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.

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.

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.

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.

A One Health Genomic Investigation of Gentamicin Resistance in Escherichia coli from Human and Chicken Sources in Canada, 2014 to 2017.

We investigated whether gentamicin resistance (Genr) in Escherichia coli isolates from human infections was related to Genr E. coli in chicken and whether resistance may be due to coselection from use of lincomycin-spectinomycin in chickens on farms. Whole-genome sequencing was performed on 483 Genr E. coli isolates isolated between 2014 and 2017. These included 205 human-source isolates collected by the Canadian Ward (CANWARD) program and 278 chicken-source isolates: 167 from live/recently slaughtered chickens (animals) and 111 from retail chicken meat collected by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). The predominant Genr gene was different in human and chicken sources; however, both sources carried aac(3)-IId, aac(3)-VIa, and aac(3)-IVa. Forty-one percent of human clinical isolates of Genr E. coli contained a blaCTX-M extended-spectrum beta-lactamase (ESBL) gene (84/205), and 53% of these were sequence type 131 (ST131). Phylogenomic analysis revealed a high diversity of Genr isolates; however, there were three small clusters of closely related isolates from human and chicken sources. Genr and spectinomycin resistance (Specr) genes were colocated in 148/167 (89%) chicken animal isolates, 94/111 (85%) chicken retail meat isolates, and 137/205 (67%) human-source isolates. Long-read sequencing of 23 isolates showed linkage of the Genr and Specr genes on the same plasmid in 14/15 (93%) isolates from chicken(s) and 6/8 (75%) isolates from humans. The use of lincomycin-spectinomycin on farms may be coselecting for gentamicin-resistant plasmids in E. coli in broiler chickens; however, Genr isolates and plasmids were mostly different in chickens and humans.

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.

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.

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.

Pseudomonas aeruginosa Pneumonia: Evolution of Antimicrobial Resistance and Implications for Therapy.

Pseudomonas aeruginosa (PA), a non-lactose-fermenting gram-negative bacillus, is a common cause of nosocomial infections in critically ill or debilitated patients, particularly ventilator-associated pneumonia (VAP), and infections of urinary tract, intra-abdominal, wounds, skin/soft tissue, and bloodstream. PA rarely affects healthy individuals, but may cause serious infections in patients with chronic structural lung disease, comorbidities, advanced age, impaired immune defenses, or with medical devices (e.g., urinary or intravascular catheters, foreign bodies). Treatment of pseudomonal infections is difficult, as PA is intrinsically resistant to multiple antimicrobials, and may acquire new resistance determinants even while on antimicrobial therapy. Mortality associated with pseudomonal VAP or bacteremias is high (> 35%) and optimal therapy is controversial. Over the past three decades, antimicrobial resistance (AMR) among PA has escalated globally, via dissemination of several international multidrug resistant "epidemic" clones. We discuss the importance of PA as a cause of pneumonia including health care-associated pneumonia, hospital-acquired pneumonia, VAP, the emergence of AMR to this pathogen, and approaches to therapy (both empirical and definitive).

Infections Due to Acinetobacter baumannii-calcoaceticus Complex: Escalation of Antimicrobial Resistance and Evolving Treatment Options.

Bacteria within the genus Acinetobacter (principally A. baumannii-calcoaceticus complex [ABC]) are gram-negative coccobacilli that most often cause infections in nosocomial settings. Community-acquired infections are rare, but may occur in patients with comorbidities, advanced age, diabetes mellitus, chronic lung or renal disease, malignancy, or impaired immunity. Most common sites of infections include blood stream, skin/soft-tissue/surgical wounds, ventilator-associated pneumonia, orthopaedic or neurosurgical procedures, and urinary tract. Acinetobacter species are intrinsically resistant to multiple antimicrobials, and have a remarkable ability to acquire new resistance determinants via plasmids, transposons, integrons, and resistance islands. Since the 1990s, antimicrobial resistance (AMR) has escalated dramatically among ABC. Global spread of multidrug-resistant (MDR)-ABC strains reflects dissemination of a few clones between hospitals, geographic regions, and continents; excessive antibiotic use amplifies this spread. Many isolates are resistant to all antimicrobials except colistimethate sodium and tetracyclines (minocycline or tigecycline); some infections are untreatable with existing antimicrobial agents. AMR poses a serious threat to effectively treat or prevent ABC infections. Strategies to curtail environmental colonization with MDR-ABC require aggressive infection-control efforts and cohorting of infected patients. Thoughtful antibiotic strategies are essential to limit the spread of MDR-ABC. Optimal therapy will likely require combination antimicrobial therapy with existing antibiotics as well as development of novel antibiotic classes.

A Clostridioides difficile surveillance study of Canadian retail meat samples from 2016-2018.

In this study, we isolated and molecularly characterized 10 (1.6%) C. difficile isolates from 644 commercially available raw meat samples. Molecular typing by PFGE and ribotyping revealed NAP and ribotypes commonly associated with human clinical cases, suggesting retail meat could be a possible source of transmission warranting further investigation.

Characterization of Proteobacterial Plasmid Integron-Encoded qac Efflux Pump Sequence Diversity and Quaternary Ammonium Compound Antiseptic Selection in Escherichia coli Grown Planktonically and as Biofilms.

Qac efflux pumps from proteobacterial multidrug-resistant plasmids are integron encoded and confer resistance to quaternary ammonium compound (QAC) antiseptics; however, many are uncharacterized and misannotated. A survey of >2,000 plasmid-carried qac genes identified 37 unique qac sequences that correspond to one of five representative motifs: QacE, QacEΔ1, QacF/L, QacH/I, and QacG. Antimicrobial susceptibility testing of each cloned qac member in Escherichia coli highlighted distinctive antiseptic susceptibility patterns that were most prominent when cells grew as biofilms.

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.

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.

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%.

Risk versus Benefit of Using Hydroxychloroquine to Treat Patients with COVID-19.

Hydroxychloroquine (HCQ), also known by its trade name Plaquenil®, has been used for over 50 years as a treatment for malaria, systemic lupus erythematosus, and rheumatoid arthritis. As the COVID-19 pandemic emerged in the United States and globally in early 2020, HCQ began to garner attention as a potential treatment and as prophylaxis against COVID-19. Preliminary data indicated that HCQ as well as chloroquine (CQ) possessed in vitro antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Early clinical data from China and France reported that HCQ and CQ were associated with viral load reduction and clinical improvement in patients with COVID-19 compared to control groups; however, an overwhelming number of randomized controlled trials, meta-analyses, and systematic reviews have since concluded that HCQ used alone, or in combination with azithromycin (AZ), provides no mortality or time-to-recovery benefit in hospitalized patients with COVID-19. Additionally, these same trials reported adverse events including cardiac, neuropsychiatric, hematologic, and hepatobiliary manifestations in patients with COVID-19 whom had been treated with HCQ. This review article summarizes the available data pertaining to the adverse events associated with HCQ use, alone or in combination with azithromycin, in patients with COVID-19 in order to fully assess the risk versus benefit of treating COVID-19 patients with these agents. The results of this review lead us to conclude that the risks of adverse events associated with HCQ use (with or without AZ) outweigh the potential clinical benefits and thus recommend against its use in the treatment or prevention of COVID-19.

Riboswitch-Associated Guanidinium-Selective Efflux Pumps Frequently Transmitted on Proteobacterial Plasmids Increase Escherichia coli Biofilm Tolerance to Disinfectants.

Members of the small multidrug resistance (SMR) efflux pump family known as SugE (recently renamed Gdx) are known for their narrow substrate selectivity to small guanidinium (Gdm+) compounds and disinfectant quaternary ammonium compounds (QACs). Gdx members have been identified on multidrug resistance plasmids in Gram-negative bacilli, but their functional role remains unclear, as few have been characterized. Here, we conducted a survey of sequenced proteobacterial plasmids that encoded one or more SugE/Gdx sequences in an effort to (i) identify the most frequently represented Gdx member(s) on these plasmids and their sequence diversity, (ii) verify if Gdx sequences possess a Gdm+ riboswitch that regulates their translation similarly to chromosomally encoded Gdx members, and (iii) determine the antimicrobial susceptibility profile of the most predominate Gdx member to various QACs and antibiotics in Escherichia coli strains BW25113 and KAM32. The results of this study determined 14 unique SugE sequences, but only one Gdx sequence, annotated as "SugE(p)," predominated among the >140 plasmids we surveyed. Enterobacterales plasmids carrying sugE(p) possessed a guanidine II riboswitch similar to the upstream region of E. coligdx Cloning and expression of sugE(p), gdx, and emrE sequences into a low-copy-number expression vector (pMS119EH) revealed significant increases in QAC resistance to a limited range of detergent-like QACs only when gdx and sugE(p) transformants were grown as biofilms. These findings suggest that sugE(p) presence on proteobacterial plasmids may be driven by species that frequently encounter Gdm+ and QAC exposure.IMPORTANCE This study characterized the function of antimicrobial-resistant phenotypes attributed to plasmid-encoded guanidinium-selective small multidrug resistance (Gdm/SugE) efflux pumps. These sequences are frequently monitored as biocide resistance markers in antimicrobial resistance surveillance studies. Our findings reveal that enterobacterial gdm sequences transmitted on plasmids possess a guanidine II riboswitch, which restricts transcript translation in the presence of guanidinium. Cloning and overexpression of this gdm sequence revealed that it confers higher resistance to quaternary ammonium compound (QAC) disinfectants (which possess guanidium moieties) when grown as biofilms. Since biofilms are commonly eradicated with QAC-containing compounds, the presence of this gene on plasmids and its biofilm-specific resistance are a growing concern for clinical and food safety prevention measures.