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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.

BACKGROUND: Antimicrobial use (AMU) has decreased significantly in Dutch pig farms since 2009. However, this decrease has stagnated recently, with relatively high AMU levels persisting mainly among weaners. The aim of this study was to identify farm-level characteristics associated with: i) total AMU and ii) use of specific antimicrobial classes.METHODS: In 2020, cross-sectional data from 154 Dutch pig farms were collected, including information on AMU and farm characteristics. A mixed-effects conditional Random Forest analysis was applied to select the subset of features that was best associated with AMU.RESULTS: The main risk factors for total AMU in weaners were vaccination for PRRS in sucklings, being a conventional farm (vs. not), high within-farm density, and early weaning. The main protective factors for total AMU in sows/sucklings were E. coli vaccination in sows and having boars for estrus detection from own production. Regarding antimicrobial class-specific outcomes, several risk factors overlapped for weaners and sows/sucklings, such as farmer's non-tertiary education, not having free-sow systems during lactation, and conventional farming. An additional risk factor for weaners was having fully slatted floors. For fatteners, the main risk factor for total AMU was PRRS vaccination in sucklings.CONCLUSIONS: Several factors found here to be associated with AMU. Some were known but others were novel, such as farmer's tertiary education, low pig aggression and free-sow systems which were all associated with lower AMU. These factors provide targets for developing tailor-made interventions, as well as an evidence-based selection of features for further causal assessment and mediation analysis.

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.

The transmission rate per hour between hosts is a key parameter for simulating transmission dynamics of antibiotic-resistant bacteria, and might differ for antibiotic resistance genes, animal species, and antibiotic usage. We conducted a Bayesian meta-analysis of resistant Escherichia coli (E. coli) transmission in broilers and piglets to obtain insight in factors determining the transmission rate, infectious period, and reproduction ratio. We included blaCTX-M-1, blaCTX-M-2, blaOXA-162, catA1, mcr-1, and fluoroquinolone resistant E. coli. The Maximum a Posteriori (MAP) transmission rate in broilers without antibiotic treatment ranged from 0.4∙10 -3 to 2.5∙10 -3 depending on type of broiler (SPF vs conventional) and inoculation strains. For piglets, the MAP in groups without antibiotic treatment were between 0.7∙10 -3 and 0.8∙10 -3, increasing to 0.9∙10 -3 in the group with antibiotic treatment. In groups without antibiotic treatment, the transmission rate of resistant E. coli in broilers was almost twice the transmission rate in piglets. Amoxicillin increased the transmission rate of E. coli carrying bla CTX-M-2 by three-fold. The MAP infectious period of resistant E. coli in piglets with and without antibiotics is between 971 and 1065 hours (40 - 43 days). The MAP infectious period of resistant E. coli in broiler without antibiotics is between 475 and 2306 hours (20 - 96 days). The MAP infectious period of resistant E. coli in broiler with antibiotics is between 2702 and 3462 hours (113 - 144 days) which means a lifelong colonization. The MAP basic reproduction ratio in piglets of infection with resistant E. coli when using antibiotics is 27.70, which is higher than MAP in piglets without antibiotics between 15.65 and 18.19. The MAP basic reproduction ratio in broilers ranges between 3.46 and 92.38. We consider three possible explanations for our finding that in the absence of antibiotics the transmission rate is higher among broilers than among piglets: i) due

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.

Fluoroquinolone resistance in E. coli isolates from livestock in Europe remains high even though the EMA restricted fluoroquinolone use in animals. However, flumequine, a quinolone similar to fluoroquinolones, is still used in livestock in the Netherland, Belgium, Greece or France. Therefore, we investigated whether flumequine selects for the same resistance mechanisms in E. coli.To accomplish this, we investigated and enumerated resistant and non-resistant E. coli isolates obtained from caecal fermentation assays and from broilers exposed to different concentrations of flumequine and enrofloxacin.Flumequine usage leads to approximately 3-fold increase of resistant E. coli in the caecal fermentation, comparable with enrofloxacin. After in vitro exposure to flumequine and enrofloxacin we detected the same SNPs (S83L, D87G) in gyrA. Furthermore, the same resistance-causing SNPs were found in phenotypic resistant E. coli isolates from broilers treated with enrofloxacin and flumequine.Flumequine induces similar resistance mechanisms as enrofloxacin and restriction for use should be the same.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.

Background: Antimicrobial use (AMU) has decreased significantly in Dutch pig farms since 2009. However, this decrease has stagnated recently, with relatively high AMU levels persisting mainly among weaners. The aim of this study was to identify farm-level characteristics associated with: i) total AMU and ii) use of specific antimicrobial classes. Methods: In 2020, cross-sectional data from 154 Dutch pig farms were collected, including information on AMU and farm characteristics. A mixed-effects conditional Random Forest analysis was applied to select the subset of features that was best associated with AMU. Results: The main risk factors for total AMU in weaners were vaccination for PRRS in sucklings, being a conventional farm (vs. not), high within-farm density, and early weaning. The main protective factors for total AMU in sows/sucklings were E. coli vaccination in sows and having boars for estrus detection from own production. Regarding antimicrobial class-specific outcomes, several risk factors overlapped for weaners and sows/sucklings, such as farmer's non-tertiary education, not having free-sow systems during lactation, and conventional farming. An additional risk factor for weaners was having fully slatted floors. For fatteners, the main risk factor for total AMU was PRRS vaccination in sucklings. Conclusions: Several factors found here to be associated with AMU. Some were known but others were novel, such as farmer's tertiary education, low pig aggression and free-sow systems which were all associated with lower AMU. These factors provide targets for developing tailor-made interventions, as well as an evidence-based selection of features for further causal assessment and mediation analysis.

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. IMPORTANCE Understanding 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 anim

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.

The transmission rate per hour between hosts is a key parameter for simulating transmission dynamics of antibiotic-resistant bacteria, and might differ for antibiotic resistance genes, animal species, and antibiotic usage. We conducted a Bayesian meta-analysis of resistant Escherichia coli (E. coli) transmission in broilers and piglets to obtain insight in factors determining the transmission rate, infectious period, and reproduction ratio. We included blaCTX-M-1, blaCTX-M-2, blaOXA-162, catA1, mcr-1, and fluoroquinolone resistant E. coli. The Maximum a Posteriori (MAP) transmission rate in broilers without antibiotic treatment ranged from 0.4∙10−3 to 2.5∙10−3 depending on type of broiler (SPF vs conventional) and inoculation strains. For piglets, the MAP in groups without antibiotic treatment were between 0.7∙10−3 and 0.8∙10−3, increasing to 0.9∙10−3 in the group with antibiotic treatment. In groups without antibiotic treatment, the transmission rate of resistant E. coli in broilers was almost twice the transmission rate in piglets. Amoxicillin increased the transmission rate of E. coli carrying blaCTX-M-2 by three-fold. The MAP infectious period of resistant E. coli in piglets with and without antibiotics is between 971 and 1065 hours (40 – 43 days). The MAP infectious period of resistant E. coli in broiler without antibiotics is between 475 and 2306 hours (20 – 96 days). The MAP infectious period of resistant E. coli in broiler with antibiotics is between 2702 and 3462 hours (113 – 144 days) which means a lifelong colonization. The MAP basic reproduction ratio in piglets of infection with resistant E. coli when using antibiotics is 27.70, which is higher than MAP in piglets without antibiotics between 15.65 and 18.19. The MAP basic reproduction ratio in broilers ranges between 3.46 and 92.38. We consider three possible explanations for our finding that in the absence of antibiotics the transmission rate is higher among broilers than among piglets: i) due to the g

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 animal

Antimicrobial resistance is a persistent challenge in human and veterinary medicine, which is often encoded on plasmids which are transmissible between bacterial cells. Incompatibility is the inability of two plasmids to be stably maintained in one cell which is caused by the presence of identical or closely related shared determinants between two plasmids originating from partition or replication mechanisms. For I-complex plasmids in Enterobacteriacae, replication- based incompatibility is caused by the small antisense RNA stem-loop structure called RNAI. The I-complex plasmid group IncK consists of two compatible subgroups, IncK1 and IncK2, for which the RNAI differs only by five nucleotides. In this study we focussed on the interaction of the IncK1 and IncK2 RNAI structures by constructing minireplicons containing the replication region of IncK1 or IncK2 plasmids coupled with a kanamycin resistance marker. Using minireplicons excludes involvement of incompatibility mechanisms other than RNAI. Additionally, we performed single nucleotide mutagenesis targeting the five nucleotides that differ between the IncK1 and IncK2 RNAI sequences of these minireplicons. The obtained results show that a single nucleotide change in the RNAI structure is responsible for the compatible phenotype of IncK1 with IncK2 plasmids. Only nucleotides in the RNAI top loop and interior loop have an effect on minireplicon incompatibility with wild type plasmids, while mutations in the stem of the RNAI structure had no significant effect on incompatibility. Understanding the molecular basis of incompatibility is relevant for future in silico predictions of plasmid incompatibility.

Antibiotic usage in livestock has been suggested as a driver of antimicrobial resistance in human and livestock populations. This has contributed to the implementation of stewardship programs to curtail usage of antibiotics in livestock. However, the consequences of antibiotic curtailment in livestock on human health are poorly understood. There is the potential for increases in the carriage of pathogens such as Salmonella spp. in livestock, and subsequent increases in human foodborne disease. We use a mathematical model fitted to four case studies, ampicillin and tetracycline usage in fattening pig and broiler poultry populations, to explore the impact of curtailing antibiotic usage in livestock on salmonellosis in humans. Increases in the daily incidence of salmonellosis and a decrease in the proportion of resistant salmonellosis were identified following curtailment of antibiotic usage in livestock. The extent of these increases in human foodborne disease ranged from negligible, to controllable through interventions to target the farm-to-fork pathway. This study provides a motivating example of one plausible scenario following curtailment of antibiotic usage in livestock and suggests that a focus on ensuring good farm-to-fork hygiene and livestock biosecurity is sufficient to mitigate the negative human health consequences of antibiotic stewardship in livestock populations.

Animal rehabilitation centres provide a unique opportunity to study the microbiome of wild animals because subjects will be handled for their treatment and can therefore be sampled longitudinally. However, rehabilitation may have unintended consequences on the animals' microbiome because of a less varied and suboptimal diet, possible medical treatment and exposure to a different environment and human handlers. Our study describes the gut microbiome of two large seal cohorts, 50 pups (0-30 days old at arrival) and 23 weaners (more than 60 days old at arrival) of stranded harbour seals admitted for rehabilitation at the Sealcentre Pieterburen in the Netherlands, and the effect of rehabilitation on it. Faecal samples were collected from all seals at arrival, two times during rehabilitation and before release. Only seals that did not receive antimicrobial treatment were included in the study. The average time in rehabilitation was 95 days for the pups and 63 days for the weaners. We observed that during rehabilitation, there was an increase in the relative abundance of some of the Campylobacterota spp and Actinobacteriota spp. The alpha diversity of the pups' microbiome increased significantly during their rehabilitation (p-value <0.05), while there were no significant changes in alpha diversity over time for weaners. We hypothesize that aging is the main reason for the observed changes in the pups' microbiome. At release, the sex of a seal pup was significantly associated with the microbiome's alpha (i.e., Shannon diversity was higher for male pups, p-value <0.001) and beta diversity (p-value 0.001). For weaners, variation in the microbiome composition (beta diversity) at release was partly explained by sex and age of the seal (p-values 0.002 and 0.003 respectively). We mainly observed variables known to change the gut microbiome composition (e.g., age and sex) and conclude that rehabilitation in itself had only minor effects on the gut microbiome of seal pups

The emergence of carbapenemase-producing Enterobacteriaceae (CPE) is a threat to public health, because of their resistance to clinically important carbapenem antibiotics. The emergence of CPE in meat-producing animals is particularly worrying because consumption of meat contaminated with resistant bacteria comparable to CPE, such as extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, contributed to colonization in humans worldwide. Currently, no data on the transmission of CPE in livestock is available. We performed a transmission experiment to quantify the transmission of CPE between broilers to fill this knowledge gap and to compare the transmission rates of CPE and other antibiotic-resistant E. coli. A total of 180 Ross 308 broiler chickens were distributed over 12 pens on the day of hatch (day 0). On day 5, half of the 10 remaining chickens in each pen were orally inoculated with 5·10 2 colony-forming units of CPE, ESBL, or chloramphenicol-resistant E. coli (catA1). To evaluate the effect of antibiotic treatment, amoxicillin was given twice daily in drinking water in 6 of the 12 pens from days 2-6. Cloacal swabs of all animals were taken to determine the number of infectious broilers. We used a Bayesian hierarchical model to quantify the transmission of the E. coli strains. E. coli can survive in the environment and serve as a reservoir. Therefore, the susceptible-infectious transmission model was adapted to account for the transmission of resistant bacteria from the environment. In addition, the caecal microbiome was analyzed on day 5 and at the end of the experiment on day 14 to assess the relationship between the caecal microbiome and the transmission rates. The transmission rates of CPE were 52 - 68 per cent lower compared to ESBL and catA1, but it is not clear if these differences were caused by differences between the resistance genes or by other differences between the E. coli strains. Differences between the groups in transmission r

Campylobacteriosis is a frequently diagnosed disease in humans. Most infections are considered foodborne and are caused by Campylobacter jejuni and C. coli. The animal reservoirs of these Campylobacter species, and the sources and routes of transmission, are described and discussed in this chapter. Most warm-blooded animals can be colonized by Campylobacter, but avian species, and in particular poultry, are preferred hosts. Much of the world’s poultry production is colonized by Campylobacter. Source attribution studies estimate that 20–40% of cases are attributed to the handling and consumption of chicken meat, while up to 80% of cases are due to Campylobacter found in the chicken reservoir. The difference suggests that routes other than through the food chain, i.e., environmental contamination, are important. The epidemiology of infections in humans differs between industrialized and low- and middle-income countries. Thus, the most effective interventions would be targeted to primary production. To date, only improved biosecurity is available. If effectively implemented, strict biosecurity can reduce the number of Campylobacter-positive flocks, but implementation to this level has proved difficult for the poultry industry. Available interventions in chicken processing plants can substantially reduce Campylobacter numbers on carcasses and consequently reduce the risk to humans. Public health strategies therefore utilize control programs, which aim at reducing the level of Campylobacter by measures along the food chain. It is now recognized that commercially acceptable complementary interventions for primary production, such as vaccines and feed additives, are urgently needed. Once Campylobacter in poultry is controlled then other minor sources of Campylobacter including contaminated drinking water, direct contact with (pet) animals, and other food items (e.g., red meat and milk) can be addressed.

The gut microbiome of humans and animals acts as a reservoir of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC). Dogs are known for having a high prevalence of ESBL-EC in their gut microbiota, although their ESBL-EC carrier status often shifts over time. We hypothesized that the gut microbiome composition of dogs is implicated in ESBL-EC colonization status. Therefore, we assessed whether ESBL-EC carriage in dogs is associated with changes in the gut microbiome and resistome. Fecal samples were collected longitudinally from 57 companion dogs in the Netherlands every 2 weeks for a total of 6 weeks ( n = 4 samples/dog). Carriage of ESBL-EC was determined through selective culturing and PCR and in line with previous studies, we observed a high prevalence of ESBL-EC carriage in dogs. Using 16s rRNA gene profiling we found significant associations between detected ESBL-EC carriage and an increased abundance of Clostridium sensu stricto 1, Enterococcus, Lactococcus, and the shared genera of Escherichia -Shigella in the dog microbiome. A resistome capture sequencing approach (ResCap) furthermore, revealed associations between detected ESBL-EC carriage and the increased abundance of the antimicrobial resistance genes: cmlA, dfrA, dhfR, floR, and sul3. In summary, our study showed that ESBL-EC carriage is associated with a distinct microbiome and resistome composition. IMPORTANCE The gut microbiome of humans and animals is an important source of multidrug resistant pathogens, including beta-lactamase-producing Escherichia coli (ESBL-EC). In this study, we assessed if the carriage of ESBL-EC in dogs was associated with changes in gut composition of bacteria and antimicrobial resistant genes (ARGs). Therefore, stool samples from 57 dogs were collected every 2 weeks for a total of 6 weeks. Sixty eight percent of the dogs carried ESBL-EC during at least one of the time points analyzed. By investigating the gut microbiome and resistome composition, we ob

Stray cats can host (zoonotic) viral pathogens and act as a source of infection for domestic cats or humans. In this cross-sectional (sero)prevalence study, sera from 580 stray cats living in 56 different cat groups in rural areas in The Netherlands were collected from October 2020 to July 2022. These were used to investigate the prevalence of the cat-specific feline leukemia virus (FeLV, n = 580), the seroprevalence of the cat-specific feline viruses feline immunodeficiency virus (FIV, n = 580) and feline coronavirus (FCoV, n = 407), and the zoonotic virus severe acute respiratory coronavirus-2 (SARS-CoV-2, n = 407) using enzyme-linked immunosorbent assays (ELISAs). ELISA-positive results were confirmed using Western blot (FIV) or pseudovirus neutralization test (SARS-CoV-2). The FIV seroprevalence was 5.0% (95% CI (Confidence Interval) 3.4–7.1) and ranged from 0–19.0% among groups. FIV-specific antibodies were more often detected in male cats, cats ≥ 3 years and cats with reported health problems. No FeLV-positive cats were found (95% CI 0.0–0.6). The FCoV seroprevalence was 33.7% (95% CI 29.1–38.5) and ranged from 4.7–85.7% among groups. FCoV-specific antibodies were more often detected in cats ≥ 3 years, cats with reported health problems and cats living in industrial areas or countryside residences compared to cats living at holiday parks or campsites. SARS-CoV-2 antibodies against the subunit 1 (S1) and receptor binding domain (RBD) protein were detected in 2.7% (95% CI 1.4–4.8) of stray cats, but sera were negative in the pseudovirus neutralization test and therefore were considered SARS-CoV-2 suspected. Our findings suggest that rural stray cats in The Netherlands can be a source of FIV and FCoV, indicating a potential risk for transmission to other cats, while the risk for FeLV is low. However, suspected SARS-CoV-2 infections in these cats were uncommon. We found no evidence of SARS-CoV-2 cat-to-cat spread in the studied stray cat groups and consider the li

INTRODUCTION: Indonesia has a large broiler industry with extensive antimicrobial use (AMU) according to empirical evidence. However, there are no quantitative data of on-farm AMU. Quantification of AMU at farm level is crucial to guide interventions on antimicrobial stewardship (AMS). The objective of this study was to compare on-farm AMU monitoring methods, to assess which monitoring method is best suited to gain insight in the quantitative AMU at farm level in medium-scale Indonesian broiler farms.METHOD: AMU was calculated using four different indicators-mg/PCU (mass-based), TF UDDindo (Treatment Frequency of Used Daily Dose, dose-based), TF DDDvet (Treatment Frequency of Defined Daily Dose, dose-based), and TF count - based (count-based)-for the total AMU of 98 production cycles with an average length of 30 days. RESULTS: Broilers were exposed to an average of 10 days of antimicrobial treatments per production cycle, whereas 60.8% of the antimicrobials belonged to the Highest Priority Critically Important Antimicrobials (HPCIAs). For each pair of indicators, the Spearman rank correlation coefficient was calculated to assess if the production cycles were ranked consistently in increasing AMU across the different indicators. The correlation varied between 0.4 and 0.8.DISCUSSION: This study illustrates the considerable difference in the ranking of AMU between the different indicators. In a setting comparable to medium-scale broiler farms in Indonesia, where resources are scarce and there is no professional oversight, the TF count - based method is best suitable. Before implementing an AMU monitoring method, careful consideration of the use-indicators is paramount to achieve fair benchmarking.

The emergence of bacterial strains resistant to topical antimicrobials in both human and veterinary medicine has raised concerns over retaining the efficacy of these preparations. Yet, little information is available regarding the use of topical antimicrobials in either sector for planning targeted interventions. This study aims to quantify the use of topical antimicrobials in 44 Dutch companion animal clinics before and during their participation in an antimicrobial stewardship programme (ASP), to explore the effect of the intervention on topical antimicrobial use (AMU). Hence, prescription and clinic animal population data, collected from July 2012 until June 2018 were used. Specifically, the period from July 2012 until June 2015 was defined as pre-intervention period, whereas clinics started to participate in the ASP from March 2016 onwards. As quantification metric, the Defined Daily Dose for Animals (DDDA) was used and a mixed effect times series model with auto-regression was applied to monthly topical AMU data. The intervention effect was modelled using a step function with a change in (linear) time trend and clinic characteristics, as potential determinants of topical AMU, were assessed using a multivariable regression model. A seasonal pattern was identified, in the pre-intervention period, where topical AMU was highest in July-August and lowest in February-March. In addition, total topical AMU appeared to significantly decrease over time in the pre-intervention period and the proportion of dogs in the clinic was positively associated with topical AMU. The intervention effect was significant only for second line and for skin product AMU. This study demonstrates that during participation in an ASP, second line and skin product AMU decreased in Dutch companion animal clinics. Additionally, this study demonstrates the existence of a seasonal effect and a decrease in topical AMU over time already before introduction of a targeted intervention.

Vibrio parahaemolyticus is an important food-borne human pathogen and presents immunogenic surface polysaccharides, which can be used to distinguish problematic and disease-causing lineages. V. parahaemolyticus is divided in 16 O-serotypes (O-antigen) and 71 K-serotypes (K-antigen). Agglutination tests are still the gold standard for serotyping, but many V. parahaemolyticus isolates are not typable by agglutination. An alternative for agglutination tests is genotyping using whole-genome sequencing data, by which K- and O- genotypes have been curated and identified previously for other clinically relevant organisms with the software tool Kaptive. In this study, V. parahaemolyticus isolates were serotyped and sequenced, and all known and several novel O- and K-loci were identified. We developed Kaptive databases for all O- and K-loci after manual curation of the loci. In our study, we could genotype the O- and K-loci of 98 and 93 % of the genomes, respectively, with a Kaptive confidence score higher than 'none'. The newly developed Kaptive databases with the identified V. parahaemolyticus O- and K-loci can be used to identify the O- and K-genotypes of V. parahaemolyticus isolates from genome sequences.

The Netherlands has been very successful in the last decade in reducing antimicrobial use in animals. On about a quarter of farms, antimicrobial use in weaned pigs remains relatively high. As Streptococcus suis ( S. suis) infections are responsible for a high consumption of antimicrobials, a specific veterinary guideline to control S. suis was developed, but seemed to be poorly adopted by veterinarians. Guided by the theoretical domains framework, the aim of this study was to identify determinants influencing veterinarians' adherence to this guideline. We interviewed 13 pig veterinarians. Interviewees described multiple approaches to managing S. suis problems and adherence to the guideline. Mentioned determinants could be categorized into 12 theoretical domains. The following six domains were mentioned in all interviews: knowledge, skills, beliefs about capabilities, beliefs about consequences, social influences, and environmental context and resources. The insights derived from this study are relevant for understanding factors influencing veterinarians' adoption of scientific evidence and guidelines and can be used to develop evidence-based implementation strategies for veterinary guidelines.

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

Staphylococcus schleiferi is an opportunistic pathogen in humans and dogs. Recent taxonomic reassignment of its subspecies (S. schleiferi subsp. schleiferi and S. schleiferi subsp. coagulans) into two separate species (S. schleiferi and S. coagulans) lacks supporting data for diagnostic implications and clinical relevance. We aimed to confirm the reclassification of S. schleiferi by using genomic and matrix-Assisted laser desorption ionization-time of flight (MALDI-TOF) data for a large set of isolates from humans and animals to investigate their molecular epidemiology and clinical relevance. Routine MALDI-TOF analysis and Illumina sequencing were performed on 165 S. schleiferi isolates from the Netherlands. With 33 publicly available genomes, the study included 198 genomes from 149 dogs, 34 humans, and 15 other sources. The Type Strain Genome Server was used to identify species in the genomes, and the MALDI-TOF MS database was extended to improve species differentiation. MALDI-TOF did not discriminate between S. schleiferi and S. coagulans. Genome phylogeny distinguished the two species in two monophyletic clusters. S. schleiferi isolates originated from humans, while S. coagulans isolates were found in animals and three human isolates clustering with the animal isolates. The sialidase B gene (nanB) was a unique marker gene for S. schleiferi, whereas the chrA gene was exclusive for S. coagulans. The mecA gene was exclusively detected in S. coagulans, as were the lnu(A), blaZ, erm(B/C), tet(O/M), and aac(69)-Aph(299) genes. The MALDI-TOF database extension did not improve differentiation between the two species. Even though our whole-genome sequencing- based approach showed clear differentiation between these two species, it remains critical to identify S. schleiferi and S. coagulans correctly in routine diagnostics. IMPORTANCE This study clearly shows that S. schleiferi is a concern in human hospital settings, whereas S. coagulans predominantly causes infections

Broiler chickens are among the main livestock sectors worldwide. With individual treatments being inapplicable, contrary to many other animal species, the need for antimicrobial use (AMU) is relatively high. AMU in animals is known to drive the emergence and spread of antimicrobial resistance (AMR). High farm biosecurity is a cornerstone for animal health and welfare, as well as food safety, as it protects animals from the introduction and spread of pathogens and therefore the need for AMU. The goal of this study was to identify the main biosecurity practices associated with AMU in broiler farms and to develop a statistical model that produces customised recommendations as to which biosecurity measures could be implemented on a farm to reduce its AMU, including a cost-effectiveness analysis of the recommended measures. AMU and biosecurity data were obtained cross-sectionally in 2014 from 181 broiler farms across nine European countries (Belgium, Bulgaria, Denmark, France, Germany, Italy, the Netherlands, Poland and Spain). Using mixed-effects random forest analysis (Mix-RF), recursive feature elimination was implemented to determine the biosecurity measures that best predicted AMU at the farm level. Subsequently, an algorithm was developed to generate AMU reduction scenarios based on the implementation of these measures. In the final Mix-RF model, 21 factors were present: 10 about internal biosecurity, 8 about external biosecurity and 3 about farm size and productivity, with the latter showing the largest (Gini) importance. Other AMU predictors, in order of importance, were the number of depopulation steps, compliance with a vaccination protocol for non-officially controlled diseases, and requiring visitors to check in before entering the farm. K-means clustering on the proximity matrix of the final Mix-RF model revealed that several measures interacted with each other, indicating that high AMU levels can arise for various reasons depending on the situation. The alg

Background: Hospitalisation and antimicrobial treatment are common in horses and significantly impact the intestinal microbiota. Antimicrobial treatment might also increase levels of resistant bacteria in faeces, which could spread to other ecological compartments, such as the environment, other animals and humans. In this study, we aimed to characterise the short- and long-term effects of transportation, hospitalisation and trimethoprim-sulfadiazine (TMS) administration on the faecal microbiota and resistome of healthy equids. Methods: In a longitudinal experimental study design, in which the ponies served as their own control, faecal samples were collected from six healthy Welsh ponies at the farm (D0–D13-1), immediately following transportation to the hospital (D13-2), during 7 days of hospitalisation without treatment (D14–D21), during 5 days of oral TMS treatment (D22–D26) and after discharge from the hospital up to 6 months later (D27–D211). After DNA extraction, 16S rRNA gene sequencing was performed on all samples. For resistome analysis, shotgun metagenomic sequencing was performed on selected samples. Results: Hospitalisation without antimicrobial treatment did not significantly affect microbiota composition. Oral TMS treatment reduced alpha-diversity significantly. Kiritimatiellaeota, Fibrobacteres and Verrucomicrobia significantly decreased in relative abundance, whereas Firmicutes increased. The faecal microbiota composition gradually recovered after discontinuation of TMS treatment and discharge from the hospital and, after 2 weeks, was more similar to pre-treatment composition than to composition during TMS treatment. Six months later, however, microbiota composition still differed significantly from that at the start of the study and Spirochaetes and Verrucomicrobia were less abundant. TMS administration led to a significant (up to 32-fold) and rapid increase in the relative abundance of resistance genes sul2, tetQ, ant6-1a, and aph(3”)-lb. lnuC s

Antimicrobial resistance (AMR) is an emerging worldwide problem. Exposure to antimicrobials selects for resistant bacteria which are a health threat for humans and animals. The concentration at which selection for resistant bacteria occurs is often lower than the minimum inhibitory concentration (MIC) and also differs between environments. Defining this minimal selective concentration (MSC) under natural conditions is essential to understand the selective window for resistant bacteria which are exposed to residual antimicrobials in humans, animals and the environment.In this study we estimated the MSCs of three antimicrobial compounds, amoxicillin, doxycycline and enrofloxacin in a complex microbial community by conducting fermentation assays with cecal material derived from broilers. We examined the phenotypic resistance of Escherichia coli, resistome and microbiome after 6 and 30 hours of fermenting in the presence of antimicrobials of interest. The concentrations are 10 to 100 times lower than the epidemiological cut-off values in E. coli for the respective antimicrobials as determined by EUCAST (https://mic.eucast.org/). In contrast to the amoxicillin and doxycycline exposure we could not determine any molecular resistance mechanism in the resistome analysis for the enrofloxacin exposure, likely because they are the result of point mutations.Our findings show at which concentrations there still is selection for AMR bacteria. This knowledge can be used to manage the risk of the emergence of AMR bacteria.Importance Antimicrobial resistance is an emerging threat to the health of humans and animals; it might affect economic prosperity in the future. The rise of antimicrobial resistant bacteria is a consequence of the use of antimicrobial compounds in humans and animals which selects for resistant bacteria. It is critical to understand the relation between the concentrations of antimicrobial compounds and their selection for antimicrobial resistant bacteria. In our s