In September 2020, an unexpected increase in Salmonella Muenchen patient isolates and notifications was observed. We investigated the outbreak to identify the vehicle of infection. RKI defined cases as patients with laboratory-confirmed S. Muenchen infections reported between September 2020 and July 2021. Genomes of clinical, food, and animal S. Muenchen isolates were analysed using cgMLST. We conducted interviews and performed a frequency-matched case-control study. We calculated frequencies and adjusted odds ratios (aOR) using logistic regression. We identified 301 cases in eight federal states in Germany. Hypothesis-generating interviews did not provide a conclusive hint of a possible vehicle. S. Muenchen strains were detected in dried coconut pieces, milk powder used for chocolate production, and a wild swan, all with a cgMLST profile indistinguishable from the prominent node comprising 116 patient isolates. Cases included in the case-control study more often consumed dried coconut pieces (22/30) than controls (2/116) (aOR: 176 (95% confidence interval: 32-954)). In this investigation, cgMLST analysis presented identical strains in three different isolate sources. The case-control study supported dried coconut pieces as vehicle of infection demonstrating the importance of interdisciplinary investigations and underscoring the potential impact of unusual vehicles.

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is the second most common serovar causing human salmonellosis in Aotearoa New Zealand (NZ). Sequence type (ST)183, which includes phage types 9a and 11, was the second most frequently isolated S. Enteritidis strain from human cases between 2020 and 2023. This ST is considered endemic in NZ as well as in mainland Europe and Great Britain, where the European hedgehog (Erinaceus europaeus) is a recognized wildlife reservoir. Hedgehogs were introduced to NZ in the late 19th century; however, their role in the ecology of ST183 in NZ has not been formally evaluated. The aim of this study was to investigate whether hedgehogs act as a reservoir for S. Enteritidis ST183 in NZ and to assess the evolutionary history and epidemiology of this strain across human, animal and environmental contexts. We analysed human, animal and environmental ST183 isolates, including hedgehog carcasses opportunistically sampled in NZ, using Bayesian phylogenetic methods integrated with national epidemiological data. Although S. Enteritidis ST183 was isolated from three of 45 hedgehog carcasses during our study, consistent with recent prior detections of ST183 in NZ hedgehogs, Bayesian phylogenetic analysis supports a most recent common ancestor for currently circulating ST183 strains in the late 20th century, ~100 years after the introduction of hedgehogs into NZ, providing no evidence that the strain was introduced concomitantly with hedgehogs. Epidemiological analysis revealed that, unlike in Europe, ST183 infections in NZ are more common in people aged over 60 years compared with non-ST183 S. Enteritidis infections, with rural residence and contact with farm animals identified as key risk factors. Together, these findings suggest that S. Enteritidis ST183 is established within the NZ rural environment, with evidence of interspecies transmission. While hedgehogs may contribute to the maintenance of ST183, they are unlikely to represent the original source of introduction, indicating a complex, multi-host ecology.

Salmonella serotyping is shifting from slide agglutination toward whole-genome sequencing (WGS). While WGS allows for comprehensive analyses, phenotypic information about lipopolysaccharide-deficient ("LPS-rough") isolates obtained from slide agglutination is lost. This discrepancy represents a challenge for Salmonella control in livestock because in the European Union, LPS-rough Salmonella isolated from food-producing animals that are untypable using slide agglutination alone are not subject to control measures, whereas isolates of certain serovars in certain matrices would be, when based on geno-serotyping. Here, we provide an account of the relevance of this phenotype in the context of routine diagnostics and food safety by characterizing the occurrence, diversity, and isolation matrices of LPS-rough isolates among non-human Salmonella enterica subsp. enterica isolates from Germany. Using available WGS data, we examined phylogenetic relationships and associations with certain genomic features. On average, 5% of isolates exhibited an LPS-rough phenotype across 46 serovars, but clonal distribution of this phenotype along the food chain was not evident. LPS-rough isolates were more commonly found in S. Choleraesuis isolated from wild boar and in S. Typhimurium isolated from pork products, compared with other matrices. We also found associations with two virulence factors, an AMR gene, and a plasmid marker. The present work lays the foundation for future research into the role of certain matrices, environments, or genomic factors in the development of LPS-rough Salmonella isolates and will facilitate the elucidation of the genomic basis of this phenotype, which may improve recommendations regarding risk management and control measures.IMPORTANCEThe present work highlights some of the challenges associated with the recent shift from serology- to sequence-based typing of Salmonella enterica serovars and provides a national perspective on the presence and relevance of the lipopolysaccharide-deficient ("LPS-rough") phenotype in samples obtained from food, animals, and the environment, including considerations regarding the choice of typing method. We provide evidence that clonal distribution of isolates with this phenotype is unlikely, but that certain environments may favor its development, and certain genomic factors may increase survival rates of LPS-rough isolates when exposed to environmental stressors. These findings could have important implications for regulations regarding the surveillance and management of Salmonella isolated from food, feed, and animals in the future, in particular in the context of using different typing methods, and warrant further detailed research.

Nontyphoidal Salmonella (NTS) poses a significant burden of bacteremia in sub-Saharan Africa (SSA). Antimicrobial resistance (AMR) complicates the management of NTS infections. The NTS carriage of mobile genetic elements harboring AMR genes can result in the spread of resistance. We investigated the AMR profiles and genomic characteristics of 74 NTS isolates recovered from children under 5 years of age during acute disease, post-treatment shedding, and in healthy carriers. Whole genome sequencing data were used to identify AMR genes, plasmid replicons, and virulence determinants. Of the 74 NTS isolates, 63.5% (47) were Salmonella Enteritidis, the rest being Salmonella Typhimurium. More than a third, 37.8% (2874), were resistant to at least one antibiotic. A small proportion, 8.1% (6/74), were multidrug resistant, with a majority (5/6) being S. Typhimurium. Resistance to azithromycin, one of the current drugs of choice for treating invasive nontyphoidal Salmonella (iNTS), was observed in 13.5% (10/74) of the isolates. Notably, the macrolide resistance gene mph(A) was detected in one isolate. Extended-spectrum β-lactamase (ESBL) genes (blaCTX-M-3 and blaTEM) were identified in 8.1% of the isolates, all of which were S. Typhimurium. Six plasmid replicons were detected, with epidemic plasmids IncFIB(S)_1 and IncFII(S)_1 observed in all isolates. The ESBL gene blaCTX-M-3 was harbored in the IncI1_1_Alpha plasmid replicon. A total of 117 virulence determinants were identified, with only one isolate carrying the shdA gene associated with prolonged fecal shedding. Resistance and reduced susceptibility of NTS to antibiotics could result in treatment failure and underscore the need for continued surveillance, vaccine introduction, and antimicrobial stewardship.IMPORTANCEAntimicrobial resistance is a huge public health concern globally, particularly in low-resource settings due to a high burden of infectious diseases, limited access to quality healthcare, and misuse and/or overuse of antibiotics. Azithromycin is one of the antibiotics used in the treatment of iNTS; notably, 13.5% of the NTS isolates in this study showed resistance to azithromycin. The detection of AMR determinants and plasmids highlights the possibility of these determinants spreading, resulting in increased resistance. Resistance of NTS to azithromycin and other antibiotics in this setting could lead to treatment failure, resulting in poor patient outcomes. These findings emphasize the need for vaccine introduction and antimicrobial stewardship in health facilities in low-resource settings.

Different laboratories employ different Whole-Genome Sequencing (WGS) pipelines for Food and Waterborne disease (FWD) surveillance, casting doubt on the comparability of their results and hindering optimal communication at intersectoral and international levels. Through a collaborative effort involving eleven European institutes spanning the food, animal, and human health sectors, we aimed to assess the inter-pipeline clustering congruence across all resolution levels and perform an in-depth comparative analysis of cluster composition at outbreak level for four important foodborne pathogens: Listeria monocytogenes, Salmonella enterica, Escherichia coli, and Campylobacter jejuni. We found a general concordance between allele-based pipelines for all species, except for C. jejuni, where the different resolution power of allele-based schemas led to marked discrepancies. Still, we identified non-negligible differences in outbreak detection and demonstrated how a threshold flexibilization favors the detection of similar outbreak signals by different laboratories. These results, together with the observation that different traditional typing groups (e.g., serotypes) exhibit a remarkably different genetic diversity, represent valuable information for future outbreak case-definitions and WGS-based nomenclature design. This study reinforces the need, while demonstrating the feasibility, of conducting continuous pipeline comparability assessments, and opens good perspectives for a smoother international and intersectoral cooperation towards an efficient One Health FWD surveillance.

We report on a carbapenem-, extended spectrum β-lactam-, fluoroquinolone-, and tetracycline-resistant Salmonella enterica serovar Typhi strain in a patient returning to Germany from India. Considering the recent emergence of extensively drug-resistant Salmonella Typhi strains, further expansion of antibiotic resistance to carbapenems poses a serious threat for typhoid fever treatment.

Non-typhoidal Salmonella (NTS) presents a considerable health threat to children in low-resource settings, where clean water, sanitation, and hygiene are often inadequate. However, the environmental factors influencing NTS persistence and spread remain poorly understood. We utilized a case-control approach to investigate environmental factors associated with NTS infection in children living in Nairobi's informal settlements between August 2022 and July 2023. Stool samples were collected from febrile children, with or without diarrhea, who visited healthcare facilities. The study included 42 laboratory-confirmed NTS-positive cases and 42 NTS-negative children from the same community. Environmental samples, including drinking water, open drains, soil, and household effluent, were collected from both case and control households, in addition to raw sewage from main sewer-line convergence points. Conventional microbiological culture and quantitative Polymerase Chain Reaction techniques were employed for NTS detection, with genomic sequencing used for strain characterization. Environmental samples from case households showed a higher NTS contamination rate of 33.3% (42/126) compared to control households of 7.2% (9/126). Higher odds of NTS infection in children were associated with household environmental factors, particularly exposure to household effluent (OR = 7.7, 95% CI: 2.18-34.82, p = 0.0005), drinking water (OR = 6.4, 95% CI: 1.57-37.76, p = 0.0055), and soil (OR = 5.4, 95% CI: 1.01-54.28, p = 0.0485). Genomic analysis revealed a common strain, Salmonella Enteritidis ST11, in clinical and environmental isolates. These findings highlight the plausible role of the household environment as a reservoir for NTS, perpetuating infection cycles within the community. Addressing this challenge requires a multifaceted approach, including improved sanitation infrastructure, environmental monitoring, and integrated public health interventions to reduce NTS exposure and transmission in high-risk populations.

The Klebsiella oxytoca species complex is part of the human microbiome, especially during infancy and childhood. K. oxytoca species complex strains can produce enterotoxins, namely, tilimycin and tilivalline, while also contributing to colonization resistance (CR). The relationship between these seemingly contradictory roles is not well understood. Here, by coupling ex vivo assays with CRISPR-mutagenesis and various mouse models, we show that K. oxytoca provides CR against Salmonella Typhimurium. In vitro, the antimicrobial activity against various Salmonella strains depended on tilimycin production and was induced by various simple carbohydrates. In vivo, CR against Salmonella depended on toxin production in germ-free mice, while it was largely toxin-independent in mice with residual microbiota. This was linked to the relative levels of toxin-inducing carbohydrates in vivo. Finally, dulcitol utilization was essential for toxin-independent CR in gnotobiotic mice. Together, this demonstrates that nutrient availability is key to both toxin-dependent and substrate-driven competition between K. oxytoca and Salmonella.

Nontyphoidal Salmonella (NTS) is a predominant cause of invasive disease in sub-Saharan Africa especially among children under 5 years. Asymptomatic fecal shedding of NTS is hypothesized to contribute to the human-to-human transmission of NTS especially in low-resource settings. However, the role of pathogen shedding in invasive disease is unknown. This study aimed to investigate the prevalence and duration of fecal shedding of NTS among children under 5 years convalescing from invasive NTS disease and among healthy individuals in the community. Children presenting with fever of ≥38°C with or without diarrhea were recruited at four health facilities in Nairobi, between June 2021 and August 2023. Blood and stool samples collected were subjected to culture for the isolation of NTS (S. Enteritidis and S. Typhimurium). Children with NTS culture-positive samples (index cases) were followed up post-acute disease where household contacts and controls provided stool samples for isolation of NTS. NTS prevalence among the 3,293 individuals recruited was 1.52%. Asymptomatic shedding post-treatment was observed in almost one-third (31%) of the 42 index cases followed up. Of the 13 with intestinal shedding, 7 were shedding NTS of the same sequence type (ST) as the one recovered during acute disease. The longest duration of intestinal shedding was 3 months post-treatment. Of the 241 healthy individuals recruited, 8 had asymptomatic shedding of NTS, and 2 of these were closely related to those recovered from index cases. These findings support the hypothesis of human-to-human transmission of NTS in sub-Saharan Africa highlighting the possible benefit of vaccine introduction. IMPORTANCE:Asymptomatic fecal shedding of nontyphoidal Salmonella (NTS) is hypothesized to contribute to the human-to-human transmission of NTS especially in low-resource settings which could lead to invasive disease among high-risk populations, especially children. Our findings reiterate the hypothesis that human reservoirs could be important in the transmission of nontyphoidal Salmonella in sub-Saharan Africa. This underscores the importance of developing infection prevention measures which could include vaccine deployment and improving water, sanitation and hygiene infrastructure.

Food and waterborne disease (FWD) surveillance requires Whole-Genome Sequencing (WGS)-based systems following a One Health approach. However, different laboratories employ different WGS pipelines in their routine surveillance activities, casting doubt on the comparability of their results and hindering optimal communication at intersectoral and international levels. Through a collaborative effort involving eleven European institutes across seven countries and spanning the food, animal and human health sectors, we aimed to assess the inter-laboratory comparability of WGS clustering results for four important foodborne pathogens: Listeria monocytogenes, Salmonella enterica, Escherichia coli and Campylobacter jejuni. Each participating institute (n=9) applied its surveillance pipeline over the same WGS datasets (>2000 isolates per species), and, for each pipeline, genetic clusters were identified at each possible allele/SNP distance threshold. Inter-pipeline clustering congruence was assessed by calculating a Congruence Score (relying on Adjusted Wallace and Adjusted Rand coefficients) across all resolution levels, followed by an in-depth comparative analysis of cluster composition at outbreak level. An additional cluster congruence assessment was performed between WGS and traditional typing, which, depending on the species, included Sequence Type (ST), Clonal Complex (CC) and/or serotype. Our results revealed a general high concordance between allele-based pipelines at all resolution levels for all species, except for C. jejuni, where the different resolution power of available allele-based schemas led to marked discrepancies. Still, this study identified non-negligible differences in allele-based pipeline performance for outbreak cluster detection, suggesting that a threshold flexibilization is important for the detection of similar outbreak signals by different laboratories. These results, together with the observation that different STs, CCs and serotypes exhibit remarkably different genetic diversity, should inform future threshold selections for outbreak case definitions. In conclusion, this study provides valuable insights into the comparability of pipelines commonly used for routine genomics surveillance, and reinforces the need, while demonstrating the feasibility, of conducting continuous and comprehensive WGS pipeline comparability assessments. Ultimately, it opens good perspectives for a smoother international and intersectoral cooperation and communication towards a sustainable and efficient One Health FWD surveillance. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by co-funding from the European Union's Horizon 2020 Research and Innovation program under grant agreement No 773830: One Health European Joint Programme (2020 to 2022) (https://onehealthejp.eu/projects/foodborne-zoonoses/jrp-beone) and by the ISIDORe project (funding from the European Union's Horizon Europe Research & Innovation Programme, Grant Agreement no. 101046133). VM contribution was funded by national funds through FCT - Foundation for Science and Technology, I.P., in the frame of Individual CEEC 2022.00851.CEECIND/CP1748/CT0001 (2023 onwards). JDS contribution was supported by the project "Sustainable use and integration of enhanced infrastructure into routine genome-based surveillance and outbreak investigation activities in Portugal" (GENEO, https://www.insa.min-saude.pt/category/projectos/geneo/) on behalf of the EU4H programme (EU4H-2022-DGA-MS-IBA-1). Research at the National Veterinary Research Institute (PIWet) Poland was supported by the Polish Ministry of Education and Science from the funds for science in the years 2018-2022 allocated for the implementation of a co-financed international project. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Anonymized sequencing reads of the BeONE dataset are deposited in the European Nucleotide Archive (ENA) database under the BioProjects PRJEB57166, PRJEB57179, PRJEB57098 and PRJEB57119. Genome assemblies are deposited in the Zenodo repository (L. monocytogenes: 10.5281/ZENODO.7267486; S. enterica: 10.5281/ZENODO.7267785; E. coli: 10.5281/ZENODO.7267844; C. jejuni: 10.5281/ZENODO.7267879). The public dataset data was retrieved from Zenodo (L. monocytogenes: 10.5281/ZENODO.7116878; S. enterica: 10.5281/ZENODO.7119735; E. coli: 10.5281/ZENODO.7120057; C. jejuni: 10.5281/ZENODO.7120166). The collection of scripts used to conduct these analyses are available at the github repository https://github.com/insapathogenomics/WGS\_cluster\_congruence. Supplementary data are available in the Zenodo repository (https://doi.org/10.5281/zenodo.12805750).

Background In recent years, whole genome sequencing (WGS) in combination with bioinformatic analyses has become state of the art in evaluating the pathogenicity/resistance potential and relatedness of bacteria. WGS analysis thus represents a central tool in the investigation of the resistance and virulence potential of pathogens, as well as their dissemination via outbreak clusters and transmission chains within the framework of molecular epidemiology. In order to gain an overview of the available genotypic and phenotypic methods used for pathogen typing of Salmonella and Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany at state and federal level, along with the availability of WGS-based typing and corresponding analytical methods, a survey of laboratories was conducted.Methods An electronic survey of laboratories working for public health protection and consumer health protection was conducted from February to June 2020.Results and conclusion The results of the survey showed that many of the participating laboratories provide a wide range of phenotypic and molecular methods. Molecular typing is most commonly used for species identification of Salmonella. In many cases, WGS-based methods have already been established at federal and state institutions or are in the process of being established. The Illumina sequencing technology is the most widely used technology. The survey confirms the importance of molecular biology and whole genome typing technologies for laboratories in the diagnosis of bacterial zoonotic pathogens.

INTRODUCTION:In order to improve patient care and to increase food safety within the framework of One Health, the project "Integrated Genomic Surveillance of Zoonotic Agents (IGS-Zoo)" aims to develop concepts for a genomic surveillance of Shiga toxin(Stx)-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany. METHODS:An online survey was conducted to assess the currently available and applied STEC/EHEC typing methods in the federal laboratories of veterinary regulation, food control, and public health service. RESULTS:Twenty-six questionnaires from 33 participants were evaluated with regard to STEC/EHEC. The number of STEC/EHEC-suspected samples that the laboratories process per year ranges between 10 and 3500, and out of these they obtain between 3 and 1000 pathogenic isolates. Currently the most frequently used typing method is the determination of Stx- and intimin-coding genes using polymerase chain reaction (PCR). Whole genome sequencing (WGS) is currently used by eight federal state laboratories, and nine are planning to implement it in the future. The most common obstacle for further typing of STEC/EHEC is that isolation from sample material is often unsuccessful despite apparent PCR detection of the stx genes. DISCUSSION:The results of the survey should facilitate the integration of the analysis methods developed in the project and emphasize the target groups' individual needs for corresponding training concepts.

Zusammenfassung Einleitung Für eine Verbesserung der Patientenversorgung und die Erhöhung der Lebensmittelsicherheit im Sinne von One Health wurden im Rahmen des Projekts „Integrierte Genomische Surveillance von Zoonoseerregern (IGS-Zoo)“ Konzepte für eine sektorübergreifende genomische Surveillance von Shiga Toxin(Stx)-bildenden bzw. enterohämorrhagischen Escherichia coli (STEC/EHEC) in Deutschland entwickelt. Methoden Mittels Onlineumfrage erfolgte zunächst eine Bestandsaufnahme der aktuell in den Laboratorien im Bereich der staatlichen Untersuchungsämter im Veterinärwesen, der Lebensmittelüberwachung und des Öffentlichen Gesundheitsdienstes der Länder verfügbaren und der tatsächlich angewandten Typisierungsmethoden für diese Erreger. Ergebnisse Von 33 Teilnehmenden konnten 26 Fragebögen hinsichtlich STEC/EHEC ausgewertet werden. Pro Jahr werden in den Laboratorien zwischen 10 und 3500 Proben bearbeitet, daraus werden zwischen 3 und 1000 Erregerisolate gewonnen. Die aktuell am häufigsten verwendete Typisierungsmethode ist dabei die Bestimmung der Stx- und Intimin-kodierenden Gene mittels Polymerase-Kettenreaktion (PCR). Genomsequenzierung ( Whole Genome Sequencing, WGS) ist in 8 staatlichen Laboratorien der Länder etabliert und zusätzlich in 9 Einrichtungen geplant. Als häufigstes Hindernis für eine weiterführende STEC/EHEC-Typisierung wurde angeführt, dass auch bei eindeutigem Nachweis des stx -Gens mittels PCR eine Isolierung aus Probenmaterial oftmals nicht erfolgreich ist. Diskussion Die Ergebnisse der Befragung sollen dazu beitragen, die im Projekt entwickelten Analyseverfahren bei der Zielgruppe zu integrieren, und zudem aufzeigen, wo Schwerpunkte bei der bedarfsgerechten Entwicklung von entsprechenden Schulungskonzepten gesetzt werden sollen.

Zusammenfassung Hintergrund In den vergangenen Jahren hat sich die Gesamtgenomsequenzierung („whole genome sequencing“; WGS) in Kombination mit bioinformatischen Analysen zum Stand der Technik bei der Bewertung des Pathogenitäts- und Resistenzpotenzials sowie der Verwandtschaftsgrade zwischen Bakterien entwickelt. Die WGS-Analyse stellt somit ein zentrales Instrument bei der Typisierung von Erregern und der Untersuchung von Krankheits- und Ausbruchsclustern im Rahmen der molekularen Epidemiologie dar. Ziel der Studie war die Generierung eines Überblicks der in Deutschland auf Landes- und Bundesebene verfügbaren Erregertypisiermethoden von Salmonellen und Shiga-Toxin-bildenden bzw. enterohämorrhagischen Escherichia coli (STEC/EHEC) und den angewandten geno- und phänotypischen Methoden sowie über die Verfügbarkeit der genombasierten Typisierung und entsprechenden Analyseverfahren. Methoden Im Zeitraum vom Februar bis Juni 2020 wurde eine elektronische Umfrage bei Laboratorien durchgeführt, die für den öffentlichen Gesundheitsschutz und gesundheitlichen Verbraucherschutz tätig sind. Ergebnisse und Fazit Die Ergebnisse der Umfrage zeigten, dass viele der teilnehmenden Laboratorien über eine große Auswahl an phänotypischen und molekularbiologischen Methoden verfügen. Molekularbiologische Typisierungen werden am häufigsten für die Speziesidentifizierung von Salmonellen herangezogen. WGS-Verfahren sind vielfach schon bei Einrichtungen auf Bundes- und Landesebene etabliert oder befinden sich im Aufbau. Die Illumina-Sequenzierung ist dabei die am weitesten verbreitete Technologie. Die Umfrage bestätigt die Bedeutung von molekularbiologischen und genombasierten Typisierungstechnologien für die Laboratorien bei der Diagnostik von bakteriellen zoonotischen Erregern.

(1) Background: Resistance plasmids are under selective conditions beneficial for the bacterial host, but in the absence of selective pressure, this carriage may cause fitness costs. Compensation of this fitness burden is important to obtain competitive ability under antibiotic-free conditions. In this study, we investigated fitness effects after a conjugative transfer of plasmids containing various beta-lactamase genes transferred into Escherichia coli. (2) Methods: Fourteen beta-lactamase-encoding plasmids were transferred from clinical donor strains to E. coli J53. Growth rates were compared for all transconjugants and the recipient. Selected transconjugants were challenged in long-term growth experiments. Growth rates were assessed at different time points during growth for 500 generations. Whole-genome sequencing (WGS) of initial and evolved transconjugants was determined. Results: Most plasmid acquisitions resulted in growth differences, ranging from −4.5% to 7.2%. Transfer of a single blaCMY-16-carrying plasmid resulted in a growth burden and a growth benefit in independent mating. Long-term growth led to a compensation of fitness burdens and benefits. Analyzing WGS revealed genomic changes caused by Single Nucleotide Polymorphisms (SNPs) and insertion sequences over time. Conclusions: Fitness effects associated with plasmid acquisitions were variable. Potential compensatory mutations identified in transconjugants’ genomes after 500 generations give interesting insights into aspects of plasmid–host adaptations.

Non-typhoidal Salmonella enterica is an important gastrointestinal pathogen causing a considerable burden of disease. Resistance to third generation cephalosporins poses a serious threat for treatment of severe infections. In this study occurrence, phylogenetic relationship, and mechanisms of third generation cephalosporin resistance were investigated for clinical non-typhoidal S. enterica isolates in Germany. From 2017 to 2019, we detected 168 unique clinical S. enterica isolates with phenotypic resistance to third generation cephalosporins in a nation-wide surveillance. Compared to previous years, we observed a significant (P=0.0002) and consistent increase in resistant isolates from 0.41 % in 2005 to 1.71 % in 2019. In total, 34 different serovars were identified, most often S. Infantis (n=41; 24.4 %), S. Typhimurium (n=27; 16.1 %), S. Kentucky (n=21; 12.5 %), and S. Derby (n=17; 10.1 %). Whole genome analyses revealed extended-spectrum β-lactamase (ESBL) genes as main cause for third generation cephalosporin resistance, and most prevalent were blaCTX-M-1 (n=55), blaCTX-M-14 (n=25), and blaCTX-M-65 (n=23). There was no strict correlation between serovar, phylogenetic lineage, and ESBL type but some serovar/ESBL gene combinations were detected frequently, such as blaCTX-M-1 and blaCTX-M-65 in S. Infantis or blaCTX-M-14b in S. Kentucky. The ESBL genes were mainly located on plasmids, including IncI, IncA/C variants, emerging pESI variants, and a novel blaCTX-M-1harbouring plasmid. We conclude that third generation cephalosporin resistance is on the rise among clinical S. enterica isolates in Germany, and occurrence in various S. enterica serovars is most probably due to multiple acquisition events of plasmids.

The aim of this study was to gain an overview of the genetic diversity of Salmonella found in wildlife in Germany. We were particularly interested in exploring whether wildlife acts as a reservoir of certain serovars/subtypes or antimicrobial resistance (AMR) genes. Moreover, we wanted to explore the potential of Salmonella in spreading from wildlife to livestock and humans. To answer these questions, we sequenced 260 Salmonella enterica subsp. enterica isolates sampled between 2002 and 2020 from wildlife across Germany, using short-read whole genome sequencing. We found, consistent with previous findings, that some Salmonella sequence types are associated with certain animal species, such as S. Choleraesuis ST145 with wild boar and S. Enteritidis ST183 with hedgehogs. Antibiotic resistance was detected in 14.2% of all isolates, with resistance against important WATCH group antibiotics present in a small number of isolates. We further found that wildlife isolates do not form separate phylogenetic clusters distant to isolates from domestic animals and foodstuff, thus indicating frequent transmission events between these reservoirs. Overall, our study shows that Salmonella in German wildlife are diverse, with a low AMR burden and close links to Salmonella populations of farm and food-production environments.

Despite extensive monitoring programs and preventative measures, Salmonella spp. continue to cause tens of thousands human infections per year, as well as many regional and international food-borne outbreaks, that are of great importance for public health and cause significant socio-economic costs. In Germany, salmonellosis is the second most common cause of bacterial diarrhea in humans and is associated with high hospitalization rates. Whole-genome sequencing (WGS) combined with data analysis is a high throughput technology with an unprecedented discriminatory power, which is particularly well suited for targeted pathogen monitoring, rapid cluster detection and assignment of possible infection sources. However, an effective implementation of WGS methods for large-scale microbial pathogen detection and surveillance has been hampered by the lack of standardized methods, uniform quality criteria and strategies for data sharing, all of which are essential for a successful interpretation of sequencing data from different sources. To overcome these challenges, the national GenoSalmSurv project aims to establish a working model for an integrated genome-based surveillance system of Salmonella spp. in Germany, based on a decentralized data analysis. Backbone of the model is the harmonization of laboratory procedures and sequencing protocols, the implementation of open-source bioinformatics tools for data analysis at each institution and the establishment of routine practices for cross-sectoral data sharing for a uniform result interpretation. With this model, we present a working solution for cross-sector interpretation of sequencing data from different sources (such as human, veterinarian, food, feed and environmental) and outline how a decentralized data analysis can contribute to a uniform cluster detection and facilitate outbreak investigations.