Phenotypic and molecular characterization of Streptococcus pneumoniae serotype 3 isolates from blood and respiratory samples in Canada: CANWARD 2007-21.

BACKGROUND: Lower respiratory infections and invasive disease caused by Streptococcus pneumoniae serotype 3 remain major clinical challenges around the world, despite widespread availability of updated vaccines. METHODS: As part of CANWARD, antimicrobial susceptibility testing and serotyping were performed on all S. pneumoniae isolates from 2007 to 2021. A subset of 226/264 (85.6%) serotype 3 isolates were selected for WGS to determine sequence type (ST)/clonal cluster (CC) and correspondence of antimicrobial resistance determinants (erm, mefAE, tetM, cat, folA, folP) with resistance phenotype. RESULTS: Of the 3,039 S. pneumoniae isolates obtained from 2007 to 2021, 8.7% (n = 264) were serotype 3, with 64.0% of respiratory origin and 36.0% from blood. Of 226 sequenced serotype 3 isolates, 184 (81.4%) were ST180 (GPSC12). The proportion of ST8561 (single locus variant of ST180) increased from 7.2% to 16.6% during the study period. An increasing proportion of serotype 3 isolates had phenotypic resistance (P = 0.0007) and genetic resistance determinants (P = 0.004), comparing 2017-21 to 2007-11, largely due to a recently expanded ST180 clade with cat, tetM and mef determinants. CONCLUSIONS: S. pneumoniae serotype 3 from GPSC12 continues to dominate throughout Canada, with an increase in the proportion of ST8561. The proportion of serotype 3 isolates that are phenotypically resistant and with genetic resistance determinants is increasing over time, reflecting a global increase in GPSC12 genotypes with known resistance determinants. Phylogenomic characterization of isolates collected over time and from around the world may facilitate improved treatment and enhanced prevention strategies, including new vaccines with activity against S. pneumoniae serotype 3.

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.

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.

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.

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.

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%.

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 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.

Susceptibility of Clinical Isolates of Escherichia coli to Fosfomycin as Measured by Four In Vitro Testing Methods.

Clinical isolates of Escherichia coli (n = 554) were tested against fosfomycin using agar dilution, disk diffusion, and Etest. Agar dilution (reference method) identified few isolates with fosfomycin MICs of 64 (n = 3), 128 (n = 4), and ≥256 µg/ml (n = 2). Applying CLSI (M100, 2020) and EUCAST (v. 10.0, 2020) breakpoints, 98.9% and 98.4% (agar dilution), 99.3% and 99.1% (disk diffusion), and 99.1% and 98.9% (Etest) of isolates were fosfomycin susceptible, respectively. Essential agreement (agar dilution versus Etest) was low (40.8%); 59.3% (131/221) of isolates with agar dilution MICs of 2 to 128 µg/ml tested 2 to 4 doubling dilutions lower by Etest. Applying CLSI breakpoints, categorical agreement was >99% for both disk diffusion and Etest; no major errors (MEs) or very major errors (VMEs) were identified, and rates of minor errors (mEs) were <1%. EUCAST breakpoints yielded categorical agreements of >99% and no MEs for both disk diffusion and Etest; however, VMEs occurred at unacceptable rates of 44.4% (disk diffusion) and 33.3% (Etest). All isolates with agar dilution MICs of ≥32 µg/ml (n = 12) and a subset of isolates with MICs of ≤16 µg/ml (n = 49) were also tested using the Vitek 2 AST-N391 card and generated fosfomycin MICs 1 to ≥3 doubling dilutions lower than agar dilution for 11/12 isolates with agar dilution MICs of ≥32 µg/ml. We conclude that performing fosfomycin disk diffusion or Etest on urinary isolates of E. coli and interpreting results using CLSI breakpoints reliably identified fosfomycin-susceptible isolates regardless of differences in endpoint reading criteria. EUCAST breakpoints generated excessive rates of VMEs for our isolate collection of high fosfomycin susceptibility.

Antimicrobial susceptibility of Clostridioides difficile isolated from diarrhoeal stool specimens of Canadian patients: summary of results from the Canadian Clostridioides difficile (CAN-DIFF) surveillance study from 2013 to 2017.

OBJECTIVES: To summarize data generated by the Canadian Clostridioides difficile (CAN-DIFF) surveillance study from 2013 to 2017. METHODS: Isolates of C. difficile (n = 2158) were cultured from toxin-positive diarrhoeal stool specimens submitted by eight hospital laboratories to a coordinating laboratory. Antimicrobial susceptibility testing was performed according to the CLSI agar dilution method (M11, 2018). Isolate ribotypes were determined using an international, standardized, high-resolution capillary gel-based electrophoresis protocol. RESULTS: Of the 2158 isolates of C. difficile, 2133 (98.8%) had vancomycin MICs ≤2 mg/L [i.e. were vancomycin susceptible (EUCAST breakpoint tables, v 9.0, 2019) or WT (CLSI M100, 29th edition, 2019)]. Fidaxomicin MICs were lower than those of all other agents tested (MIC90, 0.5 mg/L); however, one isolate with a fidaxomicin MIC of >8 mg/L was identified. Metronidazole MICs ranged from 0.12 to 4 mg/L; all isolates were metronidazole susceptible by the CLSI breakpoint (≤8 mg/L) compared with 96.8% susceptible by the EUCAST breakpoint (≤2 mg/L). In total, 182 different ribotypes were identified from 2013 to 2017. The most common ribotypes identified were 027 (19.3% of isolates) and 106 (8.2%). Ribotype 027 isolates were frequently moxifloxacin resistant (87.3% of isolates) and MDR (48.6%), associated with vancomycin (10/25, 40.0%) and metronidazole (58/69, 84.1%) resistance and from patients aged ≥80 years. The prevalence of ribotype 027 decreased significantly (P < 0.0001) from 2013 (27.5%) to 2017 (9.0%) and was replaced by increases in ribotype 106 (P = 0.0003) and multiple less common ribotypes. CONCLUSIONS: Periodic surveillance is required to monitor clinical isolates of C. difficile for changes to in vitro susceptibility testing profiles and ribotype evolution.

Fosfomycin resistance mediated by fos genes remains rare among extended-spectrum beta-lactamase-producing Escherichia coli clinical isolates recovered from the urine of patients evaluated at Canadian hospitals (CANWARD, 2007-2017).

Among 162 isolates of extended-spectrum beta-lactamase-(ESBL)-producing Escherichia coli recovered from the urine of Canadian patients (2007-2017), five (3.1%) were not susceptible in vitro to fosfomycin (MIC ≥128 µg/mL). These isolates underwent whole genome sequencing to assess for the presence of fos genes. The fosA3 gene was detected in one isolate.

42936 pathogens from Canadian hospitals: 10 years of results (2007-16) from the CANWARD surveillance study.

OBJECTIVES: The CANWARD surveillance study was established in 2007 to annually assess the in vitro susceptibilities of a variety of antimicrobial agents against bacterial pathogens isolated from patients receiving care in Canadian hospitals. METHODS: 42 936 pathogens were received and CLSI broth microdilution testing was performed on 37 355 bacterial isolates. Limited patient demographic data submitted with each isolate were collated and analysed. RESULTS: Of the isolates tested, 43.5%, 33.1%, 13.2% and 10.2% were from blood, respiratory, urine and wound specimens, respectively; 29.9%, 24.8%, 19.0%, 18.1% and 8.2% of isolates were from patients in medical wards, emergency rooms, ICUs, hospital clinics and surgical wards. Patient demographics associated with the isolates were: 54.6% male/45.4% female; 13.1% patients aged ≤17 years, 44.3% 18-64 years and 42.7% ≥65 years. The three most common pathogens were Staphylococcus aureus (21.2%, both methicillin-susceptible and MRSA), Escherichia coli (19.6%) and Pseudomonas aeruginosa (9.0%). E. coli were most susceptible to meropenem and tigecycline (99.9%), ertapenem and colistin (99.8%), amikacin (99.7%) and ceftolozane/tazobactam and plazomicin (99.6%). Twenty-three percent of S. aureus were MRSA. MRSA were most susceptible to ceftobiprole, linezolid and telavancin (100%), daptomycin (99.9%), vancomycin (99.8%) and tigecycline (99.2%). P. aeruginosa were most susceptible to ceftolozane/tazobactam (98.3%) and colistin (95.0%). CONCLUSIONS: The CANWARD surveillance study has provided 10 years of reference antimicrobial susceptibility testing data on pathogens commonly causing infections in patients attending Canadian hospitals.

Comparison of antimicrobial resistance patterns in Streptococcus pneumoniae from respiratory and blood cultures in Canadian hospitals from 2007-16.

OBJECTIVES: To compare the epidemiology and antimicrobial susceptibility patterns of Streptococcus pneumoniae collected from respiratory and blood culture samples in Canada between 2007 and 2016. METHODS: S. pneumoniae strains were obtained from Canadian hospitals as part of the ongoing national surveillance study, CANWARD. Isolates were serotyped using the Quellung method. Antimicrobial susceptibility testing was performed using the CLSI broth microdilution method. MDR and XDR were defined as resistance to three or more and five or more classes of antimicrobials, respectively. RESULTS: Of the 2581 S. pneumoniae isolates collected, 1685 (65.3%) and 896 (34.7%) were obtained from respiratory and blood samples, respectively. Respiratory isolates demonstrated lower rates of antimicrobial susceptibility than blood isolates to penicillin, ceftriaxone, clarithromycin, clindamycin, doxycycline and trimethoprim/sulfamethoxazole (P ≤ 0.03). From 2007 to 2016, invasive isolates demonstrated trends towards increasing penicillin susceptibility and decreasing clarithromycin susceptibility. MDR was significantly higher in respiratory S. pneumoniae compared with blood (9.1% versus 4.5%, P < 0.0001). Serotypes 11A, 16F, 19F, 23A/B/F, 34, 35B and non-typeable strains were more commonly isolated from respiratory specimens, while 4, 5, 7F, 8, 12F, 14 and 19A were more commonly invasive serotypes. Numerous serotypes, including 3 and 22F, were isolated frequently from both specimen sources. CONCLUSIONS: S. pneumoniae from respiratory samples demonstrated lower antimicrobial susceptibilities and higher MDR in a greater diversity of serotypes than isolates obtained from blood. Many serotypes were associated with one specific specimen source, while others were associated with both; genetic characterization is necessary to elucidate the specific factors influencing the ability of these serotypes to commonly cause both invasive and non-invasive disease.

Species distribution and antifungal susceptibility of invasive Candida isolates from Canadian hospitals: results of the CANWARD 2011-16 study.

OBJECTIVES: Understanding the epidemiology of invasive Candida infections is essential to patient management decisions and antifungal stewardship practices. This study characterized the species distribution and antifungal susceptibilities of prospectively collected isolates of Candida species causing bloodstream infections (BSIs) in patients admitted to tertiary care hospitals located in 14 cities across 8 of the 10 Canadian provinces between 2011 and 2016. METHODS: Antifungal susceptibility testing was performed by broth microdilution using CLSI methods, breakpoints and epidemiological cut-off values. DNA sequencing of fks loci was performed on all echinocandin-non-susceptible isolates. RESULTS: Candida albicans (49.6%), Candida glabrata (20.8%) and Candida parapsilosis complex (12.0%) were the most common species out of 1882 isolates associated with BSIs. Candida tropicalis (5.2%), Candida krusei (4.3%), Candida dubliniensis (4.1%), Candida lusitaniae (1.4%) and Candida guilliermondii (1.1%) were less frequently isolated. Between 2011 and 2016, the proportion of C. albicans significantly decreased from 60.9% to 42.1% (P < 0.0001) while that of C. glabrata significantly increased from 16.4% to 22.4% (P = 0.023). C. albicans (n = 934), C. glabrata (n = 392) and C. parapsilosis complex (n = 225) exhibited 0.6%, 1.0% and 4.9% resistance to fluconazole and 0.1%, 2.5% and 0% resistance to micafungin, respectively. Mutations in fks hot-spot regions were confirmed in all nine micafungin non-susceptible C. glabrata. CONCLUSIONS: Antifungal resistance in contemporary isolates of Candida causing BSIs in Canada is uncommon. However, the proportion of C. glabrata isolates has increased and echinocandin resistance in this species has emerged. Ongoing surveillance of local hospital epidemiology and appropriate antifungal stewardship practices are necessary to preserve the utility of available antifungal agents.