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 at the 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.

Estimation of epidemiological cut-off values for eight antibiotics used for treatment of bovine mastitis caused by Streptococcus uberis and Streptococcus dysgalactiae subsp. dysgalactiae.

Interpretive criteria for antimicrobial susceptibility testing are lacking for most antimicrobials used for bovine streptococcal mastitis. The objectives of this study were to determine (tentative) epidemiological cut-off ((T)ECOFF) values for clinically relevant antibiotics used for treatment of bovine mastitis, and to estimate the proportion of acquired resistance (non-wild-types) in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus uberis. A total of 255 S. uberis and 231 S. dysgalactiae subsp. dysgalactiae isolates were obtained in Denmark and Norway from bovine mastitis. The isolates were tested for susceptibility to 10 antibiotics using broth microdilution. In accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard operating procedure, additional published MIC distributions were included for the estimation of ECOFFs for cloxacillin, cephapirin, lincomycin and tylosin, and TECOFFs for amoxicillin, benzylpenicillin, cephapirin and oxytetracycline. The proportion of non-wild-type (NWT) isolates for the beta-lactams was significantly higher in the Danish S. uberis (45-55%) compared to the Norwegian isolates (10-13%). For oxytetracycline, the proportion of NWT was significantly higher in the Danish isolates, both for S. uberis (28% vs. 3%) and S. dysgalactiae (22% vs. 0%). A bridging study testing in parallel MICs in a subset of isolates (n = 83) with the CLSI-specified and the EUCAST-specified broths showed excellent correlation between the MICs obtained with the two methods. The new ECOFFs and TECOFFs proposed in this study can be used for surveillance of antimicrobial resistance, and - for antimicrobials licensed for streptococcal bovine mastitis - as surrogate clinical breakpoints for predicting their clinical efficacy for this indication.

Genetic background of neomycin resistance in clinical Escherichia coli isolated from Danish pig farms.

Neomycin is the first-choice antibiotic for the treatment of porcine enteritis caused by enterotoxigenic Escherichia coli. Resistance to this aminoglycoside is on the rise after the increased use of neomycin due to the ban on zinc oxide. We identified the neomycin resistance determinants and plasmid contents in a historical collection of 128 neomycin-resistant clinical E. coli isolates from Danish pig farms. All isolates were characterized by whole-genome sequencing and antimicrobial susceptibility testing, followed by conjugation experiments and long-read sequencing of eight selected representative strains. We detected 35 sequence types (STs) with ST100 being the most prevalent lineage (38.3%). Neomycin resistance was associated with two resistance genes, namely aph(3')-Ia and aph(3')-Ib, which were identified in 93% and 7% of the isolates, respectively. The aph(3')-Ia was found on different large conjugative plasmids belonging to IncI1α, which was present in 67.2% of the strains, on IncHI1, IncHI2, and IncN, as well as on a multicopy ColRNAI plasmid. All these plasmids except ColRNAI carried genes encoding resistance to other antimicrobials or heavy metals, highlighting the risk of co-selection. The aph(3')-Ib gene occurred on a 19 kb chimeric, mobilizable plasmid that contained elements tracing back its origin to distantly related genera. While aph(3')-Ia was flanked by either Tn903 or Tn4352 derivatives, no clear association was observed between aph(3')-Ib and mobile genetic elements. In conclusion, the spread of neomycin resistance in porcine clinical E. coli is driven by two resistance determinants located on distinct plasmid scaffolds circulating within a highly diverse population dominated by ST100. IMPORTANCE Neomycin is the first-choice antibiotic for the management of Escherichia coli enteritis in pigs. This work shows that aph(3')-Ia and to a lesser extent aph(3')-Ib are responsible for the spread of neomycin resistance that has been recently observed among pig clinical isolates and elucidates the mechanisms of dissemination of these two resistance determinants. The aph(3')-Ia gene is located on different conjugative plasmid scaffolds and is associated with two distinct transposable elements (Tn903 and Tn4352) that contributed to its spread. The diffusion of aph(3')-Ib is mediated by a small non-conjugative, mobilizable chimeric plasmid that likely derived from distantly related members of the Pseudomonadota phylum and was not associated with any detectable mobile genetic element. Although the spread of neomycin resistance is largely attributable to horizontal transfer, both resistance determinants have been acquired by a predominant lineage (ST100) associated with enterotoxigenic E. coli, which accounted for approximately one-third of the strains.

Dogs Can Be Reservoirs of Escherichia coli Strains Causing Urinary Tract Infection in Human Household Contacts.

This study aimed to investigate the role played by pets as reservoirs of Escherichia coli strains causing human urinary tract infections (UTIs) in household contacts. Among 119 patients with community-acquired E. coli UTIs, we recruited 19 patients who lived with a dog or a cat. Fecal swabs from the household pet(s) were screened by antimicrobial selective culture to detect E. coli displaying the resistance profile of the human strain causing UTI. Two dogs shed E. coli isolates indistinguishable from the UTI strain by pulsed-field gel electrophoresis. Ten months later, new feces from these dogs and their owners were screened selectively and quantitatively for the presence of the UTI strain, followed by core-genome phylogenetic analysis of all isolates. In one pair, the resistance phenotype of the UTI strain occurred more frequently in human (108 CFU/g) than in canine feces (104 CFU/g), and human fecal isolates were more similar (2-7 SNPs) to the UTI strain than canine isolates (83-86 SNPs). In the other pair, isolates genetically related to the UTI strain (23-40 SNPs) were only detected in canine feces (105 CFU/g). These results show that dogs can be long-term carriers of E. coli strains causing UTIs in human household contacts.

Pilot testing the EARS-Vet surveillance network for antibiotic resistance in bacterial pathogens from animals in the EU/EEA.

Introduction: As part of the EU Joint Action on Antimicrobial Resistance (AMR) and Healthcare-Associated Infections, an initiative has been launched to build the European AMR Surveillance network in veterinary medicine (EARS-Vet). So far, activities included mapping national systems for AMR surveillance in animal bacterial pathogens, and defining the EARS-Vet objectives, scope, and standards. Drawing on these milestones, this study aimed to pilot test EARS-Vet surveillance, namely to (i) assess available data, (ii) perform cross-country analyses, and (iii) identify potential challenges and develop recommendations to improve future data collection and analysis. Methods: Eleven partners from nine EU/EEA countries participated and shared available data for the period 2016-2020, representing a total of 140,110 bacterial isolates and 1,302,389 entries (isolate-antibiotic agent combinations). Results: Collected data were highly diverse and fragmented. Using a standardized approach and interpretation with epidemiological cut-offs, we were able to jointly analyze AMR trends of 53 combinations of animal host-bacteria-antibiotic categories of interest to EARS-Vet. This work demonstrated substantial variations of resistance levels, both among and within countries (e.g., between animal host species). Discussion: Key issues at this stage include the lack of harmonization of antimicrobial susceptibility testing methods used in European surveillance systems and veterinary diagnostic laboratories, the absence of interpretation criteria for many bacteria-antibiotic combinations of interest, and the lack of data from a lot of EU/EEA countries where little or even surveillance currently exists. Still, this pilot study provides a proof-of-concept of what EARS-Vet can achieve. Results form an important basis to shape future systematic data collection and analysis.

In vitro and in vivo susceptibility to cefalexin and amoxicillin/clavulanate in canine low-level methicillin-resistant Staphylococcus pseudintermedius.

BACKGROUND: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) lineages harbouring staphylococcal cassette chromosome (SCC) mec types IV, V and ΨSCCmec57395 usually display low oxacillin MICs (0.5-2 mg/L). OBJECTIVES: To evaluate how oxacillin MICs correlate with PBP mutations and susceptibility to ß-lactams approved for veterinary use. METHODS: Associations between MICs and PBP mutations were investigated by broth microdilution, time-kill and genome sequence analyses in 117 canine MRSP strains harbouring these SCCmec types. Clinical outcome was retrospectively evaluated in 11 MRSP-infected dogs treated with ß-lactams. RESULTS: Low-level MRSP was defined by an oxacillin MIC <4 mg/L. Regardless of strain genotype, all low-level MRSP isolates (n = 89) were cefalexin susceptible, whereas no strains were amoxicillin/clavulanate susceptible according to clinical breakpoints. Exposure to 2× MIC of cefalexin resulted in complete killing within 8 h. High (≥4 mg/L) oxacillin MICs were associated with substitutions in native PBP2, PBP3, PBP4 and acquired PBP2a, one of which (V390M in PBP3) was statistically significant by multivariable modelling. Eight of 11 dogs responded to systemic therapy with first-generation cephalosporins (n = 4) or amoxicillin/clavulanate (n = 4) alone or with concurrent topical treatment, including 6 of 7 dogs infected with low-level MRSP. CONCLUSIONS: Oxacillin MIC variability in MRSP is influenced by mutations in multiple PBPs and correlates with cefalexin susceptibility. The expert rule recommending that strains with oxacillin MIC ≥0.5 mg/L are reported as resistant to all ß-lactams should be reassessed based on these results, which are highly clinically relevant in light of the shortage of effective antimicrobials for systemic treatment of MRSP infections in veterinary medicine.

Usage of Antimicrobials in Equine Veterinary Practice in Denmark - A Case-Based Survey.

Horses may be carriers of important resistant bacteria like methicillin-resistant staphylococci. Such bacteria can potentially threaten both equine and public health, but little is known about predisposing factors like antimicrobial usage patterns in equines. Objectives of this study were to investigate the antimicrobial usage practices by Danish equine practitioners as well as factors impacting usage. A total of 103 equine practitioners filled in an online questionnaire. When asked to explain their typical treatment of six clinical case scenarios, only 1% and 7% of respondents prescribed systemic antimicrobials for a cough and pastern dermatitis, respectively. More frequent usage was reported for diarrhoea (43%), extraction of a cracked tooth (44%), strangles (56%), and superficial wound near a joint (72%). Among the antibiotics indicated for treatment, enrofloxacin was the only critically important antimicrobial agent reported by two respondents. Thirty-eight (36%) respondents worked in practices with antimicrobial protocols. When asked to prioritize the most important factor influencing prescribing habits, bacterial culture (47%) and antimicrobial protocols (45%) were selected far more often than the owner´s economy (5%) and expectations (4%). Veterinarians reported limitations such as the availability of only one oral antibiotic (sulphadiazine/trimethoprim), and a need for clearer treatment guidelines. In conclusion, the study highlighted important aspects regarding antimicrobial usage among equine practitioners. Antimicrobial protocols and pre- and post-graduate education on prudent antimicrobial usage are recommended.

Neomycin resistance in clinical Escherichia coli from Danish weaner pigs is associated with recent neomycin use and presence of F4 or F18 fimbriaes.

Neomycin is a first-choice antibiotic for treatment of porcine enteritis caused by enterotoxigenic Escherichia coli (ETEC), but little is known about factors influencing resistance to this drug. The aims of this study were to assess antimicrobial resistance and virulence in 325 E. coli isolates obtained in 2020 from various infections in pigs, and to identify factors associated with neomycin resistance development. Susceptibility to 16 antimicrobial agents was determined by broth microdilution, and occurrence of ETEC-associated virulence factors was screened by PCR and hemolysis on blood agar. Univariate and multivariate logistic regression analyses were performed to determine if age group, virulence factors, or antibiotic use (neomycin and other antibiotics) were associated with neomycin resistance. STa, STb, LT, F4, and F18 were detected in 14%, 37%, 26%, 21% and 23% of the isolates, respectively. Resistance was low for antimicrobials of high public health importance (1.5% for cefotaxime, 1% for colistin and no fluoroquinolone resistance) but high for drugs used for treatment of ETEC enteritis (e.g. 20% for neomycin). Isolates with the ETEC pathotype were significantly associated with the weaner age group and intestinal/fecal origin. Multivariate analysis showed that recent neomycin use and presence of F4 or F18 were significantly associated with neomycin resistance amongst isolates from weaners. These results prove an association between neomycin resistance and use at the farm level. Further research is warranted to determine why neomycin resistance was associated with F4 and F18, and whether neomycin use may co-select for virulent strains.

Quality of MALDI-TOF mass spectra in routine diagnostics: results from an international external quality assessment including 36 laboratories from 12 countries using 47 challenging bacterial strains.

OBJECTIVES: Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it. METHODS: For baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared. RESULTS: At baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2-25]), reproducibility between technical replicates (range = [55%-86%]), and measurement error (range = [147 parts per million (ppm)-588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values <0.001, paired Wilcoxon test); for six out of ten devices, which detected a low number of marker masses, the number of detected marker masses increased (p-values <0.001, paired Wilcoxon test). DISCUSSION: We have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.

Horsing around: Escherichia coli ST1250 of equine origin harbouring epidemic IncHI1/ST9 plasmid with bla CTX-M-1 and an operon for short-chain fructooligosaccharides metabolism.

The relatedness of the equine-associated Escherichia coli ST1250 and its single- and double-locus variants (ST1250-SLV/DLV), obtained from horses in Europe, was studied by comparative genome analysis. A total of 54 isolates of E. coli ST1250 and ST1250-SLV/DLV from healthy and hospitalized horses across Europe [Czech Republic (n=23), the Netherlands (n=18), Germany (n=9), Denmark (n=3) and France (n=1)] from 2008-2017 were subjected to whole-genome sequencing. An additional 25 draft genome assemblies of E. coli ST1250 and ST1250-SLV/DLV were obtained from the public databases. The isolates were compared for genomic features, virulence genes, clade structure and plasmid content. The complete nucleotide sequences of eight IncHI1/ST9 and one IncHI1/ST2 plasmids were obtained using long-read sequencing by PacBio or MinION. In the collection of 79 isolates, only 10 were phylogenetically close (<8 SNP). The majority of isolates belonged to phylogroup B1 (73/79, 92.4%) and carried bla CTX-M-1 (58/79, 73.4%). The plasmid content of the isolates was dominated by IncHI1 of ST9 (56/62, 90.3%) and ST2 (6/62, 9.7%), while 84.5% (49/58) bla CTX-M-1 genes were associated with presence of IncHI1 replicon of ST9 and 6.9% (4/58) with IncHI1 replicon of ST2 within the corresponding isolates. The operon for the utilization of short chain fructooligosaccharides (fos operon) was present in 55 (55/79, 69.6%) isolates, and all of these carried IncHI1/ST9 plasmids. The eight complete IncHI1/ST9 plasmid sequences showed the presence of bla CTX-M-1 and the fos operon within the same molecule. Sequences of IncHI1/ST9 plasmids were highly conserved (>98% similarity) regardless of country of origin and varied only in the structure and integration site of MDR region. E. coli ST1250 and ST1250-SLV/DLV are phylogenetically-diverse strains associated with horses. A strong linkage of E. coli ST1250 with epidemic multi-drug resistance plasmid lineage IncHI1/ST9 carrying bla CTX-M-1 and the fos operon was identified.

Macrolide Resistance and In Vitro Potentiation by Peptidomimetics in Porcine Clinical Escherichia coli.

Escherichia coli is intrinsically resistant to macrolides due to outer membrane impermeability, but may also acquire macrolide resistance genes by horizontal transfer. We evaluated the prevalence and types of acquired macrolide resistance determinants in pig clinical E. coli, and we assessed the ability of peptidomimetics to potentiate different macrolide subclasses against strains resistant to neomycin, a first-line antibiotic in the treatment of pig-enteric infections. The erythromycin MIC distribution was determined in 324 pig clinical E. coli isolates, and 62 neomycin-resistant isolates were further characterized by genome sequencing and MIC testing of azithromycin, spiramycin, tilmicosin, and tylosin. The impact on potency achieved by combining these macrolides with three selected peptidomimetic compounds was determined by checkerboard assays in six strains representing different genetic lineages and macrolide resistance gene profiles. Erythromycin MICs ranged from 16 to >1,024 µg/mL. Azithromycin showed the highest potency in wild-type strains (1 to 8 µg/mL), followed by erythromycin (16 to 128 µg/mL), tilmicosin (32 to 256 µg/mL), and spiramycin (128 to 256 µg/mL). Isolates with elevated MIC mainly carried erm(B), either alone or in combination with other acquired macrolide resistance genes, including erm(42), mef(C), mph(A), mph(B), and mph(G). All peptidomimetic-macrolide combinations exhibited synergy (fractional inhibitory concentration index [FICI] < 0.5) with a 4- to 32-fold decrease in the MICs of macrolides. Interestingly, the MICs of tilmicosin in wild-type strains were reduced to concentrations (4 to 16 µg/mL) that can be achieved in the pig intestinal tract after oral administration, indicating that peptidomimetics can potentially be employed for repurposing tilmicosin in the management of E. coli enteritis in pigs. IMPORTANCE Acquired macrolide resistance is poorly studied in Escherichia coli because of intrinsic resistance and limited antimicrobial activity in Gram-negative bacteria. This study reveals new information on the prevalence and distribution of macrolide resistance determinants in a comprehensive collection of porcine clinical E. coli from Denmark. Our results contribute to understanding the correlation between genotypic and phenotypic macrolide resistance in E. coli. From a clinical standpoint, our study provides an initial proof of concept that peptidomimetics can resensitize E. coli to macrolide concentrations that may be achieved in the pig intestinal tract after oral administration. The latter result has implications for animal health and potential applications in veterinary antimicrobial drug development in view of the high rates of antimicrobial-resistant E. coli isolated from enteric infections in pigs and the lack of viable alternatives for treating these infections.

Review and Analysis of National Monitoring Systems for Antimicrobial Resistance in Animal Bacterial Pathogens in Europe: A Basis for the Development of the European Antimicrobial Resistance Surveillance Network in Veterinary Medicine (EARS-Vet).

The monitoring of antimicrobial resistance (AMR) in bacterial pathogens of animals is not currently coordinated at European level. To fill this gap, experts of the European Union Joint Action on Antimicrobial Resistance and Healthcare Associated Infections (EU-JAMRAI) recommended building the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet). In this study, we (i) identified national monitoring systems for AMR in bacterial pathogens of animals (both companion and food-producing) among 27 countries affiliated to EU-JAMRAI, (ii) described their structures and operations, and (iii) analyzed their respective strengths, weaknesses, opportunities and threats (SWOT). Twelve countries reported having at least one national monitoring system in place, representing an opportunity to launch EARS-Vet, but highlighting important gaps in AMR data generation in Europe. In total, 15 national monitoring systems from 11 countries were described and analyzed. They displayed diverse structures and operations, but most of them shared common weaknesses (e.g., data management and representativeness) and common threats (e.g., economic vulnerability and data access), which could be addressed collectively under EARS-Vet. This work generated useful information to countries planning to build or improve their system, by learning from others' experience. It also enabled to advance on a pragmatic harmonization strategy: EARS-Vet shall follow the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards, collect quantitative data and interpret AMR data using epidemiological cut-off values.

Defining the scope of the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet): a bottom-up and One Health approach.

BACKGROUND: Building the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet) was proposed to strengthen the European One Health antimicrobial resistance (AMR) surveillance approach. OBJECTIVES: To define the combinations of animal species/production types/age categories/bacterial species/specimens/antimicrobials to be monitored in EARS-Vet. METHODS: The EARS-Vet scope was defined by consensus between 26 European experts. Decisions were guided by a survey of the combinations that are relevant and feasible to monitor in diseased animals in 13 European countries (bottom-up approach). Experts also considered the One Health approach and the need for EARS-Vet to complement existing European AMR monitoring systems coordinated by the ECDC and the European Food Safety Authority (EFSA). RESULTS: EARS-Vet plans to monitor AMR in six animal species [cattle, swine, chickens (broilers and laying hens), turkeys, cats and dogs], for 11 bacterial species (Escherichia coli, Klebsiella pneumoniae, Mannheimia haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae, Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus hyicus, Streptococcus uberis, Streptococcus dysgalactiae and Streptococcus suis). Relevant antimicrobials for their treatment were selected (e.g. tetracyclines) and complemented with antimicrobials of more specific public health interest (e.g. carbapenems). Molecular data detecting the presence of ESBLs, AmpC cephalosporinases and methicillin resistance shall be collected too. CONCLUSIONS: A preliminary EARS-Vet scope was defined, with the potential to fill important AMR monitoring gaps in the animal sector in Europe. It should be reviewed and expanded as the epidemiology of AMR changes, more countries participate and national monitoring capacities improve.

A Diagnostic Survey of Aborted Equine Fetuses and Stillborn Premature Foals in Denmark.

Background: Loss of pregnancy in mares can have many different causes, including both infectious and non-infectious conditions. Extrapolation of findings from other studies is often uncertain as the significance of each cause varies across regions. Causes of pregnancy loss in mares have never been thoroughly studied in Denmark, so a prospective cross-sectional cohort study targeting the entire Danish population of pregnant mares was performed over a period of 13 months to obtain knowledge of the significance of individual causes. Fifty aborted or prematurely delivered stillborn fetuses were submitted for necropsy and examined by a panel of diagnostic laboratory methods. Results: Overall, a cause of fetal loss was established for 72% of the examined cases. Most cases (62%) were lost due to a non-infectious cause, of which obstruction of the feto-placental blood circulation due to severe torsion of the umbilical cord was most prevalent. Pregnancy loss due to a variety of opportunistic bacteria, including bacteria not previously associated with abortion in mares, accounted for 12%, while equid alphaherpesvirus (EHV) type 1 was the cause of pregnancy loss in 8% of the cases. EHV type 4 and Chlamydiaceae species were identified in some cases, but not regarded as the cause of fetal loss. Conclusion: Umbilical cord torsion was found to be the most prevalent cause of fetal loss in Danish mares, while infectious causes such as EHV type 1 and streptococci only accounted for a minor proportion of the losses. The study highlights the need for defined criteria for establishing an abortion diagnosis in mares, particularly in relation to EHV types 1 and 4.

Overview and Evaluation of Existing Guidelines for Rational Antimicrobial Use in Small-Animal Veterinary Practice in Europe.

Antimicrobial stewardship guidelines (ASGs) represent an important tool to help veterinarians optimize their antimicrobial use with the objective of decreasing antimicrobial resistance. The aim of this study was to map and qualitatively assess the ASGs for antimicrobial use in cats and dogs in Europe. Country representatives of the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT) were asked to identify ASGs published in their countries. All collated ASGs updated since January 2010 containing recommendations on antimicrobial therapy for at least three conditions affecting different organ systems in cats and dogs underwent detailed review including AGREE II analysis. Out of forty countries investigated, fifteen ASGs from eleven countries met the inclusion criteria. Several critical principles of antimicrobial use were identified, providing a framework that should assist development of stewardship guidance. The AGREE II analysis highlighted several methodological limitations of the currently available ASGs. This study sheds light on the lack of national ASGs for dogs and cats in multiple European countries and should encourage national bodies to prioritize guideline development in small animals. A greater awareness of the need to use a structured approach to guideline development could improve the quality of ASGs in the future.

Evaluating a tylosin dosage regimen for treatment of Staphylococcus delphini infection in mink (Neovison vison): a pharmacokinetic-pharmacodynamic approach.

Staphylococcus delphini is one of the most common pathogens isolated from mink infections, especially dermatitis. Tylosin (TYL) is used frequently against these infections, although no evidence-based treatment regimen exists. This study aimed to explore the dosage of TYL for infections caused by S. delphini in mink. Two animal experiments with a total of 12 minks were conducted to study the serum pharmacokinetic (PK) characteristics of TYL in mink after 10 mg/kg IV and oral dosing, respectively. The concentration of TYL in serum samples collected before and eight times during 24 h after TYL administration was quantitated with liquid chromatography quadrupole time-of-flight mass spectrometry, and the TYL disposition was analyzed using non-linear mixed effect analysis. The pharmacodynamics (PD) of TYL against S. delphini were studied using semi-mechanistic modeling of in vitro time-kill experiments. PKPD modeling and simulation were done to establish the PKPD index and dosage regimen. The disposition of TYL was described by a two-compartmental model. The area under the free concentration-time curve of TYL over the minimum inhibitory concentration of S. delphini (fAUC/MIC) was determined as PKPD index with breakpoints of 48.9 and 98.7 h for bacteriostatic and bactericidal effect, respectively. The calculated daily oral dose of TYL was 2378 mg/kg, which is 238-fold higher than the currently used TYL oral dosage regimen in mink (10 mg/kg). Accordingly, sufficient TYL concentrations are impossible to achieve in mink plasma, and use of this drug for extra-intestinal infections in this animal species must be discouraged.

Dogs are carriers of Clostridioides difficile lineages associated with human community-acquired infections.

There is an increasing concern about the role of animals as reservoirs of Clostridioides difficile. In this study, we investigated prevalence, antimicrobial resistance and zoonotic potential of C. difficile in dogs. Two-hundred and twenty-five dog faecal deposits were collected from trashcans in nine public gardens. C. difficile was isolated using selective plating and enrichment culture, identified by MALDI-TOF, tested for susceptibility to seven antibiotics by E-test, and sequenced on an Illumina NextSeq platform. Genome sequences were analysed to determine multilocus sequence types and resistance and toxin gene profiles. Zoonotic potential was assessed by measuring genetic variations of core genome (cg)MLST types between canine isolates and 216 temporally and spatially related human clinical isolates from a national database. C. difficile was isolated from 11 samples (4.9%). Seven isolates were toxigenic (tcdA+, tcdB+, cdtA/B-) and belonged to the sequence types ST2, ST6, ST10 and ST42. The four non-toxigenic isolates were assigned to ST15, ST26 and one novel ST. ST2, corresponding to PCR ribotype RT014/020, was the dominating lineage (n = 4) and, together with ST26 and ST42 isolates, showed close resemblance to human isolates, i.e. 2-5 allelic differences among the 1999 genes analysed by cgMLST. Three non-toxigenic isolates displayed resistance to clindamycin, erythromycin and tetracycline mediated by erm(B) and tet(M). Resistance to metronidazole, moxifloxacine, rifampicin or vancomycin was not detected. In conclusion, a small proportion of faecal deposits contained toxigenic C. difficile such as ST2 (RT014/020), which is a major cause of community-acquired infections. Our finding suggests that pathogenic strains can be exchanged between dogs and humans.

Building the European Antimicrobial Resistance Surveillance network in veterinary medicine (EARS-Vet).

Antimicrobial resistance (AMR) should be tackled through a One Health approach, as stated in the World Health Organization Global Action Plan on AMR. We describe the landscape of AMR surveillance in the European Union/European Economic Area (EU/EEA) and underline a gap regarding veterinary medicine. Current AMR surveillance efforts are of limited help to veterinary practitioners and policymakers seeking to improve antimicrobial stewardship in animal health. We propose to establish the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet) to report on the AMR situation, follow AMR trends and detect emerging AMR in selected bacterial pathogens of animals. This information could be useful to advise policymakers, explore efficacy of interventions, support antimicrobial stewardship initiatives, (re-)evaluate marketing authorisations of antimicrobials, generate epidemiological cut-off values, assess risk of zoonotic AMR transmission and evaluate the burden of AMR in animal health. EARS-Vet could be integrated with other AMR monitoring systems in the animal and medical sectors to ensure a One Health approach. Herein, we present a strategy to establish EARS-Vet as a network of national surveillance systems and highlight challenges of data harmonisation and bias. Strong political commitment at national and EU/EEA levels is required for the success of EARS-Vet.

Validating an empiric sulfadiazine-trimethoprim dosage regimen for treatment of Escherichia coli and Staphylococcus delphini infections in mink (Neovison vison).

Antimicrobial agents are used extensively off-label in mink, as almost no agents are registered for this animal species. Pharmacokinetic (PK) and pharmacodynamic (PD) data are required to determine antimicrobial dosages specifically targeting mink bacterial pathogens. The aims of this study were to assess, in a PKPD framework, the empirical dosage regimen for a combination of trimethoprim (TMP) and sulfadiazine (SDZ) in mink, and secondarily to produce data for future setting of clinical breakpoints. TMP and SDZ PK parameters were obtained experimentally in 22 minks following IV or oral administration of TMP/SDZ (30 mg/kg, i.e. 5 mg/kg TMP and 25 mg/kg SDZ). fAUC/MIC with a target value of 24 hr was selected as the PKPD index predictive of TMP/SDZ efficacy. Using a modeling approach, PKPD cutoffs for TMP and SDZ were determined as 0.062 and 16 mg/L, respectively. By incorporating an anticipated potentiation effect of SDZ on TMP against Escherichia coli and Staphylococcus delphini, the PKPD cutoff of TMP was revised to 0.312 mg/L, which is above the tentative epidemiological cutoffs (TECOFF) for these species. The current empirical TMP/SDZ dosage regimen (30 mg/kg, PO, once daily) therefore appears adequate for treatment of wild-type E. coli and S. delphini infections in mink.

Corrigendum to 'quantitative determination of 64Cu-liposome accumulation at inflammatory and infectious sites: Potential for future theranostic system': [journal of controlled release 327 (2020) 737-746].

BACKGROUND: Therapeutic interventions for infectious and inflammatory diseases are becoming increasingly challenging in terms of therapeutic resistance and side-effects. Theranostic systems to ameliorate diagnosis and therapy are therefore highly warranted. The pathophysiological changes in inflammatory lesions provide an attractive basis for extravasation and accumulation of PEGylated liposomes. The objective of this study was to provide direct quantitative information on the theranostic potential of radiolabeled liposome for accumulation in inflammatory models using position emission tomography (PET). METHOD: Preclinical murine models of inflammation (turpentine and LPS), infection (Staphylococcus aureus) and collagen-induced arthritis (CIA) was established and monitored using bioluminescence imaging (BLI). Across all models PET imaging using radiolabeled PEGylated liposomes (64Cu-liposomes) were performed and evaluated in terms of accumulation properties in inflammatory and infectious lesions. RESULTS: BLI demonstrated that the inflammatory and infectious models were successfully established and provided information on lesion pathology. Activity of 64Cu-liposomes were increased in inflammatory and infectious lesions between early (10-min or 3-h) and late (24-h) PET scans, which validates that a continuous extravasation and accumulation of long circulation PEGylated liposomes occurs. CONCLUSION: The theranostic potential of long circulating PEGylated radiolabeled liposomes was shown in multiple preclinical models. Impressive accumulation was seen in both inflammatory and infectious lesions. These results are encouraging towards advancing PEGylated liposomes as imaging and drug delivery systems in inflammatory and infectious diseases.