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.

In vitro susceptibility of urinary Escherichia coli isolates to first- and second-line empirically prescribed oral antimicrobials: CANWARD surveillance study results for Canadian outpatients, 2007-2016.

Escherichia coli isolates (n = 2035) from urine specimens of outpatients presenting to Canadian medical clinics and hospital emergency departments from 2007-2016 were collected as part of the CANWARD surveillance study. Isolate identification and antimicrobial susceptibility testing (AST) were performed at a central site (Health Sciences Centre, Winnipeg, Canada). AST of first- and second-line oral antimicrobial agents was performed using CLSI methods (M07, 11th ed, 2018); fosfomycin was tested by agar dilution and all other agents by broth microdilution. Minimum inhibitory concentrations (MICs) were interpreted using CLSI M100 (2018) criteria. Fosfomycin (99.2% of isolates susceptible), nitrofurantoin (97.5%) and cefalexin (93.6%) were the most active agents tested; amoxicillin/clavulanic acid (AMC) (85.6%), ciprofloxacin (83.0%) and trimethoprim/sulfamethoxazole (SXT) (77.0%) were less active. Annual percentages of isolates positive for extended-spectrum ß-lactamases (ESBLs) or demonstrating multidrug-resistant (MDR) phenotypes increased from 0.8% (2007) to 10.1% (2016), and from 9.7% (2007) to 16.5% (2016), respectively, whilst the annual frequency of AmpC-positive isolates decreased from a high of 3.2% in 2008 to 0.7% in 2016. The most common MDR phenotype of E. coli was non-susceptibility to AMC, ciprofloxacin, and SXT, accounting for 12.7% (26/205) of all MDR isolates. Rates of susceptibility were higher for fosfomycin than for the five other oral agents tested against ESBL-positive (96.1% susceptible) and MDR (95.1%) isolates and were equal to nitrofurantoin (96.4%) against AmpC-positive isolates. Prudent use of antimicrobials and close monitoring of antimicrobial susceptibilities of clinical uropathogenic E. coli isolates are imperative to help preserve the utility of oral antimicrobials.

PCR ribotyping and antimicrobial susceptibility testing of isolates of Clostridium difficile cultured from toxin-positive diarrheal stools of patients receiving medical care in Canadian hospitals: the Canadian Clostridium difficile Surveillance Study (CAN-DIFF) 2013-2015.

Clostridium difficile toxin-positive diarrheal stool specimens submitted to eight Canadian hospital laboratories from 2013 to 2015 were cultured. Polymerase chain reaction ribotyping of isolates was performed using an internationally standardized, high-resolution capillary gel-based electrophoresis protocol and antimicrobial susceptibility testing conducted by CLSI-defined agar dilution (M11-A8, 2012). Among the 1310 isolates of C. difficile cultured, 141 different ribotypes were identified; the most common ribotypes were 027 (24.5% of isolates), 014 (7.7%), 020 (6.6%), 106 (6.1%), and 002 (4.6%). Ribotype 027 was the commonest ribotype in all geographic regions of Canada and was more frequently isolated from patients aged ≥80 years (40.6%) than younger patients (P<0.00001). Ribotype 027 isolates were frequently moxifloxacin-resistant (92.2% of isolates) and multidrug-resistant (49.5%). Fidaxomicin demonstrated the greatest in vitro potency (lowest MIC90, 0.5 µg/mL; lowest maximum MIC, 2 µg/mL) of eight antimicrobial agents tested and was the most active agent against each of the five commonest ribotypes (MIC90, 0.25-1 µg/mL).

In vitro activity of nemonoxacin, a novel nonfluorinated quinolone, against 2,440 clinical isolates.

The in vitro activity of nemonoxacin (TG-873870), a novel nonfluorinated quinolone, was tested against 2,440 clinical isolates. Nemonoxacin was at least fourfold more active than levofloxacin and moxifloxacin against most gram-positive cocci tested (shown by the following MIC(90)/range [microg/ml] values; community-associated methicillin [meticillin]-resistant Staphylococcus aureus, 0.5/0.015 to 2; Staphylococcus epidermidis, 0.5/0.015 to 4 for methicillin-susceptible staphylococci and 2/0.12 to 2 for methicillin-resistant staphylococci; Streptococcus pneumoniae, 0.015/< or = 0.008 to 0.25; Enterococcus faecalis, 1/0.03 to 128). Nemonoxacin activity against gram-negative bacilli was similar to levofloxacin and moxifloxacin (MIC(90)/range [microg/ml]; Escherichia coli, 32/< or = 0.015 to > or = 512; Klebsiella pneumoniae, 2/< or = 0.015 to 128; K. oxytoca, 0.5/0.06 to 1; Proteus mirabilis, 16/0.25 to > or = 512; Pseudomonas aeruginosa, 32/< or = 0.015 to > or = 512; Acinetobacter baumannii, 1/0.12 to 16).

In vitro activity of OPT-80 tested against clinical isolates of toxin-producing Clostridium difficile.

Agar dilution antimicrobial susceptibility testing (CLSI, M11-A7, 2007) performed for 208 toxin-producing clinical isolates of Clostridium difficile resulted in OPT-80 MICs ranging from 0.06 to 1 microg/ml, with 90% of the isolates inhibited by a concentration of 0.5 microg/ml. The in vitro activity of OPT-80 was independent of the susceptibilities of isolates to nine other antimicrobial agents.

Molecular characterization of increasing fluoroquinolone resistance in Streptococcus pneumoniae isolates in Canada, 1997 to 2005.

Molecular characterization of fluoroquinolone-resistant Streptococcus pneumoniae in Canada was conducted from 1997 to 2005. Over the course of the study, 205 ciprofloxacin-resistant isolates were evaluated for ParC and GyrA quinolone resistance-determining region (QRDR) substitutions, substitutions in the full genes of ParC, ParE, and GyrA, reserpine sensitivity, and serotype and by pulsed-field gel electrophoresis. Rates of ciprofloxacin resistance of S. pneumoniae increased significantly, from less than 1% in 1997 to 4.2% in 2005. Ciprofloxacin resistance was greatest in people >64 years of age and least in those <16 years of age. Significant increases were also noted in rates of resistance to gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin, to the current rates of 1.6%, 1.0%, 1.1%, and 1.0%, respectively. The most common genotype observed consisted of QRDR substitutions in GyrA (Ser81Phe) and ParC (Ser79Phe). Substitutions outside the QRDR of GyrA, ParC, and ParE were not associated with fluoroquinolone resistance in this study. Overall, 21% of isolates were reserpine-sensitive and were thus assumed to be efflux positive. The ciprofloxacin-resistant isolates belonged to 35 different serotypes, but 10 (19F, 11A, 23F, 6B, 22F, 12F, 6A, 14, 9V, and 19A) accounted for 72% of all isolates. The majority of the isolates were found to be genetically unrelated by pulsed-field gel electrophoresis. Within the observed clusters, there was considerable genetic heterogeneity with regard to fluoroquinolone resistance mechanisms and serotypes. Continued surveillance and molecular analysis of fluoroquinolone-resistant S. pneumoniae in Canada are essential for appropriate empirical treatment of infections and early detection of novel resistance mechanisms.

Fluoroquinolone-resistant urinary isolates of Escherichia coli from outpatients are frequently multidrug resistant: results from the North American Urinary Tract Infection Collaborative Alliance-Quinolone Resistance study.

Ciprofloxacin-resistant Escherichia coli isolates (n = 1,858) from outpatient midstream urine specimens at 40 North American clinical laboratories in 2004 to 2005 were frequently resistant to ampicillin (79.8% of isolates) and trimethoprim-sulfamethoxazole (66.5%); concurrent resistance to cefdinir (9.0%) or nitrofurantoin (4.0%) was less common. Only 10.8% of isolates were resistant to ciprofloxacin alone. Fluoroquinolone-resistant isolates of E. coli from urine were frequently multidrug resistant.

Antibiotic resistance in Escherichia coli outpatient urinary isolates: final results from the North American Urinary Tract Infection Collaborative Alliance (NAUTICA).

The North American Urinary Tract Infection Collaborative Alliance (NAUTICA) study determined the antibiotic susceptibility to commonly used agents for urinary tract infections of outpatient Escherichia coli urinary isolates obtained from various geographic regions in the USA and Canada. NAUTICA involved 40 medical centres (30 from the USA and 10 from Canada). From April 2003 to June 2004 inclusive, each centre submitted up to 50 consecutive outpatient midstream urine isolates. All isolates were identified to species level by each laboratory's existing protocol. Susceptibility testing was determined using the Clinical and Laboratory Standards Institute (CLSI) microdilution method. Ampicillin (resistant>or=32 microg/mL), sulphamethoxazole/trimethoprim (SMX/TMP) (resistant>or=4 microg/mL), nitrofurantoin (resistant>or=128 microg/mL), ciprofloxacin (resistant>or=4 microg/mL) and levofloxacin (resistant>or=8 microg/mL) resistance breakpoints used were those published by the CLSI. Of the 1142 E. coli collected, 75.5% (862) were collected from the USA and 280 (24.5%) were from Canada. Patient demographics revealed a mean age of 48.1 years (range, 2 months to 99 years), with female patients representing 79.4% of patients and males representing 20.6%. Overall, resistance to ampicillin was 37.7%, followed by SMX/TMP (21.3%), nitrofurantoin (1.1%), ciprofloxacin (5.5%) and levofloxacin (5.1%). Resistance rates for all antimicrobials were higher in US medical centres compared with Canadian centres (P<0.05). Fluoroquinolone resistance was highest in patients>or=65 years of age (P<0.05). Resistance rates demonstrated considerable geographic variability both in the USA and Canada. This study reports higher rates of antibiotic resistance in US versus Canadian outpatient urinary isolates of E. coli and demonstrates the continuing evolution of resistance to antimicrobial agents.

Molecular epidemiology of urinary tract isolates of vancomycin-resistant Enterococcus faecium from North America.

Vancomycin-resistant Enterococcus faecium (VRE) are a common cause of nosocomial infections and are important agents of gastrointestinal colonisation. As the prevalence of VRE in hospitalised patients continues to increase, implementation of appropriate infection control measures requires routine surveillance of VRE transmission patterns. The purpose of the present study was to investigate the molecular epidemiology of VRE isolates within the USA and Canada. Two hundred and eighty-three urinary tract isolates of VRE were collected in the year 2000 from ten Canadian hospitals and 28 US tertiary care medical centres representing seven of the nine geographic regions of the United States Bureau of the Census. The in vitro activity of vancomycin, teicoplanin and nine comparators was determined by broth microdilution. The genetic relatedness among isolates was evaluated by pulsed-field gel electrophoresis (PFGE). Resistance rates (intermediate and resistant) were 100% for vancomycin and 78.7% for teicoplanin. Resistance was lowest with linezolid, chloramphenicol and nitrofurantoin at 0.3%, 0.3% and 0.5%, respectively. PFGE revealed that genetic heterogeneity amongst isolates from each of the medical centres varied considerably. Despite some intracentre and intercentre VRE dissemination, the present study found no evidence for the emergence of a dominant clonal strain. This suggests that the spread of VRE within North America is a complex process involving both the horizontal transfer of glycopeptide resistance determinants and polyclonal dissemination.

Antibiotic resistance in outpatient urinary isolates: final results from the North American Urinary Tract Infection Collaborative Alliance (NAUTICA).

The goal of the North American Urinary Tract Infection Collaborative Alliance (NAUTICA) study was to determine antibiotic susceptibility to commonly used agents for urinary tract infections against outpatient urinary isolates obtained in various geographic regions in the USA and Canada. Forty-one medical centres (30 from the USA and 11 from Canada) participated, with each centre submitting up to 50 consecutive outpatient midstream urine isolates. Isolates were identified to species level by the standard protocol of each laboratory. Susceptibility testing was determined using the National Committee for Clinical Laboratory Standards (NCCLS) microdilution method. Resistance breakpoints used were those published by the NCCLS, including: ampicillin (resistant > or = 32 microg/mL), sulphamethoxazole/trimethoprim (SMX/TMP) (resistant > or = 4 microg/mL), nitrofurantoin (resistant > or = 128 microg/mL), ciprofloxacin (resistant > or = 4 microg/mL) and levofloxacin (resistant > or = 8 microg/mL). Of the 1990 isolates collected, 75.1% (1494) were collected from the USA and 24.9% (496) were collected from Canada. The mean age of the patients was 48.3 years (range 1 month to 99 years), and 79.5% and 20.5% of isolates were obtained from women and men, respectively. The most common organisms were Escherichia coli (57.5%), Klebsiella pneumoniae (12.4%), Enterococcus spp. (6.6%), Proteus mirabilis (5.4%), Pseudomonas aeruginosa (2.9%), Citrobacter spp. (2.7%), Staphylococcus aureus (2.2%), Enterobacter cloacae (1.9%), coagulase-negative staphylococci (1.3%), Staphylococcus saprophyticus (1.2%), Klebsiella spp. (1.2%), Enterobacter aerogenes (1.1%) and Streptococcus agalactiae (1.0%). Among all 1990 isolates, 45.9% were resistant to ampicillin, 20.4% to SMX/TMP, 14.3% to nitrofurantoin, 9.7% to ciprofloxacin and 8.1% to levofloxacin. Fluoroquinolone resistance was highest in patients > or = 65 years of age. For the 1142 E. coli isolates, resistance rates were: ampicillin 37.7%, SMX/TMP 21.3%, ciprofloxacin 5.5%, levofloxacin 5.1% and nitrofurantoin 1.1%. For all 1990 isolates and for the 1142 E. coli only, resistance rates were significantly higher in US compared with Canadian medical centres. This study reports higher rates of antibiotic resistance in US versus Canadian outpatient urinary isolates and demonstrates the continuing evolution of resistance to antimicrobial agents.

Antibiotic activity against urinary tract infection (UTI) isolates of vancomycin-resistant enterococci (VRE): results from the 2002 North American Vancomycin Resistant Enterococci Susceptibility Study (NAVRESS).

BACKGROUND: The purpose of this study was to assess the prevalence of vancomycin-resistant enterococci (VRE) in urinary isolates in North America, and the activity of various antibiotics against VRE. MATERIALS AND METHODS: Twenty-eight medical centres in the United States and 10 centres in Canada assessed the prevalence of VRE in urinary isolates in 2002. Each study site was asked to collect up to a maximum of 50 consecutive VRE (Enterococcus faecium, Enterococcus faecalis only) urinary isolates. Susceptibility was determined by NCCLS broth microdilution. The prevalence of vanA and vanB resistance genotypes was determined by multiplex PCR. RESULTS: From the 28 US medical centres, a total of 697 VRE (616 [88.4%] E. faecium and 81 [11.6%] E. faecalis) were received. Approximately 75% of all VRE (E. faecium and E. faecalis) isolates demonstrated a VanA phenotype (resistance to both vancomycin and teicoplanin). PCR detection of vanA and vanB resistance determinants showed that the vanA genotype was present in 584 of 697 (83.8%) VRE isolates, whereas 113 (16.2%) isolates possessed the vanB gene. The most active agents were linezolid, nitrofurantoin and chloramphenicol, with 0.3%, 0.6% and 2.4% resistance, respectively. The majority (77.8%) of vancomycin-resistant E. faecium isolates displayed the VanA phenotype, and 538 of these 616 (87.3%) isolates were PCR-positive for vanA; the vanB genotype was detected in 78 (12.7%) isolates. Resistance was lowest with linezolid, chloramphenicol and nitrofurantoin at 0.3%, 0.3% and 0.5%, respectively. Only three genetically indistinguishable vanA-positive E. faecium were isolated from the 10 Canadian medical centres. CONCLUSION: VRE urinary isolates are common in the United States, are primarily of the vanA genotype and are very susceptible to linezolid, nitrofurantoin and chloramphenicol. In Canada, VRE urinary isolates remain uncommon.