The effects of photoactivated ciprofloxacin and bile acids on biofilms on bile duct catheters.

OBJECTIVES: This study examined the potential of a novel photoactivatable ciprofloxacin to act against bacterial infections and microbiomes related to biliary diseases. It also evaluated treatment by combining the impact of bile acids and antibiotics on biofilms. Innovative strategies were evaluated to address the elusive bile duct microbiome resulting in biofilm-related infections linked to biliary catheters. The healthy biliary system is considered sterile, but bile microbiomes can occur in disease, and these correlate with hepatobiliary diseases. Causes include biofilms that form on internal-external biliary drainage catheters. These biliary catheters were used to noninvasively study the otherwise elusive bile microbiome for a pilot study. METHODS: A new photoactivatable antibiotic was tested for efficacy against human-derived pathogenic bacterial isolates - Salmonella enterica and Escherichia coli - and catheter-derived bile duct microbiomes. In addition, the effect of bile acids on the antibiotic treatment of biofilms was quantified using crystal violet staining, confocal laser scanning microscopy, and biofilm image analysis. Two novel approaches for targeting biliary biofilms were tested. RESULTS: A photoactivated antibiotic based on ciprofloxacin showed efficacy in preventing biofilm formation and reducing bacterial viability without harming eukaryotic cells. Furthermore, combination treatment of antibiotics with bile acids, such as ursodesoxycholic acid, mildly influenced biofilm biomass but reduced bacterial survival within biofilms. CONCLUSION: Bile acids, in addition to their endocrine and paracrine functions, may enhance antibiotic killing of bacterial biofilms compared with antibiotics alone. These approaches hold promise for treating biliary infections such as cholangitis.

Gut microbiome in atypical depression.

BACKGROUND: Recent studies showed that immunometabolic dysregulation is related to unipolar major depressive disorder (MDD) and that it more consistently maps to MDD patients endorsing an atypical symptom profile, characterized by energy-related symptoms including increased appetite, weight gain, and hypersomnia. Despite the documented influence of the microbiome on immune regulation and energy homeostasis, studies have not yet investigated microbiome differences among clinical groups in individuals with MDD. METHODS: Fifteen MDD patients with atypical features according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5)-5, forty-four MDD patients not fulfilling the DSM-5 criteria for the atypical subtype, and nineteen healthy controls were included in the study. Participants completed detailed clinical assessment and stool samples were collected. Samples were sequenced for the prokaryotic 16S rRNA gene, in the V3-V4 variable regions. Only samples with no antibiotic exposure in the previous 12 months and a minimum of >2000 quality-filtered reads were included in the analyses. RESULTS: There were no statistically significant differences in alpha- and beta-diversity between the MDD groups and healthy controls. However, within the atypical MDD group, there was an increase in the Verrucomicrobiota phylum, with Akkermansia as the predominant bacterial genus. LIMITATIONS: Cross-sectional data, modest sample size, and significantly increased body mass index in the atypical MDD group. CONCLUSIONS: There were no overall differences among the investigated groups. However, differences were found at several taxonomic levels. Studies in larger longitudinal samples with relevant confounders are needed to advance the understanding of the microbial influences on the clinical heterogeneity of depression.

NanopoReaTA: a user-friendly tool for nanopore-seq real-time transcriptional analysis.

SUMMARY: Oxford Nanopore Technologies' (ONT) sequencing platform offers an excellent opportunity to perform real-time analysis during sequencing. This feature allows for early insights into experimental data and accelerates a potential decision-making process for further analysis, which can be particularly relevant in the clinical context. Although some tools for the real-time analysis of DNA-sequencing data already exist, there is currently no application available for differential transcriptome data analysis designed for scientists or physicians with limited bioinformatics knowledge. Here, we introduce NanopoReaTA, a user-friendly real-time analysis toolbox for RNA-sequencing data from ONT. Sequencing results from a running or finished experiment are processed through an R Shiny-based graphical user interface with an integrated Nextflow pipeline for whole transcriptome or gene-specific analyses. NanopoReaTA provides visual snapshots of a sequencing run in progress, thus enabling interactive sequencing and rapid decision making that could also be applied to clinical cases. AVAILABILITY AND IMPLEMENTATION: Github https://github.com/AnWiercze/NanopoReaTA; Zenodo https://doi.org/10.5281/zenodo.8099825.

Impaired flux of bile acids from the liver to the gut reveals microbiome-immune interactions associated with liver damage.

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.

Microbiome and immuno-metabolic dysregulation in patients with major depressive disorder with atypical clinical presentation.

Depression is highly prevalent (6% 1-year prevalence) and is the second leading cause of disability worldwide. Available treatment options for depression are far from optimal, with response rates only around 50%. This is most likely related to a heterogeneous clinical presentation of major depression disorder (MDD), suggesting different manifestations of underlying pathophysiological mechanisms. Poorer treatment outcomes to first-line antidepressants were reported in MDD patients endorsing an "atypical" symptom profile that is characterized by preserved reactivity in mood, increased appetite, hypersomnia, a heavy sensation in the limbs, and interpersonal rejection sensitivity. In recent years, evidence has emerged that immunometabolic biological dysregulation is an important underlying pathophysiological mechanism in depression, which maps more consistently to atypical features. In the last few years human microbial residents have emerged as a key influencing variable associated with immunometabolic dysregulations in depression. The microbiome plays a critical role in the training and development of key components of the host's innate and adaptive immune systems, while the immune system orchestrates the maintenance of key features of the host-microbe symbiosis. Moreover, by being a metabolically active ecosystem commensal microbes may have a huge impact on signaling pathways, involved in underlying mechanisms leading to atypical depressive symptoms. In this review, we discuss the interplay between the microbiome and immunometabolic imbalance in the context of atypical depressive symptoms. Although research in this field is in its infancy, targeting biological determinants in more homogeneous clinical presentations of MDD may offer new avenues for the development of novel therapeutic strategies for treatment-resistant depression. This article is part of the Special Issue on "Microbiome & the Brain: Mechanisms & Maladies".

Characterisation of a Staphylococcus aureus Isolate Carrying Phage-Borne Enterotoxin E from a European Badger (Meles meles).

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.

beeRapp: an R shiny app for automated high-throughput explorative analysis of multivariate behavioral data.

Summary: Animal behavioral studies typically generate high-dimensional datasets consisting of multiple correlated outcome measures across distinct or related behavioral domains. Here, we introduce the BEhavioral Explorative analysis R shiny APP (beeRapp) that facilitates explorative and inferential analysis of behavioral data in a high-throughput fashion. By employing an intuitive and user-friendly graphical user interface, beeRapp empowers behavioral scientists without programming and data science expertise to perform clustering, dimensionality reduction, correlational and inferential statistics and produce up to thousands of high-quality output plots visualizing results in a standardized and automated way. Availability and implementation: The code and data underlying this article are available at https://github.com/anmabu/beeRapp.

First Comparative Analysis of Clostridium septicum Genomes Provides Insights Into the Taxonomy, Species Genetic Diversity, and Virulence Related to Gas Gangrene.

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.

Testing assembly strategies of Francisella tularensis genomes to infer an evolutionary conservation analysis of genomic structures.

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.

An integrative understanding of the large metabolic shifts induced by antibiotics in critical illness.

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.

Bacterial gastroenteritis caused by the putative zoonotic pathogen Campylobacter lanienae: First reported case in Germany.

Foodborne campylobacteriosis is the most common cause of human bacterial enteritis in Germany. Campylobacter jejuni and Campylobacter coli are the main causative agents for enteric disease, but a number of other species are involved, including rare ones. These rare Campylobacter spp. are emerging zoonotic pathogens in humans due to increasing international movement of supplies, livestock and people. Campylobacter lanienae was first isolated from healthy abattoir workers in Switzerland and at first its pathogenic potential for humans was considered to be low. Recently, the first case of Campylobacter lanienae -associated human enteritis was reported in Canada. Here, we describe a case of mild Campylobacter lanienae -associated enteritis with subsequent asymptomatic excretion in a butcher. The isolate is available at the TLV strain collection (no. TP00333/18). This first reported case of human Campylobacter lanienae campylobacteriosis in Germany demonstrates the agent's likely zoonotic pathogenicity.

Antimicrobial Susceptibility and Genomic Analysis of Aliarcobacter cibarius and Aliarcobacter thereius, Two Rarely Detected Aliarcobacter Species.

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.

Draft Genome Sequence of Persistent Klebsiella grimontii AT013-Mero-001, Isolated from Human Feces.

Here, we report the draft genome sequence of Klebsiella grimontii AT013-Mero-001, which was isolated from feces from a sepsis patient treated with meropenem. This isolate is an antibiotic-susceptible but persistent Enterobacteriaceae strain.

Newly Isolated Animal Pathogen Corynebacterium silvaticum Is Cytotoxic to Human Epithelial Cells.

Corynebacterium silvaticum is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen Corynebacterium ulcerans and the widely distributed animal pathogen Corynebacterium pseudotuberculosis. In this study, Corynebacterium silvaticum strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of C. silvaticum to different human epithelial cell lines and to an invertebrate animal model, Galleria mellonella larvae, comparable to diphtheria toxin-secreting C. ulcerans. Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.

Comparative in silico genome analysis of Clostridium perfringens unravels stable phylogroups with different genome characteristics and pathogenic potential.

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.

Using affinity propagation clustering for identifying bacterial clades and subclades with whole-genome sequences of Francisella tularensis.

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.

Complete genome and plasmid sequences of a multidrug-resistant Salmonella enterica subsp. enterica serovar Panama isolate from a German cattle farm.

We describe a rare isolate of Salmonella enterica subsp. enterica serovar Panama with an extended-spectrum ß-lactamase (ESBL) profile from a German cattle-fattening farm. Applying two next-generation sequencing methods we generated sequences of the genome as well as the plasmids; assembled the draft genome sequence of Salmonella enterica subsp. enterica serovar Panama isolate 18PM0209. Antimicrobial resistance genes, virulence-associated genes and plasmids were analyzed using bioinformatics. Occurrence of multidrug-resistant Salmonella serovars at cattle-fattening farms indicate the need of enhanced surveillance to prevent further spread of these organisms.

Draft genome sequence of multi-resistant Salmonella enterica subsp. enterica serovar Rissen strain 19CS0416 isolated from Vietnam reveals mcr-1 plasmid mediated resistance to colistin already in 2013.

We report the first draft genome sequence of a Salmonella strain with plasmid-mediated resistance to colistin encoded by mcr-1 gene in Vietnam. Salmonella enterica subsp. enterica serovar Rissen was isolated from a Vietnamese pig slaughterhouse in 2013. We can confirm that mcr-1 gene is identical to the first reported mcr-1 gene of the Escherichia coli strain SHP45, isolated in 2015 from a Chinese pig. The plasmid containing this gene in the strain 19CS0416 was highly related (96.86% identity) to the plasmid (pHNSHP45) contained in this Chinese strain. Moreover, this plasmid was determined to be 100% identical to a plasmid (p13P477T-7) belonging to an Escherichia coli (13P477T) found in Hong Kong during the same year in pigs. Our results will aid in understanding the dissemination of mcr-1 gene in East Asia, dating back to as early as 2013.

Rapid and Culture Free Identification of Francisella in Hare Carcasses by High-Resolution Tandem Mass Spectrometry Proteotyping.

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.