Search

IntroductionVeterinary clinical microbiology laboratories play a key role in antimicrobial stewardship, surveillance of antimicrobial resistance and prevention of healthcare associated-infections. However, there is a shortage of international harmonized guidelines covering all steps of veterinary bacterial culture from sample receipt to reporting.MethodsIn order to gain insights, the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT) designed an online survey focused on the practices and interpretive criteria used for bacterial culture and identification (C&ID), and antimicrobial susceptibility testing (AST) of animal bacterial pathogens.ResultsA total of 241 microbiology laboratories in 34 European countries completed the survey, representing a mixture of academic (37.6%), governmental (27.4%), and private (26.5%) laboratories. The C&ID turnaround varied from 1 to 2 days (77.8%) to 3–5 days (20%), and 6– 8 days (1.6%), with similar timeframes for AST. Individual biochemical tests and analytical profile index (API) biochemical test kits or similar were the most frequent tools used for bacterial identification (77% and 56.2%, respectively), followed by PCR (46.6%) and MALDI-TOF MS (43.3%). For AST, Kirby-Bauer disk diffusion (DD) and minimum inhibitory concentration (MIC) determination were conducted by 43.8% and 32.6% of laboratories, respectively, with a combination of EUCAST and CLSI clinical breakpoints (CBPs) preferred for interpretation of the DD (41.2%) and MIC (47.6%) results. In the absence of specific CBPs, laboratories used human CBPs (53.3%) or veterinary CBPs representing another body site, organism or animal species (51.5%). Importantly, most laboratories (47.9%) only report the qualitative interpretation of the result (S, R, and I). As regards testing for AMR mechanisms, 48.5% and 46.7% of laboratories routinely screened isolates for methicillin resistance and ESBL production, respectively. Notably, selective reporting of AST results (i.e. excluding highest priority critically important antimicrobials from AST reports) was adopted by 39.5% of laboratories despite a similar proportion not taking any approach (37.6%) to guide clinicians towards narrower-spectrum or first-line antibiotics.DiscussionIn conclusion, we identified a broad variety of methodologies and interpretative criteria used for C&ID and AST in European veterinary microbiological diagnostic laboratories. The observed gaps in veterinary microbiology practices emphasize a need to improve and harmonize professional training, innovation, bacterial culture methods and interpretation, AMR surveillance and reporting strategies.

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.

Background: Hip and knee osteoarthritis (OA) patients demonstrate distinct gait patterns, yet detecting subtle abnormalities with wearable sensors remains uncertain. This study aimed to assess a predictive model's efficacy in distinguishing between hip and knee OA gait patterns using accelerometer data. Method: Participants with hip or knee OA underwent overground walking assessments, recording lower limb accelerations for subsequent time and frequency domain analyses. Logistic regression with regularization identified associations between frequency domain features of acceleration signals and OA, and k-nearest neighbor classification distinguished knee and hip OA based on selected acceleration signal features. Findings: We included 57 knee OA patients (30 females, median age 68 [range 49–89], median BMI 29.7 [range 21.0–45.9]) and 42 hip OA patients (19 females, median age 70 [range 47–89], median BMI 28.3 [range 20.4–37.2]). No significant difference could be found in the time domain's averaged shape of acceleration signals. However, in the frequency domain, five selected features showed a diagnostic ability to differentiate between knee and hip OA. Using these features, a model achieved a 77 % accuracy in classifying gait cycles into hip or knee OA groups, with average precision, recall, and F1 score of 77 %, 76 %, and 78 %, respectively. Interpretation: The study demonstrates the effectiveness of wearable sensors in differentiating gait patterns between individuals with hip and knee OA, specifically in the frequency domain. The results highlights the promising potential of wearable sensors and advanced signal processing techniques for objective assessment of OA in clinical settings.

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.

IntroductionAs 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.MethodsEleven 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).ResultsCollected 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).DiscussionKey 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.

Abstract Background Quantitative bacterial culture (QBC) is the gold standard for diagnosing canine urinary tract infection. Current guidelines recommend QBC within 24 h of urine collection and that unpreserved urine is refrigerated until culture. However, temperature-controlled transport is rarely feasible, indicating a need for alternative storage during transport of urine from primary veterinary practices to the microbiology laboratory. The objective was to investigate the effect of storage temperature and boric acid sponge-preservation on quantitative bacterial culture of canine urine. Results Significant bacteriuria was detected in 72 out of 179 samples (40%) collected from 141 dogs. Overall accuracy was 94–98% for both storage conditions and time points. Non-inferiority (15% margin) to reference quantitative bacterial culture was evident for sensitivity, specificity and predictive values for both storage methods and time points, except for the negative predictive value for 48 h boric acid preservation (NPV: 89, 95% CI [79;95]). There was no significant difference between the sensitivity and specificity for either of the time-points (p-value = 0.07–1). Conclusions Boric acid sponge-preservation using Uriswab™ is a useful alternative to refrigeration of urine samples during transport. Reliable quantitative bacterial culture results can be obtained from canine urine up to 48 h after collection if urine is refrigerated, and for at least 24 h if urine is stored using a boric acid-containing urine transport system.

ABSTRACT 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]

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.

Abstract 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.

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.

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.

Globally, there is an increased demand for sustainable protein sources for animal feed. Grass and forage legumes have the yield potential to become such alternatives, but the protein needs to be separated from the fibres. Red clover, white clover, lucerne and perennial ryegrass were fractionated into a green juice and a fibrous pulp in a screw-press and protein was subsequently precipitated. The nitrogen (N) and amino acid composition of the produced fractions was analysed and the digestibility of dry matter (DM) and N was evaluated using a rat digestibility trial. The aim was to determine the effect of fractionation on composition and digestibility in order to evaluate the four plants as potential protein sources for monogastrics. Protein concentrates with CP concentrations of 240 to 388 g/kg DM and fibrous pulps with CP concentrations of 111 to 216 g/kg DM were produced. The sum of all analysed amino acids was highest in the protein concentrates corresponding to a low concentration of non-protein nitrogen ranging from 4.9% to 10.4%. Only small variations were seen in the amino acid compositions of the different plants and fractions. The concentration of the essential lysine and methionine in the protein concentrate ranged from 6.27 to 6.67 g/16 g N and 1.54 to 2.09 g/16 g N for lysine and methionine, respectively. For all plants species, total tract digestibility of DM and standardised N digestibility was significantly higher in the protein concentrates (60.8% to 76.5% and 75.4% to 85.0% for DM and N, respectively) compared to pulp (21.2% to 43.4% and 52.1% to 72.5% for DM and N, respectively). Digestibility of lucerne protein concentrate (76.5% and 85.0% for DM and N, respectively) was higher than of the unprocessed plant (39.6% and 74.9% for DM and N, respectively), whereas for red and white clover no difference was found. The amino acids methionine and cysteine were limiting for pigs and broilers in all fractions regardless of plant origin, and low scores were also found for lysine. The study demonstrated great potential of using green plants as a protein source for monogastrics because of high protein content, balanced amino acid composition and high digestibility of DM and N. The effects of processing and protein precipitation were pronounced in lucerne where significantly improved digestibility was observed in the protein concentrate. The results from the study provide valuable and enhanced knowledge to the production of alternative and sustainable protein sources for monogastric feed.

Preterm infants are at risk of multiple morbidities including necrotizing enterocolitis (NEC). Suspected NEC patients receive intravenous antibiotics (AB) to prevent sepsis, although enteral AB is arguably more effective at reducing NEC but is rarely used due to the risk of AB resistance. Fecal microbiota transplantation (FMT) has shown protective effects against NEC in animal experiments, but the interaction between AB and FMT has not been investigated in neonates. We hypothesized that administration of enteral AB followed by rectal FMT would effectively prevent NEC with negligible changes in AB resistance and systemic immunity. Using preterm piglets, we examined host and gut microbiota responses to AB, FMT, or a sequential combination thereof, with emphasis on NEC development. In a saline-controlled experiment, preterm piglets (n = 67) received oro-gastric neomycin (50 mg/kg/d) and amoxicillin-clavulanate (50/12.5 mg/kg/d) (hereafter AB) for four days after cesarean delivery, and were subsequently given rectal FMT from healthy suckling piglet donors. Whereas AB protected the stomach and small intestine, and FMT primarily protected the colon, the sequential combination treatment surprisingly provided no NEC protection. Furthermore, minor changes in the gut microbiota composition were observed in response to either treatment, although AB treatment decreased species diversity and increased AB resistance among coliform bacteria and Enterococci, which were both partly reversed by FMT. Besides, enteral AB treatment suppressed cellular and functional systemic immune development, which was not prevented by subsequent FMT. We discovered an antagonistic relationship between enteral AB and FMT in terms of NEC development. The outcome may depend on choice of AB compounds, FMT composition, doses, treatment duration, and administration routes, but these results challenge the applicability of enteral AB and FMT in preterm infants.

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.

Background Clinical signs of urinary tract disease in dogs often lead to prescription of antibiotics. Appropriate diagnostic work‐up could optimize treatment and reduce the risk of inappropriate use of antibiotics. Hypothesis/Objectives To describe and evaluate the impact of diagnostic work‐up on decision to treat (DTT) and choice of antibiotic treatment (COT) for dogs presenting with clinical signs of urinary tract disease. Animals One hundred and fifty‐one dogs presenting to 52 Danish veterinary practices. Methods Prospective, observational study. Clinical signs, diagnostic work‐up, and prescriptions were recorded. Urine samples were submitted to a reference laboratory for quantitative bacterial culture (QBC) and susceptibility testing. The laboratory results were used as reference for assessing the appropriateness of DTT and COT. Results In the majority of dogs, veterinarians performed dipstick (99%), microscopic examination of urine (80%) and bacterial culture (56%). Fifty‐one percent of dogs had urinary tract infection (UTI) based on reference QBC. Appropriate DTT was made for 62% of the dogs, while 36% were over‐prescribed and 2% under‐prescribed. Inappropriate use of second‐line agents was found in 57% of the UTI cases. Performing microscopy—but not culture—significantly impacted DTT (P = 0.039) while no difference was seen in COT (P = 0.67). The accuracy of in‐house microscopy and culture were 64.5 and 77%, respectively. Conclusions and Clinical Importance: Over‐prescription of antibiotics was common among dogs with suspected UTI, regardless of the diagnostic work‐up performed. Test inaccuracy under practice conditions and incoherence between diagnostic test results and decision‐making both explained inappropriate and unnecessary use of antibiotics.

Despite inducing strong T cell responses, Mycobacterium tuberculosis (Mtb) infection fails to elicit protective immune memory. As such latently infected or successfully treated Tuberculosis (TB) patients are not protected against recurrent disease. Here, using a mouse model of aerosol Mtb infection, we show that memory immunity to H56/CAF01 subunit vaccination conferred sustained protection in contrast to the transient natural immunity conferred by Mtb infection. Loss of protection to re-infection in natural Mtb memory was temporally linked to an accelerated differentiation of ESAT-6- and to a lesser extent, Ag85B-specific CD4 T cells in both the lung parenchyma and vasculature. This phenotype was characterized by high KLRG1 expression and low, dual production of IFN-γ and TNF. In contrast, H56/CAF01 vaccination elicited cells that expressed low levels of KLRG1 with copious expression of IL-2 and IL-17A. Co-adoptive transfer studies revealed that H56/CAF01 induced memory CD4 T cells efficiently homed into the lung parenchyma of mice chronically infected with Mtb. In comparison, natural Mtb infection- and BCG vaccine-induced memory CD4 T cells exhibited a poor ability to home into the lung parenchyma. These studies suggest that impaired lung migratory capacity is an inherent trait of the terminally differentiated memory responses primed by mycobacteria/mycobacterial vectors.

Klebsiella pneumoniae causes a variety of infections in both humans and animals. In this study, we characterised the genomes of human and animal isolates from two diagnostic laboratories on St. Kitts, a small Caribbean island inhabited by a large population of vervet monkeys. In view of the increased chances of direct or indirect contact with humans and other animal species, we used the One Health approach to assess transmission of K. pneumoniae across host species by sequencing 82 presumptive K. pneumoniae clinical isolates from humans (n = 51), vervets (n = 21), horses (n = 5), dogs (n = 4) and a cat (n = 1). Whole genome sequencing (WGS) was carried out using Illumina technology. De novo assembly was performed in CLC Genomics Workbench v.11.0. Single nucleotide polymorphisms were detected using NASP followed by phylogenetic analysis using IQ-TREE. Virulence and antimicrobial resistance gene contents were analysed using the Kleborate and CGE pipelines. WGS-based analysis showed that 72 isolates were K. pneumoniae sensu stricto and five K. quasipneumoniae and five K. variicola. K. pneumoniae isolates belonged to 35 sequence types (ST), three of which were occasionally shared between humans and animals: ST23, ST37 and ST307. The ST23 strains from vervets formed a separate cluster amongst publicly available sequenced ST23 strains, indicating the presence of a specific vervet sublineage. Animal strains harbored fewer resistance genes and displayed distinct virulence traits that appeared to be host-specific in vervet isolates. Our results show that K. pneumoniae infections on this Caribbean island are usually caused by host-specific lineages.

Abstract Background The Danish antibiotic use guidelines for companion animal practice were published by the Danish Veterinary Association in 2012. Since then, national surveillance data indicate a 10% reduction in the total use of antibiotics for companion animals, particularly a marked reduction in the use of third generation cephalosporins. The aim of the study was to assess if and how the guidelines have impacted diagnostic and antibiotic prescription habits of the users, and to identify user perceived barriers to implementation. Results An online questionnaire was sent to all 882 members of the Danish Small Animal Veterinary Association in October 2015. The survey was completed by 151 veterinarians. Respondents most frequently consulted the recommendations on skin and urinary tract infections (UTI), and users generally reported a high degree of adherence to the recommendations. Sixty-five per cent indicated that the guidelines had influenced their habits in one or more of the areas being investigated, i.e. perioperative use of antibiotics, use of first line antibiotics for the treatment of pyoderma or UTI, and/or use of microbiological diagnostics. Perioperative use of antibiotics for clean surgeries was uncommon, irrespective of whether respondents had consulted the relevant recommendations or not. On the contrary, significant differences in the prescribing habits between guideline users and non-users were observed for pyoderma and UTI, suggesting an impact of the guidelines towards more prudent antimicrobial use. The diagnostic habits were examined in a subgroup of 63 guideline users. Of those, 19 and 39% reported frequent use of culture and susceptibility (C&S) testing prior to treating pyoderma and UTI respectively, whereas 68–84% reported C&S testing in the event of poor response to treatment or recurrence of infections. The main barriers for implementation of therapeutic recommendations were confidence in old prescribing practices and unavailability of recommended drugs. The main barriers for C&S testing were good experience with empiric treatment, and the owners’ financial situation. Conclusions The findings suggest a positive influence of the national antibiotic guidelines on prescription patterns among companion animal practitioners in Denmark. Sustained campaign activity is encouraged and should include promotion of bacteriological testing.

Abstract Background Individual differences of mink, including color type, are speculated to affect the course of wound healing, thereby impacting wound assessment and management on the farms, as well as the assessment of wounds in forensic cases. In this study, we examined the effect of color type on early wound healing in farmed mink. Full thickness excisional wounds (2 × 2 cm) were made on the back in 18 mink of the color types Brown, Silverblue and Blue Iris. Gross and microscopic pathology of the wounds was evaluated 2 days post-wounding together with degree of wound size reduction, presence of bacteria and blood analyses. Results Pathological examination on day 2 showed the greatest mean wound size reduction in Brown mink (11.0%) followed by Blue Iris (7.9%) and Silverblue (1.6%). Bacteria were cultured from all wounds, and predominantly Staphylococcus species were recovered in mixed or pure culture. Histopathology from day 2 wounds showed a scab overlying necrotic wound edges, which were separated from underlying vital tissue by a demarcation zone rich in polymorphonuclear leukocytes. Fibroblasts and plump endothelial cells were more numerous in the deeper tissues. Complete blood count parameters were within normal ranges in most cases, however, the mink showed mildly to markedly decreased hematocrit and six mink of the color types Silverblue and Blue Iris showed moderately elevated numbers of circulating segmented neutrophils on day 2. There was a marked increase in concentration of serum amyloid A from day 0 to day 2 in all color types. Conclusions We have described differences in early wound healing between mink of the color types Brown, Silverblue and Blue Iris by use of an experimental wound model in farmed mink. The most pronounced difference pertained to the degree of wound size reduction which was greatest in Brown mink, followed by Blue Iris and Silverblue, respectively.

Limited information is available on antimicrobial susceptibility and clonal distribution of Staphylococcus aureus in the Caribbean region. The aims of this study were to determine the prevalence of antimicrobial resistance among S. aureus isolates and to reveal the frequency and population structure of methicillin-resistant S. aureus (MRSA) in St. Kitts and Nevis, a small island country in the West Indies. A total of 152 S. aureus isolates were collected from consecutive samples submitted to the clinical microbiology laboratory of the main referral hospital from March 2017 to January 2018. Samples came from all units in the hospital and a small number came from external submissions, and comprised a total of 119 clinical specimens and 33 nasal swabs collected from staff and patients. All S. aureus isolates were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Minimal Inhibitory Concentrations (MICs) of clinically relevant antimicrobials were determined by broth microdilution, and diversity of MRSA isolates was assessed by whole genome sequencing (WGS) analysis. MRSA accounted for 45% (69/152) of the isolates. The highest rates of resistance to non-β-lactam agents were observed for erythromycin (55%), moxifloxacin (41%), and levofloxacin (40%), whereas resistance to the other drugs tested was ≤6%. All isolates were susceptible to ceftaroline, linezolid, teicoplanin, telavancin, and vancomycin. WGS-based multilocus sequence typing (MLST) showed that approximately 88% of the MRSA isolates belonged to ST8. Phylogenetic analysis on 801 single-nucleotide polymorphisms (SNPs) identified among the MRSA ST8 isolates indicates a large degree of genetic diversity. However, all ST8 strains clustered within the distinct clade that defines the USA300 North American Epidemic lineage (Panton-Valentine Leukocidin (PVL) +, arginine catabolic mobile element (ACME) +, Staphylococcal cassettes chromosome mec IVa (SCCmec IVa)). Our data show high levels of methicillin, macrolide, and fluoroquinolone resistance among S. aureus on St. Kitts and Nevis. The USA300 North American epidemic lineage is responsible for the vast majority of MRSA infections on this Caribbean island.

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.

VetCAST is the EUCAST sub-committee for Veterinary Antimicrobial Susceptibility Testing. Its remit is to define clinical breakpoints (CBPs) for antimicrobial drugs (AMDs) used in veterinary medicine in Europe. This position paper outlines the procedures and reviews scientific options to solve challenges for the determination of specific CBPs for animal species, drug substances and disease conditions. VetCAST will adopt EUCAST approaches: the initial step will be data assessment; then procedures for decisions on the CBP; and finally the release of recommendations for CBP implementation. The principal challenges anticipated by VetCAST are those associated with the differing modalities of AMD administration, including mass medication, specific long-acting product formulations or local administration. Specific challenges comprise mastitis treatment in dairy cattle, the range of species and within species breed considerations and several other variable factors not relevant to human medicine. Each CBP will be based on consideration of: (i) an epidemiological cut-off value (ECOFF) – the highest MIC that defines the upper end of the wild-type MIC distribution; (ii) a PK/PD breakpoint obtained from pre-clinical pharmacokinetic data [this PK/PD break-point is the highest possible MIC for which a given percentage of animals in the target population achieves a critical value for the selected PK/PD index (fAUC/MIC or fT > MIC)] and (iii) when possible, a clinical cut-off, that is the relationship between MIC and clinical cure. For the latter, VetCAST acknowledges the paucity of such data in veterinary medicine. When a CBP cannot be established, VetCAST will recommend use of ECOFF as surrogate. For decision steps, VetCAST will follow EUCAST procedures involving transparency, consensus and independence. VetCAST will ensure freely available dissemination of information, concerning standards, guidelines, ECOFF, PK/PD breakpoints, CBPs and other relevant information for AST implementation. Finally, after establishing a CBP, VetCAST will promulgate expert comments and/or recommendations associated with CBPs to facilitate their sound implementation in a clinical setting.

The epidemiologically most important mechanism of antibiotic resistance in Staphylococcus aureus is associated with mecA-an acquired gene encoding an extra penicillin-binding protein (PBP2a) with low affinity to virtually all β-lactams. The introduction of mecA into the S. aureus chromosome has led to the emergence of methicillin-resistant S. aureus (MRSA) pandemics, responsible for high rates of mortality worldwide. Nonetheless, little is known regarding the origin and evolution of mecA. Different mecA homologues have been identified in species belonging to the Staphylococcus sciuri group representing the most primitive staphylococci. In this study we aimed to identify evolutionary steps linking these mecA precursors to the β-lactam resistance gene mecA and the resistance phenotype. We sequenced genomes of 106 S. sciuri, S. vitulinus and S. fleurettii strains and determined their oxacillin susceptibility profiles. Single-nucleotide polymorphism (SNP) analysis of the core genome was performed to assess the genetic relatedness of the isolates. Phylogenetic analysis of the mecA gene homologues and promoters was achieved through nucleotide/amino acid sequence alignments and mutation rates were estimated using a Bayesian analysis. Furthermore, the predicted structure of mecA homologue-encoded PBPs of oxacillin-susceptible and -resistant strains were compared. We showed for the first time that oxacillin resistance in the S. sciuri group has emerged multiple times and by a variety of different mechanisms. Development of resistance occurred through several steps including structural diversification of the non-binding domain of native PBPs; changes in the promoters of mecA homologues; acquisition of SCCmec and adaptation of the bacterial genetic background. Moreover, our results suggest that it was exposure to β-lactams in human-created environments that has driven evolution of native PBPs towards a resistance determinant. The evolution of β-lactam resistance in staphylococci highlights the numerous resources available to bacteria to adapt to the selective pressure of antibiotics.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) constitutes an emerging health problem for companion animals in veterinary medicine. Therefore, discovery of novel antimicrobial agents for treatment of Staphylococcus-associated canine infections is urgently needed to reduce use of human antibiotics in veterinary medicine. In the present work, we characterized the antimicrobial activity of the peptoid D2 against S. pseudintermedius and Pseudomonas aeruginosa, which is another common integumentary pathogen in dogs. Furthermore, we performed a structure–activity relationship study of D2, which included 19 peptide/peptoid analogs. Our best compound D2D, an all d-peptide analogue, showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius (2–4 µg/mL) and P. aeruginosa (4 µg/mL) isolates as well as other selected dog pathogens (2–16 µg/mL). Time–kill assays demonstrated that D2D was able to inhibit MRSP in 30 min at 1× MIC, significantly faster than D2. Our results suggest that at high concentrations D2D is rapidly lysing the bacterial membrane while D2 is inhibiting macromolecular synthesis. We probed the mechanism of action at sub-MIC concentrations of D2, D2D, the l-peptide analog and its retro analog by a macromolecular biosynthesis assay and fluorescence spectroscopy. Our data suggest that at sub-MIC concentrations D2D is membrane inactive and primarily works by cell wall inhibition, while the other compounds mainly act on the bacterial membrane.

Antibiotic discovery is vital when considering the increasing antimicrobial resistance threat. The aim of this work was to provide a high-throughput screen (HTS) assay using multidrug-resistant Escherichia coli strains to enable further research into antimicrobial lead discovery and identify novel antimicrobials. This study describes a primary HTS of a diverse library of 7884 small molecules against a susceptible E. coli strain. A secondary screening of 112 molecules against four E. coli strains with different susceptibility profiles revealed NSC319726 as a potential antimicrobial lead serving as a novel template. NSC319726 is a good candidate for an analoguing program.

Staphylococci are associated with both humans and animals. While most are non-pathogenic colonizers, Staphylococcus aureus is an opportunistic pathogen capable of causing severe infections. S. aureus virulence is controlled by the agr quorum sensing system responding to secreted auto-inducing peptides (AIPs) sensed by AgrC, a two component histidine kinase. agr loci are found also in other staphylococcal species and for Staphylococcus epidermidis, the encoded AIP represses expression of agr regulated virulence genes in S. aureus. In this study we aimed to better understand the interaction between staphylococci and S. aureus, and show that this interaction may eventually lead to the identification of new anti-virulence candidates to target S. aureus infections. Here we show that culture supernatants of 37 out of 52 staphylococcal isolates representing 17 different species inhibit S. aureus agr. The dog pathogen, Staphylococcus schleiferi, expressed the most potent inhibitory activity and was active against all four agr classes found in S. aureus. By employing a S. aureus strain encoding a constitutively active AIP receptor we show that the activity is mediated via agr. Subsequent cloning and heterologous expression of the S. schleiferi AIP in S. aureus demonstrated that this molecule was likely responsible for the inhibitory activity, and further proof was provided when pure synthetic S. schleiferi AIP was able to completely abolish agr induction of an S. aureus reporter strain. To assess impact on S. aureus virulence, we co-inoculated S. aureus and S. schleiferi in vivo in the Galleria mellonella wax moth larva, and found that expression of key S. aureus virulence factors was abrogated. Our data show that the S. aureus agr locus is highly responsive to other staphylococcal species suggesting that agr is an inter-species communication system. Based on these results we speculate that interactions between S. aureus and other colonizing staphylococci will significantly influence the ability of S. aureus to cause infection, and we propose that other staphylococci are potential sources of compounds that can be applied as anti-virulence therapy for combating S. aureus infections.

Background and rationale: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a major cause of infections in dogs, also posing a zoonotic risk to humans. This systematic review aimed to determine the global epidemiology of MRSP and provide new insights into the population structure of this important veterinary pathogen.Methodology: Web of Science was searched systematically for articles reporting data on multilocus sequence typing (MLST) of S. pseudintermedius isolates from dogs or other animal or human patients and carriers. Data from the eligible studies were then integrated with data from the MLST database for this species. Analysis of MLST data was performed with eBURST and ClonalFrame, and the proportion of MRSP isolates resistant to selected antimicrobial drugs was determined for the most predominant clonal complexes.Results: Fifty-eight studies published over the last 10 years were included in the review. MRSP represented 76% of the 1428 isolates characterized by the current MLST scheme. The population of S. pseudintermedius was highly diverse and included five major MRSP clonal complexes (CCs). CC71, previously described as the epidemic European clone, is now widespread worldwide. In Europe, CC258, which is more frequently susceptible to enrofloxacin and aminoglycosides, and more frequently resistant to sulphonamides/trimethoprim than CC71, is increasingly reported in various countries. CC68, previously described as the epidemic North American clone, is frequently reported in this region but also in Europe, while CC45 (associated with chloramphenicol resistance) and CC112 are prevalent in Asia. It was estimated that clonal diversification in this species is primarily driven by homologous recombination (r/m=7.52).Conclusion: This study provides evidence that S. pseudintermedius has an epidemic population structure, in which five successful MRSP clones with specific traits regarding antimicrobial resistance, genetic diversity and geographical distribution have emerged upon a weakly clonal background through acquisition of SCCmec and other mobile genetic elements.

Antifolates targeting folate biosynthesis within the shikimate-chorismate-folate metabolic pathway are ideal and selective antimicrobials, since higher eukaryotes lack this pathway and rely on an exogenous source of folate. Resistance to the available antifolates, inhibiting the folate pathway, underlines the need for novel antibiotic scaffolds and molecular targets. While para-aminobenzoic acid synthesis within the chorismate pathway constitutes a novel molecular target for antifolates, abyssomicins are its first known natural inhibitors. This review describes the abyssomicin family, a novel spirotetronate polyketide Class I antimicrobial. It summarizes synthetic and biological studies, structural, biosynthetic, and biological properties of the abyssomicin family members. This paper aims to explain their molecular target, mechanism of action, structure–activity relationship, and to explore their biological and pharmacological potential. Thirty-two natural abyssomicins and numerous synthetic analogues have been reported. The biological activity of abyssomicins includes their antimicrobial activity against Gram-positive bacteria and mycobacteria, antitumor properties, latent human immunodeficiency virus (HIV) reactivator, anti-HIV and HIV replication inducer properties. Their antimalarial properties have not been explored yet. Future analoging programs using the structure–activity relationship data and synthetic approaches may provide a novel abyssomicin structure that is active and devoid of cytotoxicity. Abyssomicin J and atrop-o-benzyl-desmethylabyssomicin C constitute promising candidates for such programs.

Antimicrobial peptides (AMPs) hold promise as the next generation of antimicrobial agents, but often suffer from rapid degradation in vivo. Modifying AMPs with non-proteinogenic residues such as peptoids (oligomers of N-alkylglycines) provides the potential to improve stability. We have identified two novel peptoid-based compounds, B1 and D2, which are effective against the canine skin pathogen Staphylococcus pseudintermedius, the main cause of antibiotic use in companion animals. We report on their potential to treat infections topically by characterizing their release from formulation and in vitro ADME properties. In vitro ADME assays included skin penetration profiles, stability to proteases and liver microsomes, and plasma protein binding. Both B1 and D2 were resistant to proteases and >98% bound to plasma proteins. While half-lives in liver microsomes for both were >2 h, peptoid D2 showed higher stability to plasma proteases than the peptide-peptoid hybrid B1 (>2 versus 0.5 h). Both compounds were suitable for administration in an oil-in-water cream formulation (50% release in 8 h), and displayed no skin permeation, in the absence or presence of skin permeability modifiers. Our results indicate that these peptoid-based drugs may be suitable as antimicrobials for local treatment of canine superficial pyoderma and that they can overcome the inherent limitations of stability encountered in peptides.