EUCAST Methodology Research Articles

Wild-type MIC distributions and epidemiological cutoff values for 5-flucytosine and Candida species as determined by EUCAST broth microdilution.

EUCAST has established clinical breakpoints and epidemiological cutoff values (ECOFFs) for Candida spp. However, limited data are available for 5-flucytosine (5-FC). We assessed the in vitro susceptibility of 5-FC against a large collection of clinical Candida species using EUCAST methodology and determined the associated ECOFFs. A total of 5622 Candida isolates were collected from patients across the Netherlands between 2008 and 2024. 5-FC MICs were determined using the EUCAST microbroth dilution reference method. Furthermore, MICs were extracted from the EUCAST website. The MICs from this study and those extracted were used to determine ECOFFs and local ECOFFs (L-ECOFFs). 5-FC exhibited potent in vitro activity against C. albicans, N. glabratus and C. parapsilosis, while decreased susceptibility was observed for C. tropicalis, Pichia species, K. marxianus, Y. lipolytica, and C. auris. The ECOFFs (mg/L) and the percentages of WT isolates for 5-FC were: C. albicans: 0.5 (97.2%), N. glabratus: 0.5 (96.6%), C. parapsilosis: 0.5 (99.5%) and P. kudriavzevii: 8 (99.4%). The L-ECOFF (mg/L) and the percentages of WT isolates for 5-FC were: C. dubliniensis: 0.25 (96.8%), C. tropicalis: 0.25 (67.2%), K. marxianus: 0.25 (48.0%), C. lusitaniae: 0.25 (86.5%), M. guillermondii: 0.125 (95.9%) and P. norvegiensis: 8 (94.2%). 5-FC remains a valuable drug to manage difficult-to-treat invasive Candida infections. In vitro susceptibility cannot be predicted based on species identification for most Candida species, but requires MIC-testing. ECOFFs will help to interpret the MICs to support treatment decisions.

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

Evidence-based clinical susceptibility breakpoints have been lacking for antimicrobial agents used for diphtheria. 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. 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. 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. 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.

Ampicillin susceptibility testing of Haemophilus influenzae in the routine clinical laboratory by the EUCAST methodology compared to broth microdilution and the presence of ftsI gene mutations

ObjectivesTo investigate the prevalence of ampicillin resistance in Haemophilus influenzae and the diagnostic accuracy of the EUCAST recommended disc diffusion method to detect the increasingly prevalent ampicillin resistance due to the presence of PBP3 alterations based on mutations in the ftsI gene. MethodsDuring a 6-month period all consecutive non-duplicate H. influenzae isolates were prospectively collected and stored. MICs of ampicillin were determined by broth microdilution (BMD). PCR was performed to detect mutations in the ftsI gene. Results of routine disc diffusion susceptibility testing, including the penicillin screening test in accordance with the current EUCAST methodology, as well as additional Etest results, were compared to the BMD as the reference method. ResultsIn 102 isolates, the prevalence of ampicillin resistance was 28% (29/102) by BMD. There was a good correlation between MICs of ampicillin and the presence of a β-lactamase and/or an ftsI gene mutation. The prevalence of ampicillin resistance was overestimated using the EUCAST method (33% (34/102)) and underestimated when an additional Etest was used (24% (24/102)) (not significant). The sensitivity and specificity of the EUCAST methodology for the detection of ampicillin resistance were 97% ((28/29); 95% CI, 82–100%) and 92% ((67/73); 95% CI, 83–97%), respectively. ConclusionsThe prevalence of ampicillin resistance was 28%, as determined by BMD. Although the overall diagnostic accuracy of the EUCAST ampicillin disc diffusion was high, misclassification of ampicillin susceptibility may still occur.

Molecular analysis of metallo-beta-lactamase-producing Pseudomonas aeruginosa in Switzerland 2022–2023

ObjectivesThe occurrence of metallo-beta-lactamase-producing Pseudomonas aeruginosa (MBL-PA) isolates is increasing globally, including in Switzerland. The aim of this study was to characterise, phenotypically and genotypically, the MBL-PA isolates submitted to the Swiss National Reference Center for Emerging Antibiotic Resistance (NARA) reference laboratory over a 12-month period from July 2022 to July 2023.MethodsThirty-nine non-duplicate MBL-PA Isolates were submitted to NARA over the study period from across Switzerland. Susceptibility was determined by broth microdilution according to EUCAST methodology. Whole-genome sequencing was performed on 34 isolates. Sequence types (STs) and resistance genes were ascertained using the Centre for Genomic Epidemiology platform. MBL genes, blaNDM-1, blaIMP-1, and blaVIM-2, were cloned into vector pUCP24 and transformed into P. aeruginosa PA14.ResultsThe most prevalent MBL types identified in this study were VIM (21/39; 53.8%) followed by NDM (11/39; 28.2%), IMP (6/39; 15.4%), and a single isolate produced both VIM and NDM enzymes. WGS identified 13 different STs types among the 39 isolates. They all exhibited resistance to cephalosporins, carbapenems, and the beta-lactam-beta-lactamase inhibitor combinations, ceftolozane-tazobactam, ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam, and 8 isolates were cefiderocol (FDC) resistant. Recombinant P. aeruginosa strains producing blaNDM-1, blaIMP-1, and blaVIM-2 exhibited FDC MICs of 16, 8, and 1 mg/L, respectively.ConclusionsThis study showed that the MBL-PA in Switzerland could be attributed to the wide dissemination of high-risk clones that accounted for most isolates in this study. Although FDC resistance was only found in 8 isolates, MBL carriage was shown to be a major contributor to this phenotype.

Comparison Between Moxifloxacin and Chloramphenicol for the Treatment of Bacterial Eye Infections

Background: Moxifloxacin is a bactericidal methoxyquinolone used for the treatment of conjunctivitis and prophylactic therapy in cataract and refractive surgeries. Chloramphenicol is a bacteriostatic organochlorine introduced into clinical practice in 1948 and used mainly in topical preparations because of its known toxicity. Objectives: The study aimed to evaluate the in vitro antibacterial effect and the ocular cytotoxicity of these broad-spectrum antibiotics. Methods: Antimicrobic activity was tested on four bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis), and determined through calculation of Minimum Inhibitory Concentration (MIC) and IC50 for each microorganism. Antibacterial activity was determined by microdilution method after 24 hours incubation with 2-fold serial dilutions (2.5 mg/ml to 4.883 µg/ml) of moxifloxacin and chloramphenicol. Disk diffusion test were performed according to EUCAST methodology. Biofilm formation inhibition (BIC) and biofilm Eradication Concentration (BEC) assay were conducted for P. aeruginosa and S. epidermidis using the microdilution method. Cytotoxicity of antibiotics was evaluated by MTT colorimetric assay on human corneal cell (HCE-2). Results: Cytotoxicity of antibiotics was evaluated on human epithelial corneal cells after 4 hours treatment by viability assay. Results showed that corneal cell viability was significantly higher after moxifloxacin treatment compared to Chloramphenicol (p<0.01). Moxifloxacin is characterized by a significantly lower MIC and IC50 values and a larger inhibition zone for all the strain tested, with high performance in controlling gram-negative growth, compared to Chloramphenicol. Moreover, Moxifloxacin showed higher activity compared to Chloramphenicol in the inhibition of biofilm formation and in the disruption of biofilm, especially against S. epidermidis biofilm. Conclusions: The lower corneal cell toxicity and the broader spectrum of antibacterial activity observed with Moxifloxacin suggests its use in ophthalmic solution for the treatment of bacterial eye infections.

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

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. 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. 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. 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.

Development of an agar-based screening method for terbinafine, itraconazole, and amorolfine susceptibility testing of Trichophyton spp.

Resistance in dermatophytes is an emerging global public health issue. We, therefore, developed an agar-based method for screening Trichophyton spp. susceptibility to terbinafine (TRB), itraconazole (ITC), and amorolfine (AMF) and validated it using molecularly characterized isolates. Α total of 40 Trichophyton spp. isolates, 28 TRB wild type (WT) (13 T. rubrum, 10 T. mentagrophytes, 5 T. interdigitale) and 12 TRB non-WT (7 T. rubrum, 5 T. indotineae) with different alterations in the squalene epoxidase (SQLE) gene, were used. The optimal test conditions (inoculum and drug concentrations, incubation time, and temperature) and stability over time were evaluated. The method was then applied for 86 WT Trichophyton spp. clinical isolates (68 T. rubrum, 7 T. interdigitale, 6 T. tonsurans, 5 T. mentagrophytes) and 4 non-WT T. indotineae. Optimal growth of drug-free controls was observed using an inoculum of 20 µL 0.5 McFarland after 5-7 days of incubation at 30°C. The optimal concentrations that prevented the growth of WT isolates were 0.016 mg/L of TRB, 1 mg/L of ITC, and 0.25 mg/L of AMF, whereas 0.125 mg/L of TRB was used for the detection of Trichophyton strong SQLE mutants (MIC ≥0.25 mg/L). The agar plates were stable up to 4 months. Inter-observer and inter-experimental agreement were 100%, and the method successfully detected TRB non-WT Trichophyton spp. strains showing 100% agreement with the reference EUCAST methodology. An agar-based method was developed for screening Trichophyton spp. in order to detect TRB non-WT weak and strong mutant isolates facilitating their detection in non-expert routine diagnostic laboratories.

Approaches to the antimicrobial susceptibility testing of Campylobacter to monitor the spread of antibiotic-resistant strains

Introduction. Campylobacteriosis is one of the most common diarrhea-associated infections over the world. The situation is getting worse along with increasing cases of the disease caused by the Campylobacter spp. pathogen resistant to antimicrobials (AMPs). Preventing the disease requires monitoring the spread of resistant Campylobacter strains isolated from both sick people and animals, food, and water. &#x0D; Aim of the study is to evaluate the results of the antimicrobial susceptibility testing of Campylobacter according to the requirements of the EUCAST and CLSI methodologies using Russia-made nutrient media. &#x0D; Materials and methods. Collected and freshly isolated strains of C. jejuni, C. coli, C. fetus and C. lari were used. Campylobacter cultures were subcultured on Campylobacter Agar Base (HiMedia), Preston laboratory-produced medium and iron-erythritol blood agar (Obolensk). Their sensitivity to AMPs was determined by three methods as ffollows: disc diffusion, gradient diffusion and microdilutions using Mueller-Hinton agar, and broth of two manufacturers (BD BBL and Obolensk) according to EUCAST and CLSI.&#x0D; Results. Using Russian-made and imported Mueller-Hinton agar and broth allowed obtaining identical results for AMPs susceptibility of Campylobacter spp. The campylobacter strains were attributed to the same susceptibility category by all three methods in frame of any methodology (EUCAST or CLSI) when interpreting results. Due to differences in cutoffs of MIC and inhibition zone diameters in the EUCAST and CLSI standards, there were some differences in the interpretation of the results.&#x0D; Limitations. Eight strains of four species Campylobacter were tested for their susceptibility to three antimicrobials by three methods according to EUCAST and CLSI methodology.&#x0D; Conclusion. The results obtained confirm the possibility of applying a complex of domestic nutrient media for cultivating and monitoring the spread of antibiotic resistant strains of Campylobacter spp. This is especially important in view of implementing the import substitution program.

Multifactorial resistance mechanisms associated with resistance to ceftazidime-avibactam in clinical Pseudomonas aeruginosa isolates from Switzerland.

Increasing reports of multidrug resistance (MDR) in clinical Pseudomonas aeruginosa have led to a necessity for new antimicrobials. Ceftazidime-avibactam (CZA) is indicated for use against MDR P. aeruginosa across a broad range of infection types and particularly those that are carbapenem resistant. This study sought to determine the molecular mechanisms of CZA and imipenem (IPM)-resistance in clinical P. aeruginosa isolates obtained from Swiss hospitals. Clinical P. aeruginosa isolates were obtained from inpatients in three hospitals in Switzerland. Susceptibility was determined by either antibiotic disc testing or broth microdilution according to EUCAST methodology. AmpC activity was determined using cloxacillin and efflux activity was determined using phenylalanine-arginine β-naphthylamide, in agar plates. Whole Genome Sequencing was performed on 18 clinical isolates. Sequence types (STs) and resistance genes were ascertained using the Centre for Genomic Epidemiology platform. Genes of interest were extracted from sequenced isolates and compared to reference strain P. aeruginosa PAO1. Sixteen different STs were identified amongst the 18 isolates in this study indicating a high degree of genomic diversity. No carbapenemases were detected but one isolate did harbor the ESBL bla PER-1. Eight isolates were CZA-resistant with MICs ranging from 16-64 mg/L, and the remaining ten isolates had either low/wildtype MICs (n=6; 1-2 mg/L) or elevated, but still susceptible, MICs (n=4; 4-8 mg/L). Ten isolates were IPM-resistant, seven of which had mutations resulting in truncations of OprD, and the remaining nine IPM-susceptible isolates had intact oprD genes. Within CZA-R isolates, and those with reduced susceptibility, mutations resulting in ampC derepression, OprD loss, mexAB overexpression and ESBL (bla PER-1) carriage were observed in various combinations and one harbored a truncation of the PBP4 dacB gene. Within the six isolates with wildtype-resistance levels, five had no mutations that would affect any antimicrobial resistance (AMR) genes of interest when compared to PAO1. This preliminary study highlights that CZA-resistance in P. aeruginosa is multifactorial and could be caused by the interplay between different resistance mechanisms including ESBL carriage, increased efflux, loss of permeability and derepression of its intrinsic ampC.

Development of an in vitro pharmacokinetic/pharmacodynamic model in the presence of serum for studying micafungin activity against Candida albicans: a need for revision of CLSI susceptibility breakpoints.

The CLSI breakpoint for micafungin and Candida albicans is 0.25 mg/L, higher than the CLSI epidemiological cut-off value (0.03 mg/L) whereas the EUCAST values are identical (0.016 mg/L). We developed a novel in vitro dialysis-diffusion pharmacokinetic/pharmacodynamic (PK/PD) model, confirmed correlation to in vivo outcome and studied micafungin pharmacodynamics against Canida albicans. Four C. albicans isolates, including a weak (F641L) and a strong (R647G) fks1 mutants, were studied using a 104 cfu/mL inoculum and RPMI medium with and without 10% pooled human serum. The exposure-effect relationship fAUC0-24/MIC was described for CLSI and EUCAST methodology. Monte Carlo simulation analysis included standard (100 mg i.v.) and higher (150-300 mg) doses q24h to determine the corresponding probability of target attainment (PTA). The in vitro PK/PD targets for stasis/1-log kill were 36/57 fAUC0-24/MIC in absence and 2.8/9.2 fAUC0-24/MIC in the presence of serum, and similar for wild-type and fks mutant isolates. The PTAs for both PK/PD targets were high (>95%) for EUCAST susceptible isolates but not for CLSI susceptible non-wild-type isolates (CLSI MICs 0.06-0.25 mg/L). 300 mg q24h was needed to attain PK/PD targets for non-wild-type isolates with CLSI MICs 0.06-0.125 mg/L and EUCAST MICs 0.03-0.06 mg/L. The in vitro 1-log kill effect corresponded to stasis in animal model and mycological response in patients with invasive candidiasis, thereby validating the model for studying pharmacodynamics of echinocandins in vitro. EUCAST breakpoints were well supported by our findings but our data questions whether the current CLSI breakpoint, which is higher than the epidemiological cut-off values, is appropriate.

Susceptibility of Aerococcus urinae and Aerococcus sanguinicola to Standard Antibiotics and to Nitroxoline.

Aerococcus urinae and Aerococcus sanguinicola have been increasingly recognized as causative agents of urinary tract infection (UTI) during the last decade. Nitroxoline achieves high urinary concentrations after oral administration and is recommended in uncomplicated UTI in Germany, but its activity against Aerococcus spp. is unknown. The aim of this study was to assess the in vitro susceptibility of clinical Aerococcus species isolates to standard antibiotics and to nitroxoline. Between December 2016 and June 2018, 166 A. urinae and 18 A. sanguinicola isolates were recovered from urine specimens sent to the microbiology laboratory of the University Hospital of Cologne, Germany. Susceptibility to standard antimicrobials was analyzed by disk diffusion (DD) according to EUCAST methodology, nitroxoline was tested by DD and agar dilution. Susceptibility of Aerococcus spp. to benzylpenicillin, ampicillin, meropenem, rifampicin, nitrofurantoin, and vancomycin was 100% and resistance was documented only against ciprofloxacin (20 of 184; 10.9%). MICs of nitroxoline in A. urinae isolates were low (MIC50/90 1/2 mg/L) while significantly higher MICs were observed in A. sanguinicola (MIC50/90 64/128 mg/L). If the EUCAST nitroxoline breakpoint for E. coli and uncomplicated UTI was applied (16 mg/L), 97.6% of A. urinae isolates would be interpreted as susceptible while all A. sanguinicola isolates would be considered resistant. Nitroxoline demonstrated high activity against clinical A. urinae isolates, but low activity against A. sanguinicola. Nitroxoline is an approved antimicrobial for UTI and could be an alternative oral drug to treat A. urinae urinary tract infection, yet clinical studies are needed to demonstrate this potential in vivo. IMPORTANCE A. urinae and A. sanguinicola have been increasingly recognized as causative agents in urinary tract infections. Currently, there are few data available on the activity of different antibiotics against these species and no data on nitroxoline. We demonstrate that clinical isolates in Germany are highly susceptible to ampicillin, while resistance to ciprofloxacin was common (10.9%). Additionally, we show that nitroxoline is highly active against A. urinae, but not against A. sanguinicola, which based on the presented data, should be considered intrinsically resistant. The presented data will help to improve the therapy of urinary tract infections by Aerococcus species.

Towards clinical breakpoints for non-tuberculous mycobacteria – Determination of epidemiological cut off values for the Mycobacterium avium complex and Mycobacterium abscessus using broth microdilution

ObjectiveFor non-tuberculous mycobacteria (NTM), minimum inhibitory concentration (MIC) distributions of wild-type isolates have not been systematically evaluated despite their importance for establishing antimicrobial susceptibility testing (AST) breakpoints. MethodsWe gathered MIC distributions for drugs used against the Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) obtained by commercial broth microdilution (SLOMYCOI and RAPMYCOI) from 12 laboratories. Epidemiological cut-off values (ECOFFs) and tentative ECOFFs (TECOFFs) were determined by EUCAST methodology including quality control (QC) strains. ResultsThe clarithromycin ECOFF was 16 mg/L for M. avium (n = 1271) whereas TECOFFs were 8 mg/L for M. intracellulare (n = 415) and 1 mg/L for MAB (n = 1014) confirmed by analysing MAB subspecies without inducible macrolide resistance (n = 235). For amikacin, the ECOFFs were 64 mg/L for MAC and MAB. For moxifloxacin, the WT spanned >8 mg/L for both MAC and MAB. For linezolid, the ECOFF and TECOFF were 64 mg/L for M. avium and M. intracellulare, respectively. Current CLSI breakpoints for amikacin (16 mg/L), moxifloxacin (1 mg/L) and linezolid (8 mg/L) divided the corresponding WT distributions. For QC M. avium and M. peregrinum, ≥95% of MIC values were well within recommended QC ranges. ConclusionAs a first step towards clinical breakpoints for NTM, (T)ECOFFs were defined for several antimicrobials against MAC and MAB. Broad wild-type MIC distributions indicate a need for further method refinement which is now under development within the EUCAST subcommittee for anti-mycobacterial drug susceptibility testing. In addition, we showed that several CLSI NTM breakpoints are not consistent in relation to the (T)ECOFFs.

P185 Epidemiology of human fusariosis in Greece: results from a 16-year nationwide multicenter survey

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesFusariosis in humans comprises a vast array of rare but serious and difficult-to-treat infections, ranging from keratitis and onychomycosis in immunocompetent hosts to life-threatening systemic infections in immunocompromised patients such as those with hematologic malignancies. We aimed to assess the disease burden and baseline epidemiology of fusariosis in Greece.MethodsFrom 2004 through 2020 a prospective, nationwide, multicenter survey took place. Demographic and clinical data of fusariosis cases were recorded. Fusarium strains isolated were identified to species level with molecular methods and/or MALDI-TOF MS, and tested for antifungal susceptibility in vitro with the EUCAST methodology.ResultsA total of 54 cases were registered. The most frequent infection was keratitis (n = 21, 39%), followed by bloodstream infections in patients with hematologic malignancy (n = 12, 22.2%). Other infections involved the respiratory tract (n = 3, 5.5%), and sinuses (n = 3, 5.5%) in immunocompromised patients, soft tissues after trauma (n = 5, 9.3%), or diabetic foot (n = 2, 3.7%), and onychomycosis (n = 8, 14.8%). The estimated incidence of invasive Fusarium infections was 2.9 cases/year (0.027 cases/100 000 population). The most frequently isolated species were: F. solani species complex (SC) (n = 13, 24%), F. oxysporum SC (13, 24%), F. fujikuroi SC (n = 12, 22.2%, of which 6 F. verticillioides and 6 F. proliferatum). Other SCs included F. brachygibbosum, F. chlamydosporum, and F. dimerum. In keratitis cases, F. solani was associated with infection secondary to injury with plant material, whereas F. fujikuroi with soft contact lens wear. Fusarium oxysporum was more frequently isolated from nails or soft tissue infections (53.8%). Antifungal MICs were high, with no clear interspecies differences (geometric mean 1.6, 2.5, 3.2 mg/l for amphotericin B, voriconazole, and posaconazole, respectively, median values 2, 4, 8 mg/L, respectively). Fluconazole and the echinocandins showed no activity (MIC >32 mg/l). The most frequently used antifungals were amphotericin B and voriconazole, usually in combination. Treatment failure in keratitis was 38.5%. In patients with hematologic malignancy the crude mortality rate was 71.4%, usually related to the underlying disease. Soft tissue infections complicating diabetic foot or trauma were treated surgically, with favorable outcomes.Conclusion Fusarium infections in Greece remain rare, but with considerable morbidity and mortality in the immunocompromised. Early diagnosis and initiation of the appropriate treatment were critical for a successful outcome in keratitis cases, despite moderately high MICs of the antifungals used.

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

ObjectivesMueller-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. MethodsAST 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. ResultsThe 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. DiscussionThe 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.

Ralstonia pickettii bacteremia in a cardiac surgery patient in Belgrade, Serbia.

Ralstonia pickettii is an opportunistic bacterium found in the water environment with an increasing incidence as a nosocomial pathogen. The objectives of this study were to describe R. pickettii bacteremia in a cardiac surgery patient and to evaluate its ability to grow in a saline solution and to form biofilm. The patient in this study underwent mitral and aortic valve replacement surgery with two aortocoronary bypasses. She developed signs of respiratory and renal failure, therefore hemodialysis was started. After 25 days in an intensive care unit, the patient had recurrent episodes of fever with signs of bacteremia. R. pickettii was identified from blood cultures by MALDI-TOF MS. Antimicrobial susceptibility testing was performed using disc diffusion and broth microdilution methods in accordance with EUCAST methodology and results were interpreted following clinical breakpoints for Pseudomonas spp. The isolate was susceptible to all tested antimicrobial agents except aminoglycosides and colistin. Survival of R. pickettii was analyzed in saline solution with four different starting concentrations at 25 °C and 37 °C for six days. Biofilm capacity was tested using the microtiter plate method. R. pickettii showed substantial growth in saline solution, with starting concentration of 2 CFU ml-1 reaching 107 CFU ml-1 after six days. There was no significant difference between growth at 25 °C and 37 °C. This indicates that storage of contaminated solutions at room temperature can enhance the count of R. pickettii. Our strain did not show the capacity to form biofilm. The patient responded well to adequate treatment with ceftazidime, and after 48 days in ICU she was discharged to convalesce.

Cefiderocol: EUCAST criteria for disc diffusion and broth microdilution for antimicrobial susceptibility testing.

ObjectivesThe 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.MethodsCefiderocol 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.ResultsMIC 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.ConclusionsDD 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.

P44 Nosocomial neurosurgical meningitis due to XDR Pseudomonas aeruginosa: clinical experience with cefiderocol

BackgroundCarbapenem resistance due to MBL production is relatively uncommon in Pseudomonas aeruginosa infections in the UK. We report a case of post-operative meningitis caused by a New-Delhi MBL-producing (NDM-1) strain, outlining the microbiological and management challenges.Patient caseThe patient was admitted to intensive care after craniotomy and evacuation for an intracerebral haemorrhage. Three weeks later she was febrile due to nosocomial meningitis. Purulent material evacuated from extradural and subdural spaces cultured P. aeruginosa, along with blood cultures and CSF. Susceptibility testing as per EUCAST methodology indicated the isolate was resistant to all β-lactams, aminoglycosides and quinolones but susceptible to colistin. Susceptibility to cefiderocol by disc diffusion was in the area of technical uncertainty at 21 mm (S≥22 mm). Immunochromatographic flow testing (CARBA5 NG Biotech) detected an NDM carbapenemase. WGS (Illumina) confirmed the presence of blaNDM-1 and a plethora of AMR genes responsible for the XDR phenotype {cephalosporins (blaPDC-10, blaOXA-488), aminoglycosides [aph(3')-VIa], quinolones (crpP), macrolides [mph(E), msr(E)], phenicols (catB7) and fosfomycin (fosA)}. No mutations in PBPs associated with resistance to β-lactams or iron acquisition systems involved in uptake of siderophore conjugated antibiotics were identified. The isolate belonged to ST1047, serotype 07, previously identified as a high-risk epidemic clone originating in Myanmar.ResultsTreatment was commenced with IV and intrathecal colistin as well as IV cefiderocol (Figure 1); a novel siderophore cephalosporin licensed for Gram-negative infections with limited treatment options. No synergy between the two drugs could be demonstrated in chequerboard assays. Although CSF sterility was achieved on three subsequent cultures with clinical response, the patient sadly died due to the underlying intracerebral injury. Four other patients subsequently became colonized with this strain and infection control measures (twice weekly screening of all patients, environmental cleaning, water testing, isolation of cases) were instituted, bringing the outbreak under control. Variable number tandem repeat analysis linked these strains to that of another patient discharged from the unit 9 months prior, but not previously reported in the UK.Figure 1.Clinical timeline, culture results, antimicrobial regimens and cefiderocol levels. BC, blood culture; Resp, respiratory specimen; Swab, craniotomy swab; Pus, subdural and extradural pus; Cro, ceftriaxone; Mero, meropenem; Cefid, cefiderocol; Vanc, vancomycin; IT, intrathecal; MU, million units; bd, two times a day; od, once daily.ConclusionsThe first problem encountered was a lack of evidence for the CNS penetration of cefiderocol and efficacy in the treatment of meningitis. The external ventricular drain required to administer intrathecal colistin enabled us to monitor CSF cefiderocol levels. Five CSF samples were shipped at −80°C for analysis in Germany by mass spectrometry. CSF cefiderocol levels of 1.22 μg/mL, 1.34 μg/mL, 2.39 μg/mL, 3.53 μg/mL and 3.90 μg/mL were disappointingly close to the pharmacodynamic breakpoint of 2 μg/mL and only available after treatment withdrawal. Dosing of cefiderocol was initially cautious starting at 1 g/8 h due to a creatinine clearance of 53 mL/min but later increased to 1.5 g/8 h. Further dose increases guided by serum and CSF monitoring could feasibly lead to therapeutic CSF cefiderocol levels. Clinical and pharmacological studies are needed in this area. To our knowledge this is the first attempt to treat a case of XDR neurosurgical meningitis using cefiderocol with precision treatment and achieving microbiological cure.

Antifungal activity of liriodenine on clinical strains of Cryptococcus neoformans and Cryptococcus gattii species complexes.

Background: Cryptoccocal meningitis continues to present high incidence among AIDS patients. The treatment of choice is the synergistic combination of flucytosine (5-FC) with amphotericin B deoxycholate (AmBd) or its lipid formulations. However, 5-FC is unavailable in many countries and AmB demands hospitalization. The combination of AmB with the fungistatic fluconazole (FLC) or the use of high FLC daily doses alone became the choice. Nonetheless, sterilization of cerebrospinal fluid is delayed with FLC monotherapy, mainly with high fungal burden. These findings suggest the search for new antifungal compounds, such as liriodenine.Methods: Liriodenine antifungal activity was evaluated by three procedures: determining the minimum inhibitory concentration (MIC) on 30 strains of the Cryptococcus neoformans (C. neoformans) complex and 30 of the Cryptococcus gattii (C. gattii) complex, using EUCAST methodology and amphotericin B deoxycholate as control; performing the time-kill methodology in two strains of the C. neoformans complex and one of the C. gattii complex; and injury to cryptococcal cells, evaluated by transmission electron microscopy (TEM). Liriodenine absorption and safety at 0.75 and 1.50 mg.kg-1 doses were evaluated in BALB/c mice.Results: Liriodenine MICs ranged from 3.9 to 62.5 μg.mL-1 for both species complexes, with no differences between them. Time-kill methodology confirmed its concentration-dependent fungicidal effect, killing all the strains below the limit of detection (33 CFU.mL-1) at the highest liriodenine concentration (32-fold MIC), with predominant activity during the first 48 hours. Liriodenine induced severe Cryptococcus alterations - cytoplasm with intense rarefaction and/or degradation, injury of organelles, and presence of vacuoles. Liriodenine was better absorbed at lower doses, with no histopathological alterations on the digestive tract.Conclusion: The fungicidal activity confirmed by time-kill methodology, the intense Cryptococcus injury observed by TEM, the absorption after gavage administration, and the safety at the tested doses indicate that the liriodenine molecule is a promising drug lead for development of anticryptococcal agents.

Interaction in vitro of pulmonary surfactant with antifungal agents used for treatment and prevention of invasive aspergillosis.

Optimizing antifungal therapy is important to improve outcomes in severely immunocompromised patients. We analysed the in vitro interaction between pulmonary surfactant and antifungal agents used for management of invasive pulmonary aspergillosis. Amphotericin B formulations, mould-active triazoles and echinocandins were tested in vitro against 24 clinical isolates of different Aspergillus spp. with and without the addition of a commercial porcine surfactant (Curosurf®; Poractant alfa, Nycomed, Austria). The data are presented as MIC or minimum effective concentration (MEC) ranges, as MIC or MEC values that inhibited 90% of the isolates (MIC90 or MEC90) and as geometric mean (GM) MIC or MEC values. For amphotericin B products, addition of surfactant to a final concentration of 10% led to a statistically significant reduction of the GM MIC for all Aspergillus isolates tested after 24 h (0.765 versus 0.552 mg/L; P < 0.05). For the mould-active triazoles, addition of 10% surfactant resulted in a significantly higher GM MIC at 48 h (0.625 versus 0.898 mg/L; P < 0.05). For the echinocandins, the addition of 10% surfactant led to a significantly higher GM MEC after both 24 h (0.409 versus 0.6532 mg/L; P < 0.01) and 48 h (0.527 versus 0.9378 mg/L; P < 0.01). There were no meaningful differences between individual members of the three existing classes of antifungal agents or between the different Aspergillus spp. tested. Using EUCAST methodology, addition of porcine surfactant up to a concentration of 10% had a minor, and presumably non-relevant, impact on the in vitro activity of antifungal agents used in prophylaxis and treatment of invasive pulmonary aspergillosis.

Spectrophotometric azole and amphotericin B MIC readings against Aspergillus fumigatus sensu lato using the EUCAST 9.3.2 methodology. Are ≥90 and ≥95% fungal growth inhibition endpoints equally suitable?

Spectrophotometrically obtained MIC readings using either≥90% or≥95% fungal growth inhibition endpoints and following the EUCAST methodology are suitable against A. fumigatus sensu stricto. However, the≥95% fungal growth inhibition endpoint is preferred against cryptic species.