Microbiota of the Gut-Lymph Node Axis: Depletion of Mucosa-Associated Segmented Filamentous Bacteria and Enrichment of Methanobrevibacter by Colistin Sulfate and Linco-Spectin in Pigs

Microorganisms are translocated from the gut to lymphatic tissues via immune cells, thereby challenging and training the mammalian immune system. Antibiotics alter the gut microbiome and consecutively might also affect the corresponding translocation processes, resulting in an imbalanced state between the intestinal microbiota and the host. Hence, understanding the variant effects of antibiotics on the microbiome of gut-associated tissues is of vital importance for maintaining metabolic homeostasis and animal health. In the present study, we analyzed the microbiome of (i) pig feces, ileum, and ileocecal lymph nodes under the influence of antibiotics (Linco-Spectin and Colistin sulfate) using 16S rRNA gene sequencing for high-resolution community profiling and (ii) ileocecal lymph nodes in more detail with two additional methodological approaches, i.e., cultivation of ileocecal lymph node samples and (iii) metatranscriptome sequencing of a single lymph node sample. Supplementation of medicated feed showed a local effect on feces and ileal mucosa-associated microbiomes. Pigs that received antibiotics harbored significantly reduced amounts of segmented filamentous bacteria (SFB) along the ileal mucosa (p = 0.048; 199.17-fold change) and increased amounts of Methanobrevibacter, a methanogenic Euryarchaeote in fecal samples (p = 0.005; 20.17-fold change) compared to the control group. Analysis of the porcine ileocecal lymph node microbiome exposed large differences between the viable and the dead fraction of microorganisms and the microbiome was altered to a lesser extent by antibiotics compared with feces and ileum. The core microbiome of lymph nodes was constituted mainly of Proteobacteria. RNA-sequencing of a single lymph node sample unveiled transcripts responsible for amino acid and carbohydrate metabolism as well as protein turnover, DNA replication and signal transduction. The study presented here is the first comparative study of microbial communities in feces, ileum, and its associated ileocecal lymph nodes. In each analyzed site, we identified specific phylotypes susceptible to antibiotic treatment that can have profound impacts on the host physiological and immunological state, or even on global biogeochemical cycles. Our results indicate that pathogenic bacteria, e.g., enteropathogenic Escherichia coli, could escape antibiotic treatment by translocating to lymph nodes. In general ileocecal lymph nodes harbor a more diverse and active community of microorganisms than previously assumed

Microbiota of the Gut-Lymph Node Axis: Depletion of Mucosa-Associated Segmented Filamentous Bacteria and Enrichment of Methanobrevibacter by Colistin Sulfate and Linco-Spectin in Pigs

Microorganisms are translocated from the gut to lymphatic tissues via immune cells, thereby challenging and training the mammalian immune system. Antibiotics alter the gut microbiome and consecutively might also affect the corresponding translocation processes, resulting in an imbalanced state between the intestinal microbiota and the host. Hence, understanding the variant effects of antibiotics on the microbiome of gut-associated tissues is of vital importance for maintaining metabolic homeostasis and animal health. In the present study, we analyzed the microbiome of (i) pig feces, ileum, and ileocecal lymph nodes under the influence of antibiotics (Linco-Spectin and Colistin sulfate) using 16S rRNA gene sequencing for high-resolution community profiling and (ii) ileocecal lymph nodes in more detail with two additional methodological approaches, i.e., cultivation of ileocecal lymph node samples and (iii) metatranscriptome sequencing of a single lymph node sample. Supplementation of medicated feed showed a local effect on feces and ileal mucosa-associated microbiomes. Pigs that received antibiotics harbored significantly reduced amounts of segmented filamentous bacteria (SFB) along the ileal mucosa (p = 0.048; 199.17-fold change) and increased amounts of Methanobrevibacter, a methanogenic Euryarchaeote in fecal samples (p = 0.005; 20.17-fold change) compared to the control group. Analysis of the porcine ileocecal lymph node microbiome exposed large differences between the viable and the dead fraction of microorganisms and the microbiome was altered to a lesser extent by antibiotics compared with feces and ileum. The core microbiome of lymph nodes was constituted mainly of Proteobacteria. RNA-sequencing of a single lymph node sample unveiled transcripts responsible for amino acid and carbohydrate metabolism as well as protein turnover, DNA replication and signal transduction. The study presented here is the first comparative study of microbial communities in feces, ileum, and its associated ileocecal lymph nodes. In each analyzed site, we identified specific phylotypes susceptible to antibiotic treatment that can have profound impacts on the host physiological and immunological state, or even on global biogeochemical cycles. Our results indicate that pathogenic bacteria, e.g., enteropathogenic Escherichia coli, could escape antibiotic treatment by translocating to lymph nodes. In general ileocecal lymph nodes harbor a more diverse and active community of microorganisms than previously assumed

Development of a DNA Metabarcoding Method for the Identification of Bivalve Species in Seafood Products

The production of bivalve species has been increasing in the last decades. In spite of strict requirements for species declaration, incorrect labelling of bivalve products has repeatedly been detected. We present a DNA metabarcoding method allowing the identification of bivalve species belonging to the bivalve families Mytilidae (mussels), Pectinidae (scallops), and Ostreidae (oysters) in foodstuffs. The method, developed on Illumina instruments, targets a 150 bp fragment of mitochondrial 16S rDNA. We designed seven primers (three primers for mussel species, two primers for scallop species and a primer pair for oyster species) and combined them in a triplex PCR assay. In each of eleven reference samples, the bivalve species was identified correctly. In ten DNA extract mixtures, not only the main component (97.0–98.0%) but also the minor components (0.5–1.5%) were detected correctly, with only a few exceptions. The DNA metabarcoding method was found to be applicable to complex and processed foodstuffs, allowing the identification of bivalves in, e.g., marinated form, in sauces, in seafood mixes and even in instant noodle seafood. The method is highly suitable for food authentication in routine analysis, in particular in combination with a DNA metabarcoding method for mammalian and poultry species published recently

Development of an analytical DNA metabarcoding method for species differentiation in food (focus on seafood)

Dissertation - Veterinärmedizinische Universität Wien - 2021Die Deklaration der Meeresfrüchte sowie die Haftung der Lebensmittelhersteller für die Authentizität ihrer Produkte werden mittels Verordnungen der Europäischen Union geregelt. Jedoch zeigen vergangene Lebensmittelskandale, dass nicht immer die Zutaten in den Lebensmittelprodukten enthalten sind, die auf den Produkten angegeben werden. Optisch ist es sehr schwierig eine Aussage darüber zu treffen, welche Bestandteile in einem verarbeiteten Lebensmittel enthalten sind. Authentische Lebensmittel (ausreichende und korrekte Kennzeichnung) sind aus verschiedenen persönlichen Gründen (Allergien, etc.) für Endkonsumenten, sowie zur Erfassung globaler Auswirkungen (illegale Fischerei, Rückverfolgbarkeit und Schutz bedrohter Arten) wichtig. Es existieren zahlreiche Nachweismethoden, die verschiedene Meeresfrüchte in verarbeiteten Lebensmitteln identifizieren können, wie zum Beispiel die Polymerasekettenreaktion (PCR) und PCR mit spezifischen Sonden (real-time PCR). Der Nachteil dieser Analysemethoden ist die Beschränkung auf nur wenige Spezies pro Analyselauf. Bei der Barcoding Methode wird ein spezifischer Teilbereich der Desoxyribonukleinsäure (DNA) mittels Sanger Sequenzierung sequenziert. Nachteilig ist hier, dass die Lebensmittel nur auf eine Tierspezies gleichzeitig untersucht werden können. Durch die Erweiterung dieser Methode, „Metabarcoding“, ist es möglich, Lebensmittel, die verschiede Spezies enthalten, mittels Next Generation Sequenzierung gleichzeitig zu sequenzieren. In dieser vorgestellten Arbeit über die Entwicklung eines Metabarcodingsystems der Muscheln, wurden drei konventionelle Muschelfamilien (Mytilidae (Miesmuscheln), Pectinidae (Kammmuscheln) und Ostreidae (Austern)) genutzt. Dazu wurden neue, universelle Primer für jede Muschelfamilie entwickelt und zu einem Triplexsystem zusammengefügt. Das 150 bp lange Ziel-Gen ist auf der 16S rDNA lokalisiert und erlaubt es eine Reihe verschiedener Muschelspezies voneinander zu unterscheiden. Die Sequenzierung erfolgte auf der Illumina MiSeq© und iSeq© Plattform. Es wurden 75 verschiedene Lebensmittel (unterschiedlichen Verarbeitungsgrad) mit der neuen Analysemethode untersucht. Dabei zeigte sich, dass es möglich ist Muscheln in hochverarbeiteten Lebensmitteln zu detektieren und auf Spezieslevel zu unterscheiden. Eine Vielzahl an Lebensmittelprodukten kann so zur selben Zeit analysiert werden. Somit ist dieses Metabarcodingsystem für Routineuntersuchungen geeignet und trägt dazu bei Lebensmittelkriminalität zu bekämpfen, wodurch das Vertrauen der Verbraucher in die Industrie gestärkt wird und Nahrungsmittel authentisch bleiben.Dissertation - University of Veterinary Medicine Vienna - 2021The appropriate declaration of seafood is stipulated by European regulations which also determine the responsibility of food producers for a correct authentication of their products. However, many pecuniary motivations do exist to manipulate food origin (e.g., food of regional versus global origin), food quality (e.g., organic vs. conventional production) or food composition (replacement of higher priced food components by other cheaper ones). Past food scandals show that ingredients in seafood products are not always what they claim to be. From a consumer`s perspective, seafood is seen as a luxury food and they are willing to accept higher prices than for more classical protein sources. Although accurate species assignment is already difficult when a seafood source is optically intact, assessment of the composition of ingredients in processed seafood is almost impossible. Traceable food authentication (sufficient and correct labelling) is important for a variety of reasons, personal ones (allergies, etc), food fraud related ones as well as for the sake of avoidance of illegal fishing and protection of endangered species. Numerous analytical methods exist which are able to distinguish different seafood sources in processed conditions. Polymerase chain reaction (PCR) based assays are among the most widespread and PCR assays were developed either without or with specific probes (real time PCR). The disadvantage of these analytical methods is the limitation of a few species per assay. As an advancement, a barcoding method was developed that sequences a specific part of the deoxyribonucleic acid (DNA) using Sanger technology. The disadvantage of this method is the limitation of analysing only one DNA target strand of a species. In the metabarcoding method, an extension of the barcoding approach, it is possible to sequence DNA containing different DNA target strands of multiple species simultaneously by using next generation sequencing. In this presented work on the development of a metabarcoding system of bivalves, three common bivalve families (Mytilidae (mussels), Pectinidae (scallops), and Ostreidae (oysters)) were used. For this purpose, new universal primers for each bivalve´s family were designed and combined into a triplex system. The 150 bp long target gene is localised on the 16S rDNA and allows to distinguish between a number of different bivalve species. Sequencing was performed on the Illumina MiSeq and iSeq platforms. Seventy-five different foods were analysed using the new method. It was demonstrated that it is possible to detect bivalves in even highly processed foods and to identify them at the species level. This allows a large number of food products to be analysed at the same time. This metabarcoding method is suitable for routine investigations and therefore supports fighting against food crime, strengthening consumer confidence in the industry, and keeping food authentic

Microbiota of the Gut-Lymph Node Axis: Depletion of Mucosa-Associated Segmented Filamentous Bacteria and Enrichment of Methanobrevibacter by Colistin Sulfate and Linco-Spectin in Pigs

Microorganisms are translocated from the gut to lymphatic tissues via immune cells, thereby challenging and training the mammalian immune system. Antibiotics alter the gut microbiome and consecutively might also affect the corresponding translocation processes, resulting in an imbalanced state between the intestinal microbiota and the host. Hence, understanding the variant effects of antibiotics on the microbiome of gut-associated tissues is of vital importance for maintaining metabolic homeostasis and animal health. In the present study, we analyzed the microbiome of (i) pig feces, ileum, and ileocecal lymph nodes under the influence of antibiotics (Linco-Spectin and Colistin sulfate) using 16S rRNA gene sequencing for high-resolution community profiling and (ii) ileocecal lymph nodes in more detail with two additional methodological approaches, i.e., cultivation of ileocecal lymph node samples and (iii) metatranscriptome sequencing of a single lymph node sample. Supplementation of medicated feed showed a local effect on feces and ileal mucosa-associated microbiomes. Pigs that received antibiotics harbored significantly reduced amounts of segmented filamentous bacteria (SFB) along the ileal mucosa (p = 0.048; 199.17-fold change) and increased amounts of Methanobrevibacter, a methanogenic Euryarchaeote in fecal samples (p = 0.005; 20.17-fold change) compared to the control group. Analysis of the porcine ileocecal lymph node microbiome exposed large differences between the viable and the dead fraction of microorganisms and the microbiome was altered to a lesser extent by antibiotics compared with feces and ileum. The core microbiome of lymph nodes was constituted mainly of Proteobacteria. RNA-sequencing of a single lymph node sample unveiled transcripts responsible for amino acid and carbohydrate metabolism as well as protein turnover, DNA replication and signal transduction. The study presented here is the first comparative study of microbial communities in feces, ileum, and its associated ileocecal lymph nodes. In each analyzed site, we identified specific phylotypes susceptible to antibiotic treatment that can have profound impacts on the host physiological and immunological state, or even on global biogeochemical cycles. Our results indicate that pathogenic bacteria, e.g., enteropathogenic Escherichia coli, could escape antibiotic treatment by translocating to lymph nodes. In general ileocecal lymph nodes harbor a more diverse and active community of microorganisms than previously assumed.This article is published as Zwirzitz B, Pinior B, Metzler-Zebeli B, Handler M, Gense K, Knecht C, Ladinig A, Dzieciol M, Wetzels SU, Wagner M, Schmitz-Esser S and Mann E (2019) Microbiota of the Gut-Lymph Node Axis: Depletion of Mucosa-Associated Segmented Filamentous Bacteria and Enrichment of Methanobrevibacter by Colistin Sulfate and Linco-Spectin in Pigs. Front. Microbiol. 10:599. doi: 10.3389/fmicb.2019.00599.</p