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Abstract Currently, there is evidence that alteration in the gut ecosystem contributes to the development of liver diseases, however, the complex mechanisms involved are still unclear. We induced cholestasis in mice by bile duct ligation (BDL), mirroring the phenotype of a bile duct obstruction, to understand how gut microbiota alterations caused by an impaired flow of bile acid to the gut contribute to the pathogenesis and progression of liver disease. We performed longitudinal stool, heart, and liver sampling using mice receiving BDL and controls receiving sham operation (ShamOP). Shotgun metagenomics profiling using fecal samples taken before and on day 1, day 3, and day 7 after surgery was performed, and the cytokines and clinical chemistry profiles from heart blood, as well as the liver bile acids profile, were measured. The BDL surgery reshaped the microbiome of mice, resulting in highly distinct characteristics compared to the ShamOP. Our analysis of the microbiome pathways and ECs revealed that BDL reduces the production of hepatoprotective compounds in the gut, such as biotin, spermidine, arginine, and ornithine, which were negatively associated with inflammatory cytokines (IL-6, IL-23, MCP-1). The reduction of the functional potential of the gut microbiota in producing those hepatoprotective compounds is associated with the decrease of beneficial bacteria species from Anaerotruncus, Blautia, Eubacterium, and Lachnoclostridium genera, as well as the increase of disease-associated bacteria e.g., Escherichia coli and Entercoccus faecalis. Our findings advances our knowledge of the gut microbiome-bile acids-liver triangle, which may serve as a potential therapeutic strategy for liver diseases.

Abstract Background We benchmarked sequencing technology and assembly strategies for short-read, long-read, and hybrid assemblers in respect to correctness, contiguity, and completeness of assemblies in genomes of Francisella tularensis. Benchmarking allowed in-depth analyses of genomic structures of the Francisella pathogenicity islands and insertion sequences. Five major high-throughput sequencing technologies were applied, including next-generation “short-read” and third-generation “long-read” sequencing methods. Results We focused on short-read assemblers, hybrid assemblers, and analysis of the genomic structure with particular emphasis on insertion sequences and the Francisella pathogenicity island. The A5-miseq pipeline performed best for MiSeq data, Mira for Ion Torrent data, and ABySS for HiSeq data from eight short-read assembly methods. Two approaches were applied to benchmark long-read and hybrid assembly strategies: long-read-first assembly followed by correction with short reads (Canu/Pilon, Flye/Pilon) and short-read-first assembly along with scaffolding based on long reads (Unicyler, SPAdes). Hybrid assembly can resolve large repetitive regions best with a “long-read first” approach. Conclusions Genomic structures of the Francisella pathogenicity islands frequently showed misassembly. Insertion sequences (IS) could be used to perform an evolutionary conservation analysis. A phylogenetic structure of insertion sequences and the evolution within the clades elucidated the clade structure of the highly conservative F. tularensis.

Staphylococcus (S.) aureus colonizes up to 30% of all humans and can occasionally cause serious infections. It is not restricted to humans as it can also often be found in livestock and wildlife. Recent studies have shown that wildlife strains of S. aureus usually belong to other clonal complexes than human strains and that they might differ significantly with regard to the prevalence of genes encoding antimicrobial resistance properties and virulence factors. Here, we describe a strain of S. aureus isolated from a European badger (Meles meles). For molecular characterisation, DNA microarray-based technology was combined with various next-generation sequencing (NGS) methods. Bacteriophages from this isolate were induced with Mitomycin C and characterized in detail by transmission electron microscopy (TEM) and NGS. The S. aureus isolate belonged to ST425 and had a novel spa repeat sequence (t20845). It did not carry any resistance genes. The uncommon enterotoxin gene see was detected in one of its three temperate bacteriophages. It was possible to demonstrate the induction of all three prophages, although only one of them was expected to be capable of excision based on its carriage of the excisionase gene xis. All three bacteriophages belonged to the family Siphoviridae. Minor differences in size and shape of their heads were noted in TEM images. The results highlight the ability of S. aureus to colonize or infect different host species successfully, which can be attributed to a variety of virulence factors on mobile genetic elements, such as bacteriophages. As shown in the strain described herein, temperate bacteriophages not only contribute to the fitness of their staphylococcal host by transferring virulence factors, but also increase mobility among themselves by sharing genes for excision and mobilization with other prophages.

Abstract Clostridium perfringens causes a plethora of devastating infections, with toxin production being the underlying mechanism of pathogenicity in various hosts. Genomic analyses of 206 public-available C. perfringens strains´ sequence data identified a substantial degree of genomic variability in respect to episome content, chromosome size and mobile elements. However, the position and order of the local collinear blocks on the chromosome showed a considerable degree of preservation. The strains were divided into five stable phylogroups (I–V). Phylogroup I contained human food poisoning strains with chromosomal enterotoxin (cpe) and a Darmbrand strain characterized by a high frequency of mobile elements, a relatively small genome size and a marked loss of chromosomal genes, including loss of genes encoding virulence traits. These features might correspond to the adaptation of these strains to a particular habitat, causing human foodborne illnesses. This contrasts strains that belong to phylogroup II where the genome size points to the acquisition of genetic material. Most strains of phylogroup II have been isolated from enteric lesions in horses and dogs. Phylogroups III, IV and V are heterogeneous groups containing a variety of different strains, with phylogroup III being the most abundant (65.5%). In conclusion, C. perfringens displays five stable phylogroups reflecting different disease involvements, prompting further studies on the evolution of this highly important pathogen.

Clostridium septicum is a Gram-positive, toxin-producing, and spore-forming bacterium that is recognized, together with C. perfringens, as the most important etiologic agent of progressive gas gangrene. Clostridium septicum infections are almost always fatal in humans and animals. Despite its clinical and agricultural relevance, there is currently limited knowledge of the diversity and genome structure of C. septicum. This study presents the complete genome sequence of C. septicum DSM 7534T type strain as well as the first comparative analysis of five C. septicum genomes. The taxonomy of C. septicum, as revealed by 16S rRNA analysis as well as by genomic wide indices such as protein-based phylogeny, average nucleotide identity, and digital DNA–DNA hybridization indicates a stable clade. The composition and presence of prophages, CRISPR elements and accessory genetic material was variable in the investigated genomes. This is in contrast to the limited genetic variability described for the phylogenetically and phenotypically related species Clostridium chauvoei. The restriction-modification (RM) systems between two C. septicum genomes were heterogeneous for the RM types they encoded. C. septicum has an open pangenome with 2,311 genes representing the core genes and 1,429 accessory genes. The core genome SNP divergence between genome pairs varied up to 4,886 pairwise SNPs. A vast arsenal of potential virulence genes was detected in the genomes studied. Sequence analysis of these genes revealed that sialidase, hemolysin, and collagenase genes are conserved compared to the α-toxin and hyaluronidase genes. In addition, a conserved gene found in all C. septicum genomes was predicted to encode a leucocidin homolog (beta-channel forming cytolysin) similar (71.10% protein identity) to Clostridium chauvoei toxin A (CctA), which is a potent toxin. In conclusion, our results provide first, valuable insights into strain relatedness and genomic plasticity of C. septicum and contribute to our understanding of the virulence mechanisms of this important human and animal pathogen.

Aliarcobacter cibarius and Aliarcobacter thereius are two rarely detected Aliarcobacter species. In the study, we analyzed the antimicrobial susceptibility and provide detailed insights into the genotype and phylogeny of both species using whole-genome sequencing. Thermophilic Campylobacter species are the most common bacterial foodborne pathogens causing gastroenteritis in humans worldwide. The genus Aliarcobacter is part of the Campylobacteraceae family and includes the species Aliarcobacter butzleri, Aliarcobacter cryaerophilus, Aliarcobacter skirrowii, and the rarely described Aliarcobacter cibarius, Aliarcobacter faecis, Aliarcobacter lanthieri, Aliarcobacter thereius, and Acrobarter trophiarum. Aliarcobacter are emergent enteropathogens and potential zoonotic agents. Here, we generated, analyzed, and characterized whole-genome sequences of Aliarcobacter cibarius and Aliarcobacter thereius. They were isolated from water poultry farms in Germany, cultured and identified by MALDI-TOF MS. With PCR the identity was verified. Antibiotic susceptibility testing was carried out with erythromycin, ciprofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, ampicillin, and cefotaxime using the gradient strip method (E-test). Whole-genome sequences were generated including those of reference strains. Complete genomes for six selected strains are reported. These provide detailed insights into the genotype. With these, we predicted in silico known AMR genes, virulence-associated genes, and plasmid replicons. Phenotypic analysis of resistance showed differences between the presence of resistance genes and the prediction of phenotypic resistance profiles. In Aliarcobacter butzleri, the nucleotide sequence of the gyrA gene (DQ464331) can show a signature mutation resulting in an amino acid change T85>I. Acrobarter cibarius and Acrobarter thereius showed the same gene as assessed by similarity annotation of the mutations 254C>G. Most of the isolates were found to be sensitive to ciprofloxacin. The ciprofloxacin-resistant Aliarcobacter thereius isolate was associated with the amino acid change T85>I. But this was not predicted with antibiotic resistance databases, before. Ultimately, a phylogenetic analysis was done to facilitate in future outbreak analysis.

Antibiotics are commonly used in the Intensive Care Unit (ICU); however, several studies showed that the impact of antibiotics to prevent infection, multi-organ failure, and death in the ICU is less clear than their benefit on course of infection in the absence of organ dysfunction. We characterized here the compositional and metabolic changes of the gut microbiome induced by critical illness and antibiotics in a cohort of 75 individuals in conjunction with 2,180 gut microbiome samples representing 16 different diseases. We revealed an “infection-vulnerable” gut microbiome environment present only in critically ill treated with antibiotics (ICU+). Feeding of Caenorhabditis elegans with Bifidobacterium animalis and Lactobacillus crispatus, species that expanded in ICU+ patients, revealed a significant negative impact of these microbes on host viability and developmental homeostasis. These results suggest that antibiotic administration can dramatically impact essential functional activities in the gut related to immune responses more than critical illness itself, which might explain in part untoward effects of antibiotics in the critically ill.

By combining a reference-independent SNP analysis and average nucleotide identity (ANI) with affinity propagation clustering (APC), we developed a significantly improved methodology allowing resolving phylogenetic relationships, based on objective criteria. These bioinformatics tools can be used as a general ruler to determine phylogenetic relationships and clustering of bacteria, exemplary done with Francisella (F.) tularensis. Molecular epidemiology of F. tularensis is currently assessed mostly based on laboratory methods and molecular analysis. The high evolutionary stability and the clonal nature makes Francisella ideal for subtyping with single nucleotide polymorphisms (SNPs). Sequencing and real-time PCR can be used to validate the SNP analysis. We investigate whole-genome sequences of 155 F. tularensis subsp. holarctica isolates. Phylogenetic testing was based on SNPs and average nucleotide identity (ANI) as reference independent, alignment-free methods taking small-scale and large-scale differences within the genomes into account. Especially the whole genome SNP analysis with kSNP3.0 allowed deciphering quite subtle signals of systematic differences in molecular variation. Affinity propagation clustering (APC) resulted in three clusters showing the known clades B.4, B.6, and B.12. These data correlated with the results of real‐time PCR assays targeting canSNPs loci. Additionally, we detected two subtle sub-clusters. SplitsTree was used with standard-setting using the aligned SNPs from Parsnps. Together APC, HierBAPS, and SplitsTree enabled us to generate hypotheses about epidemiologic relationships between bacterial clusters and describing the distribution of isolates. Our data indicate that the choice of the typing technique can increase our understanding of the pathogenesis and transmission of diseases with the eventual for prevention. This is opening perspectives to be applied to other bacterial species. The data provide evidence that Germany might be the collision zone where the clade B.12, also known as the East European clade, overlaps with the clade B.6, also known as the Iberian clade. Described methods allow generating a new, more detailed perspective for F. tularensis subsp. holarctica phylogeny. These results may encourage to determine phylogenetic relationships and clustering of other bacteria the same way. Author summary By combining a reference-independent SNP analysis and ANI (average nucleotide identity) with affinity propagation clustering (APC), we tested a methodology allowing resolving phylogenetic relationships, based on objective criteria. These bioinformatics tools can be used as a general ruler to determine phylogenetic relationships and clustering of bacteria, exemplary done with Francisella tularensis. Francisella tularensis causes the zoonosis tularemia. We analyzed the relationships between Francisella tularensis subsp. holarctica isolates from Germany using whole-genome sequences. We chose open source, reference independent methods to optimize the level of discrimination. Using a recently described clustering algorithm, we exploit a novel approach to the clustering of bacteria. APC can be used for assigning clades and can be used for rapidly typing strains when they arise. Additionally, we detected two sub-clusters. The data provide evidence that Germany is the collision zone where the clade B.12, also known as the East European clade, overlaps with the clade B.6, also known as the Iberian clade.

Zoonotic pathogens that can be transmitted via food to humans have a high potential for large-scale emergencies, comprising severe effects on public health, critical infrastructures, and the economy. In this context, the development of laboratory methods to rapidly detect zoonotic bacteria in the food supply chain, including high-resolution mass spectrometry proteotyping are needed. In this work, an optimized sample preparation method for liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteome profiling was established for Francisella isolates, and a cluster analysis, as well as a phylogenetic tree, was generated to shed light on evolutionary relationships. Furthermore, this method was applied to tissues of infected hare carcasses from Germany. Even though the non-informative data outnumbered by a manifold the information of the zoonotic pathogen in the resulting proteome profiles, the standardized evaluation of MS data within an established automated analysis pipeline identified Francisella (F.) tularensis and, thus, could be, in principle, an applicable method to monitor food supply chains.

Abstract Objectives Corynebacterium ulcerans can colonize a wide variety of animals and also humans are infected, typically by zoonotic transmission. Symptoms range from skin ulcers or systemic infections to diphtheria-like illness. In contrast, Corynebacterium pseudotuberculosis is widely distributed among herds of sheep, goats and other farm animals, where it causes high economic losses due to caseous lymphadenitis. Here we describe the genome sequence of an atypical C. ulcerans strain isolated from a wild boar with necrotizing lymphadenitis. This strain has similarities to C. pseudotuberculosis. Data description Genome sequence data of C. ulcerans isolate W25 were generated, analyzed and taxonomical relationship to other Corynebacterium species as well as growth properties of the isolate were characterized. The genome of C. ulcerans W25 comprises 2,550,924 bp with a G+C content of 54.41% and a total of 2376 genes.

Abstract In humans, stress can be contagiously transmitted via chemosignals on a subconscious level. This study investigates how pregnancy affects neural responses to anxiety chemosignals. Using cotton pads, 28 men donated axillary sweat immediately before an academic examination (anxiety sweat) and during ergometer training (control). Via a constant-flow olfactometer, samples were presented (oddball paradigm) to 12 non-pregnant (NP) women, 14 women in their first (T1), and 18 in their third (T3) trimester of pregnancy. Chemosensory event-related potentials and current source densities (CSD) were analysed (60 electrode setup). Compared to NP-women, pregnant women display diminished evaluative processing of the sweat samples (targets; P3-1/ P3-2 amplitudes) and delayed evaluative processing of the anxiety sweat (targets; P3-2 latency). T3-women show attenuated early processing (targets; N1 amplitude) compared to NP-women, and reduced evaluative processing compared to T1-women (standards; P3-2 amplitude). CSDs (P3-1/ P3-2 latency ranges) reveal that T1- and T3-women show an atypical activation distribution to anxiety sweat. Most participants were unable to detect the sweat samples (anxiety sweat: 79.5%, sport sweat 88.6%). The results demonstrate that the processing of anxiety chemosignals progressively vanishes during pregnancy. This effect is likely to occur without any cognitive control.

In food safety the detection of food contaminations with pathogenic microorganisms is a race against time and often outpaced by error-prone epidemiological approaches. For evidence-based outbreak investigations fast and reliable techniques and procedures are required to identify the source of infection. Metagenomics has the potential to become a powerful tool in the field of modern food safety, since it allows the detection, identification and characterization of a broad range of pathogens in a single experiment without pre-cultivation within a couple of days. Nevertheless, sample handling, sequencing and data analysis are challenging and can introduce errors and biases into the analysis. In order to evaluate the potential of metagenomics in food safety, we generated a mock community containing DNA of foodborne bacteria. Herewith, we compare the aptitude of the two prevalent approaches – 16S rDNA amplicon sequencing and whole genome shotgun sequencing – for the detection of foodborne bacteria using different parameters during sample preparation, sequencing and data analysis. 16S rDNA sequencing did not only result in high deviations from the expected sample composition on genus and species level, but more importantly lacked the detection of several pathogenic species. While shotgun sequencing is more suitable for species detection, abundance estimation, genome assembly and species characterization, the performance can vary depending on the library preparation kit, which was confirmed for a naturally Francisella tularensis contaminated game meat sample. The application of the Nextera XT DNA Library Preparation Kit for shotgun sequencing did not only result in lower reference genome recovery and coverage, but also in distortions of the mock community composition. For data analysis, we propose a publicly available workflow for pathogen detection and characterization and demonstrate its benefits on the usability of metagenomic sequencing in food safety by analyzing an authentic metagenomic sample.

Aims/hypothesis: It has been suggested that air pollution may increase the risk of type 2 diabetes but data on particulate matter with diameter

Campylobacter spp. are considered the most common bacterial cause of foodborne gastroenteritis in the world. The family Campylobacteraceae includes the genus Arcobacter with the three species Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii as emergent enteropathogens and potential zoonotic agents. Here, we characterized genome sequences of Arcobacter that were isolated from water poultry on farms in Germany. Isolates were cultured, identified by MALDI-TOF MS and identification was verified with PCR assays. Antibiotic susceptibility testing of isolates was carried out with erythromycin, ciprofloxacin, doxycycline, tetracycline, gentamicin, and streptomycin using the gradient strip method (E-test). We also sequenced whole genomes and predicted antibiotic resistance determinants, virulence factors, performed a phylogenetic analysis to determine the genetic relatedness of these isolates and searched for plasmids.

Francisella (F.) tularensis is a highly virulent, Gram-negative bacterial pathogen and the causative agent of the zoonotic disease tularemia. Here, we generated, analyzed and characterized a high quality circular genome sequence of the F. tularensis subsp. holarctica strain 12T0050 that caused fatal tularemia in a hare. Besides the genomic structure, we focused on the analysis of oriC, unique to the Francisella genus and regulating replication in and outside hosts and the first report on genomic DNA methylation of a Francisella strain. The high quality genome was used to establish and evaluate a diagnostic whole genome sequencing pipeline. A genotyping strategy for F. tularensis was developed using various bioinformatics tools for genotyping. Additionally, whole genome sequences of F. tularensis subsp. holarctica isolates isolated in the years 2008–2015 in Germany were generated. A phylogenetic analysis allowed to determine the genetic relatedness of these isolates and confirmed the highly conserved nature of F. tularensis subsp. holarctica.

Based upon research on “coupled careers”, the partnership is seen as an important context where not only career opportunities but also barriers are generated. Using data from the Socio-Economic Panel Study (SOEP), this article pays particular attention to status homogamy and partners’ social support as beneficial aspects of partnerships for career advancement. The analysis of dual-earner couples reveals that men are more likely to move up into high occupational positions when they live in a highly qualified status homogameous partnership where both partners are highly educated and work in similar occupations. This is in line with arguments from social capital theory assuming an exchange of knowledge and understanding between partners especially in homogamous partnerships. Furthermore, the analysis shows that for women partners’ social support is of significant importance for gaining a high occupational position: Women are more likely to advance their careers if they report that their partners support their career advancement, and if the partner carries out the higher amount of domestic work. Because in Germany the division of domestic labor is still traditional, with women carrying out the lion’s share of domestic work, this “support gap” plays a significant role in explaining the underrepresentation of women in high occupational positions, as the analysis shows. Zusammenfassung Als wichtiger Kontext, in dem Aufstiegschancen, aber auch -hindernisse generiert werden, gilt die Partnerschaft. Der Artikel knüpft mit Daten des Sozio-ökonomischen Panels an Forschung zu „coupled careers“ an und fokussiert insbesondere auf Status-Homogamie und soziale Unterstützungsleistungen als mögliche karriereförderliche Aspekte von Partnerschaften. Die Analyse von erwerbstätigen Frauen und Männern in Doppelverdiener- Partnerschaften verdeutlicht, dass insbesondere Männer häufig in hohe berufliche Positionen aufsteigen, wenn sie und ihre Partnerin im gleichen Berufsfeld arbeiten und beide eine hohe Bildung aufweisen. Dies kann mit sozialkapitaltheoretischen Überlegungen erklärt werden, die von einem partnerschaftlichen Austausch beruflicher Netzwerke, Wissenstransfers und Verständnis gerade bei dem Vorliegen einer solchen Status- Homoganie. Darüber hinaus zeigen die Analyseergebnisse, dass insbesondere für Frauen die soziale Unterstützung durch den Partner wichtig ist, um eine hohe berufliche Position zu erreichen. So steigen Frauen signifikant häufiger auf, wenn sie angeben, dass ihr Partner sie im beruflichen Fortkommen unterstützt, und wenn er den höheren Anteil an Hausarbeit verrichtet. Da die partnerschaftliche Hausarbeitsteilung in Deutschland nach wie vor stark zu Lasten der Frauen geht, spielt ein solcher „support gap“ zur Erklärung der Unterrepräsentanz von Frauen in hohen beruflichen Positionen eine entscheidende Rolle, wie die Analyse verdeutlicht.

Atypical neuropathological and molecular phenotypes of bovine spongiform encephalopathy (BSE) have recently been identified in different countries. One of these phenotypes, named bovine "amyloidotic" spongiform encephalopathy (BASE), differs from classical BSE for the occurrence of a distinct type of the disease-associated prion protein (PrP), termed PrP(Sc), and the presence of PrP amyloid plaques. Here, we show that the agents responsible for BSE and BASE possess different biological properties upon transmission to transgenic mice expressing bovine PrP and inbred lines of nontransgenic mice. Strikingly, serial passages of the BASE strain to nontransgenic mice induced a neuropathological and molecular disease phenotype indistinguishable from that of BSE-infected mice. The existence of more than one agent associated with prion disease in cattle and the ability of the BASE strain to convert into the BSE strain may have important implications with respect to the origin of BSE and spongiform encephalopathies in other species, including humans.

The persistence of infectious biomolecules in soil constitutes a substantial challenge. This holds particularly true with respect to prions, the causative agents of transmissible spongiform encephalopathies (TSEs) such as scrapie, bovine spongiform encephalopathy (BSE), or chronic wasting disease (CWD). Various studies have indicated that prions are able to persist in soil for years without losing their pathogenic activity. Dissemination of prions into the environment can occur from several sources, e.g., infectious placenta or amniotic fluid of sheep. Furthermore, environmental contamination by saliva, excrements or non-sterilized agricultural organic fertilizer is conceivable. Natural transmission of scrapie in the field seems to occur via the alimentary tract in the majority of cases, and scrapie-free sheep flocks can become infected on pastures where outbreaks of scrapie had been observed before. These findings point to a sustained contagion in the environment, and notably the soil. By using outdoor lysimeters, we simulated a contamination of standard soil with hamster-adapted 263K scrapie prions, and analyzed the presence and biological activity of the soil-associated PrP(Sc) and infectivity by Western blotting and hamster bioassay, respectively. Our results showed that 263K scrapie agent can persist in soil at least over 29 months. Strikingly, not only the contaminated soil itself retained high levels of infectivity, as evidenced by oral administration to Syrian hamsters, but also feeding of aqueous soil extracts was able to induce disease in the reporter animals. We could also demonstrate that PrP(Sc) in soil, extracted after 21 months, provides a catalytically active seed in the protein misfolding cyclic amplification (PMCA) reaction. PMCA opens therefore a perspective for considerably improving the detectability of prions in soil samples from the field.

In the past, natural scrapie and bovine spongiform encephalopathy (BSE) infections have essentially not been diagnosed in sheep homozygous for the A136R154R171 haplotype of the prion protein. This genotype was therefore assumed to confer resistance to BSE and classic scrapie under natural exposure conditions. Hence, to exclude prions from the human food chain, massive breeding efforts have been undertaken in the European Union to amplify this gene. We report the identification of 2 natural scrapie cases in ARR/ARR sheep that have biochemical and transmission characteristics similar to cases of classic scrapie, although the abnormally folded prion protein (PrPSc) was associated with a lower proteinase-K resistance. PrPSc was clearly distinct from BSE prions passaged in sheep and from atypical scrapie prions. These findings strongly support the idea that scrapie prions are a mosaic of agents, which harbor different biologic properties, rather than a unique entity.