Highly pathogenic avian influenza (HPAI) H5 virus exposure in domestic cats and rural stray cats, the Netherlands, October 2020 to June 2023.

BackgroundHighly pathogenic avian influenza (HPAI) H5Nx and human H1N1pdm2009 influenza viruses can infect cats. Infections in cats may result in viral adaptations or recombinant viruses, which may facilitate zoonotic transfer.AimWe aimed to investigate the presence of HPAI H5 clade 2.3.4.4 and H1 influenza viruses and antibodies to these viruses in domestic and rural stray cats in the Netherlands and factors associated with exposure.MethodsSera from stray and domestic cats, sampled 2020-2023, were analysed by ELISA and confirmed by hemagglutination inhibition assay (HAI) and pharyngeal swabs and lung tissue for influenza A virus by RT-qPCR.ResultsIn 701 stray cats, 83 (11.8%; 95% confidence interval (CI): 9.5-14.5) sera were positive for HPAI H5 and 65 findings were confirmed. In HAI, two sera were positive for both HPAI H5 and H1. In 871 domestic cats, four (0.46%; 95% CI: 0.13-1.2) sera were HPAI H5 positive and none were confirmed but 40 (4.6%; 95% CI: 3.3-6.2) sera were seropositive for H1 and 26 were confirmed. Stray cats living in nature reserves (odds ratio (OR) = 5.4; 95% CI: 1.5-20.1) and older cats (OR = 3.8; 95% CI: 2.7-7.1) were more likely to be HPAI H5 seropositive. No influenza A virus was detected in 230 cats.ConclusionThe higher HPAI H5 seroprevalence in stray cats compared with domestic cats suggests more frequent viral exposure, most likely due to foraging on wild birds. In contrast, exposure to H1 was more common in domestic cats compared with stray cats.

Campylobacter fetus Plasmid Diversity: Comparative Analysis of Fully Sequenced Plasmids and Proposed Classification Scheme.

Campylobacter fetus is an animal pathogen that contains 2 mammal-associated subspecies: Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius that exhibit different biochemical traits and differences in pathogenicity. Although plasmids are important in the horizontal transfer of antimicrobial resistance genes and virulence factors, C. fetus plasmids are understudied. Here, the closed sequences of 12 plasmids from Spanish C. fetus isolates were compared with the publicly available DNA sequences of C. fetus plasmids and other members of the Campylobacterales order. Sizes of C. fetus plasmids from Spanish isolates ranged between 4 and 50 kb and most of them (10/12) were potentially conjugative. Comparative analysis of the plasmids' gene content revealed a close genetic relationship between the plasmids of C. fetus isolated in Spain and those from other geographical regions, while being clearly distinct from plasmids of other Campylobacter species. Furthermore, C. fetus plasmids were grouped into two main clusters regardless of their geographic location or lineage. The distribution pattern of relaxase, replicase, and single-stranded DNA binding SSB protein encoding genes showed a clustering comparable to that resulting from plasmid whole gene content analysis, suggesting its potential use for the classification of C. fetus plasmids. Most of the larger plasmids harbored mobile genetic elements. These results can help to better understand the evolutionary dynamics and pathogenic implications of C. fetus plasmids.

The developing pig respiratory microbiome harbors strains antagonistic to common respiratory pathogens.

In the global efforts to combat antimicrobial resistance and reduce antimicrobial use in pig production, there is a continuous search for methods to prevent and/or treat infections. Within this scope, we explored the relationship between the developing piglet nasal microbiome and (zoonotic) bacterial pathogens from birth until 10 weeks of life. The nasal microbiome of 54 pigs was longitudinally studied over 16 timepoints on 9 farms in 3 European countries (Germany, Ireland, and the Netherlands) using amplicon sequencing targeting the V3-V4 16S rRNA region as well as the tuf gene for its staphylococcal discrimination power. The piglets' age, the farm, and the litter affected the nasal microbiome, with piglets' age explaining 19% of the variation in microbial composition between samples. Stabilization of the microbiome occurred around 2 weeks post-weaning. Notably, while opportunistic pathogens were ubiquitously present, they did not cause disease. The piglet nasal microbiome often carried species associated with gut, skin, or vagina, which suggests that contact with the vaginal and fecal microbiomes shapes the piglet nasal microbiome. We identified bacterial co-abundance groups of species that were present in the nasal microbiomes in all three countries over time. Anti-correlation between these species and known bacterial pathogens identified species that might be exploited for pathogen reduction. Further experimental evidence is required to confirm these findings. Overall, this study advances our understanding of the piglet nasal microbiome, the factors influencing it, and its longitudinal development, providing insights into its role in health and disease. IMPORTANCE: Our study on the nasal microbiota development in piglets across farms in three European countries found that the microbiomes developed similarly in all locations. Additionally, we observed that the colonization of porcine pathogens was either positively or negatively associated with the presence of other bacterial species. These findings enhance our knowledge of co-colonizing species in the nasal cavity and the identified microbial interactions that can be explored for the development of interventions to control pathogens in porcine husbandry.

Selection for amoxicillin-, doxycycline-, and enrofloxacin-resistant Escherichia coli at concentrations lower than the ECOFF in broiler-derived cecal fermentations.

Antimicrobial resistance (AMR) is an emerging worldwide problem and a health threat for humans and animals. Antimicrobial usage in human and animal medicine or in agriculture results in selection for AMR. The selective concentration of antimicrobial compounds can be lower than the minimum inhibitory concentration and differs between environments, which can be a reason for bacterial resistance. Therefore, knowledge of the minimal selective concentration (MSC), under natural conditions, is essential to understand the selective window of bacteria when exposed to residual antimicrobials. In this study, we estimated the MSCs of three antimicrobials, 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 the antimicrobials of interest. The concentrations were estimated to be 10-100 times lower than the epidemiological cut-off values in E. coli for the respective antimicrobials as determined by EUCAST, resulting in an MSC between 0.08 and 0.8 mg/L for amoxicillin, 0.4 and 4 mg/L for doxycycline, and 0.0125 and 0.125 mg/L for enrofloxacin. Additionally, resistome analysis provided an MSC for doxycycline between 0.4 and 4 mg/L, but amoxicillin and enrofloxacin exposure did not induce a significant difference. Our findings indicate at which concentrations there is still selection for antimicrobial-resistant bacteria. This knowledge can be used to manage the risk of the emergence of antimicrobial-resistant bacteria.IMPORTANCEAntimicrobial resistance possibly affects human and animal health, as well as economic prosperity in the future. The rise of antimicrobial-resistant bacteria is a consequence of using antimicrobial compounds in humans and animals selecting for antimicrobial-resistant bacteria. Concentrations reached during treatment are known to be selective for resistant bacteria. However, at which concentrations residues are still selective is important, especially for antimicrobial compounds that remain in the environment at low concentrations. The data in this paper might inform decisions regarding guidelines and regulations for the use of specific antimicrobials. In this study, we are providing these minimal selective concentrations for amoxicillin, doxycycline, and enrofloxacin in complex environments.

Campylobacter fetus subspecies specific PCR assays inferred from comparative genomic analysis for accurate subspecies identification.

Bovine Genital Campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis and is a notifiable disease to the WOAH (World Organisation for Animal Health). For an effective BGC control program, the reliable differentiation of Campylobacter fetus subsp. venerealis (Cfv) from the closely related Campylobacter fetus subsp. fetus (Cff) is required. However, the available molecular C. fetus subspecies identification assays lack sensitivity and specificity to differentiate C. fetus isolates based on their phenotypic or genotypic differences. Furthermore, the current biochemical subspecies identification is not fully congruent with the genomic differentiation of C. fetus strains. In this study, the genome sequences of 41C. fetus strains with well identified subspecies, were analyzed with the large-scale BLAST score ratio (LS-BSR) pipeline to identify Cff and Cfv specific sequences. With this analysis, the asd gene encoding an aspartate-semialdehyde dehydrogenase was identified, which contained a 6-bp Cff-specific sequence, and this 6-bp sequence was absent in the asd gene of Cfv strains. This sequence was used for the development of PCR assays to differentiate Cff and Cfv strains. The C. fetus subspecies identification of the developed asd PCR assays was in full congruence with the genomic classification of strains and are recommended for molecular identification of C. fetus subspecies in BGC control programs. The asd PCR can be assessed on sequenced genomes using a web interface containing the Cfvcatch tool, which includes placement of the tested genome in a phylogenetic tree with reference C. fetus genomes to distinguish the two subspecies and to detect antimicrobial resistance genes.

A coaching approach to strengthen farm management teams to reduce antimicrobial use in Dutch high usage pig farms: a 2 year intervention study.

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.

Methicillin-resistant Staphylococcus aureus outbreak in a Dutch equine referral clinic.

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.

Extended period of selection for antimicrobial resistance due to recirculation of persistent antimicrobials in broilers.

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.

Azole Resistance in Veterinary Clinical Aspergillus fumigatus Isolates in the Netherlands.

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.

Risk factors for antimicrobial use in Dutch pig farms: A cross-sectional study.

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.

Using Implementation Mapping to develop an intervention program to support veterinarians' adherence to the guideline on Streptococcus suis clinical practice in weaned pigs.

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 stepped-wedge design. The Implementation Mapping approach ensured that all relevant adopters and implementers were involved, and that outcomes, determinants (influencing factors), and objectives were systematically discussed.

The European livestock resistome.

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 populations across Europe finding that the resistome and AMR levels are also shaped by factors related to bacterial diversity, as well as dispersal limitations. Thus, future studies and interventions aimed at reducing AMR should not only address antimicrobial usage but also consider other epidemiological and ecological factors.

Transmission rates of veterinary and clinically important antibiotic resistant Escherichia coli: A meta- ANALYSIS.

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 gut microbiome of animals, ii) fitness costs of bacteria, and iii) differences in experimental set-up between the studies. Regarding infectious period and reproduction ratio, the effect of the resistance gene, antibiotic treatment, and animal species are inconclusive due to limited data.

Global diversity of enterococci and description of 18 previously unknown species.

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.

Characterising the gut microbiome of stranded harbour seals (Phoca vitulina) in rehabilitation.

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 and seal weaners.

Understanding the genetic basis of the incompatibility of IncK1 and IncK2 plasmids.

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.

Modelling the effects of antibiotic usage in livestock on human salmonellosis.

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.

Safety Evaluation of an Intranasally Applied Cocktail of Lactococcus lactis Strains in Pigs.

Three Lactococcus lactis strains from the nasal microbiota of healthy pigs were identified as candidates for reducing MRSA in pigs. The safety of nasal administration of a cocktail of these strains was examined in new-born piglets. Six days pre-farrowing, twelve sows were assigned to the placebo or cocktail group (n = 6/group). After farrowing, piglets were administered with either 0.5 mL of the placebo or the cocktail to each nostril. Health status and body weight were monitored at regular time points. Two piglets from three sows/treatment group were euthanised at 24 h, 96 h and 14 d after birth, and conchae, lung and tonsil samples were collected for histopathological and gene expression analysis. Health scores were improved in the cocktail group between d1-5. Body weight and daily gains did not differ between groups. Both groups displayed histological indications of euthanasia and inflammation in the lungs, signifying the findings were not treatment related. The expression of pBD2, TLR9 and IL-1ß in the nasal conchae differed between groups, indicating the cocktail has the potential to modulate immune responses. In summary, the L. lactis cocktail was well tolerated by piglets and there was no negative impact on health scores, growth or lung histopathology indicating that it is safe for administration to new-born piglets.

Comparing the transmission of carbapenemase-producing and extended-spectrum beta-lactamase-producing Escherichia coli between broiler chickens.

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 5·102 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 rates and microbiome diversity did not correspond to each other, indicating that differences in transmission rates were probably not caused by major differences in the community structure in the caecal microbiome. Amoxicillin treatment from day 2-6 increased the transmission rate more than three-fold in all inoculums. It also increased alpha-diversity compared to untreated animals on day 5, but not on day 14, suggesting only a temporary effect. Future research could incorporate more complex transmission models with different species of resistant bacteria into the Bayesian hierarchical model.

Gut Colonization by ESBL-Producing Escherichia coli in Dogs Is Associated with a Distinct Microbiome and Resistome Composition.

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 observed specific changes at time points when dogs were colonized with ESBL-EC compared to time points whenESBL-EC were not detected. In conclusion, our study highlights the importance to study the microbial diversity in companion animals, as gut colonization of particular antimicrobial resistant bacteria might be an indication of a changed microbial composition that is associated with the selection of particular ARGs.