 to 2020, with 30.7% of isolates covered by the PCV13 vaccine, 43.6% of isolates covered by the PCV15 vaccine (including 12.9% non-PCV13 serotypes 22F and 33F), and 62.6% of isolates covered by the PCV20 vaccine (including 19.0% non-PCV15 serotypes 8, 10A, 11A, 12F and 15B/C). Non-PCV20 serotypes 2, 9N, 17F and 20, but not 6A (present in PPSV23) represented 8.8% of all IPD isolates. Higher-valency vaccine formulations covered significantly more isolates by age, sex, region and resistance phenotype including MDR isolates. Coverage of XDR isolates did not significantly differ between vaccine formulations. CONCLUSIONS: When compared with PCV13 and PCV15, PCV20 covered significantly more IPD isolates stratified by patient age, region, sex, individual antimicrobial resistance phenotypes and MDR phenotype.

Antimicrobial resistance patterns of bacterial pathogens recovered from the urine of patients at Canadian hospitals from 2009 to 2020.

Objectives: To investigate in vitro susceptibility patterns of bacterial pathogens recovered from the urine of outpatients (isolates from outpatient clinics or emergency departments) and hospital inpatients across Canada from 2009 to 2020 as part of the CANWARD study. Methods: Canadian hospital microbiology laboratories submitted bacterial pathogens cultured from urine to the CANWARD study coordinating laboratory on an annual basis (January 2009 to December 2020). Antimicrobial susceptibility testing was performed by CLSI broth microdilution, with MICs interpreted by current CLSI breakpoints. Results: In total, 4644 urinary pathogens were included in this study. Escherichia coli was recovered most frequently (53.3% of all isolates), followed by Enterococcus faecalis, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. Together, these six species accounted for 84.2% of study isolates. Nitrofurantoin demonstrated excellent in vitro activity versus E. coli, with 97.6% of outpatient and 96.1% of inpatient isolates remaining susceptible. In contrast, E. coli susceptibility rates were lower for ciprofloxacin (outpatient 79.5%, inpatient 65.9%) and trimethoprim/sulfamethoxazole (outpatient 75.2%, inpatient 73.5%). The percentage of E. coli isolates that were phenotypically positive for ESBL production significantly increased from 4.2% (2009-11) to 11.3% (2018-20). A similar although less pronounced temporal trend was observed with ESBL-producing K. pneumoniae. Conclusions: E. coli was the pathogen most frequently recovered from the urine of Canadian patients, and the proportion of isolates that were ESBL producers increased over time. Susceptibility data presented here suggest that ciprofloxacin and trimethoprim/sulfamethoxazole may be suboptimal for the empirical treatment of complicated urinary infections.

In vitro activity of sulopenem against 1880 bacterial pathogens isolated from Canadian patients with urinary tract infections (CANWARD, 2014-21).

INTRODUCTION: There are limited oral antimicrobial options for the treatment of urinary infections caused by ESBL-producing and MDR Enterobacterales. Sulopenem is an investigational thiopenem antimicrobial that is being developed as both an oral and IV formulation. The purpose of this study was to evaluate the in vitro activity of sulopenem versus bacterial pathogens recovered from the urine of patients admitted to or assessed at hospitals across Canada (CANWARD). MATERIALS AND METHODS: The in vitro activity of sulopenem and clinically relevant comparators was determined for 1880 Gram-negative and Gram-positive urinary isolates obtained as part of the CANWARD study (2014 to 2021) using the CLSI broth microdilution method. RESULTS: Sulopenem demonstrated excellent in vitro activity versus members of the Enterobacterales, with MIC90 values ranging from 0.06 to 0.5 mg/L for all species tested. Over 90% of ESBL-producing, AmpC-producing and MDR (not susceptible to ≥1 antimicrobial from ≥3 classes) Escherichia coli were inhibited by ≤0.25 mg/L of sulopenem. Sulopenem had an identical MIC90 to meropenem for ESBL-producing and MDR E. coli. The MIC90 of sulopenem and meropenem versus MSSA was 0.25 mg/L. Sulopenem was not active in vitro versus Pseudomonas aeruginosa (similar to ertapenem), and it demonstrated poor activity versus Enterococcus faecalis (similar to meropenem). CONCLUSIONS: Sulopenem demonstrated excellent in vitro activity versus bacterial pathogens recovered from the urine of Canadian patients. These data suggest that sulopenem may have a role in the treatment of urinary infections caused by antimicrobial-resistant Enterobacterales, but additional clinical studies are required.

In vitro activity of fosfomycin against bacterial pathogens isolated from urine specimens in Canada from 2007 to 2020: CANWARD surveillance study.

BACKGROUND: Multiple susceptible breakpoints are published to interpret fosfomycin MICs: ≤64 mg/L for Escherichia coli and Enterococcus faecalis grown from urine (CLSI M100); ≤32 mg/L for Enterobacterales and staphylococci when parenteral fosfomycin is prescribed (EUCAST); and ≤8 mg/L for uncomplicated urinary tract infection with E. coli when oral fosfomycin is used (EUCAST). Clinical laboratories are frequently requested to test fosfomycin against antimicrobial-resistant urinary isolates not included in standard documents. METHODS: The in vitro activity of fosfomycin was determined using the CLSI agar dilution method for a 2007-20 collection of clinically significant Gram-negative (3656 Enterobacterales; 140 Pseudomonas aeruginosa) and Gram-positive (346 E. faecalis; 94 Staphylococcus aureus) urinary isolates from the CANWARD surveillance study. Comparator agents were tested using CLSI broth microdilution. RESULTS: Using the CLSI MIC breakpoint (≤64 mg/L), 99.2% of E. coli (n = 2871; MIC90, 4 mg/L), including 96.7% of ESBL-positive isolates, were fosfomycin susceptible. Similarly, 95.8% of E. coli, including 95.2% of ESBL-positive isolates, were fosfomycin susceptible at ≤8 mg/L (EUCAST oral susceptible MIC breakpoint). All other species of Enterobacterales (except Citrobacter freundii) and P. aeruginosa had higher fosfomycin MICs (MIC90s, 64 to >512 mg/L) than E. coli. Using published breakpoints, 88.4% of E. faecalis (MIC ≤64 mg/L) and 97.9% of S. aureus (MIC ≤32 mg/L) isolates were fosfomycin susceptible. CONCLUSIONS: Fosfomycin demonstrated in vitro activity against frequently encountered Gram-positive and Gram-negative urinary pathogens; however, the extrapolation of current CLSI and EUCAST MIC breakpoints to pathogens not specified by standard methods requires further study and is currently not recommended.

In Vitro Activity of Cefiderocol against Extensively Drug-Resistant Pseudomonas aeruginosa: CANWARD, 2007 to 2019.

Cefiderocol was evaluated by broth microdilution versus 1,050 highly antimicrobial-resistant Pseudomonas aeruginosa clinical isolates from the CANWARD study (2007 to 2019). Overall, 98.3% of isolates remained cefiderocol susceptible (MIC, ≤4 µg/mL), including 97.4% of extensively drug-resistant (XDR) (n = 235) and 97.9% of multidrug-resistant (MDR) (n = 771) isolates. Most isolates testing not susceptible to ceftolozane-tazobactam, ceftazidime-avibactam, and imipenem-relebactam remained susceptible to cefiderocol. In vitro data suggest that cefiderocol may be a treatment option for infections caused by MDR and XDR P. aeruginosa. IMPORTANCE After testing cefiderocol against a large collection of clinical isolates of highly antimicrobial-resistant Pseudomonas aeruginosa, we report that cefiderocol is active versus 97.4% of extensively drug-resistant (XDR) and 97.9% of multidrug-resistant (MDR) (n = 771) isolates. These data show that cefiderocol may be a treatment option for infections caused by MDR and XDR P. aeruginosa.

Activity of cefepime/taniborbactam and comparators against whole genome sequenced ertapenem-non-susceptible Enterobacterales clinical isolates: CANWARD 2007-19.

OBJECTIVES: This study assessed in vitro activities of cefepime/taniborbactam and comparator antimicrobial agents against ertapenem-non-susceptible Enterobacterales (ENSE) clinical isolates collected from the CANWARD study 2007-19, and associations between MIC and various mechanisms of ß-lactam resistance identified using WGS. METHODS: A total of 179 ENSE (MIC ≥ 1 mg/L) isolates underwent susceptibility testing using reference CLSI broth microdilution. WGS was performed using the Illumina NextSeq platform. Carbapenemases, ESBLs and other ß-lactamases were identified using ResFinder 4.0. Alterations in ompC/F and ftsI (PBP3) were identified by comparing extracted sequences to the appropriate NCBI reference gene. Porin alterations were analysed with Provean v1.1.3. Specific alterations of interest in PBP3 included a YRIN or YRIK insertion after P333. RESULTS: Cefepime/taniborbactam was highly active (MIC50/MIC90, 0.5/2 mg/L; 177/179 isolates inhibited at ≤ 8 mg/L) against ENSE with various antimicrobial resistance phenotypes. Thirteen (7.3%) of the 179 ENSE isolates demonstrated cefepime/taniborbactam MIC values ≥ 4 mg/L and possessed combinations of ß-lactam resistance mechanisms, including a carbapenemase and/or ESBL and/or other ß-lactamase genes, as well as alterations in OmpC and/or OmpF and/or PBP3. Of the two Escherichia coli isolates that demonstrated a cefepime/taniborbactam MIC of 32 mg/L, one possessed NDM-5, OXA-181 and TEM-1B, an OmpC alteration and P333_Y334insYRIN in PBP3, while the second contained CTX-M-71, a truncated OmpF and a large alteration in OmpC (F182_R195delinsMTTNGRDDVFE). CONCLUSIONS: Cefepime/taniborbactam was highly active against ENSE with various antimicrobial resistance phenotypes/genotypes. ENSE isolates with cefepime/taniborbactam MIC values ≥ 4 mg/L possessed combinations of ß-lactam resistance mechanisms, including ß-lactamase genes, as well as alterations in OmpC and/or OmpF and/or PBP3.

PCV-15 and PPSV-23 coverage of invasive and respiratory tract Streptococcus pneumoniae, including MDR and XDR isolates: CANWARD 2007-20.

OBJECTIVES: To compare the proportion of invasive and respiratory tract isolates of Streptococcus pneumoniae, including MDR and XDR strains, that demonstrated PCV-15 and PPSV-23 serotypes in Canada from 2007 to 2020. METHODS: The CANWARD study collected 2984 S. pneumoniae isolates from 2007 to 2020 (1054 invasive, 1930 respiratory). Serotyping was performed using the Quellung reaction. Antimicrobial susceptibility testing was performed using CLSI methods. MDR/XDR was defined as resistance to ≥3/≥5 antimicrobial classes, respectively. RESULTS: Overall, the proportion of vaccine serotypes demonstrating a PCV-15/PPSV-23 serotype was significantly higher in blood isolates (54.6%/76.2%, respectively) than respiratory isolates (38.9%/55.3%; P < 0.0001). Similarly, PCV-15 and PPSV-23 vaccine coverage was higher for blood isolates for all demographic categories, including both genders, all regions and all age groups (P ≤ 0.0213). PCV-15/PPSV-23 coverage was also significantly higher for blood isolates demonstrating clarithromycin resistance (60.4/75.1% blood, 47.8/57.4% respiratory; P ≤ 0.009) and penicillin resistance (68.9/63.0% blood, 45.2/43.0% respiratory; P < 0.0001) and trimethoprim/sulfamethoxazole-resistant isolates for PPSV-23 only (82.6% blood, 64.3% respiratory; P = 0.0057). Vaccine coverage was numerically higher but not significantly different between specimen source for children <2 years of age, as well as ceftriaxone-, doxycycline- and levofloxacin-resistant isolates. PCV-15/PPSV-23 vaccine coverage for MDR isolates (61.8%/67.3% blood, 52.2%/56.2% respiratory) and XDR isolates (93.3% blood, 89.6% respiratory for both vaccines) was not significantly different between specimen sources. CONCLUSIONS: PCV-15 and PPSV-23 serotype coverage is generally greater for blood versus respiratory isolates but not for MDR and XDR isolates. Continued pneumococcal surveillance is warranted to determine future trends in vaccine coverage, serotype distribution and antimicrobial susceptibilities under the pressure of vaccine use.

Use of Fosfomycin Etest To Determine In Vitro Susceptibility of Clinical Isolates of Enterobacterales Other than Escherichia coli, Nonfermenting Gram-Negative Bacilli, and Gram-Positive Cocci.

Clinical isolates of Enterobacterales other than Escherichia coli (EOTEC), nonfermenting Gram-negative bacilli, and Gram-positive cocci were tested for susceptibility to fosfomycin using Etest and reference agar dilution. Applying EUCAST (v. 11.0, 2021) intravenous fosfomycin breakpoints, Etest MICs for EOTEC showed essential agreement (EA), categorical agreement (CA), major error (ME), and very major error (VME) rates of 70.4%, 88.4%, 4.1%, and 32.1%, respectively. No species of EOTEC tested with acceptable rates for all of EA (≥90%), CA (≥90%), ME (≤3%), and VME (≤3%). Etest MICs for Enterococcus faecalis, interpreted using CLSI oral/urine criteria (M100, 2021) showed EA, CA, minor error, ME, and VME rates of 98.5%, 81.2%, 18.8%, 0%, and 0%. Against Staphylococcus aureus, EA, CA, and ME rates were 84.1%, 98.7%, and 1.3% (EUCAST intravenous criteria). S. aureus isolates with fosfomycin MICs of >32 µg/ml (resistant) were not identified by agar dilution. We conclude that performing fosfomycin Etest on isolates of S. aureus will reliably identify fosfomycin-susceptible isolates with low, acceptable rates of MEs and VMEs. Testing of urinary isolates of E. faecalis by Etest is associated with an unacceptably high rate of minor errors (18.8%) but low, acceptable rates of MEs and VMEs when results are interpreted using CLSI criteria. Isolates of EOTEC tested by Etest with resulting MICs interpreted by EUCAST criteria were associated with an unacceptably high VME rate (32.1%). In vitro testing of clinical isolates beyond E. coli, E. faecalis, and S. aureus to determine susceptibility to fosfomycin is problematic with current methods and breakpoints.

Whole genome characterization of Streptococcus pneumoniae from respiratory and blood cultures collected from Canadian hospitals before and after PCV-13 implementation in Canada: Focus on serotypes 22F and 33F from CANWARD 2007-2018.

The population of pneumococci circulating in Canada is constantly shifting under the pressures of antimicrobial and conjugate vaccine use. A new 15-valent pneumococcal conjugate vaccine (PCV), containing PCV-13 serotypes plus additional serotypes 22F and 33F, is currently undergoing clinical trials. The purpose of this study was to utilize whole genome sequencing to characterize invasive and respiratory Streptococcus pneumoniae isolates collected from Canadian hospitals pre- (2007-2011) and post-PCV-13 implementation (2012-2018) in Canada, particularly serotypes 22F and 33F. Isolates were obtained from the CANWARD 2007 to 2018 study. Overall, 597 S. pneumoniae isolates were sequenced using the Illumina MiSeq platform: 180 (101 respiratory, 79 blood) isolates of serotype 22F, 74 (41 respiratory, 33 blood) isolates of serotype 33F and 343 isolates randomly selected to broadly encompass pneumococci in Canada. Genomes were clustered using PopPUNK v2.0.2 and assigned to a Global Pneumococcal Sequencing Cluster (GPSC) and MLST sequence type (ST), and visualized using Cytoscape v3.8.0. Acquired resistance genes were identified using ResFinder 2.1, and genes with chromosomal mutations conferring resistance were extracted and compared to standard reference genome R6. PopPUNK clustering suggests that a clone of S. pneumoniae serotype 22F/ST433/GPSC19 demonstrating mefA-mediated macrolide resistance is emerging in Canada post-PCV-13 introduction, collected from both invasive and respiratory sources. Similarly, there is evidence to support a post-PCV-13 shift towards macrolide- and trimethoprim/sulfamethoxazole-resistant serotype 33F/ST100/GPSC3, including a cluster associated with invasive isolates. While some lineages containing vaccine serotypes were predominantly identified pre-PCV-13 implementation (serotype 5/GPSC8, serotype 7F/GPSC15), others (serotype 19A/GPSC1 and 4, serotype 3/GPSC12) continue to maintain a significant presence over time despite inclusion in PCV-13. Further genomic surveillance is necessary to determine additional trends over time in these upcoming vaccine serotypes, as well as the overall pneumococcal population in Canada.

ESBL-positive Escherichia coli and Klebsiella pneumoniae isolates from across Canada: CANWARD surveillance study, 2007-18.

OBJECTIVES: ESBL-producing Escherichia coli and Klebsiella pneumoniae are pathogens of increasing importance in Canada and elsewhere in the world. The purpose of this study was to phenotypically and molecularly characterize ESBL-producing E. coli and K. pneumoniae clinical isolates obtained from patients attending Canadian hospitals over a 12 year period. METHODS: Isolates were collected between January 2007 and December 2018 as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the CLSI (M100) phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent WGS. RESULTS: In total, 671 E. coli and 141 K. pneumoniae were confirmed to be ESBL producers. The annual proportion of ESBL-producing isolates increased for both E. coli (from 3.3% in 2007 to 11.2% in 2018; P < 0.0001) and K. pneumoniae (from 1.3% in 2007 to 9.3% in 2018; P < 0.0001). The most frequent STs were ST131 for E. coli [62.4% (419/671) of isolates] and ST11 [7.8% (11/141)] and ST147 [7.8% (11/141)] for K. pneumoniae. Overall, 97.2% of ESBL-producing E. coli and K. pneumoniae isolates were MDR. blaCTX-M-15 predominated in both ESBL-producing E. coli (62.3% of isolates) and ESBL-producing K. pneumoniae (48.9% of isolates). CONCLUSIONS: The proportion of ESBL-producing E. coli, especially ST131, and K. pneumoniae, especially ST11 and ST147, in Canada increased significantly from 2007 to 2018. Continued prospective surveillance of these evolving MDR and at times XDR pathogens is imperative.

Comparison of phenotypic antimicrobial susceptibility testing results and WGS-derived genotypic resistance profiles for a cohort of ESBL-producing Escherichia coli collected from Canadian hospitals: CANWARD 2007-18.

OBJECTIVES: To determine whether the genotypic resistance profile inferred from WGS could accurately predict phenotypic resistance for ESBL-producing Escherichia coli isolated from patient samples in Canadian hospital laboratories. METHODS: As part of the ongoing CANWARD study, 671 E. coli were collected and phenotypically confirmed as ESBL producers using CLSI M100 disc testing criteria. Isolates were sequenced using the Illumina MiSeq platform, resulting in 636 high-quality genomes for comparison. Using a rules-based approach, the genotypic resistance profile was compared with the phenotypic resistance interpretation generated using the CLSI broth microdilution method for ceftriaxone, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole. RESULTS: The most common genes associated with non-susceptibility to ceftriaxone, gentamicin and trimethoprim/sulfamethoxazole were CTX-M-15 (n = 391), aac(3)-IIa + aac(6')-Ib-cr (n = 121) and dfrA17 + sul1 (n = 169), respectively. Ciprofloxacin non-susceptibility was most commonly attributed to alterations in both gyrA (S83L + D87N) and parC (S80I + E84V), with (n = 187) or without (n = 197) aac(6')-Ib-cr. Categorical agreement (susceptible or non-susceptible) between actual and predicted phenotype was 95.6%, 98.9%, 97.6% and 88.8% for ceftriaxone, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole, respectively. Only ciprofloxacin results (susceptible or non-susceptible) were predicted with major error (ME) and very major error (VME) rates of <3%: ciprofloxacin (ME, 1.5%; VME, 1.1%); gentamicin (ME, 0.8%-31.7%; VME, 4.8%); ceftriaxone (ME, 81.8%; VME, 3.0%); and trimethoprim/sulfamethoxazole (ME, 0.9%-23.0%; VME, 5.2%-8.5%). CONCLUSIONS: Our rules-based approach for predicting a resistance phenotype from WGS performed well for ciprofloxacin, with categorical agreement of 98.9%, an ME rate of 1.5% and a VME rate of 1.1%. Although high categorical agreements were also obtained for gentamicin, ceftriaxone and trimethoprim/sulfamethoxazole, ME and/or VME rates were ≥3%.

Invasive pneumococcal disease caused by serotypes 22F and 33F in Canada: the SAVE study 2011-2018.

A 15-valent conjugate vaccine that provides protection against Streptococcus pneumoniae serotypes 22F and 33F is in development. Here we report on the prevalence, antimicrobial susceptibility, and clonal structure of these serotypes in Canada. From 2011 to 2018, the SAVE study collected 11,044 invasive S. pneumoniae isolates. Of these, 9.3% (1024/11,044) and 3.8% (416/11,044) were 22F and 33F, respectively. Serotype 22F isolates were susceptible to most antimicrobials tested except clarithromycin, where susceptibility significantly decreased over time (2011: 80.4%, 2018: 52.9%, P < 0.0001). Only 1.6% of serotype 22F isolates were multidrug-resistant (MDR), while 96% of typed strains were clonal cluster (CC) 433. Serotype 33F isolates demonstrated low susceptibility to clarithromycin and trimethoprim/sulfamethoxazole (22.4% and 24.6%, respectively) and 4.8% MDR. Most serotype 33F isolates were CC100, CC673 and CC717. CC100 prevalence increased significantly over time (2011: 50.0%, 2018: 84.8%, P < 0.006). Continued surveillance of these serotypes is crucial to identify further changes in prevalence, antimicrobial susceptibility, and clonal spread.

In vitro activity of imipenem-relebactam against various resistance phenotypes/genotypes of Enterobacterales and Pseudomonas aeruginosa isolated from patients across Canada as part of the CANWARD study, 2016-2019.

Broth microdilution was used to determine the in vitro activities of imipenem-relebactam and comparators versus 4260 Enterobacterales and 1324 Pseudomonas aeruginosa clinical isolates. Excluding Serratia marcescens, 96.7% to 100% of Enterobacterales species were susceptible to imipenem-relebactam. Susceptibility of P. aeruginosa isolates to imipenem-relebactam and imipenem was 91.3% and 59.1%, respectively.

In vitro activity and resistance rates of topical antimicrobials fusidic acid, mupirocin and ozenoxacin against skin and soft tissue infection pathogens obtained across Canada (CANWARD 2007-18).

BACKGROUND: Current antimicrobial susceptibility/resistance data versus skin and soft tissue infection (SSTI) pathogens help to guide empirical treatment using topical antimicrobials. OBJECTIVES: To assess the in vitro activity and resistance rates of fusidic acid, mupirocin, ozenoxacin and comparator agents against pathogens isolated from patients with SSTIs in Canada. METHODS: SSTI isolates of MSSA (n = 422), MRSA (n = 283) and Streptococcus pyogenes (n = 46) obtained from CANWARD 2007-18 were tested using CLSI broth microdilution. Fusidic acid low-level resistance was defined as an MIC of ≥2 mg/L and high-level resistance as an MIC ≥512 mg/L. Mupirocin high-level resistance was defined as an MIC ≥512 mg/L and low-level resistance was an MIC of 2-256 mg/L. RESULTS: Low-level and high-level fusidic acid resistance in MSSA was 10.9% and 1.7%, respectively. Low-level and high-level fusidic acid resistance in MRSA was 10.6% and 3.5%, respectively. High-level mupirocin resistance was identified in 1.4% of MSSA and 14.1% of MRSA, respectively. Versus MSSA, ozenoxacin demonstrated MIC50 and MIC90 of 0.004 and 0.25 mg/L, respectively. Against MRSA, ozenoxacin inhibited all isolates at an MIC of ≤0.5 mg/L, including isolates with ciprofloxacin MICs >2 mg/L, clarithromycin-resistant, clindamycin-resistant, high-level fusidic acid-resistant and high-level mupirocin-resistant isolates. CONCLUSIONS: We conclude that fusidic acid low-level resistance exceeded 10% for both MSSA and MRSA while fusidic acid high-level resistance was ≤3.5%. Mupirocin high-level resistance exceeded 10% in MRSA. Ozenoxacin is active versus SSTI pathogens including MRSA resistant to fluoroquinolones, macrolides, clindamycin, fusidic acid and mupirocin.

In vitro susceptibility of common bacterial pathogens causing respiratory tract infections in Canada to lefamulin, a new pleuromutilin.

Background: Community-acquired pneumonia (CAP) is a significant global health concern. Pathogens causing CAP demonstrate increasing resistance to commonly prescribed empiric treatments. Resistance in Streptococcus pneumoniae, the most prevalent bacterial cause of CAP, has been increasing worldwide, highlighting the need for improved antibacterial agents. Lefamulin, a novel pleuromutilin, is a recently approved therapeutic agent highly active against many lower respiratory tract pathogens. However, to date minimal data are available to describe the in vitro activity of lefamulin against bacterial isolates associated with CAP. Methods: Common bacterial causes of CAP obtained from both lower respiratory and blood specimen isolates cultured by hospital laboratories across Canada were submitted to the annual CANWARD study's coordinating laboratory in Winnipeg, Canada, from January 2015 to October 2018. A total of 876 bacterial isolates were tested against lefamulin and comparator agents using the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution method, and minimum inhibitory concentrations (MICs) were interpreted using accepted breakpoints. Results: All S. pneumoniae isolates tested from both respiratory (n = 315) and blood specimens (n = 167) were susceptible to lefamulin (MIC ≤0.5 µg/mL), including isolates resistant to penicillins, clarithromycin, doxycycline, and trimethoprim-sulfamethoxazole. Lefamulin also inhibited 99.0% of Haemophilus influenzae isolates (regardless of ß-lactamase production) (99 specimens; MIC ≤2 µg/mL) and 95.7% of methicillin-susceptible Staphylococcus aureus (MSSA) (MIC ≤0.25 µg/mL; 70 specimens) at their susceptible breakpoints. Conclusions: Lefamulin demonstrated potent in vitro activity against all respiratory isolates tested and may represent a significant advancement in empiric treatment options for CAP.

Historique: La pneumonie communautaire est une préoccupation sanitaire importante dans le monde. Les agents pathogènes qui en sont responsables démontrent une résistance croissante envers des traitements empiriques souvent prescrits. La résistance du Streptococcus pneumoniae, la principale cause bactérienne de la pneumonie communautaire, augmente au Canada et dans le monde, ce qui fait ressortir l'importance d'agents antibactériens nouveaux et améliorés. La léfamuline, une nouvelle pleuromutiline, est un agent thérapeutique récemment homologué qui est très actif contre de nombreux agents pathogènes des voies respiratoires inférieures. Jusqu'à maintenant, peu de données sont toutefois disponibles pour décrire l'activité in vitro de la léfamuline contre les isolats bactériens associés à la pneumonie communautaire. Méthodologie: Les causes bactériennes courantes de la pneumonie communautaire déterminées à partir d'isolats des voies respiratoires inférieures et d'hémocultures dans des laboratoires canadiens mis en culture par des laboratoires hospitaliers du Canada et soumis à l'étude de surveillance canadienne annuelle dans les services hospitaliers du laboratoire coordonnateur de Winnipeg, au Canada, entre janvier 2015 et octobre 2018. Au total, les chercheurs ont testé 876 isolats bactériens au regard de la lémafuline et des agents comparatifs à l'aide de la méthode de référence de la microdilution dans un milieu de culture du Clinical and Laboratory Standards Institute (CLSI) et ont interprété les concentrations minimales inhibitrices (CMI) d'après les seuils acceptés. Résultats: La totalité des isolats de S. pneumoniae testés à partir de prélèvements des voies respiratoires (n = 315) et d'hémocultures (n = 167) était susceptible à la léfamuline (CMI ≤0,5 µg/mL), y compris les isolats résistants aux pénicillines, à la clarithromycine, à la doxycycline, au triméthoprime-sulfaméthoxazole et à des isolats multirésistants. La léfamuline inhibait également 99,0 % des isolats d'Haemophilus influenzae (quelle que soit leur production de ß-lactamases; n = 99; CMI ≤2 µg/mL) et 95,7 % de ceux de Staphylococcus aureus susceptibles à la méthicilline (SASM; n = 70; CMI ≤0,25 µg/mL) à leurs seuils susceptibles. La léfamuline a démontré des valeurs de CMI90 (concentration inhibant 90 % des isolats) de 0,25 µg/mL par rapport au SASM et au S. aureus résistant à la méthicilline (n = 130). Conclusion: La léfamuline a démontré une puissante activité in vitro au regard de tous les isolats respiratoires testés et peut représenter une avancée importante des traitements empiriques de la pneumonie communautaire.

Lefamulin: A Novel Oral and Intravenous Pleuromutilin for the Treatment of Community-Acquired Bacterial Pneumonia.

Lefamulin is a novel oral and intravenous (IV) pleuromutilin developed as a twice-daily treatment for community-acquired bacterial pneumonia (CABP). It is a semi-synthetic pleuromutilin with a chemical structure that contains a tricyclic core of five-, six-, and eight-membered rings and a 2-(4-amino-2-hydroxycyclohexyl)sulfanylacetate side chain extending from C14 of the tricyclic core. Lefamulin inhibits bacterial protein synthesis by binding to the 50S bacterial ribosomal subunit in the peptidyl transferase center (PTC). The pleuromutilin tricyclic core binds to a pocket close to the A site, while the C14 side chain extends to the P site causing a tightening of the rotational movement in the binding pocket referred to as an induced-fit mechanism. Lefamulin displays broad-spectrum antibacterial activity against Gram-positive and Gram-negative aerobic and anaerobic bacteria as well as against atypical bacteria that commonly cause CABP. Pleuromutilin antibiotics exhibit low rates of resistance development and lack cross-resistance to other antimicrobial classes due to their unique mechanism of action. However, pleuromutilin activity is affected by mutations in 23S rRNA, 50S ribosomal subunit proteins rplC and rplD, ATP-binding cassette (ABC)-F transporter proteins such as vga(A), and the methyltransferase cfr. The pharmacokinetic properties of lefamulin include: volume of distribution (Vd) ranging from 82.9 to 202.8 L, total clearance (CLT) of 19.5 to 21.4 L/h, and terminal elimination half-life (t1/2) of 6.9-13.2 h; protein binding of lefamulin is high and non-linear. The oral bioavailability of lefamulin has been estimated as 24% in fasted subjects and 19% in fed subjects. A single oral dose of lefamulin 600 mg administered in fasted patients achieved a maximum plasma concentration (Cmax) of 1.2-1.5 mg/L with a time of maximum concentration (Tmax) ranging from 0.8 to 1.8 h, and an area under the plasma concentration-time curve from 0 to infinity (AUC0-∞) of 8.5-8.8 mg h/L. The pharmacodynamic parameter predictive of lefamulin efficacy is the free plasma area under the concentration-time curve divided by the minimum inhibitory concentration (fAUC24h/MIC). Lefamulin efficacy has been demonstrated using various animal models including neutropenic murine thigh infection, pneumonia, lung infection, and bacteremia. Lefamulin clinical safety and efficacy was investigated through a Phase II clinical trial of acute bacterial skin and skin structure infection (ABSSSI), as well as two Phase III clinical trials of CABP. The Phase III trials, LEAP 1 and LEAP 2 established non-inferiority of lefamulin to moxifloxacin in both oral and IV formulations in the treatment of CABP. The United States Food and Drug Administration (FDA), European Medicines Agency (EMA), and Health Canada have each approved lefamulin for the treatment of CABP. A Phase II clinical trial has been completed for the treatment of ABSSSI, while the pediatric program is in Phase I. The most common adverse effects of lefamulin include mild-to-moderate gastrointestinal-related events such as nausea and diarrhea. Lefamulin represents a safe and effective option for treating CABP in cases of antimicrobial resistance to first-line therapies, clinical failure, or intolerance/adverse effects to currently used agents. Clinical experience and ongoing clinical investigation will allow clinicians and antimicrobial stewardship programs to optimally use lefamulin in the treatment of CABP.