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.

Global Distribution of O Serotypes and Antibiotic Resistance in Extraintestinal Pathogenic Escherichia coli Collected From the Blood of Patients With Bacteremia Across Multiple Surveillance Studies.

BACKGROUND: Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of bacteremia worldwide, with older populations having increased risk of invasive bacterial disease. Increasing resistance to first-line antibiotics and emergence of multidrug-resistant (MDR) strains represent major treatment challenges. ExPEC O serotypes are key targets for potential multivalent conjugate vaccine development. Therefore, we evaluated the O serotype distribution and antibiotic resistance profiles of ExPEC strains causing bloodstream infections across 4 regions. METHODS: Blood culture isolates from patients aged ≥60 years collected during 5 retrospective E. coli surveillance studies in Europe, North America, Asia-Pacific, and South America (2011-2017) were analyzed. Isolates were O serotyped by agglutination; O genotyping was performed for nontypeable isolates. Antimicrobial susceptibility testing was also conducted. RESULTS: Among 3217 ExPEC blood culture isolates, the most ubiquitous O serotype was O25 (n = 737 [22.9%]), followed by O2, O6, O1, O75, O15, O8, O16, O4, O18, O77 group, O153, O9, O101/O162, O86, and O13 (prevalence of ≥1%). The prevalence of these O serotypes was generally consistent across regions, apart from South America; together, these 16 O serotypes represented 77.6% of all ExPEC bacteremia isolates analyzed. The overall MDR frequency was 10.7%, with limited variation between regions. Within the MDR subset (n = 345), O25 showed a dominant prevalence of 63.2% (n = 218). CONCLUSIONS: Predominant O serotypes among ExPEC bacteremia isolates are widespread across different regions. O25 was the most prevalent O serotype overall and particularly dominant among MDR isolates. These findings may inform the design of multivalent conjugate vaccines that can target the predominant O serotypes associated with invasive ExPEC disease in older adults.

Determination and quantification of microbial communities and antimicrobial resistance on food through host DNA-depleted metagenomics.

Food products carry bacteria unless specifically sterilised. These bacteria can be pathogenic, commensal or associated with food spoilage, and may also be resistant to antimicrobials. Current methods for detecting bacteria on food rely on culturing for specific bacteria, a time-consuming process, or 16S rRNA metabarcoding that can identify different taxa but not their genetic content. Directly sequencing metagenomes of food is inefficient as its own DNA vastly outnumbers the bacterial DNA present. We optimised host DNA depletion enabling efficient sequencing of food microbiota, thereby increasing the proportion of non-host DNA sequenced 13-fold (mean; range: 1.3-40-fold) compared to untreated samples. The method performed best on chicken, pork and leafy green samples which had high mean prokaryotic read proportions post-depletion (0.64, 0.74 and 0.74, respectively), with lower mean prokaryotic read proportions in salmon (0.50) and prawn samples (0.19). We show that bacterial compositions and concentrations of antimicrobial resistance (AMR) genes differed by food type, and that salmon metagenomes were influenced by the production/harvesting method. The approach described in this study is an efficient and effective method of identifying and quantifying the predominant bacteria and AMR genes on food.

Genome-wide association studies of Shigella spp. and Enteroinvasive Escherichia coli isolates demonstrate an absence of genetic markers for prediction of disease severity.

BACKGROUND: We investigated the association of symptoms and disease severity of shigellosis patients with genetic determinants of infecting Shigella and entero-invasive Escherichia coli (EIEC), because determinants that predict disease outcome per individual patient could be used to prioritize control measures. For this purpose, genome wide association studies (GWAS) were performed using presence or absence of single genes, combinations of genes, and k-mers. All genetic variants were derived from draft genome sequences of isolates from a multicenter cross-sectional study conducted in the Netherlands during 2016 and 2017. Clinical data of patients consisting of binary/dichotomous representation of symptoms and their calculated severity scores were also available from this study. To verify the suitability of the methods used, the genetic differences between the genera Shigella and Escherichia were used as control. RESULTS: The isolates obtained were representative of the population structure encountered in other Western European countries. No association was found between single genes or combinations of genes and separate symptoms or disease severity scores. Our benchmark characteristic, genus, resulted in eight associated genes and > 3,000,000 k-mers, indicating adequate performance of the algorithms used. CONCLUSIONS: To conclude, using several microbial GWAS methods, genetic variants in Shigella spp. and EIEC that can predict specific symptoms or a more severe course of disease were not identified, suggesting that disease severity of shigellosis is dependent on other factors than the genetic variation of the infecting bacteria. Specific genes or gene fragments of isolates from patients are unsuitable to predict outcomes and cannot be used for development, prioritization and optimization of guidelines for control measures of shigellosis or infections with EIEC.

Antimicrobial Resistance in Salmonella enterica Serovar Paratyphi B Variant Java in Poultry from Europe and Latin America.

Salmonella enterica serovar Paratyphi B variant Java sequence type 28 is prevalent in poultry and poultry meat. We investigated the evolutionary relatedness between sequence type 28 strains from Europe and Latin America using time-resolved phylogeny and principal component analysis. We sequenced isolates from Colombia, Guatemala, Costa Rica, and the Netherlands and complemented them with publicly available genomes from Europe, Africa, and the Middle East. Phylogenetic time trees and effective population sizes (Ne) showed separate clustering of strains from Latin America and Europe. The separation is estimated to have occurred during the 1980s. Ne of strains increased sharply in Europe around 1995 and in Latin America around 2005. Principal component analysis on noncore genes showed a clear distinction between strains from Europe and Latin America, whereas the plasmid gene content was similar. Regardless of the evolutionary separation, similar features of resistance to ß-lactams and quinolones/fluoroquinolones indicated parallel evolution of antimicrobial resistance in both regions.

Incompatibility and phylogenetic relationship of I-complex plasmids.

Plasmid incompatibility is the inability of two plasmids to be stably maintained in one cell, resulting in loss of one of the plasmids in daughter cells. Dislodgement is a phenotypically distinct form of incompatibility, described as an imperfect reproduction, manifesting in rapid exclusion of a resident plasmid after superinfection. The relationship between plasmids of the phenotypic incompatibility groups IncB/O and IncZ is unclear. Their inability to co-exist was initially referred to as dislodgement while other research reached the conclusion that IncB/O and IncZ plasmids are incompatible. In this manuscript we re-evaluated the relationship between IncB/O and IncZ plasmids to settle these conflicting conclusions. We performed dislodgement testing of R16Δ (IncB/O) and pSFE-059 (IncZ) plasmids by electroporation in a bacterial cell and checked their stability. Stability tests of the obtained plasmid pair showed that the IncB/O plasmid was exclusively and almost completely lost from the heteroplasmid Escherichia coli population. Other IncB/O - IncZ pairs could not form a heteroplasmid population, using conjugation or electroporation. Our data supports the previous suggestion that IncB/O and IncZ plasmids may be considered phenotypically incompatible.

The Contribution of Genetic Variation of Streptococcus pneumoniae to the Clinical Manifestation of Invasive Pneumococcal Disease.

Background: Different clinical manifestations of invasive pneumococcal disease (IPD) have thus far mainly been explained by patient characteristics. Here we studied the contribution of pneumococcal genetic variation to IPD phenotype. Methods: The index cohort consisted of 349 patients admitted to 2 Dutch hospitals between 2000-2011 with pneumococcal bacteremia. We performed genome-wide association studies to identify pneumococcal lineages, genes, and allelic variants associated with 23 clinical IPD phenotypes. The identified associations were validated in a nationwide (n = 482) and a post-pneumococcal vaccination cohort (n = 121). The contribution of confirmed pneumococcal genotypes to the clinical IPD phenotype, relative to known clinical predictors, was tested by regression analysis. Results: Among IPD patients, the presence of pneumococcal gene slaA was a nationwide confirmed independent predictor of meningitis (odds ratio [OR], 10.5; P = .001), as was sequence cluster 9 (serotype 7F: OR, 3.68; P = .057). A set of 4 pneumococcal genes co-located on a prophage was a confirmed independent predictor of 30-day mortality (OR, 3.4; P = .003). We could detect the pneumococcal variants of concern in these patients' blood samples. Conclusions: In this study, knowledge of pneumococcal genotypic variants improved the clinical risk assessment for detrimental manifestations of IPD. This provides us with novel opportunities to target, anticipate, or avert the pathogenic effects related to particular pneumococcal variants, and indicates that information on pneumococcal genotype is important for the diagnostic and treatment strategy in IPD. Ongoing surveillance is warranted to monitor the clinical value of information on pneumococcal variants in dynamic microbial and susceptible host populations.

Phylogenomic Investigation of IncI1-Iγ Plasmids Harboring blaCMY-2 and blaSHV-12 in Salmonella enterica and Escherichia coli in Multiple Countries.

The objective of this study was to elucidate the genetic and evolutionary relatedness of blaCMY-2- and blaSHV-12-carrying IncI1-Iγ plasmids. Phylogenomic analysis based on core genome alignments and gene presence/absence was performed for different IncI1-Iγ sequence types (STs). Most IncI1-Iγ/ST12 and IncI1-Iγ/ST231 plasmids had near-identical core genomes. The data suggest that widely occurring blaCMY-2-carrying IncI1-Iγ/ST12 plasmids originate from a common ancestor. In contrast, blaSHV-12 was inserted independently into different IncI1-Iγ/ST231-related plasmids.

Streptococcus pneumoniae PspC Subgroup Prevalence in Invasive Disease and Differences in Contribution to Complement Evasion.

The pneumococcal capsular serotype is an important determinant of complement resistance and invasive disease potential, but other virulence factors have also been found to contribute. Pneumococcal surface protein C (PspC), a highly variable virulence protein that binds complement factor H to evade C3 opsonization, is divided into two subgroups: choline-bound subgroup I and LPxTG-anchored subgroup II. The prevalence of different PspC subgroups in invasive pneumococcal disease (IPD) and functional differences in complement evasion are unknown. The prevalence of PspC subgroups in IPD isolates was determined in a collection of 349 sequenced strains of Streptococcus pneumoniae isolated from adult patients. pspC deletion mutants and isogenic pspC switch mutants were constructed to study differences in factor H binding and complement evasion in relation to capsule thickness. Subgroup I pspC was far more prevalent in IPD isolates than subgroup II pspC The presence of capsule was associated with a greater ability of bound factor H to reduce complement opsonization. Pneumococcal subgroup I PspC bound significantly more factor H and showed more effective complement evasion than subgroup II PspC in isogenic encapsulated pneumococci. We conclude that variation in the PspC subgroups, independent of capsule serotypes, affects pneumococcal factor H binding and its ability to evade complement deposition.

Campylobacter blaseri sp. nov., isolated from common seals (Phoca vitulina).

During a study to assess the faecal microbiome of common seals (Phoca vitulina) in a Dutch seal rehabilitation centre, 16S rRNA gene sequences of an unknown Campylobacter taxon were identified. Campylobacter isolates, which differed from the established Campylobacter taxa, were cultured and their taxonomic position was determined by a polyphasic study based on ten isolates. The isolates were characterized by 16S rRNA and atpA gene sequence analyses and by conventional phenotypic testing. Based on the whole genome sequences, the average nucleotide identity and core genome phylogeny were determined. The isolates formed a separate phylogenetic clade, divergent from all other Campylobacter taxa and most closely related to Campylobacter corcagiensis, Campylobacter geochelonis and Campylobacter ureolyticus. The isolates can be distinguished phenotypically from all other Campylobacter taxa based on their lack of motility, growth at 25 °C and growth on MacConkey agar. This study shows that these isolates represent a novel species within the genus Campylobacter, for which the name Campylobacter blaseri sp. nov. is proposed. The type strain for this novel species is 17S00004-5T (=LMG 30333T=CCUG 71276T).

Plasmids of Distinct IncK Lineages Show Compatible Phenotypes.

IncK plasmids are some of the main carriers of blaCTX-M-14 and blaCMY-2 genes and show high similarity to other plasmids belonging to the I complex, including IncB/O plasmids. Here, we studied the phylogenetic relationship of 37 newly sequenced IncK and IncB/O plasmids. We show that IncK plasmids can be divided into two compatible lineages named IncK1 and IncK2.

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.

Activation of the l-fucose utilization cluster in Campylobacter jejuni induces proteomic changes and enhances Caco-2 cell invasion and fibronectin binding.

Most Campylobacter jejuni isolates carry the fucose utilization cluster (Cj0480c-Cj0489) that supports the metabolism of l-fucose and d-arabinose. In this study we quantified l-fucose and d-arabinose metabolism and metabolite production, and the impact on Caco-2 cell interaction and binding to fibronectin, using C. jejuni NCTC11168 and the closely related human isolate C. jejuni strain 286. When cultured with l-fucose and d-arabinose, both isolates showed increased survival and production of acetate, pyruvate and succinate, and the respective signature metabolites lactate and glycolic acid, in line with an overall upregulation of l-fucose cluster proteins. In vitro Caco-2 cell studies and fibronectin-binding experiments showed a trend towards higher invasion and a significantly higher fibronectin binding efficacy of C. jejuni NCTC11168 cells grown with l-fucose and d-arabinose, while no significant differences were found with C. jejuni 286. Both fibronectin binding proteins, CadF and FlpA, were detected in the two isolates, but were not significantly differentially expressed in l-fucose or d-arabinose grown cells. Comparative proteomics analysis linked the C. jejuni NCTC11168 phenotypes uniquely to the more than 135-fold upregulated protein Cj0608, putative TolC-like component MacC, which, together with the detected Cj0606 and Cj0607 proteins, forms the tripartite secretion system MacABC with putative functions in antibiotic resistance, cell envelope stress response and virulence in Gram negative pathogenic bacteria. Further studies are required to elucidate the role of the MacABC system in C. jejuni cell surface structure modulation and virulence.

Host associations of Campylobacter jejuni and Campylobacter coli isolates carrying the L-fucose or d-glucose utilization cluster.

Campylobacter was considered asaccharolytic, but is now known to carry saccharide metabolization pathways for L-fucose and d-glucose. We hypothesized that these clusters are beneficial for Campylobacter niche adaptation and may help establish human infection. We investigated the distribution of d-glucose and L-fucose clusters among ∼9600 C. jejuni and C. coli genomes of different isolation sources in the Netherlands, the United Kingdom, the United States of America and Finland. The L-fucose utilization cluster was integrated at the same location in all C. jejuni and C. coli genomes, and was flanked by the genes rpoB, rpoC, rspL, repsG and fusA, which are associated with functions in transcription as well as translation and in acquired drug resistance. In contrast, the flanking regions of the d-glucose utilization cluster were variable among the isolates, and integration sites were located within one of the three different 16S23S ribosomal RNA areas of the C. jejuni and C. coli genomes. In addition, we investigated whether acquisition of the L-fucose utilization cluster could be due to horizontal gene transfer between the two species and found three isolates for which this was the case: one C. jejuni isolate carrying a C. coli L-fucose cluster, and two C. coli isolates which carried a C. jejuni L-fucose cluster. Furthermore, L-fucose utilization cluster alignments revealed multiple frameshift mutations, most of which were commonly found in the non-essential genes for L-fucose metabolism, namely, Cj0484 and Cj0489. These findings support our hypothesis that the L-fucose cluster was integrated multiple times across the C. coli/C. jejuni phylogeny. Notably, association analysis using the C. jejuni isolates from the Netherlands showed a significant correlation between human C. jejuni isolates and C. jejuni isolates carrying the L-fucose utilization cluster. This correlation was even stronger when the Dutch isolates were combined with the isolates from the UK, the USA and Finland. No such correlations were observed for C. coli or for the d-glucose cluster for both species. This research provides insight into the spread and host associations of the L-fucose and d-glucose utilization clusters in C. jejuni and C. coli, and the potential benefits in human infection and/or proliferation in humans, conceivably after transmission from any reservoir.

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.

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.

Development of Kaptive databases for Vibrio parahaemolyticus O- and K-antigen genotyping.

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.

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.

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.

Antimicrobial resistance in Campylobacter fetus: emergence and genomic evolution.

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 presence of ARGs in multiple mobile elements distributed among different Cff lineages highlights the risk for spread and further emergence of AMR in C. fetus. Surveillance for these resistances requires the establishment of ECOFFs for C. fetus.