Corynebacterium diphtheriae and Corynebacterium ulcerans: development of EUCAST methods and generation of data on which to determine breakpoints.

BACKGROUND: Evidence-based clinical susceptibility breakpoints have been lacking for antimicrobial agents used for diphtheria. OBJECTIVES: We aimed to evaluate broth microdilution and disc diffusion methods and create a dataset of MIC values and inhibition zone diameters (ZDs) from which breakpoints could be determined. METHODS: We included 400 recent clinical isolates equally distributed by species (Corynebacterium diphtheriae and Corynebacterium ulcerans) and by national surveillance programmes (France and Germany). Non-duplicate toxigenic and non-toxigenic isolates were chosen to enable the inclusion of a diversity of susceptibility levels for the 13 agents tested. Broth microdilution and disc diffusion, using EUCAST methodology for fastidious organisms, were used. RESULTS: The distributions of MIC and ZD values were largely in agreement among methods and countries. Breakpoints to allow categorization of WT isolates as susceptible, i.e. susceptible (S) or susceptible, increased exposure (I) were determined for 12 agents. The data supported a breakpoint for benzylpenicillin and amoxicillin of resistant (R) > 1 mg/L since WT isolates were inhibited by 1 mg/L or less. WT isolates were categorized as I (S ≤ 0.001 mg/L) for benzylpenicillin, emphasizing the need for increased exposure, and S (S ≤ 1 mg/L) for amoxicillin. Erythromycin breakpoints were set at S ≤ 0.06 mg/L and R > 0.06 mg/L. The corresponding ZD breakpoints were determined for all agents except amoxicillin, for which categorization was based on benzylpenicillin results. CONCLUSIONS: This work provided a large set of antimicrobial susceptibility data for C. diphtheriae and C. ulcerans, using a harmonized methodology. The dataset allowed EUCAST and experts in the diphtheria field to develop evidence-based breakpoints in January 2023.

Vibrio species: development of EUCAST susceptibility testing methods and MIC and zone diameter distributions on which to determine clinical breakpoints.

OBJECTIVES: Most human infections caused by Vibrio spp. do not warrant antimicrobial treatment but in severe cases, targeted antimicrobial treatment can be lifesaving. For Vibrio spp., standardized antimicrobial susceptibility testing (AST) guidelines with EUCAST methodology are lacking. In this study, we aimed to produce data suitable for EUCAST to establish clinical MIC breakpoints and zone diameter correlates for Vibrio spp. METHODS: An intercontinental collection (N = 524) comprising five important Vibrio spp. (V. alginolyticus, V. cholerae, V. fluvialis, V. parahaemolyticus and V. vulnificus) was organized. All isolates were subjected to broth microdilution (BMD) against 11 antimicrobial agents according to ISO 20776-1 using unsupplemented Mueller-Hinton broth on freeze-dried Sensititre panels (Thermo Scientific, UK), and most isolates (n = 371) were also tested with disc diffusion according to EUCAST methodology for non-fastidious organisms. RESULTS: Aggregated results were used to generate MIC and zone diameter distributions and to prepare graphs of MIC-zone diameter correlation. Based on these results, the EUCAST Steering Committee determined clinical susceptible (S) and resistant (R) MIC (mg/L) breakpoints (S≤/R>) for the five Vibrio spp. for piperacillin/tazobactam (1/1), cefotaxime (0.25/0.25), ceftazidime (1/1), meropenem (0.5/0.5), ciprofloxacin (0.25/0.25), levofloxacin (0.25/0.25), azithromycin (4/4), doxycycline (0.5/0.5) and trimethoprim/sulfamethoxazole (0.25/0.25). The corresponding zone diameter breakpoints were identified. CONCLUSIONS: We demonstrated the validity of using standard BMD and EUCAST disc diffusion methodology for AST of five Vibrio spp., and generated suitable data to allow EUCAST to determine clinical MIC and zone diameter breakpoints for five pathogenic Vibrio spp., including both non-toxigenic and toxigenic V. cholerae.

Assessing the quality of the anaerobic environment - a method developed to support EUCAST disk diffusion of anaerobic bacteria.

The purpose of this study is to establish a method for assessing the anaerobic environment for EUCAST disk diffusion antimicrobial susceptibility testing (AST) of anaerobic bacteria on fastidious anaerobe agar with 5% mechanically defibrinated horse blood (FAA-HB). The method utilizes the association between a decrease in the metronidazole disk zone diameter and increasing oxygen levels with an aerotolerant Clostridium perfringens strain DSM 25589 (CCUG 75076 and NCTC 14679). The C. perfringens strain was tested on FAA-HB with a McFarland 1 inoculum and a metronidazole 5 µg disk. FAA-HB was incubated for 16-20 h at 35-37°C. The association between oxygen levels (0, 0.16, 1, 2, and 4% oxygen) and the metronidazole zone diameter was determined. Reproducibility at 0% oxygen was investigated as part of a European multi-centre study of disk diffusion of anaerobic bacteria. The median zone diameters (n=12) at each oxygen level were 29 mm (0%), 21 mm (0.16%), 16 mm (1%), 15 mm (2%), and 15 mm (4%). The metronidazole zone diameters at 0% oxygen from the multi-centre reproducibility-study had a median of 29 mm and a 95%-percentile range of 25-33 mm (n=236). Only one reading was below 25 mm. Based on our results, a zone diameter of ≥25 mm using a metronidazole 5 µg disk and the C. perfringens strain, tested with EUCAST recommendations, can be used to indicate that the anaerobic environment is of sufficient quality for culture and disk diffusion AST. EUCAST has included the method as part of the quality control for AST of anaerobic bacteria.

The European committee on antimicrobial susceptibility testing disc diffusion susceptibility testing method for frequently isolated anaerobic bacteria.

OBJECTIVES: Antimicrobial resistance in anaerobic bacteria is increasing and there is a link between inappropriate antimicrobial therapy and poor clinical outcome in the treatment of infections caused by anaerobic bacteria. Accurate and timely antimicrobial susceptibility testing of anaerobic bacteria is therefore of critical importance. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) has recently described a disc diffusion susceptibility testing method for anaerobic bacteria using fastidious anaerobe agar (FAA) supplemented with 5% defibrinated horse blood (HB). This method was previously validated for Bacteroides spp. only. The aim of this study was to determine the suitability of FAA-HB for disc diffusion and also for frequently isolated anaerobic bacteria. METHODS: Clinical isolates, including 54 Bacteroides/Phocaeicola/Parabacteroides spp., 49 Prevotella spp., 51 Fusobacterium necrophorum, 58 Clostridium perfringens, and 54 Cutibacterium acnes were evaluated against six antimicrobial agents. MICs were determined by agar dilution following Clinical and Laboratory Standards Institute methodology, modified to use FAA-HB as recommended by EUCAST, instead of supplemented Brucella agar, and disc diffusion was performed on FAA-HB following EUCAST methodology. RESULTS: Results for quality control strains were reproducible, with 99.3% of zones within range. Disc diffusion by EUCAST methodology was able to distinguish between susceptible and resistant isolates of anaerobic bacteria for benzylpenicillin, piperacillin-tazobactam, meropenem, clindamycin, and metronidazole (98.7% correct categorization). No isolates resistant to vancomycin were tested, but zone diameters correctly categorized the susceptible isolates, and there was a logical relationship between MICs and inhibition zones. DISCUSSION: The recently published EUCAST method for disc diffusion for anaerobic bacteria based on FAA-HB is a reproducible and accurate method for susceptibility testing of frequently isolated anaerobic bacteria.

Evaluation of ten brands of pre-poured Mueller-Hinton agar plates for EUCAST disc diffusion testing.

OBJECTIVES: Mueller-Hinton agar (MHA) is recommended by EUCAST and CLSI for disc diffusion antimicrobial susceptibility testing (AST). We have previously investigated the quality of dehydrated MHA from several manufacturers. In this study, we evaluated the performance of ten commercial brands of pre-poured MHA plates. METHODS: AST was performed according to EUCAST methodology and results analyzed against targets and ranges in EUCAST quality control (QC) tables. MHA plates from different brands were tested in triplicate against four non-fastidious QC strains. The agar depth and pH were measured for all products. RESULTS: The best performance was observed for MHA from Becton Dickinson (BBL MHA II), bioMérieux (MHE agar) and Hardy Diagnostics, for which >97% of zone diameters were within QC ranges and >60% on target ±1 mm. The poorest performance was seen for plates from HiMedia (MHA and MHA no. 2), where 20% and 18% of readings were outside the QC ranges, respectively. The differences in pH and agar depth of the products were small and mostly within EUCAST specifications. DISCUSSION: The accuracy and reproducibility of disc diffusion AST depends on standardised procedures and high-quality discs and media. The performance among ten brands of pre-poured MHA plates differed significantly. The results indicate a poorer performance for pre-poured commercial plates as compared to in-house prepared plates from dehydrated powder of corresponding brands in our previous study. Manufacturers and clinical laboratories have a shared responsibility for the quality of AST. EUCAST provides QC criteria to be used both by manufacturers and laboratories.

Aerococcus urinae and Aerococcus sanguinicola: Susceptibility Testing of 120 Isolates to Six Antimicrobial Agents Using Disk Diffusion (EUCAST), Etest, and Broth Microdilution Techniques.

BACKGROUND: Aerococcus urinae and Aerococcus sanguinicola are relatively newcomers and emerging organisms in clinical and microbiological practice. Both species have worldwide been associated with urinary tract infections. More rarely cases of bacteremia/septicemia and infective endocarditis have been reported. Treatment options are therefore important. Just recently, European recommendations on susceptibility testing and interpretive criteria have been released. OBJECTIVE: In this investigation 120 A. urinae and A. sanguinicola isolates were tested for susceptibility to six antimicrobial agents: Penicillin, cefotaxime, meropenem, vancomycin, linezolid, and rifampicin. METHODS: Three susceptibility testing methods were used; disk diffusion according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) standardized disk diffusion methodology and MIC determination with Etest and broth microdilution (BMD). All testing was performed with EUCAST media for fastidious organisms. RESULTS: Data obtained in this study were part of the background data for establishing EUCAST breakpoints. MIC values obtained by Etest and BMD were well correlated with disk diffusion results. CONCLUSION: All isolates were found susceptible to all six antimicrobial agents: penicillin, cefotaxime, meropenem, vancomycin, linezolid, and rifampicin.

Whole-Genome Sequencing for Routine Pathogen Surveillance in Public Health: a Population Snapshot of Invasive Staphylococcus aureus in Europe.

UNLABELLED: The implementation of routine whole-genome sequencing (WGS) promises to transform our ability to monitor the emergence and spread of bacterial pathogens. Here we combined WGS data from 308 invasive Staphylococcus aureus isolates corresponding to a pan-European population snapshot, with epidemiological and resistance data. Geospatial visualization of the data is made possible by a generic software tool designed for public health purposes that is available at the project URL (http://www.microreact.org/project/EkUvg9uY?tt=rc). Our analysis demonstrates that high-risk clones can be identified on the basis of population level properties such as clonal relatedness, abundance, and spatial structuring and by inferring virulence and resistance properties on the basis of gene content. We also show that in silico predictions of antibiotic resistance profiles are at least as reliable as phenotypic testing. We argue that this work provides a comprehensive road map illustrating the three vital components for future molecular epidemiological surveillance: (i) large-scale structured surveys, (ii) WGS, and (iii) community-oriented database infrastructure and analysis tools. IMPORTANCE: The spread of antibiotic-resistant bacteria is a public health emergency of global concern, threatening medical intervention at every level of health care delivery. Several recent studies have demonstrated the promise of routine whole-genome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control. However, as this technology becomes more widely adopted, the key challenges of generating representative national and international data sets and the development of bioinformatic tools to manage and interpret the data become increasingly pertinent. This study provides a road map for the integration of WGS data into routine pathogen surveillance. We emphasize the importance of large-scale routine surveys to provide the population context for more targeted or localized investigation and the development of open-access bioinformatic tools to provide the means to combine and compare independently generated data with publicly available data sets.

Antimicrobial Resistance of Escherichia coli Causing Uncomplicated Urinary Tract Infections: A European Update for 2014 and Comparison with 2000 and 2008.

INTRODUCTION: The objective of this study was to provide an update on the resistance of Escherichia coli in women with acute uncomplicated urinary tract infections (UTIs) in France, Germany, Spain, Sweden, and the United Kingdom (UK) to mecillinam [amdinocillin (United States Adopted Name)], amoxicillin-clavulanic acid, cefadroxil, nitrofurantoin, ciprofloxacin, and trimethoprim, and to compare the results with resistance in the ECO.SENS I and II surveys in 2000 and 2008, respectively. METHODS: The susceptibility of E. coli in France (166 isolates), Germany (133 isolates), Spain (169 isolates), Sweden (137 isolates), and the UK (124 isolates) was determined by disc diffusion according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints and methodology. Resistance rates were compared using Fisher's exact test, 2-tailed, with P < 0.05 indicating statistical significance. RESULTS: Since 2000, there has been a significant increase in resistance to cefadroxil in Germany (1% to 12%) and Spain (3% to 8%), to ciprofloxacin in Germany (2% to 21%), Spain (15% to 31%), Sweden (0% to 7%), and the UK (1% to 15%), to trimethoprim in Germany (23% to 37%), Spain (25% to 37%), Sweden (9% to 17%), and the UK (13% to 46%), to mecillinam in Spain (1% to 6.5%), and to nitrofurantoin in the UK (0% to 6%); there was also a significant decrease in resistance to nitrofurantoin in Spain (4% to 0%). Since 2008, there has been a significant increase in resistance to ciprofloxacin in Sweden (3% to 15%) and the UK (1% to 15%), and to trimethoprim (13% to 46%) and nitrofurantoin (0% to 6%) in the UK. CONCLUSION: E. coli isolates from women with acute uncomplicated UTIs have increasing antimicrobial resistance, particularly to ciprofloxacin and trimethoprim. However, resistance to mecillinam and nitrofurantoin mostly remains low. FUNDING: LEO Pharma.

Development of a Pefloxacin Disk Diffusion Method for Detection of Fluoroquinolone-Resistant Salmonella enterica.

Fluoroquinolones (FQs) are among the drugs of choice for treatment of Salmonella infections. However, fluoroquinolone resistance is increasing in Salmonella due to chromosomal mutations in the quinolone resistance-determining regions (QRDRs) of the topoisomerase genes gyrA, gyrB, parC, and parE and/or plasmid-mediated quinolone resistance (PMQR) mechanisms including qnr variants, aac(6')-Ib-cr, qepA, and oqxAB. Some of these mutations cause only subtle increases in the MIC, i.e., MICs ranging from 0.12 to 0.25 mg/liter for ciprofloxacin (just above the wild-type MIC of ≤0.06 mg/liter). These isolates are difficult to detect with standard ciprofloxacin disk diffusion, and plasmid-mediated resistance, such as qnr, is often not detected by the nalidixic acid screen test. We evaluated 16 quinolone/fluoroquinolone disks for their ability to detect low-level-resistant Salmonella enterica isolates that are not serotype Typhi. A total of 153 Salmonella isolates characterized for the presence (n = 104) or absence (n = 49) of gyrA and/or parC topoisomerase mutations, qnrA, qnrB, qnrD, qnrS, aac(6')-Ib-cr, or qepA genes were investigated. All isolates were MIC tested by broth microdilution against ciprofloxacin, levofloxacin, and ofloxacin and by disk diffusion using EUCAST or CLSI methodology. MIC determination correctly categorized all isolates as either wild-type isolates (MIC of ≤0.06 mg/liter and absence of resistance genes) or non-wild-type isolates (MIC of >0.06 mg/liter and presence of a resistance gene). Disk diffusion using these antibiotics and nalidixic acid failed to detect some low-level-resistant isolates, whereas the 5-µg pefloxacin disk correctly identified all resistant isolates. However, pefloxacin will not detect isolates having aac(6')-Ib-cr as the only resistance determinant. The pefloxacin disk assay was approved and implemented by EUCAST (in 2014) and CLSI (in 2015).