ENOVAT: the European Network for Optimization of Veterinary Antimicrobial Treatment.

The global antimicrobial resistance crisis has been the driver of several international strategies on antimicrobial stewardship. For their implementation on field level, the veterinary sector encounters several specific challenges and in particular: (i) a shortage of experts in key disciplines related to antimicrobial stewardship, (ii) a lack of evidence-based antimicrobial treatment guidelines, and (iii) inferior diagnostic tests available compared to human medicine. The present white paper describes how the COST Action ENOVAT (the European Network for Optimization of Veterinary Antimicrobial Treatment, CA18217), comprising 332 persons from 51 countries, worked towards solutions to these challenges. Initially, surveys were conducted to explore the present state in Europe in terms of existing antimicrobial use guidelines and microbiology practices performed. Concurrently, various research activities were launched to optimize diagnostics, including development of epidemiological cut-offs, clinical breakpoints and matrix-assisted laser desorption ionization time of flight mass spectrometry interpretive criteria. Also, guidelines drafting groups working towards evidence-based antimicrobial treatment guidelines for six conditions in food-producing and companion animals were established. The processes and outcomes, also in terms of capacity building, are summarized in this white paper where emphasis is placed on sustainability of the activities. Although several ENOVAT initiatives and spin-off projects will continue beyond the Action, we recommend that a new European veterinary research agenda is launched focusing on research and funding leading to long-term impacts on veterinary antimicrobial use.

Antimicrobial resistance is an urgent global public health threat that is amplified by over- and misuse of antimicrobials. As a result of antimicrobial resistance, antibiotics and other antimicrobial medicines become ineffective and infections become difficult or impossible to treat. This goes for human infections, but also for infections in animals. In a recently finished European project called ENOVAT we tried to tackle the problem of antimicrobial resistance in animals. We focused on two topics. First we optimized and harmonized diagnostics of bacterial infections in the laboratory, and second we developed evidence-based treatment guidelines to support veterinary practitioners on how and when to use antibiotics in the best way. Improved diagnostics and new treatment guidelines can help veterinary practitioners to a more sensible antibiotic choice and with that less over- and misuse of antimicrobials. This article summarizes the process and progress of the work done in the ENOVAT project. Emphasis is also put on how the project benefitted from a unique consortium encompassing 332 professionals with diverse backgrounds, from 51 countries.

Apramycin has high in vitro activity against Mycobacterium tuberculosis.

Introduction. Aminoglycoside antibiotics such as amikacin and kanamycin are important components in the treatment of Mycobacterium tuberculosis (Mtb) infection. However, more and more clinical strains are found to be aminoglycoside antibiotic-resistant. Apramycin is another kind of aminoglycoside antibiotic that is commonly used to treat infections in animals.Hypothesis. Apramycin may have in vitro activity against Mtb.Aim. This study aims to evaluate the efficacy of apramycin against Mtb in vitro and determine its epidemiological cut-off (ECOFF) value.Methodology. One hundred Mtb isolates, including 17 pansusceptible and 83 drug-resistant tuberculosis (DR-TB) strains, were analysed for apramycin resistance using the MIC assay.Results. Apramycin exhibited significant inhibitory activity against Mtb clinical isolates, with an MIC50 of 0.5 µg ml-1 and an MIC90 of 1 µg ml-1. We determined the tentative ECOFF value as 1 µg ml-1 for apramycin. The resistant rates of multidrug-resistant tuberculosis (MDR-TB), pre-extensively drug-resistant (pre-XDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains were 12.12 % (4/33), 20.69 % (6/29) and 66.67 % (14/21), respectively. The rrs gene A1401G is associated with apramycin resistance, as well as the cross-resistance between apramycin and other aminoglycosides.Conclusion. Apramycin shows high in vitro activity against the Mtb clinical isolates, especially the MDR-TB clinical isolates. This encouraging discovery calls for more research on the functions of apramycin in vivo and as a possible antibiotic for the treatment of drug-resistant TB.

European multi-centre study to establish MIC and zone diameter epidemiological cut-off values for Bacillusanthracis.

OBJECTIVES: 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. METHODS: About 335 B. anthracis isolates were tested by broth microdilution and disc diffusion methodologies. MIC and inhibition zone diameters were curated according to EUCAST SOP 10.2 and the results were submitted to EUCAST for ECOFFs and clinical breakpoint determination. RESULTS: Broth microdilution and disc diffusion data distributions revealed putative wild-type distributions for the tested agents. For each antimicrobial agent, ECOFFs were defined. Three highly resistant strains with MIC values of 32 mg/L benzylpenicillin were found. MIC values slightly above the defined ECOFFs were observed in a few isolates, indicating the presence of resistance mechanisms to doxycycline, tetracycline, and amoxicillin. DISCUSSION: B. anthracis antimicrobial susceptibility testing results were used by EUCAST to determine ECOFFs for ten antimicrobial agents. The MIC distributions were used in the process of determining clinical breakpoints. The ECOFFs can be used for the sensitive detection of isolates with resistance mechanisms, and for monitoring resistance development. Genetic changes causing phenotypic shifts in isolates displaying slightly elevated MICs remain to be investigated.

Wild-type distributions of minimum inhibitory concentrations and epidemiological cut-off values-laboratory and clinical utility.

The characterization of wild-type minimum inhibitory concentration (MIC) and zone diameter distributions with the setting of epidemiological cut-off values (ECOFFs or ECVs) provides a reference for the otherwise relative MIC values in the international system for antimicrobial susceptibility testing. Distributions of MIC values for a species and an agent follow a log-normal distribution, which in the absence of resistance mechanisms is monomodal and designated wild type (WT). The upper end of the WT distribution, the ECOFF, can be identified with statistical methods. In the presence of phenotypically detectable resistance, the distribution has at least one more mode (the non-WT), but despite this, the WT is most often identifiable using the same methods. The ECOFF provides the most sensitive measure of resistance development in a species against an agent. The WT and non-WT modes are independent of the organism´s response to treatment, but when the European Committee on Antimicrobial Susceptibility Testing (EUCAST) determines the clinical breakpoints, the committee avoids breakpoints that split WT distributions of target species. This is to avoid the poorer reproducibility of susceptibility categorization when breakpoints split major populations but also because the EUCAST has failed to identify different clinical outcomes for isolates with different MIC values inside the wild-type distribution. In laboratory practice, the ECOFF is used to screen for and exclude resistance and allows the comparison of resistance between systems with different breakpoints from different breakpoint organizations, breakpoints evolving over time, and different breakpoints between human and animal medicine. The EUCAST actively encourages colleagues to question MIC distributions as presented on the website (https://www.eucast.org/mic_and_zone_distributions_and_ecoffs) and to contribute MIC and inhibition zone diameter data.

Antimicrobial Resistance in Escherichia coli and Its Correlation with Antimicrobial Use on Commercial Poultry Farms in Bangladesh.

Antimicrobial resistance is a global concern, posing risks to human and animal health. This research quantified antimicrobial resistance (AMR) in E. coli isolates from poultry fecal and environmental samples in Bangladesh and explored their association with antimicrobial use (AMU). We screened 725 fecal and 250 environmental samples from 94 conventional broilers and 51 Sonali farms for E. coli presence using MALDI-TOF mass spectrometry. AMU data were collected at flock levels, expressed as treatment incidence (TI), while minimum inhibitory concentrations (MIC) for 14 antibiotics were determined on five fecal E. coli isolates per farm and on all environmental isolates. MIC results were interpreted using human clinical breakpoints and EUCAST epidemiological cut-off values (ECOFFs). Acquired resistance against commonly used antimicrobial agents such as ciprofloxacin, tetracycline and ampicillin, was extremely high and predominantly clinically relevant. There was a moderate correlation between fecal and environmental antibiotic resistance index (ARI), but there was no significant correlation between AMU and AMR, suggesting that the observed AMR prevalence is unrelated to current AMU in poultry, but may be due to high historical AMU. A high level of multidrug resistance, including against critically important antimicrobials, was found in both farm types. Therefore, an AMR/AMU surveillance program is urgently needed in the poultry production sector of Bangladesh.

Clinical application of Vitek-derived minimum inhibitory concentration values: Proof of concept study.

Background: Minimum inhibitory concentration (MIC) values are useful in guiding appropriate antimicrobial therapy however, routine provision and interpretation of MIC values to guide clinical decision-making is challenging. Objectives: This proof of concept study aims to demonstrate the clinical utility and application of Vitek®-derived MIC values through categorisation of clinical isolates as wild type. Method: A random selection of clinically relevant Gram negative isolates routinely tested on the Vitek® instrument were included. The Vitek® MIC values, for selected antimicrobials at the lowest calling range of that card, were compared to the broth microdilution reference method. The specified end-point was concordance between the two results if the reference MIC was less than or equal to the EUCAST-defined epidemiological cut-off value (ECOFF) for that drug-bug combination. Results: A total of 525 isolates were included (468 Enterobacterales and 57 Pseudomonas aeruginosa), with an overall concordance rate of 96.4% (508/525). Correct ECOFF categorisation by the Vitek® was highest for ceftazidime and piperacillin (100%, n = 48 and n = 55, respectively) and lowest for cefepime (81.8%, n = 66). Conclusion: Vitek®-derived MIC values can be used to categorise organisms as wild-type if the MIC is reported at the card's lowest calling range (≤) as there is high likelihood that the MIC is at or below the ECOFF. This has important implications for antimicrobial management, assisting in choice of agent and in improving probability of target attainment for desired pharmacodynamic targets which can translate into improved clinical outcomes. Contribution: Minimum inhibitory concentration data from an automated antimicrobial susceptibility testing instrument can be used to guide clinical decisions.

Antimicrobial resistance in Campylobacter fetus: emergence and genomic evolution.

Campylobacter fetus is a pathogen, which is primarily associated with fertility problems in sheep and cattle. In humans, it can cause severe infections that require antimicrobial treatment. However, knowledge on the development of antimicrobial resistance in C. fetus is limited. Moreover, the lack of epidemiological cut-off values (ECOFFs) and clinical breakpoints for C. fetus hinders consistent reporting about wild-type and non-wild-type susceptibility. The aim of this study was to determine the phenotypic susceptibility pattern of C. fetus and to determine the C. fetus resistome [the collection of all antimicrobial resistance genes (ARGs) and their precursors] to describe the genomic basis of antimicrobial resistance in C. fetus isolates over time. Whole-genome sequences of 295 C. fetus isolates, including isolates that were isolated in the period 1939 till the mid 1940s, before the usage of non-synthetic antimicrobials, were analysed for the presence of resistance markers, and phenotypic antimicrobial susceptibility was obtained for a selection of 47 isolates. C. fetus subspecies fetus (Cff) isolates showed multiple phenotypic antimicrobial resistances compared to C. fetus subspecies venerealis (Cfv) isolates that were only intrinsic resistant to nalidixic acid and trimethoprim. Cff isolates showed elevated minimal inhibitory concentrations for cefotaxime and cefquinome that were observed in isolates from 1943 onwards, and Cff isolates contained gyrA substitutions, which conferred resistance to ciprofloxacin. Resistances to aminoglycosides, tetracycline and phenicols were linked to acquired ARGs on mobile genetic elements. A plasmid-derived tet(O) gene in a bovine Cff isolate in 1999 was the first mobile genetic element observed, followed by detection of mobile elements containing tet(O)-aph(3')-III and tet(44)-ant(6)-Ib genes, and a plasmid from a single human isolate in 2003, carrying aph(3')-III-ant(6)-Ib and a chloramphenicol resistance gene (cat). The presence of ARGs in multiple mobile elements distributed among different Cff lineages highlights the risk for spread and further emergence of AMR in C. fetus. Surveillance for these resistances requires the establishment of ECOFFs for C. fetus.

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

OBJECTIVE: For 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. METHODS: We 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. RESULTS: The 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. CONCLUSION: As 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.

EUCAST Ibrexafungerp MICs and Wild-Type Upper Limits for Contemporary Danish Yeast Isolates.

Ibrexafungerp is a novel triterpenoid antifungal that inhibits glucan synthase and thus fungal cell wall synthesis. We examined the in vitro activity against contemporary clinical yeast, investigated inter-laboratory and intra-laboratory variability, suggested wild-type upper-limit values (WT-UL), and compared in vitro activity of ibrexafungerp to five licensed antifungals. Susceptibility to ibrexafungerp and comparators was investigated prospectively for 1965 isolates (11,790 MICs) and repetitively for three QC strains (1764 MICs) following the EUCAST E.Def 7.3.2 method. Elevated ibrexafungerp/echinocandin MICs prompted FKS sequencing. Published ibrexafungerp EUCAST MIC-distributions were retrieved and aggregated for WT-UL determinations following EUCAST principles. Ibrexafungerp MICs were ≤2 mg/L except against C. pararugosa, Cryptococcus and some rare yeasts. Modal MICs (mg/L) were 0.06/0.125/0.25/0.5/0.5/0.5/0.5/1/2 for C. albicans/C. dubliniensis/C. glabrata/C. krusei/C. parapsilosis/C. tropicalis/S. cerevisiae/C. guilliermondii/C. lusitaniae and aligned within ±1 dilution with published values. The MIC ranges for QC strains were: 0.06-0.25/0.5-1/0.125-0.5 for CNM-CL-F8555/ATCC6258/ATCC22019. The WT-UL (mg/L) were: 0.25/0.5/1/1/2 for C. albicans/C. glabrata/C. krusei/C. parapsilosis/C. tropicalis. Adopting these, non-wild-type rates were 0.3%/0.6%/0%/8%/3% for C. albicans/C. glabrata/C. krusei/C. parapsilosis/C. tropicalis and overall lower than for comparators except amphotericin B. Five/six non-wild-type C. albicans/C. glabrata were echinocandin and Fks non-wild-type (F641S, F659del or F659L). Eight C. parapsilosis and three C. tropicalis non-wild-type isolates were echinocandin and Fks wild-type. Partial inhibition near 50% in the supra-MIC range may explain variable MICs. Ibrexafungerp EUCAST MIC testing is robust, although the significance of paradoxical growth for some species requires further investigation. The spectrum is broad and will provide an oral option for the growing population with azole refractory infection.

The mysteries of target site concentrations of antibiotics in bone and joint infections: what is known? A narrative review.

INTRODUCTION: Currently, antibiotic treatment is often a standard dosing regimen in bone and joint infections (BJI). However, it remains unknown if exposure at the target-site is adequate. The aim of this review is to gain more insight in the relationship between the target site concentration of antibiotic and the minimal inhibitory concentration to target the bacteria in bone and joint infections (BJI). AREAS COVERED: A literature search was performed by Erasmus MC Medical library. Bone, bone tissue and synovial concentration of antibiotics were covered in humans. In addition, we reported number of patients, dose, sampling method, analytical method and tissue and plasma concentrations. We used the epidemiological cutoff value (ECOFF) values of the targeted micro-organisms. If more than 3 publications were available on the antibiotic, we graphically presented ECOFFS values against reported antibiotic concentrations. EXPERT OPINION: For most antibiotics, the literature is sparse. In addition, a lot of variable and total antibiotic concentrations are published. Ciprofloxacin, cefazolin, cefuroxime, vancomycin and linezolid seem to have adequate average exposure if correlating total concentration to ECOFF, when standard dosing is used. With regard to other antibiotics, results are inconclusive. More extensive pharmacokinetic/pharmacodynamic modeling in BJI is needed.

Antimicrobial resistance monitoring in commensal and clinical Escherichia coli from broiler chickens: Differences and similarities.

BACKGROUND: In the Netherlands, antimicrobial resistance (AMR) is monitored in commensal indicator Escherichia coli from healthy broilers at slaughter as part of a European monitoring programme. In a separate programme for poultry health, AMR is monitored in veterinary pathogens from diseased broilers. So far, it is unknown how the outcomes of these two AMR monitoring approaches in the same animal population are associated. AIMS: This study aims to investigate the association between the outcomes of monitoring non-wildtype susceptibility (using epidemiological cut-off values, ECOFF, as prescribed by EU legislation) in commensal E. coli isolated from healthy broilers (i.e. active surveillance) with the outcomes of monitoring clinical resistance (using clinical breakpoints, to determine susceptibility for antibiotic treatment in veterinary practice) in E. coli isolated from diseased broilers (i.e. passive surveillance). METHODS: Data acquired by broth microdilution was analysed for commensal indicator E. coli and clinical E. coli from the Netherlands, 2014-2019. A generalized linear multivariable model (Poisson regression) was used to determine time trends and identify differences in mean resistant proportions. RESULTS: Observed resistant proportions of the monitored commensal E. coli and clinical E. coli were similar with overlapping confidence intervals for most time points for ampicillin, gentamicin, cefotaxime, tetracycline, colistin and trimethoprim/sulfonamide. The statistical analysis showed that only for cefotaxime and tetracycline, mean resistant proportions were different. In commensal E. coli, a decrease of resistant proportions over time was observed, except for gentamicin. In clinical E. coli, no time trend was detected in resistant proportions, except for cefotaxime and colistin. CONCLUSIONS: Generally, the resistant proportions monitored in commensal and clinical E. coli were similar. However, some relevant differences were found, which can be explained by the type of monitoring approach, i.e. active or passive surveillance. The random sample of commensal E. coli isolated from healthy animals (active surveillance), was more suitable to monitor AMR time trends. The sample of clinical isolates from diseased animals (passive surveillance), resulted in a higher chance to detect low-prevalent resistance: i.e. cefotaxime and colistin. The clinical E. coli data showed more fluctuation over time, and data from a longer period of time would be needed to determine the association. This study shows the value of both an active and a passive surveillance component for AMR monitoring.

In Vitro Bedaquiline and Clofazimine Susceptibility Testing in Mycobacterium abscessus.

Bedaquiline and clofazimine are increasingly used to treat infections with Mycobacterium abscessus. We determined distributions of MICs by broth microdilution for bedaquiline and clofazimine for 61 M. abscessus clinical isolates using different media and incubation times. We show that incubation time and growth media critically influence the MIC. Our data will aid in defining future clinical breakpoints for in vitro susceptibility testing for bedaquiline and clofazimine in M. abscessus.

A Pragmatic Approach to Susceptibility Classification of Yeasts without EUCAST Clinical Breakpoints.

EUCAST has established clinical breakpoints for the six most common Candida species and Cryptococcus neoformans but not for less common yeasts because sufficient evidence is lacking. Consequently, the question "How to interpret the MIC?" for other yeasts often arises. We propose a pragmatic classification for amphotericin B, anidulafungin, fluconazole, and voriconazole MICs against 30 different rare yeasts. This classification takes advantage of MIC data for more than 4000 isolates generated in the EUCAST Development Laboratory for Fungi validated by alignment to published EUCAST MIC data. The classification relies on the following two important assumptions: first, that when isolates are genetically related, pathogenicity and intrinsic susceptibility patterns may be similar; and second, that even if species are not phylogenetically related, the rare yeasts will likely respond to therapy, provided the MIC is comparable to that against wild-type isolates of more prevalent susceptible species because rare yeasts are most likely "rare" due to a lower pathogenicity. In addition, the treatment recommendations available in the current guidelines based on the in vivo efficacy data and clinical experience are taken into consideration. Needless to say, it is of utmost importance (a) to ascertain that the species identification is correct (using MALDI-TOF or sequencing), and (b) to re-test the isolate once or twice to confirm that the MIC is representative for the isolate (because of the inherent variability in MIC determinations). We hope this pragmatic guidance is helpful until evidence-based EUCAST breakpoints can be formally established.

How to: ECOFFs-the why, the how, and the don'ts of EUCAST epidemiological cutoff values.

BACKGROUND: Identifying the MIC wild-type distribution and its delineation of species targeted for receiving antimicrobial agent breakpoints is an important first step for determining clinical breakpoints. Having the main responsibility in the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for characterizing the wild-type distributions and setting epidemiological cut-off values (ECOFFs), we explain the why, the how, and frequent misconceptions of wild-type MIC distributions and ECOFFs. OBJECTIVES: To clarify how wild-type MIC distributions and ECOFFs for agents and important target organisms are defined and determined and why these are important tools in microbiology, as well as to point to common misunderstandings and inappropriate use. SOURCES: The EUCAST database of >40 000 MIC distributions; publications addressing the definition of wild-type MIC distributions, and ECOFFs in bacteria and fungi; and the EUCAST Standard Operating Procedure 10 Documents published by the European Centre for Disease Control and the European Food Safety Agency. CONTENT: The rationale for defining wild-type distributions and ECOFFs is explained. Setting breakpoints that bisect wild-type MIC distributions leads to poor methodological reproducibility and poor correlation between clinical outcome and susceptibility testing results. The methods applied by EUCAST to select distributions for aggregation and website display are described, highlighting the importance of incorporating data from multiple sources and methods. The methods used by EUCAST to estimate ECOFFs are outlined. Finally, the common misunderstandings of these processes are addressed. IMPLICATIONS: The international community needs to agree on the phenotypic definitions of wild-type distributions. Systematic methods for developing and applying ECOFFs are essential to the conduct of phenotypic antimicrobial susceptibility testing and interpretation, which will remain the dominant laboratory method for the foreseeable future.

Determination of a Tentative Epidemiological Cut-Off Value (ECOFF) for Dalbavancin and Enterococcus faecium.

Dalbavancin is a lipoglycopeptide antibiotic that shows potent activity against Gram-positive bacteria. It circumvents vanB-type glycopeptide resistance mechanisms; however, data on the in vitro activity of dalbavancin for Enterococcus faecium (E. faecium) are scarce, and thus, no breakpoints are provided. In recent years, there has been a continuing shift from vanA-type to vanB-type vancomycin-resistance in enterococci in Central Europe. Therefore, we aimed to investigate the in vitro activity of dalbavancin against different van-genotypes, with particular focus on vanB-type E. faecium. Dalbavancin susceptibility was determined for 25 van-negative, 50 vanA-positive, and 101 vanB-positive clinical E. faecium isolates (typed by cgMLST). Epidemiological Cut-Off Values (ECOFFs) were determined using ECOFFinder. For vanB-type E. faecium isolates, dalbavancin MICs were similar to those of vancomycin-susceptible isolates reaching values no higher than 0.125 mg/L. ECOFFs for van-negative and vanB-positive isolates were 0.5 mg/l and 0.25 mg/L respectively. In contrast, E. faecium possessing vanA predominantly showed dalbavancin MICs >8 mg/L, therefore preventing the determination of an ECOFF. We demonstrated the potent in vitro activity of dalbavancin against vancomycin-susceptible and vanB-type E. faecium. On the basis of the observed wildtype distribution, a dalbavancin MIC of 0.25 mg/L can be suggested as a tentative ECOFF for E. faecium.

Employing MIC Data for Mink Pathogens to Propose Tentative Epidemiological Cut-Off Values: A Step Toward Rationalizing Antimicrobial Use in Mink.

Optimizing antimicrobial dosage regimens and development of breakpoints for antimicrobial susceptibility testing are important prerequisites for rational antimicrobial use. The objectives of the study were (1) to produce MIC data for four mink pathogens and (2) to employ these MIC data to support the development of tentative epidemiological cut-off values (TECOFFs), which may be used for future development of mink-specific antimicrobial dosages and breakpoints. Broth microdilution was used to establish MIC distributions for 322 mink bacterial isolates of clinical origin from six European mink-producing countries. The included species were E. coli (n = 162), S. delphini (n = 63), S. canis (n = 42), and P. aeruginosa (n = 55). Sixty-four E. coli isolates and 34 S. delphini isolates were whole-genome sequenced and analyzed for antimicrobial resistance genes. No EUCAST MIC data are available on S. delphini and S. canis, hence tentative ECOFFs were suggested for the majority of the tested antimicrobials. For E. coli and P. aeruginosa, the wildtype distributions were in accordance with EUCAST data. Overall, the genotypes of the sequenced isolates were in concordance with the phenotypes. These data constitute an important piece in the puzzle of developing antimicrobial dosages and clinical breakpoints for mink. Until pharmacokinetic and clinical data become available, the (tentative) ECOFFs can be used for monitoring resistance development and as surrogates for clinical breakpoints.

Burkholderia pseudomallei multi-centre study to establish EUCAST MIC and zone diameter distributions and epidemiological cut-off values.

OBJECTIVES: Melioidosis, caused by Burkholderia pseudomallei, requires intensive antimicrobial treatment. However, standardized antimicrobial susceptibility testing (AST) methodology based on modern principles for determining breakpoints and ascertaining performance of methods are lacking for B. pseudomallei. This study aimed to establish MIC and zone diameter distributions on which to set epidemiological cut-off (ECOFF) values for B. pseudomallei using standard EUCAST methodology for non-fastidious organisms. METHODS: Non-consecutive, non-duplicate clinical B. pseudomallei isolates (9-70 per centre) were tested at eight study centres against eight antimicrobials by broth microdilution (BMD) and the EUCAST disc diffusion method. Isolates without and with suspected resistance mechanisms were deliberately selected. The EUCAST Development Laboratory ensured the quality of study materials, and provided guidance on performance of the tests and interpretation of results. Aggregated results were analysed according to EUCAST recommendations to determine ECOFFs. RESULTS: MIC and zone diameter distributions were generated using BMD and disc diffusion results obtained for 361 B. pseudomallei isolates. MIC and zone diameter ECOFFs (mg/L; mm) were determined for amoxicillin-clavulanic acid (8; 22), ceftazidime (8; 22), imipenem (2; 29), meropenem (2; 26), doxycycline (2; none), tetracycline (8; 23), chloramphenicol (8; 22) and trimethoprim-sulfamethoxazole (4; 28). CONCLUSIONS: We have validated the use of standard BMD and disc diffusion methodology for AST of B. pseudomallei. The MIC and zone diameter distributions generated in this study allowed us to establish MIC and zone diameter ECOFFs for the antimicrobials studied. These ECOFFs served as background data for EUCAST to set clinical MIC and zone diameter breakpoints for B. pseudomallei.

Where have all the susceptible gonococci gone? A historical review of changes in MIC distribution over the past 75 years.

BACKGROUND: Does the emergence of antimicrobial resistance in Neisseria gonorrhoeae include the erasure of highly susceptible strains or does it merely involve a stretching of the MIC distribution? If it was the former this would be important to know as it would increase the probability that the loss of susceptibility is irreversible. METHODS: We conducted a historical analysis based on a literature review of changes of N. gonorrhoeae MIC distribution over the past 75 years for 3 antimicrobials (benzylpenicillin, ceftriaxone and azithromycin) in five countries (Denmark, Japan, South Africa, the United Kingdom and the United States). RESULTS: Changes in MIC distribution were most marked for benzylpenicillin and showed evidence of a right shifting of MIC distribution that was associated with a reduction/elimination of susceptible strains in all countries. In the case of ceftriaxone and azithromycin, where only more recent data was available, right shifting was also found in all countries but the extent of right shifting varied and the evidence for the elimination of susceptible strains was more mixed. CONCLUSIONS: The finding of right shifting of MIC distribution combined with reduction/elimination of susceptible strains is of concern since it suggests that this shifting may not be reversible. Since excess antimicrobial consumption is likely to be responsible for this right shifting, this insight provides additional impetus to promote antimicrobial stewardship.

Single-strain regression analysis evaluating disc potencies of flumequine and enrofloxacin for testing Aeromonas sobria and Vibrio anguillarum.

Two quite different disc contents are used for antimicrobial susceptibility testing of two fluoroquinolone drugs, flumequine and enrofloxacin, in the disc diffusion test, 30 and 5 µg, respectively. Using the SRA method, single-strain regression analysis, we studied the impact of disc content when testing two relevant bacterial species, Aeromonas sobria and Vibrio anguillarum. There were no major differences between the antimicrobial regression lines for the two species. Wild-type strains produced acceptable zones of inhibition over a wide range of disc contents. The flumequine 30 µg disc should be lowered in its drug content. No rational reasons for choosing so different disc contents for the two antimicrobials were apparent. At present, the choice of disc content for new antimicrobials are outside the realm of clinical microbiologists. It is recommended that reference authorities, such as EUCAST, CLSI and USCAST, are consulted for the choice of disc contents in the future.

Proposed Epidemiological Cutoff Values for Ceftriaxone, Cefepime, and Colistin in Salmonella.

We tested a diverse set of 500 isolates of nontyphoidal Salmonella enterica subsp. enterica from various animal, food, and human clinical sources for susceptibility to antimicrobials currently lacking epidemiological cutoff values (ECOFFs) set by the European Committee on Antimicrobial Susceptibility Testing. A consortium of five different laboratories each tested 100 isolates, using broth microdilution panels containing twofold dilutions of ceftriaxone, cefepime, and colistin to determine the minimum inhibitory concentrations of each drug when tested against the Salmonella isolates. Based on the resulting data, new ECOFFs of 0.25 µg/mL for ceftriaxone, 0.12 µg/mL for cefepime, and 2 µg/mL for colistin have been proposed. These thresholds will aid in the identification of Salmonella that have phenotypically detectable resistance mechanisms to these important antimicrobials.