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Aspergillus fumigatus is a saprophytic fungal pathogen that causes opportunistic infections in animals and humans. Azole resistance has been reported globally in human A. fumigatus isolates, but the prevalence of resistance in isolates from animals is largely unknown. A retrospective resistance surveillance study was performed using a collection of clinical A. fumigatus isolates from various animal species collected between 2015 and 2020. Agar-based azole resistance screening of all isolates was followed by in vitro antifungal susceptibility testing and cyp51A gene sequencing of the azole-resistant isolates. Over the 5 year period 16 (11.3%) of 142 A. fumigatus culture-positive animals harbored an azole-resistant isolate. Resistant isolates were found in birds (15%; 2/13), cats (21%; 6/28), dogs (8%; 6/75) and free-ranging harbor porpoise (33%; 2/6). Azole-resistance was cyp51A mediated in all isolates: 81.3% (T-67G/)TR 34/L98H, 12.5% TR 46/Y121F/T289A. In one azole-resistant A. fumigatus isolate a combination of C(-70)T/F46Y/C(intron7)T/C(intron66)T/M172V/E427K single-nucleotide polymorphisms in the cyp51A gene was found. Of the animals with an azole-resistant isolate and known azole exposure status 71.4% (10/14) were azole naive. Azole resistance in A. fumigatus isolates from animals in the Netherlands is present and predominantly cyp51A TR-mediated, supporting an environmental route of resistance selection. Our data supports the need to include veterinary isolates in resistance surveillance programs. Veterinarians should consider azole resistance as a reason for therapy failure when treating aspergillosis and consider resistance testing of relevant isolates.

Streptococcus suis (S. suis) infections in weaned pigs are common and responsible for a high consumption of antimicrobials, and their presence is assumed to be multi-factorial. A specific evidence-based veterinary guideline to support the control of S. suis in weaned pigs was developed for veterinary practitioners in the Netherlands in 2014. Adherence to the S. suis clinical practice guideline helps veterinary practitioners to prevent and control the disease in a systematical approach and thereby improve antimicrobial stewardship and contribute to the prevention of antimicrobial resistance in animals and humans. The impact of such a clinical practice guideline on (animal) disease management depends not only on its content, but also largely on the extent to which practitioners adhere to the clinical guideline in practice. When the S. suis guideline was published, no specific activities were undertaken to support veterinarians' uptake and implementation, thereby contributing to suboptimal adherence in clinical practice. As the S. suis guideline was comprehensively written by veterinary experts following an evidence-based approach, our aim was not to judge the (scientific) quality of the guideline but to study the possibility to improve the currently low adherence of this guideline in veterinary practice. This paper describes the systematic development, using Implementation Mapping, of a theory-based intervention program to support swine veterinarians' adherence to the S. suis guideline. The knowledge, skills, beliefs about capabilities, and beliefs about consequences domains are addressed in the program, which includes seven evidence-based methods (modelling, tailoring, feedback, discussion, persuasive communication, active learning, and self-monitoring) for use in program activities such as a peer-learning meeting and an e-learning module. The intervention program has been developed for practicing swine veterinarians, lasts eight months, and is evaluated through a

Metagenomic sequencing has proven to be a powerful tool in the monitoring of antimicrobial resistance (AMR). Here, we provide a comparative analysis of the resistome from pigs, poultry, veal calves, turkey, and rainbow trout, for a total of 538 herds across nine European countries. We calculated the effects of per-farm management practices and antimicrobial usage (AMU) on the resistome in pigs, broilers, and veal calves. We also provide an in-depth study of the associations between bacterial diversity, resistome diversity, and AMR abundances as well as co-occurrence analysis of bacterial taxa and antimicrobial resistance genes (ARGs) and the universality of the latter. The resistomes of veal calves and pigs clustered together, as did those of avian origin, while the rainbow trout resistome was different. Moreover, we identified clear core resistomes for each specific food-producing animal species. We identified positive associations between bacterial alpha diversity and both resistome alpha diversity and abundance. Network analyses revealed very few taxa-ARG associations in pigs but a large number for the avian species. Using updated reference databases and optimized bioinformatics, previously reported significant associations between AMU, biosecurity, and AMR in pig and poultry farms were validated. AMU is an important driver for AMR; however, our integrated analyses suggest that factors contributing to increased bacterial diversity might also be associated with higher AMR load. We also found that dispersal limitations of ARGs are shaping livestock resistomes, and future efforts to fight AMR should continue to emphasize biosecurity measures.IMPORTANCEUnderstanding the occurrence, diversity, and drivers for antimicrobial resistance (AMR) is important to focus future control efforts. So far, almost all attempts to limit AMR in livestock have addressed antimicrobial consumption. We here performed an integrated analysis of the resistomes of five important farmed ani

Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedentedly broad and deep view of the genus Enterococcus, including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found.

Streptococcus suis (S. suis) infections in weaned pigs are common and responsible for a high consumption of antimicrobials, and their presence is assumed to be multi-factorial. A specific evidence-based veterinary guideline to support the control of S. suis in weaned pigs was developed for veterinary practitioners in the Netherlands in 2014. Adherence to the S. suis clinical practice guideline helps veterinary practitioners to prevent and control the disease in a systematical approach and thereby improve antimicrobial stewardship and contribute to the prevention of antimicrobial resistance in animals and humans. The impact of such a clinical practice guideline on (animal) disease management depends not only on its content, but also largely on the extent to which practitioners adhere to the clinical guideline in practice. When the S. suis guideline was published, no specific activities were undertaken to support veterinarians' uptake and implementation, thereby contributing to suboptimal adherence in clinical practice. As the S. suis guideline was comprehensively written by veterinary experts following an evidence-based approach, our aim was not to judge the (scientific) quality of the guideline but to study the possibility to improve the currently low adherence of this guideline in veterinary practice. This paper describes the systematic development, using Implementation Mapping, of a theory-based intervention program to support swine veterinarians' adherence to the S. suis guideline. The knowledge, skills, beliefs about capabilities, and beliefs about consequences domains are addressed in the program, which includes seven evidence-based methods (modelling, tailoring, feedback, discussion, persuasive communication, active learning, and self-monitoring) for use in program activities such as a peer-learning meeting and an e-learning module. The intervention program has been developed for practicing swine veterinarians, lasts eight months, and is evaluated through a st

In 2020 and 2022, nine cases of surgical site infections with a methicillin-resistant Staphylococcus aureus (MRSA) were diagnosed in horses in an equine referral clinic. Sixteen isolates (horses, n=9; environment, n=3; and staff members, n=4) were analysed retrospectively using Nanopore whole-genome sequencing to investigate the relatedness of two suspected MRSA outbreaks (2020 and 2022). The MRSA isolates belonged to ST398 and ST612. ST398 genomes from 2020 and 2022 formed three phylogenetic clusters. The first ST398 cluster from 2020 consisted of isolates from five horses and one staff member, and we suspected within clinic transmission. The second cluster of ST398 isolates from 2022 originated from two horses and two staff members but showed higher single nucleotide polymorphism (SNP) distances. One ST398 isolate from an individual staff member was not related to the other two clusters. The ST612 isolates were isolated in 2022 from two horses and three environmental samples and showed very low SNP distances (<7 SNPs), indicating the transmission of MRSA ST612 in this clinic in 2022. Molecular characterization revealed an abundant set of virulence genes and plasmids in the ST612 isolates in comparison to ST398 isolates. Phenotypic antimicrobial susceptibility showed that differences between the two sequence types were consistent with the genetic characteristics. MRSA ST612 has not been reported in Europe before, but it is a dominant clone in African hospitals and has been described in horses and people working with horses in Australia, indicating the importance of surveillance.

OBJECTIVES: Antimicrobials can select for antimicrobial-resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable, recirculation of subinhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens.METHODS: The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (unstable), doxycycline or enrofloxacin (stable). Antimicrobials were extracted from the faecal samples and were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism.RESULTS: Up to 37 days after treatment, doxycycline and enrofloxacin had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the amoxicillin treatment. The amoxicillin treatment showed a significant difference (P ≤ 0.01 and P ≤ 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic resistance and a significant difference (P ≤ 0.05 and P ≤ 0.0001) in genotypic resistance throughout the trial. Furthermore, enrofloxacin treatment resulted in a complete non-WT E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations.CONCLUSIONS: Based on our findings, we suggest that persistence of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.

Considering global antimicrobial resistance (AMR) prevalence, alternative or complementary strategies to antimicrobial use, are of interest. Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is of particular interest as despite significant AMU reduction, LA-MRSA prevalence in pig husbandry has not decreased. We performed targeted isolation of bacterial species with potential antagonism against LA-MRSA in pig farms. Duplicate piglet nasal swabs from three European countries (Germany, Ireland and The Netherlands) were taken longitudinally from birth up to 10 weeks, one for amplicon sequencing and qPCR, and the other was cryopreserved for culturing. We identified potential probiotic species by anticorrelation analysis of bacterial abundance from amplicon sequencing data with quantitative S. aureus estimates from qPCR data from the samples. A literature-screen was performed on the species identified, to determine their probiotic potential. Following this, 1302 isolates were grown from selected cryopreserved swabs and identified using MALDI-TOF and additional 16S rRNA gene sequencing to isolate the anticorrelating species. Ninety-five isolates of interest were screened for absence of tetracycline resistance and hemolytic activity and whole genome sequencing was conducted to verify their taxonomy and to assess their AMR and virulence gene profile. Additional phenotypic antimicrobial resistance testing selected three different Lactococcus lactis strains. During an in vitro challenge using spent medium, all three strains demonstrated inhibition against two S. aureus strains. These L. lactis strains may have the ability to be used safely to reduce LA-MRSA carriage in the nasal passages of pigs but further in vivo testing is necessary to confirm this potential.Importance An approach to tackle antimicrobial resistance in livestock is to use competitive exclusion agents. We employed a sequencing guided isolation approach to identify bacterial species wi

Antimicrobials can select for antimicrobial resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable and very persistent, recirculation of sub-inhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens.The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (non-persistent), doxycycline or enrofloxacin (persistent). Antimicrobials were extracted from the faecal samples and concentrations were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism.Up to 37 days after treatment, persistent antimicrobials (doxycycline and enrofloxacin) had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the non-persistent (amoxicillin) treatment. The amoxicillin treatment showed a significant difference (p ≤ 0.01 and p ≤ 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic and a significant difference (p ≤ 0.05 and p ≤ 0.0001) in genotypic resistance throughout the trial. Furthermore, the enrofloxacin treatment resulted in a complete enrofloxacin-resistant E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations.Based on our findings, we suggest that persistency of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.Competing Interest StatementThe authors have declared no competing interest.

The use of antimicrobials in the pig sector in the Netherlands has been reduced by more than 70% over the last decade. However, there is still a considerable number of pig farms that have not been able to lower their antimicrobial usage (AMU) to a sufficiently low level, comparable to the majority of the other pig farms. Therefore, an intervention study was initiated to lower on-farm antimicrobial use in which 45 pig farms with high AMU were recruited. These farms were coached over a period of 2 years whereby different management interventions were introduced. During the 2-year study period a significant reduction of 13 and 17% in total AMU was seen in weaned piglets and fattening pigs respectively. The introduction of coaching as well as multiple management interventions were (univariably) associated with the decrease in AMU. After mutual adjustment of coaching and individual interventions, the association between coaching and AMU became substantially weaker, indicating that coaching and interventions were interrelated and specific interventions explained the reduction in AMU. In conclusion, a coaching effect was observed in this study, with an effect on AMU through specific interventions. More insights are needed regarding the role and effects of coaching on the influence on the management team comprising the farmer, veterinarian and (feed) advisor, and interventions implemented.

Streptococcus suis (S. suis) infections in weaned pigs are common and responsible for a high consumption of antimicrobials, and their presence is assumed to be multi-factorial. A specific evidence-based veterinary guideline to support the control of S. suis in weaned pigs was developed for veterinary practitioners in the Netherlands in 2014. Adherence to the S. suis clinical practice guideline helps veterinary practitioners to prevent and control the disease in a systematical approach and thereby improve antimicrobial stewardship and contribute to the prevention of antimicrobial resistance in animals and humans. The impact of such a clinical practice guideline on (animal) disease management depends not only on its content, but also largely on the extent to which practitioners adhere to the clinical guideline in practice. When the S. suis guideline was published, no specific activities were undertaken to support veterinarians’ uptake and implementation, thereby contributing to suboptimal adherence in clinical practice. As the S. suis guideline was comprehensively written by veterinary experts following an evidence-based approach, our aim was not to judge the (scientific) quality of the guideline but to study the possibility to improve the currently low adherence of this guideline in veterinary practice. This paper describes the systematic development, using Implementation Mapping, of a theory-based intervention program to support swine veterinarians’ adherence to the S. suis guideline. The knowledge, skills, beliefs about capabilities, and beliefs about consequences domains are addressed in the program, which includes seven evidence-based methods (modelling, tailoring, feedback, discussion, persuasive communication, active learning, and self-monitoring) for use in program activities such as a peer-learning meeting and an e-learning module. The intervention program has been developed for practicing swine veterinarians, lasts eight months, and is evaluated through a st

Aspergillus fumigatus is a saprophytic fungal pathogen that causes opportunistic infections in animals and humans. Azole resistance has been reported globally in human A. fumigatus isolates, but the prevalence of resistance in isolates from animals is largely unknown. A retrospective resistance surveillance study was performed using a collection of clinical A. fumigatus isolates from various animal species collected between 2015 and 2020. Agar-based azole resistance screening of all isolates was followed by in vitro antifungal susceptibility testing and cyp51A gene sequencing of the azole-resistant isolates. Over the 5 year period 16 (11.3%) of 142 A. fumigatus culture-positive animals harbored an azole-resistant isolate. Resistant isolates were found in birds (15%; 2/13), cats (21%; 6/28), dogs (8%; 6/75) and free-ranging harbor porpoise (33%; 2/6). Azole-resistance was cyp51A mediated in all isolates: 81.3% (T-67G/)TR34/L98H, 12.5% TR46/Y121F/T289A. In one azole-resistant A. fumigatus isolate a combination of C(-70)T/F46Y/C(intron7)T/C(intron66)T/M172V/E427K single-nucleotide polymorphisms in the cyp51A gene was found. Of the animals with an azole-resistant isolate and known azole exposure status 71.4% (10/14) were azole naive. Azole resistance in A. fumigatus isolates from animals in the Netherlands is present and predominantly cyp51A TR-mediated, supporting an environmental route of resistance selection. Our data supports the need to include veterinary isolates in resistance surveillance programs. Veterinarians should consider azole resistance as a reason for therapy failure when treating aspergillosis and consider resistance testing of relevant isolates.