Abstract Background Erysipelas, caused by infection with Erysipelothrix rhusiopathiae (ER), is an emerging disease in non-cage housed laying hens. According to Swedish field experience outbreaks seem more common in older flocks and may also occur in flocks vaccinated against erysipelas. This cross-sectional study aimed to assess single-dose vaccination outcome in laying hens with respect to antibody titres to ER and to monitor selected immune parameters to identify putative risk factors for erysipelas. Blood samples were collected from laying hen flocks at 30, 50 and 75 weeks of age. At each time point, 5 unvaccinated flocks and 5 flocks that had been vaccinated against erysipelas once at placement were sampled (20 hens/flock, total n=600). Results The results showed that the majority of hens were positive for IgY to ER and that the antibody titres were higher in older hens irrespective of vaccination status. Vaccinated hens had significantly higher IgY titres to ER compared to those of unvaccinated hens. This difference was most prominent for the youngest age category. Among the different leukocyte populations studied, blood heterophil, monocyte and γ/δTCR+ T-cell counts were significantly higher in younger hens. Also, serum MBL-levels were significantly higher in younger hens and MBL-levels were positively correlated to heterophil and monocyte counts at the individual level. Conclusions Taken together, results indicate that ER or antigenically similar bacteria are common in the hen environment and that this exposure results in antibodies recognising ER that are present at higher levels in older laying hens. In addition, a higher general pathogen load/level of subclinical infections was indicated by altered leukogram patterns and increased MBL-levels in the youngest age category. Nonetheless, among the parameters studied we found no evidence to suggest that antibody responses to single-dose vaccination should fail or why older flocks seem more susceptible to erysipelas outbreaks.

Escherichia coli is predominantly an intestinal commensal; however, avian pathogenic E. coli (APEC) causes colibacillosis in poultry. The APEC pathotype lacks a clear genetic definition, further complicated by its opportunistic nature. To compare the genomic characteristics of avian pathogenic and commensal E. coli , isolates from diseased and healthy broiler flocks in Sweden were analysed, collected between 2022 and 2024. Clinical isolates ( n =202) were collected at necropsy from 40 flocks during colibacillosis outbreaks, and non-clinical isolates ( n =109) were obtained from litter using sock sampling in 60 unaffected flocks. Whole-genome sequencing was performed to determine sequence types (STs), serotypes, phylogroups, virulence-associated genes (VAGs) and to identify ColV plasmids. A five-gene APEC marker panel targeting plasmid-associated virulence genes ( iutA, hlyF, iss, iroN and ompT ) was used to classify isolates as APEC or non-APEC, and high-risk clones were identified according to the APECtyper scheme. Clinical isolates comprised 22 STs and 25 serotypes and were dominated (59%) by the ST23 O78:H4 clone within phylogroup C. Non-clinical isolates were more diverse (44 STs, 67 serotypes), primarily within phylogroups A (48%) and B1 (33%), with no clone predominating. Clinical isolates carried significantly more VAGs ( P <0.001). Overall, 97% of clinical isolates were identified as APEC, all of which carried a ColV plasmid. Among non-clinical isolates, 28% were APEC, of which 80% were ColV-positive. However, clinical APEC isolates carried significantly more ColV-associated virulence gene clusters than non-clinical APEC isolates ( P <0.001). Only 5% of non-APEC isolates were ColV-positive. High-risk clones were restricted to clinical APEC isolates (63%). These findings indicate that colibacillosis in Swedish broilers was largely driven by a dominant APEC clone during the study period, highlighting the need for coordinated surveillance and targeted control of high-risk clones. The presence of VAG reservoirs among isolates from unaffected flocks, together with the limitations of marker-based APEC typing, supports integrated frameworks combining lineage, VAG profiles and plasmid content for more reliable APEC identification and pathogenicity assessment.

RESEARCH HIGHLIGHTS:Erysipelas is an important differential diagnosis of severe viral diseases.Most of the outbreaks occurred in organic and free-range laying hen flocks.Pre-enrichment or selective culture is useful in case of suspected co-infection.Isolates from temporally clustered outbreaks on a farm are often clonal.

Abstract Erysipelothrix rhusiopathiae (ER) causes erysipelas in multiple animal species and may persist in the environment or be carried asymptomatically. It is estimated that 30–50% of apparently healthy or convalescent pigs harbour ER in their tonsils and other lymphoid tissues. This study aimed to determine the prevalence of ER in the tonsils of healthy Swedish fattening pigs and wild boars. Tonsils were collected from 200 fattening pigs at slaughter from ten abattoirs across Sweden in 2017, with one pig per herd sampled. Wild boars ( n = 180) were sampled during hunting, primarily in Östergötland County, in 2018. Cultures were performed using selective media and isolates were confirmed as ER by MALDI-TOF MS. ER was recovered from 6/200 pig tonsils (3.0%), all originating from three abattoirs in southern Sweden. ER was isolated from 76/167 (45.5%) of wild boar tonsils. Whole-genome sequencing revealed a high genetic diversity among the isolates with no dominant clones. Overall, these results indicate that Swedish pig husbandry, characterized by indoor rearing of fattening pigs, age-segregated rearing, sow vaccination, enhanced biosecurity, and restricted straw access largely prevents tonsillar colonization by ER aligning with the low occurrence of clinically diagnosed erysipelas in such herds. For wild boars, the high isolation rate suggests that wild boar could act as a reservoir and potential source of infection for domestic pigs. The potential zoonotic risk should also be considered.

The need to include wildlife in surveillance programs for antibiotic resistance has been previously highlighted. In the present study, we explored the possibility of sampling Swedish wildlife in conjunction with ongoing monitoring programs for wildlife health and disease to evaluate the occurrence of antibiotic resistance. The animals included in the study represented different trophic levels and degrees of interaction with humans, namely bears, eagles, wolves, foxes, otters, and hares. Samples were screened for ESBL-, pAmpC-, and carbapenemase-producing Escherichia coli using selective media, and indicator E. coli was also isolated from each sample. All isolates were tested for antimicrobial susceptibility, and a subset of isolates was genome sequenced. Two samples from foxes were found to carry ESBL- or pAmpC-producing isolates, while no carbapenemase-producing E. coli could be detected. Geographical information on the samples and genetic characterization of the ESBL- and pAmpC-producing isolates suggest that proximity to anthropogenic settings could play a role in the occurrence of antibiotic resistance in wildlife. In addition, the study demonstrated that the use of ongoing wildlife surveillance programs presents a cost-efficient possibility to monitor antibiotic resistance in wild animals. However, future studies and monitoring programs should take careful consideration regarding selection of animal species and distribution.

Abstract Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens, able to cause severe disease in humans. In the DiSCoVeR project ( https://onehealthejp.eu/jrp-discover/ ) a STEC inventory from human and non-human sources from 11 European countries was set up and ≥ 3500 strains were sequenced to perform comparative genomics analysis. We used this dataset to assess STEC population structure and to investigate potential associations between genomic features, host reservoirs and symptoms. Most STEC isolates analysed by Whole Genome Sequencing (WGS) in this study were collected between years 2010-2020. An ad hoc pipeline was deployed for a harmonised characterization of the STEC in the database, allowing the determination of serotyping, stx gene subtyping, 7-loci MLST, virulotyping and cgMLST. The results were analysed with Principal Component Analysis (PCoA) in relation with isolation source to assess clustering of STEC subpopulations. When human STEC data were analysed, the PCoA revealed three distinct human STEC subpopulations (STEC_1, STEC_2 and STEC_3), which were further analysed for associations between genomic features, symptoms and variance. The non-human STEC showed a more dispersed distribution, except for one subpopulation with genes linked to specific host species, and some virulence profiles overlapping with the STEC_1 population. In conclusion, our analysis identified distinct STEC subpopulations from human cases, each characterized by specific genetic features and associated with varying proportions of severe disease outcomes. These findings provide novel insights supporting the risk assessment of STEC. Impact statement [ This lay summary of your article should be no more than 200 words, and should a) provide a perspective of how this article adds to the literature in the field; b) identify breadth of interest/utility; and c) state the significance of output (incremental or step), in terms of relevance .] This study is based on the establishment of a One Health STEC genomes database, including sequences from isolates of different sources. Most of the isolates had been isolated in the ten-years’ time span 2010-2020, in 11 different countries, for surveillance and monitoring activities or specific surveys and research purposes. The final dataset included the whole genome sequencing of 3,418 STEC isolates, mainly from human cases of infections. The metadata included the host symptoms, where available, for human STEC strains and the animal source the strains had been isolated from. We set up a pipeline for the harmonized analysis of STEC WGS, called Discover, made available though ARIES webserver or GitHub. The analysis allowed a deep characterization of STEC strains circulating in Europe. We used this resource to assess STEC population structure and to investigate potential associations between genomic features, host reservoirs, and various symptoms associated with STEC infection by PCoA. This analysis highlighted the presence of subpopulation of human STEC associated with specific features. We provide new information useful for risk characterization, as well as a large dataset genome database and associated metadata compiled from STEC strains, representing a valuable resource for the scientific community, enabling further investigations into STEC diversity, evolution, source attribution and public health relevance. Data summary The authors confirm all supporting data, including sequence data accession numbers, code and protocols have been provided within the article or through supplementary data files. One supplementary method and five supplementary tables are available with the online version of this article

Multidrug-resistant Salmonella enterica subsp. enterica serovar Infantis clone, harbouring the pESI megaplasmid, first described in Israel in 2014, is consistently reported in poultry and humans worldwide. This study aimed to investigate the genomic epidemiology of S. Infantis collected by nine European Public Health Institutions from samples of different origins (human, food and animal sources) and understand the evolutionary dynamics of pESI-like in Europe. The resolved pESI-like sequences have also been compared with complete publicly available pESI-like sequences from other countries, in a One Health context. The circulation of a S. Infantis clone in Europe carrying the mosaic megaplasmid pESI-like has been associated with resistance to sulphonamides (sul), tetracycline (tet), streptomycin and spectinomycin (aadA1). In recent years, bla CTX-M-1-positive pESI-like plasmids have been increasingly detected in the extended-spectrum beta-lactamase-producing S. Infantis clone. Using a combined short- and long-read sequencing approach, two main types of pESI variants have been identified, differing in the accessory gene content, including the bla CTX-M variant, indicating a certain stability of pESI variants detected in different geographical regions and sources over time (2011-2021). Moreover, the differences were related to the acquisition of resistance, virulence or fitness-enhancing genes that would potentially benefit the Salmonella host.

Coccidiosis is a disease of major importance in poultry husbandry, and the current control measures such as chemoprophylaxis and live vaccines are not sustainable. Therefore, there is a need for sustainable alternative approaches. It has been suggested that biochar can bind to the Eimeria parasites that cause coccidiosis and inhibit their replication in the gut. The aim of this study was to evaluate the impact of dietary biochar on growth rate, infection outcome, caecal microbiota, and specific immune response of broilers experimentally infected with E. tenella. A total of 64 day-old chicks were randomly assigned to one of two dietary treatments; control or biochar. Chickens were fed the biochar diet (2% inclusion rate) from arrival and until the end of trial when they were 30 days old. Eimeria. tenella inoculation was performed when chickens were 20 days old. We found that dietary biochar did not inhibit the E. tenella infection of the caecum demonstrated by similar oocyst shedding, lesion scores and growth rates in the control and biochar groups. Furthermore, biochar did not significantly affect the different blood leukocyte populations monitored or induction of E. tenella specific immune responses. However, biochar caused decreases in the abundance of potentially beneficial bacterial families such as Lactobacillaceae and Bifidobacteriaceae, and an increase of the fungal family Aspergillaceae in the caecum. Therefore, supplementing feed with biochar may be an unsuitable or even a contraindicated approach to prevent E. tenella infection in chickens.

BACKGROUND:Urban broiler production is rapidly expanding in East Africa, raising public health concerns about zoonotic pathogens such as Campylobacter and the spread of antibiotic resistance. This study investigated the occurrence and phenotypic and genotypic antibiotic resistance of Campylobacter from broilers in urban and peri‑urban Uganda. METHODS:A total of 194 samples, including cloacal swabs (n = 157) and boot socks (n = 37), were collected from 28 randomly selected farms in three districts. Campylobacter was isolated according to ISO 10272-1 and tested for susceptibility to nalidixic acid, ciprofloxacin, tetracycline, streptomycin, and erythromycin by disk diffusion following European Committee on Antimicrobial Susceptibility Testing guidelines. Species identification and resistance genotypes were determined by whole-genome sequencing. Isolates that could not be re-cultivated were excluded from further analyses. RESULTS:Campylobacter was isolated from all 194 samples (100%). Of the 157 sequenced isolates, 54% (n = 84) were Campylobacter coli and 46% (n = 73) were Campylobacter jejuni. Of the 170 isolates tested for antibiotic susceptibility, 16% (n = 28) were resistant to all five antibiotics. No isolate was susceptible to ciprofloxacin. Multidrug resistance occurred in 61% (51/84) of C. coli and in 6% (4/72) of C. jejuni isolates. Strong concordance was observed between quinolone resistance and gyrA T86I mutation, tetracycline resistance and tet(O/32/O) or tet(O) genes, and in C. coli, streptomycin resistance and ant(6)-Ia gene. CONCLUSIONS:Broilers in urban and peri‑urban Uganda are frequently colonized with antibiotic-resistant Campylobacter, highlighting the need for strengthened biosecurity and antibiotic stewardship to mitigate public health risks.

Coccidiosis, infection with protozoan parasites of genus Eimeria, is a major problem in poultry husbandry worldwide. The disease is currently managed by coccidiostats and live vaccines, but these approaches are not sustainable. Hence, it is important to identify new means to control the infection and/or ameliorate its detrimental effects on gut health. Laminarin, a (3-glucan found in marine brown algae, has prebiotic and bioactive properties that could be beneficial in coccidiosis control. The present study aimed to examine the potential of laminarin as an immunostimulatory and microbiota-regulatory compound in broiler chickens infected with E. tenella. Chickens were continuously fed a diet supplemented with a laminarin-rich algal extract (AE) from first feed and subsequently infected with E. tenella at 19 days old. The outcome of infection including caecal microbiota and some immune parameters were monitored during the experiment. Results showed that AE supplementation affected some lymphocyte subpopulations, with increased numbers of TCR gamma/delta+CD8-, B-cells and CD4-CD8 alpha(3+ cells and lower numbers of CD4+CD8 alpha alpha+ cells in blood and increased proportions of CD4-CD8 alpha(3+ spleen cells compared to those in control chickens. The AE diet did not affect parasite excretion, lesion scores or E. tenella specific T-cell responses. However, reductions of E. tenella induced contraction of Bifidobacteriaceae and expansion of Clostridiaceae in caecal microbiota were observed for AE fed chickens compared to chickens fed the control diet. Thus, AE feed supplementation induced some immunostimulatory activity in chickens and affected some of the alterations in caecal microbiota evoked by E. tenella infection.

Non-typhoidal Salmonella is the second most frequently reported zoonotic pathogen in the European Union and European Economic Area. Most human infections are caused by serovars Enteritidis and Typhimurium. Genomic characterisation of Salmonella isolates from humans and animals has become a routine public health surveillance tool in many countries. In this study, the relative contributions of several potential sources of human infection of the five frequently reported Salmonella serovars were estimated using machine-learning methods based on a large, cross-sectional collection of genomes from human cases, and animal and environmental sources, across ten European countries. To define the population structure, core-genome Multilocus Sequence Typing was performed. A supervised machine-learning approach was applied for source attribution in the form of a Random Forest classifier. The source and country attribution models achieved moderate accuracy (F1=0.6-0.9), which is lower than in previous studies using machine-learning on Whole Genome Sequencing data. However, attributions of human clinical isolates to different sources were generally in line with previous findings for these five serovars. While the lack of clonality in some sources hindered their prediction, it is also likely that certain sources (e.g., pets) do not serve as major contributors to human infection. Therefore, in most cases attributing these sources to the livestock species they are typically associated with, is likely appropriate. Country attributions showed that substantial human cases are attributable to countries other than their own, indicating geographical interrelatedness of sources. This highlights the value of internationally harmonised Salmonella-control policies in the food production chain.

Despite being a disease known to affect poultry for well over 100 years, several aspects of erysipelas, caused by Erysipelothrix rhusiopathiae, are still poorly understood. For many years, erysipelas in poultry was considered a disease of interest primarily in turkeys, with only a few cases reported in chickens and other poultry species. However, since the change of housing systems for layers from conventional battery cages to litter-based systems, sometimes with access to the outdoors, there has been an increase in the number of outbreaks, especially in Europe, and erysipelas is now considered to be an emerging disease in layers. The aim of this review is therefore to provide a summary background on erysipelas in general, as well as in other poultry species, after which the focus is directed to the disease in chickens, with special emphasis on recent advances in knowledge on immune responses.

Campylobacter is the most commonly reported cause of bacterial gastroenteritis in humans. Although cattle are recognized as a potential reservoir for several Campylobacter spp., most detection standards primarily target thermotolerant species, notably Campylobacter jejuni and Campylobacter coli, possibly underestimating the prevalence of others. This study evaluated the performance of different culture-based methods for detecting Campylobacter spp. in fecal samples collected rectally from dairy cows in a single commercial research herd across four time points. Six combinations of analyses were tested, involving either direct culture or enrichment broths (Preston and Bolton) paired with selective agar media (modified charcoal cefoperazone deoxycholate agar or Preston). Incubation was performed at 37°C under microaerobic conditions to support growth of non-thermotolerant species. Species identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Only C. jejuni and Campylobacter hyointestinalis were detected. While C. jejuni prevalence declined over time, C. hyointestinalis remained consistently high throughout the study period (June-August 2024). The performance of tested detection methods varied significantly between species. Enrichment with Bolton broth increased the odds of detecting C. hyointestinalis by over 3,000-fold compared to the direct culture (odds ratio [OR] = 3,075; 95% confidence interval [CI]: 272-34,651). For C. jejuni, enrichment with Preston broth increased the odds of detection by more than eightfold (OR = 8.52, 95% CI: 3.16-22.9). Detection was primarily influenced by broth; selective agar had no independent effect. These findings emphasize the importance of method selection in the detection of C. hyointestinalis and C. jejuni, suggesting that C. hyointestinalis may be more prevalent in cattle than previously assumed.IMPORTANCECampylobacter bacteria commonly cause gastrointestinal illness in humans and are frequently found in animals such as cattle. Detecting these bacteria in animal samples is important for understanding their occurrence and potential relevance to food safety. Many commonly used laboratory methods focus on Campylobacter spp. that grow under specific conditions, which may limit the detection of other species. This study compared several culture-based methods for the isolation of Campylobacter spp. from fecal samples collected from dairy cattle. Species identification was subsequently performed using MALDI-TOF MS. The findings show that detection varied, depending on the culture method and the Campylobacter spp., highlighting the potential impact of method choice on surveillance outcomes.

Background Single-cell transcriptomics provides means to study cell populations at the level of individual cells. In leukocyte biology this approach could potentially aid the identification of subpopulations and functions without the need to develop species-specific reagents. The present study aimed to evaluate single-cell RNA-seq as a tool for identification of chicken peripheral blood leukocytes. For this purpose, purified and thrombocyte depleted leukocytes from 4 clinically healthy hens were subjected to single-cell 3′ RNA-seq. Bioinformatic analysis of data comprised unsupervised clustering of the cells, and annotation of clusters based on expression profiles. Immunofluorescence phenotyping of the cell preparations used was also performed. Results Computational analysis identified 31 initial cell clusters and based on expression of defined marker genes 28 cluster were identified as comprising mainly B-cells, T-cells, monocytes, thrombocytes and red blood cells. Of the remaining clusters, two were putatively identified as basophils and eosinophils, and one as proliferating cells of mixed origin. In depth analysis on gene expression profiles within and between the initial cell clusters allowed further identification of cell identity and possible functions for some of them. For example, analysis of the group of monocyte clusters revealed subclusters comprising heterophils, as well as putative monocyte subtypes. Also, novel aspects of TCRγ/δ + T-cell subpopulations could be inferred such as evidence of at least two subtypes based on e.g., different expression of transcription factors MAF , SOX13 and GATA3 . Moreover, a novel subpopulation of chicken peripheral B-cells with high SOX5 expression was identified. An overall good correlation between mRNA and cell surface phenotypic cell identification was shown. Conclusions Taken together, we were able to identify and infer functional aspects of both previously well known as well as novel chicken leukocyte populations although some cell types. e.g., T-cell subtypes, proved more challenging to decipher. Although this methodology to some extent is limited by incomplete annotation of the chicken genome, it definitively has benefits in chicken immunology by expanding the options to distinguish identity and functions of immune cells also without access to species specific reagents.

Outbreaks of erysipelas, a disease caused by infection with Erysipelothrix rhusiopathiae (ER), is a re-emerging problem in cage-free laying hen flocks. The source of ER infection in hens is usually unknown and serological evidence has indicated the presence of ER or other antigenically related bacteria also in healthy flocks. The aim of the present study was to evaluate sample collection, culture methods and DNA-based methodology to detect ER and other Erysipelotrichales in samples from healthy chickens and their environment. We used samples from a research facility with conventionally reared chickens with no history of erysipelas outbreaks where hens with high titers of IgY recognising ER previously have been observed. Microbial DNA was extracted from samples either directly or after pre-culture in nonselective or ER-selective medium. Real-time PCR was used for detection of Erysipelothrix spp. and high-throughput amplicon sequencing of 16S rRNA sequencing was used for detection of Erysipelotrichales. A pilot serological analysis of some Erysipelotrichales members with IgY from unvaccinated and ER vaccinated high biosecurity-chickens as well as conventionally reared chickens was also performed. All samples were negative for ER, E. tonsillarum and E. piscisicarius by PCR analysis. However, 16S rRNA community profiling indicated the presence of several Erysipelotrichales genera in both environmental samples and chicken intestinal samples including Erysipelothrix spp. that were detected in environmental samples. Sequences from Erysipelothrix spp. were most frequently detected in samples pre-cultured in ER-selective medium. On species level the presence of E. anatis and/or E. aquatica was indicated. Serological results indicated that IgY raised to ER showed some cross-reactivity with E. anatis. Hence, environmental samples pre-cultured in selective medium and analysis by 16S rRNA sequencing proved a useful method for detection of Erysipelotrichales including Erysipelothrix spp. in chicken flocks. The observation of such bacteria in environmental samples offers a possible explanation for the observation of high antibody titres to ER in flocks without a history of clinical erysipelas.

The impact of S. suis on Swedish pig production has increased in recent years, and characterization of the strains present in the pig population is needed to aid in surveillance and prevention. Therefore, the aim of this study was to identify and characterize differences in the genomes between Swedish S. suis isolates associated with disease and isolates from healthy animals. Isolates categorized as being pathogenic (n = 100) or non-pathogenic (n = 117) were whole-genome sequenced, serotyped in silico, and sequence-typed using traditional MLST and core-genome MLST, and a genome-wide association study was performed to identify virulence-associated genes. In decreasing order, serotypes 2, 1, and 7 were the most common in the pathogenic group, and serotypes 15 and 12 were the most common in the non-pathogenic group. Among the commonly disease-associated sequence types, ST28 and ST25 were identified, whereas ST1 was scarcely found. The majority of isolates belonged to novel sequence types, revealing differences between Swedish isolates and those reported from other countries. The genomes of the pathogenic isolates were on average smaller and less heterogenic as compared to those of the non-pathogenic isolates. Although a majority of the previously published virulence-associated genes included in the study were found in the genomes of both pathogenic and non-pathogenic isolates, several new, significantly virulence-associated genes were identified.

Background The composition of the microbial flora associated with ixodid ticks has been studied in several species, revealing the importance of geographical origin, developmental stage(s) and feeding status of the tick, as well as substantial differences between tissues and organs. Studying the microbiome in the correct context and scale is therefore necessary for understanding the interactions between tick-borne pathogens and other microorganisms as well as other aspects of tick biology. Methods In the present study the microbial flora of whole Ixodes ricinus , I. persulcatus and I. trianguliceps ticks were analyzed with 16S rRNA amplicon sequencing. Additionally, tick organs (midguts, Malpighian tubules, ovaries, salivary glands) from flat and engorged I. ricinus female ticks were examined with the same methodology. Results The most abundant bacteria belonged to the group of Proteobacteria ( Cand. Midichloria mitochondrii and Cand. Lariskella). 16S amplicon sequencing of dissected tick organs provided more information on the diversity of I. ricinus -associated microbial flora, especially when organs were collected from engorged ticks. Bacterial genera significantly associated with tick feeding status as well as genera associated with the presence of tick-borne pathogens were identified. Conclusions These results contribute to the knowledge of microbial flora associated with ixodid ticks in their northernmost distribution limit in Europe and opens new perspectives for other investigations on the function of these bacteria, including those using other approaches like in vitro cultivation and in vitro models.

Broiler cellulitis has emerged as an important cause of economic losses for farmers and slaughter plants from carcass condemnation at processing. Avian pathogenic Escherichia coli (APEC) has been identified as the main causative agent. The aim was to characterize E. coli isolated from cellulitis and organs in broilers at slaughter by whole genome sequencing analysis to study if systemic spread could be confirmed. Isolates were collected post-mortem from 101 carcasses condemned due to dermatitis/cellulitis from five commercial farms and six flocks. Forty-six isolates were characterised to determine serotypes, sequence types and virulence-associated genes. Analysis by cgMLST was performed to study the genetic similarity between isolates from the same broiler, among birds from the same flock and between flocks. Escherichia coli was isolated from 90% of birds from subcutaneous samples. In 20 broilers, E. coli was isolated from organs in pure culture or mixed with sparse growth of other bacteria. In eight of these, there were post-mortem findings suggestive of systemic bacterial spread. The majority of the isolates from the same bird and flock belonged to the same serotype and sequence type and were genetically indistinguishable, but differed when compared between flocks. Common APEC virulence genes, i.e. chuA, fyuA, hlyF, iroN, irp2, iss, ompT, sitA, TerC, TraT, were present in > 87% of the isolates. We conclude that evidence of systemic spread of E. coli from cellulitis was present in some birds at time of slaughter but cannot be reliably detected at meat inspection.

Here we report the detection of carbapenemase-producing Enterobacterales (CPE) isolated from Swedish wastewater and gull faeces. CPE have not been detected in samples from animals in Sweden preceding this report. Sampling of wastewater treatment plant (WWTP) inlet and outlet, sedimentation basins, surface seawater from key aquatic bird habitats and freshly deposited gull faeces was done on six separate occasions during May to September 2021. Following broth enrichment, selective screening of putative CPE was performed on mSuperCarba™ (CHROMagar). Species identification was done with MALDI-TOF. Antimicrobial susceptibility testing was performed according to EUCAST. In total, seventeen CPE were verified by genome sequencing carrying blaGES-5, blaIMI-3, blaOXA-181 or blaOXA-244. The blaGES-5 was carried on IncP plasmids in four different species; Escherichia coli ST10 isolated from WWTP outlet, Raoultella ornithinolytica isolated from WWTP inlet, outlet and sedimentation basins as well as gull faeces collected at the WWTP and Klebsiella spp. isolates from WWTP inlet and outlet. The genetic environment surrounding blaGES-5 was similar in two Citrobacter freundii causing human infections. The blaIMI-3 was carried on IncFII(Yp) plasmids in four Enterobacter ludwigii, isolated from WWTP outlet and gull faeces collected at a recreational city park 2 km from the WWTP. The blaOXA-181 was located on a COLKP3 plasmid found in an E. coli, while blaOXA-244 was chromosomally located in an E. coli ST10, both isolated from WWTP inlet. Phylogenetic analysis of R. ornithinolytica and E. ludwigii isolates indicate that the gulls carried strains related to those identified in the WWTP samples. The results thus add to the increasing evidence of WWTPs as anthropogenic reservoirs for mobile genetic elements with antibiotic-resistance functionality. Such environments could profoundly impact the dissemination and spread of such genetic elements via for example aquatic birds, thereby warranting further study and surveillance.

ABSTRACT Staphylococcus pseudintermedius is a commensal and an opportunistic pathogen in dogs, and is also an opportunistic pathogen in humans. Here we report about a case of bacteraemia with a fatal outcome in a 77-year-old co-morbid male likely caused by a S. pseudintermedius and the investigation into the possible transmission from the two dogs in the patient’s household. The two dogs carried the same S. pseudintermedius strain, but this dog strain was unrelated to the strain from the patient. In contrast to the patient strain, the dog strain showed reduced susceptibility to several antibiotics and both dogs had received antibiotic treatment prior to sampling. So, it is conceivable that these treatments can have eliminated the patient’s strain between the transmission event and the dog sampling. It is also worth noting that the patient strain was positive for the expA gene, which encodes an exfoliative toxin closely related to the S. aureus exfoliative toxin B. This toxin has been linked to canine pyoderma, but its effect on humans remains unknown. Transmission of S. pseudintermedius was confirmed in the household between the dogs. However, we could not verify that the dogs were the source for the S. pseudintermedius in the patient.