BACKGROUND AND OBJECTIVES:Neomycin is widely used in livestock to control colibacillosis. Resistance among clinical Escherichia coli is increasing, likely driven by the use of this aminoglycoside following the European restrictions on zinc oxide and colistin. The aim of this study was to investigate the genetic epidemiology of neomycin-resistant E. coli across six animal species and five European countries. METHODS:A total of 750 neomycin-resistant E. coli isolates were included, combining retrospectively identified genomes with prospectively isolated and sequenced strains from multiple animal species. Genomic analyses, based on short reads (n = 529), long reads (n = 177), or hybrid assemblies (n = 44), included phylogenetic comparison, and identification of neomycin resistance genes and their genetic contexts. RESULTS:Neomycin resistance was mediated by aph(3')-Ia in 98.2% of the isolates, which were distributed across 28 clonal complexes, predominantly CC10, which was frequently detected in all countries and hosts. aph(3')-Ia was primarily carried by conjugative plasmids of diverse replicon types (70.3%) and embedded in two transposons (Tn903 and Tn4352) and three newly identified genetic elements, while a chromosomally integrated element accounted for the remaining 29.7%. Plasmids carrying aph(3')-Ia frequently co-harboured tetracycline and trimethoprim-sulfamethoxazole resistance genes, with IncX1 plasmids additionally containing chloramphenicol and quinolone resistance determinants. Phylogenetic analysis revealed dissemination of plasmid-borne elements across multiple lineages, while the chromosomal element was mostly restricted to ST117, ST88, and ST189. CONCLUSIONS:The spread of neomycin resistance in E. coli from European livestock is primarily driven by the mobility of aph(3')-Ia embedded in diverse genetic contexts and plasmid backbones, promoting co-transfer of other resistance determinants.

Post-weaning diarrhoea (PWD) is a major driver of antimicrobial use in pigs. Optimising treatment duration may reduce selection pressure while maintaining clinical efficacy, but under EU Regulation (EU) 2019/6 veterinarians must adhere to authorised treatment durations, creating a need for evidence to support label changes. This study aimed to determine whether a 3-day apramycin treatment is non-inferior to the standard 7-day course for clinical cure of PWD and to assess its effect on the abundance of the apramycin resistance gene aac(3)-IV. A cluster-randomised non-inferiority field trial was conducted in 518 diarrhoeic piglets across 28 pens in a commercial herd. Piglets received apramycin for 3 or 7 days. Clinical cure was assessed by faecal scoring at end of treatment (EOT) and at a common Day 8 timepoint after treatment initiation. A non-inferiority margin of − 10

BackgroundZoonotic transmission of livestock-associated methicillin-resistantStaphylococcus aureusclonal complex 398 (LA-MRSA CC398) from industrial pig farms to farm workers and their household members and secondary transmission to elderly and immunocompromised persons has led to a growing number of serious infections and even death.ObjectiveWe tested the hypothesis that conventional pig farms confer a higher risk for LA-MRSA CC398 carriage among farm workers and their household members than specific pathogen-free (SPF) farms, which follow a set of stringent rules concerning biosecurity, health control and transportation of pigs between herds.MethodsWe conducted a cross-sectional study of farm workers and their household members from 25 Danish pig farms positive for LA-MRSA CC398. Questionnaires were designed to obtain information about each farm and participant. Nasal samples from pigs and human volunteers were screened for LA-MRSA CC398. Multivariate logistic regression analyses were used to calculate adjusted ORs (aORs) and 95% CIs.ResultsThe study included 25 pig farms, 154 farm workers screened at the farm (93 of whom were also screened at home) and 173 household members. The risk of LA-MRSA CC398 carriage among farm workers at the farm (aOR=5.40, 95% CI 1.05 to 27.80) and household members (aOR=8.32; 95% CI 2.81 to 24.63) was significantly higher in conventional pig farms than in SPF farms.ConclusionsThis study shows that strict biosecurity measures reduce the risk of LA-MRSA CC398 carriage among farm workers and their household members and thereby the disease burden in the general population.

Rifampin is an enigma among antimicrobials. Blood and tissue compartment concentrations are a "moving target" along the treatment course due to the complex pharmacodynamic interactions within the body. Rifampin concomitant therapies are for the prevention and treatment of Rhodococcus equi infection in foals, for nearly 40 years. The necessity of rifampin concomitant therapies is based on beliefs that both antimicrobials (e.g., rifampin plus macrolide) penetrate into pulmonary abscesses and intracellular compartments above R. equi minimum inhibitory concentrations (MICs), as well as better efficacy, compared with other approaches, and limiting the rate of antimicrobial resistance to either single agent. However, rifampin acts as a perpetrator drug for many co-administered drugs. This critical review evaluates the available evidence for rifampin use in foals with R. equi , concerning pharmacokinetic/pharmacodynamic characteristics of rifampin in foals, in vitro microbiological studies and selection of antimicrobial resistance, as well as an analysis of randomized clinical trials. Rifampin is a nuclear pregnane X receptor activator, which results in strong negative drug interactions towards itself and other drugs, for drug-absorption routes either by upregulation of presystemic elimination mechanisms (e.g., intestinal and hepatic CYP3A4), or functional drug-absorption carriers (e.g., intestinal P-glycoprotein) and/or inhibition of intestinal and/or hepatic drug-uptake carriers (e.g., OATP1B1, OATP2B1, MRP2). Chronic rifampin administration results in decreases in the serum and target site/s concentrations of many parent drugs, including itself. Rifampin concomitant therapies do not demonstrate a significant advantage over monotherapy with macrolides, in randomized controlled blinded and double-blinded clinical trials for subclinical, and mild-to-moderate bronchopneumonia in foals with pulmonary abscesses, regardless of initial pulmonary abscess score. Efficacy of rifampin concomitant therapies for severe Rhodococcus equi pneumonia has not been fully investigated, but there is sufficient accumulated evidence in foals to raise major concerns about the incorrect use of rifampin in equine medicine. These concerns include rifampin as a bacteriostatic antibiotic against R. equi , with changing pharmacokinetics during treatment that decreases parent/coparent concentrations as well as the risk of selecting for multi-resistant R. equi .

The aim of this study was to develop amoxicillin (AMX) dosage regimens for the treatment of Escherichia coli and Staphylococcus delphini infections in mink using a pharmacokinetic (PK) and pharmacodynamic (PD) approach. Two animal experiments with a total of 12 minks were conducted to investigate PK parameters after intravenous or oral administration of an AMX off-label dosage of 15 mg/kg. The PK characteristics of AMX were analyzed using non-linear mixed effect modeling. In vitro time kill experiments using AMX concentrations up to 16 × MIC were conducted with two E. coli and two S. delphini isolates. Semi-mechanistic modeling of time-kill experiments was subsequently done to study PD characteristics. By integrating PK and PD data, the PK/PD index and PK/PD target were predicted. Simulations were applied to determine an optimal AMX dosage. The PD model revealed higher in vitro efficacy and lower potency of AMX against E. coli compared to S. delphini. fT(%) > MIC was selected as the PK/PD index, indicating a time-dependent effect of AMX. New mink-specific AMX dosages of 50 mg/kg twice a day for E. coli infections and 7.5 mg/kg once daily for S. delphini infections were determined. The study emphasizes the need for dose optimization studies, especially for minor animal species for which antibiotics are generally used off-label.

Enterotoxigenic Escherichia coli (ETEC) of fimbriae types F4 and F18 causes post-weaning diarrhea (PWD) in pigs and susceptibility relates to host genotype. We examined if CHCF1/FUT1 genotypes were associated with ETEC-mediated PWD in seven Danish herds. Diarrhea was assessed in 1,919 pigs by evaluating perianal fecal staining in the first week after weaning. From each herd, twelve diarrheic and twelve non-diarrheic pigs were selected for genotyping, fecal dry matter (FDM) and microbiological testing. ETEC F4 diarrhea occurred only in CHCF1 heterozygous (RS) pigs. Furthermore, CHCF1 RS pigs had higher odds of diarrhea (Odds ratio (OR) 2.09; p = 0.026) and lower mean FDM (13.9%) than homozygous resistant (RR) pigs (18.6%; p = 0.0052). ETEC F4 was more frequently isolated from CHCF1 RS pigs compared to RR pigs (OR 8.88, p = 0.00005). Additionally, hemolytic E. coli shedding was higher in CHCF1 RS pigs (mean 47.4% vs. 24.1%; p = 0.00001). Too few FUT1 RR pigs were found for analyzing association between FUT1 genotypes and ETEC F18-mediated diarrhea. In conclusion, CHCF1 genotype was strongly associated with ETEC F4-mediated PWD under production settings. ETEC F4 resistance in pigs could help to reduce PWD occurrence and antibiotic use.

Objective:To investigate the in vitro efficacy of antibiotics (amikacin, ceftiofur, and gentamicin) in combination with local anesthetics (LAs; bupivacaine hydrochloride, lidocaine hydrochloride, and mepivacaine hydrochloride), a combination commonly performed for IA injectate and regional limb perfusion (RLP) in horses. Methods:17 equine clinical isolates were tested by the checkerboard method for their minimum inhibitory concentration (MIC) against a combination of concentrations of LAs and antibiotics from August 2020 through December 2023. Results:For the majority of combinations, the antibiotic efficacy was not affected. However, in a subset of combinations (n = 70), the addition of LA to the antibiotic solution displayed a synergistic (n = 14) or antagonistic (n = 56) effect, indicating that LA increased or decreased antibiotic activity, respectively. Increased MICs seen in most antagonistic combinations appeared to be without clinical relevance as MICs remained below or above clinically achievable concentrations. In contrast, antagonism observed for aminoglycoside-LA combinations resulted in MICs higher than the concentration achievable by RLP. In some synergistic combinations, MICs decreased from markedly above to below or near clinically achievable concentrations against a specific antibiotic. Conclusions:The addition of LAs to antibiotic solutions for IA injections does not compromise the in vitro antibiotic effect. Conversely, the addition for RLPs compromises the in vitro antibiotic effect at clinical concentrations. Clinical Relevance:This in vitro study suggests that LAs can be added to IA antibiotic solutions without compromising antibiotic effects against common equine pathogens. For RLP, the combination of tested aminoglycosides (amikacin and gentamicin) and LAs is discouraged without taking into account MICs of antimicrobial susceptibility testing.

Method jBile colonisation occurs with bacteria originating from the gut.Unfortunately, there is a lack of standard procedures to guide processing specimens form companion animals which may lead to variable culture results including missing relevant pathogens.The objective of this study was to compare bacterial culture protocols and results for bile samples from different laboratories aiming to generate a consensus protocol for these specimens. Objectives Results

Antimicrobial stewardship initiatives are widely regarded as a cornerstone for ameliorating the global health impact of antimicrobial resistance. Within companion animal health, such efforts have largely focused on development and dissemination of antimicrobial stewardship guidelines (ASGs). However, there have been few attempts to understand veterinarian attitudes towards and knowledge of ASGs or to determine how awareness regarding ASGs might best be increased. An online survey regarding ASGs was formulated for veterinarians who treat companion animals. The survey was distributed across 46 European and associated countries between 12 January and 30 June, 2022. In total, 2271 surveys were completed, with 64.9% of respondents (n = 1474) reporting awareness and usage of at least one ASG. Respondents from countries with greater awareness of ASGs tended to report more appropriate use of antimicrobials (Spearman's rank coefficient = 0.6084, P ≤ 0.001), with respondents from countries with country-specific ASGs tending to score highest across both awareness and appropriate use domains. Respondents prioritised guidance around antimicrobial choice (82.0%, n = 1863), duration of treatment (66.0%, n = 1499), and dosage (51.9%, n = 1179) for inclusion in future ASGs, with 78.0% (n = 1776) of respondents preferring ASGs to be integrated into their patient management system. Awareness of ASGs and their use in companion animal veterinary practice appears to be greater than previously reported, with respondents tending to report antimicrobial prescription decision making broadly in line with current clinical recommendations. However, further initiatives aimed at maximising accessibility to ASGs both within countries and individual veterinary practices are recommended.

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 255S. uberis and 231S. 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.

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.

Introduction Veterinary 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.Methods In 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.Results A 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.Discussion In 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.

Staphylococcus pseudintermedius is the most common cause of pyoderma in dogs. We validated a point-of-care (PoC) test based on colorimetric loop-mediated isothermal amplification (LAMP) for rapid S. pseudintermedius identification and susceptibility testing for first line antimicrobials for systemic treatment of canine pyoderma, i.e., lincosamides, first generation cephalosporins and amoxicillin clavulanate. Newly designed LAMP primers targeting clinically relevant resistance genes were combined with a previously validated set of primers targeting spsL for species identification. After laboratory validation on 110 clinical isolates, we assessed the performance of the test on 101 clinical specimens using routine culture and susceptibility testing as a reference standard.The average hands-on and turnaround times for the PoC test were 30 and 90minutes, respectively. The assay showed sensitivity and specificity near 100% for both species identification and susceptibility testing when performed on bacterial cultures or clinical specimens in the laboratory. However, the PoC test yielded less accurate results when performed on-site by clinical staff (92% sensitivity and 64% specificity for species identification, 67% sensitivity and 96% specificity for β‐lactam susceptibility, and 83% sensitivity and 71% specificity for lincosamide susceptibility). These results indicate that the PoC test should be adapted to a user-friendly technology to facilitate performance and interpretation of results by clinical staff. If properly developed, the test would allow veterinarians to gain rapid information on antimicrobial choice, limiting the risk of treatment failure and facilitating adherence to antimicrobial use guidelines in small animal veterinary dermatology.

Untreatable bacterial infections (“the post-antibiotic era”) are far more likely to emerge in companion animals than in food animals, and have already arrived in the form of the multi-drug resistant (MDR) ESKAPE pathogens, the scourge of hospitals. These highly resistant, and in some cases hospital-adapted pathogens, are Enterococcus faecium, methicillin-resistant Staphylococcus pseudintermedius (and S. aureus), Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species. There has however been a general increase in extendedspectrum beta-lactamase (ESBL) producing E. coli in companion animals which, like the ESKAPE pathogens, can be shared with their owners and escape into the veterinary clinic and hospital. We are moving rapidly from the antimicrobial era through the antimicrobial resistance (AMR) era into the era of antimicrobial stewardship (AMS). This shifting paradigm in our relationship with antibiotics is part of the global responsibility that all users have to preserve for the long term the rather small range of antimicrobial drugs available to treat bacterial infections.

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 β-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 β-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 β-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.

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.

ABSTRACT 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 Tn 903 or Tn 4352 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 (Tn 903 and Tn 4352 ) 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.

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.