Abstract 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.
Bacillus anthracis clinical breakpoints, representing a systematic approach to guide clinicians in selecting the most appropriate antimicrobial treatments, are not part of the guidance from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). This is because defined distributions of MIC values and of epidemiological cut-off values (ECOFFs) have been lacking. In this study, a Europe-wide network of laboratories in collaboration with EUCAST, aimed at establishing standardized antimicrobial susceptibility testing methods, wild-type MIC distributions, and ECOFFs for ten therapeutically relevant antimicrobials.
ABSTRACT Different antimicrobial susceptibility testing methods to detect low-level vancomycin resistance in enterococci were evaluated in a Scandinavian multicenter study ( n = 28). A phenotypically and genotypically well-characterized diverse collection of Enterococcus faecalis ( n = 12) and Enterococcus faecium ( n = 18) strains with and without nonsusceptibility to vancomycin was examined blindly in Danish ( n = 5), Norwegian ( n = 13), and Swedish ( n = 10) laboratories using the EUCAST disk diffusion method ( n = 28) and the CLSI agar screen ( n = 18) or the Vitek 2 system (bioMérieux) ( n = 5). The EUCAST disk diffusion method (very major error [VME] rate, 7.0%; sensitivity, 0.93; major error [ME] rate, 2.4%; specificity, 0.98) and CLSI agar screen (VME rate, 6.6%; sensitivity, 0.93; ME rate, 5.6%; specificity, 0.94) performed significantly better ( P = 0.02) than the Vitek 2 system (VME rate, 13%; sensitivity, 0.87; ME rate, 0%; specificity, 1). The performance of the EUCAST disk diffusion method was challenged by differences in vancomycin inhibition zone sizes as well as the experience of the personnel in interpreting fuzzy zone edges as an indication of vancomycin resistance. Laboratories using Oxoid agar ( P < 0.0001) or Merck Mueller-Hinton (MH) agar ( P = 0.027) for the disk diffusion assay performed significantly better than did laboratories using BBL MH II medium. Laboratories using Difco brain heart infusion (BHI) agar for the CLSI agar screen performed significantly better ( P = 0.017) than did those using Oxoid BHI agar. In conclusion, both the EUCAST disk diffusion and CLSI agar screening methods performed acceptably (sensitivity, 0.93; specificity, 0.94 to 0.98) in the detection of VanB-type vancomycin-resistant enterococci with low-level resistance. Importantly, use of the CLSI agar screen requires careful monitoring of the vancomycin concentration in the plates. Moreover, disk diffusion methodology requires that personnel be trained in interpreting zone edges.
Abstract Objectives The reproducibility of cefiderocol MIC determination using broth microdilution (BMD) in iron-depleted CAMHB (ID-CAMHB) was investigated, and the EUCAST disc diffusion (DD) method for cefiderocol susceptibility testing was developed and validated against reference BMD. Methods Cefiderocol values were determined for wild-type (WT) and non-WT isolates using BMD plates with ID-CAMHB (Thermo Scientific, Oakwood, USA) per EUCAST guidelines. DD was performed using standard EUCAST methodology on unsupplemented Mueller–Hinton agar with cefiderocol 30 μg discs. Control agents were included in all tests. MICs were correlated with zone diameters (ZD), and ZD breakpoints (BP) best corresponding to the MIC BPs were determined. Areas of technical uncertainty (ATU) were included where appropriate. External laboratory validation of cefiderocol DD was performed per the EUCAST SOP 9.2. Results MIC and ZD distributions for cefiderocol against WT isolates were established. Cefiderocol ZD BPs were set at susceptible ≥22 mm, resistant <22 mm for Enterobacterales and Pseudomonas aeruginosa and ATUs were decided. For Acinetobacter baumannii and Stenotrophomonas maltophilia, ZD cut-off values of ≥17 mm and ≥20 mm corresponded to MIC values of ≤2 and ≤0.5 mg/L, respectively. Cefiderocol ZDs for Escherichia coli ATCC 25922 (target 27 mm) and P. aeruginosa ATCC 27853 (target 26 mm) were within ±3 mm of the target values. For DD, there was no problematic variation between discs, media or laboratories. Conclusions DD is a robust and easy-to-perform method for cefiderocol susceptibility testing. For isolates with results in the ATU, an MIC test should be performed to confirm the results.
Identification and characterization of non-typeable Haemophilus influenzae (NTHi) with reduced susceptibility to β-lactam antibiotics due to mutations in penicillin binding protein 3 (PBP3) is a clinical challenge. We analyzed a blood isolate, NTHi93–57485, that was categorized as aminopenicillin resistant but lacked key amino acid substitutions in PBP3 that have previously been associated with reduced aminopenicillin susceptibility. The significance of an alternative amino acid substitution (Y528H) in this isolate was examined. Site-directed mutagenesis of a β-lactam susceptible H. influenzae (NTHi3655) was performed to introduce the Y528H substitution into wild-type ftsI (encoding for PBP3). Disc diffusion screening and broth microdilution determination of MICs for β-lactam agents were done with the NTHi3655-PBP3Y528H mutant and were compared with the NTHi3655 wild-type as well as the original clinical isolate NTHi93–57485. Introduction of the Y528H substitution in NTHi3655 resulted in positive screening for β-lactam resistance. MICs for aminopenicillins were increased in the mutant compared to the wild-type. However, the mutant remained susceptible to aminopenicillins according to EUCAST clinical breakpoints (assuming intravenous treatment) and the introduction of Y528H alone did not increase the resistance to the same level as NTHi93–57485. None of the isolates had frame shift insertions in the acrR gene regulating the AcrAB efflux pump. In parallel to the previously well-described PBP3-substitutions R517H and N526K, we demonstrate that Y528H confers reduced aminopenicillin susceptibility.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
