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

Austrian Raw-Milk Hard-Cheese Ripening Involves Successional Dynamics of Non-Inoculated Bacteria and Fungi

Cheese ripening involves successional changes of the rind microbial composition that harbors a key role on the quality and safety of the final products. In this study, we analyzed the evolution of the rind microbiota (bacteria and fungi) throughout the ripening of Austrian Vorarlberger Bergkäse (VB), an artisanal surface-ripened cheese, by using quantitative and qualitative approaches. The real-time quantitative PCR results revealed that bacteria were more abundant than fungi in VB rinds throughout ripening, although both kingdoms were abundant along the process. The qualitative investigation was performed by high-throughput gene-targeted (amplicon) sequencing. The results showed dynamic changes of the rind microbiota throughout ripening. In the fresh products, VB rinds were dominated by Staphylococcus equorum and Candida. At early ripening times (14–30 days) Psychrobacter and Debaryomyces flourished, although their high abundance was limited to these time points. At the latest ripening times (90–160 days), VB rinds were dominated by S. equorum, Brevibacterium, Corynebacterium, and Scopulariopsis. Strong correlations were shown for specific bacteria and fungi linked to specific ripening periods. This study deepens our understanding of VB ripening and highlights different bacteria and fungi associated to specific ripening periods which may influence the organoleptic properties of the final products

Whole microbial community analysis as a tool to study contamination scenarios during raw meat processing

Dissertation - University of Veterinary Medicine Vienna - 2020Microbial spoilage of food is a global health and economic concern that continues to burden public authorities. Although hygiene standards in industrial food production have been vastly improved over the last few decades, foodborne diseases are still on the rise. The surge in foodborne disease outbreaks despite increasing efforts to combat them, challenges the existing risk assessments and underlines the need for new modes of action to control and monitor microbial contamination in food processing environments. In principal, contamination of food by microorganisms is difficult to control, because of the manifold possibilities of transmission during production, processing, storage, and distribution of the product. Microbial contamination levels are routinely monitored using microbiological plating techniques, which offer only limited information, because the majority of microorganisms remain uncultivable. Therefore, these traditional methods do not capture many important microbial community members and do not reflect the actual contamination status. Recent advancements in DNA sequencing technologies now offer the opportunity to study whole microbial communities in their natural environment at an unprecedented depth. In this thesis, we apply quantitative traditional microbiology and molecular biology techniques as well as high throughput full-length 16S rRNA gene sequencing to characterize whole microbial communities throughout the raw meat processing chain. We further evaluate this method as a tool to study contamination scenarios and transmission routes of microbial populations during processing, and elucidate our findings in the broader context of microbial community ecology. The ecological aspects of microorganisms living and interacting within the context of complex communities other has been largely neglected in the field of food microbiology. Here, we show that food processing environments harbour complex microbiomes and point out specific microbial interactions that potentially promote the survival of pathogens or spoilage microorganisms in regularly disinfected environments. Specifically, we focused on Listeria monocytogenes and its putative interactions with the resilient microbiota in a meat processing plant, which are linked to a consortia of common microorganisms with known biofilm-forming capabilities. Our results demonstrate that high-throughput full-length 16S rRNA gene sequencing has great potential to uncover previously unknown microbial transmission routes and interactions that contribute to persistence. The acquired knowledge informs stakeholders with valuable information to combat microbial contamination of food to ensure a microbiologically safe food production.Dissertation - Veterinärmedizinische Universität Wien - 2020Obwohl die Hygienestandards in der industriellen Lebensmittelproduktion in den letzten Jahrzehnten erheblich verbessert wurden, nehmen durch verdorbene Lebensmittel verursachte Krankheiten immer noch zu. Diese Entwicklung stellt die bestehenden Risikobewertungen in Frage und unterstreicht die Notwendigkeit neuer Maßnahmen zur Kontrolle und Überwachung mikrobieller Übertragungswege während der Lebensmittelverarbeitung. Grundsätzlich ist die Kontamination von Lebensmitteln durch Mikroorganismen aufgrund der vielfältigen Übertragungsmöglichkeiten während der Herstellung, Verarbeitung, Lagerung und Verteilung des Produkts schwer zu kontrollieren. Bisher wird die mikrobielle Belastung routinemäßig mit mikrobiologischen Plattierungstechniken überwacht. Da der Großteil der Mikroorganismen aber nicht kultivierbar ist, liefern diese Methoden nur begrenzte Informationen. Viele potenziell wichtige Mitglieder der mikrobiellen Gemeinschaft werden damit nicht detektiert und der tatsächliche Kontaminationsstatus wird nicht komplett erfasst. Die jüngsten Fortschritte bei DNA-Sequenzierungstechnologien bieten nun die Möglichkeit, ganze mikrobielle Gemeinschaften in ihrer natürlichen Umgebung zu untersuchen. In dieser Arbeit wenden wir quantitative mikro- und molekularbiologische Techniken an und kombinieren diese mit einer innovativen Methode um das 16S-rRNA-Gen, welches alle Bakterien besitzen, mit hohem Durchsatz in voller Länge zu sequenzieren. Damit konnten wir mikrobielle Gemeinschaften in der gesamten Verarbeitungskette für rohes Fleisch in einer beispiellosen Tiefe charakterisieren. Darüber hinaus bewerten wir diese Methode weiter als Instrument zur Untersuchung von Kontaminationsszenarien und Übertragungswegen von Mikroorganismen während der Verarbeitung und erläutern unsere Ergebnisse im breiteren Kontext der mikrobiellen Ökologie. Dieser ökologische Aspekt, dass Mikroorganismen in komplexen Gemeinschaften leben die ständig miteinander interagieren, wurde im Bereich der Lebensmittelmikrobiologie bisher weitgehend vernachlässigt. Hier zeigen wir, dass Lebensmittelverarbeitungsbetriebe komplexe Mikrobiome beherbergen und weisen auf spezifische mikrobielle Interaktionen hin, die möglicherweise das Überleben von Krankheitserregern oder verdergbniserregenden Mikroorganismen in regelmäßig desinfizierten Umgebungen fördern. Insbesondere konzentrieren wir uns auf Listeria monocytogenes und seine Wechselbeziehungen mit anderen Mikroorganismen, die für gewöhnlich in lebensmittelverarbeitenden Betrieben weit verbreitet sind. Auf diese Weise konnten wir ein Konsortium prominenter Mikroorganismen mit bekannten Fähigkeiten zur Bildung von Biofilmen identifizieren, deren Einfluss auf die Überlebensfähigkeit von L. monocytogenes genauer untersucht werden soll. Unsere Ergebnisse zeigen, dass DNA- Sequenzierungstechnologien ein großes Potenzial haben, bisher unbekannte mikrobielle Übertragungswege und Wechselwirkungen aufzudecken, die zur Persistenz von potentiell gefährlichen Bakterien beitragen. Eine Integration dieser Technologien in bestehende Überwachungssysteme kann wertvolle Informationen zur Bekämpfung der mikrobiellen Kontamination von Lebensmitteln liefern und dazu beitragen eine mikrobiologisch sichere Lebensmittelproduktion zu gewährleisten

Comparing the Efficacy of MALDI-TOF MS and Sequencing-Based Identification Techniques (Sanger and NGS) to Monitor the Microbial Community of Irrigation Water

In order to intensify and guarantee the agricultural productivity and thereby to be able to feed the world’s rapidly growing population, irrigation has become very important. In parallel, the limited water resources lead to an increase in usage of poorly characterized sources of water, which is directly linked to a higher prevalence of foodborne diseases. Therefore, analyzing the microorganisms or even the complete microbiome of irrigation water used for food production can prevent the growing numbers of such cases. In this study, we compared the efficacy of MALDI-TOF Mass spectrometry (MALDI TOF MS) identification to 16S rRNA gene Sanger sequencing of waterborne microorganisms. Furthermore, we analyzed the whole microbial community of irrigation water using high-throughput 16S rRNA gene amplicon sequencing. The identification results of MALDI-TOF MS and 16S rRNA gene Sanger sequencing were almost identical at species level (66.7%; 64.3%). Based on the applied cultivation techniques, Acinetobacter spp., Enterobacter spp., Pseudomonas spp., and Brevundimonas spp. were the most abundant cultivable genera. In addition, the uncultivable part of the microbiome was dominated by Proteobacteria followed by Actinobacteria, Bacteroidota, Patescibacteria, and Verrucomicrobiota. Our findings indicate that MALDI-TOF MS offers a fast, reliable identification method and can act as an alternative to 16S rRNA gene Sanger sequencing of isolates. Moreover, the results suggest that MALDI-TOF MS paired with 16S rRNA gene amplicon sequencing have the potential to support the routine monitoring of the microbiological quality of irrigation water

Effect of Two Soybean Varieties Treated with Different Heat Intensities on Ileal and Caecal Microbiota in Broiler Chickens

Soybean products are of high importance for the protein supply of poultry. Heat treatment of soybeans is essential to ensure optimal digestibility because of intrinsic antinutritive factors typical for this feed category. However, excessive treatment promotes the Maillard reaction and reduces protein digestibility. Furthermore, Europe’s efforts are to decrease dependence on imports of soybean products and enlarge local production. This process will include an increase in the variability of soybean batches, posing great challenges to adequate processing conditions. Intrinsic soybean properties plus heat treatment intensity might be able to modulate the gut microbiota, which is of crucial importance for an animal’s health and performance. To assess the influence of heat treatment and soybean variety on gut microbiota, 2 soybean cakes from 2 varieties were processed at 110 °C or 120 °C and subsequently fed to 336 one-day-old broiler chickens. After 36 days, the animals were slaughtered, and the digesta of the ileum and caecum was collected. Next, 16S rRNA amplicon sequencing of the extracted DNA revealed a high discrepancy between gut sections, but there were no differences between male and female birds. Significant differences attributed to the different soybean varieties and heat intensity were detected for certain bacterial taxa. However, no effect on specific families or genera appeared. In conclusion, the results indicated the potential of processing conditions and soybean variety as microbiota-modulating factors

Comparing the Efficacy of MALDI-TOF MS and Sequencing-Based Identification Techniques (Sanger and NGS) to Monitor the Microbial Community of Irrigation Water

In order to intensify and guarantee the agricultural productivity and thereby to be able to feed the world’s rapidly growing population, irrigation has become very important. In parallel, the limited water resources lead to an increase in usage of poorly characterized sources of water, which is directly linked to a higher prevalence of foodborne diseases. Therefore, analyzing the microorganisms or even the complete microbiome of irrigation water used for food production can prevent the growing numbers of such cases. In this study, we compared the efficacy of MALDI-TOF Mass spectrometry (MALDI TOF MS) identification to 16S rRNA gene Sanger sequencing of waterborne microorganisms. Furthermore, we analyzed the whole microbial community of irrigation water using high-throughput 16S rRNA gene amplicon sequencing. The identification results of MALDI-TOF MS and 16S rRNA gene Sanger sequencing were almost identical at species level (66.7%; 64.3%). Based on the applied cultivation techniques, Acinetobacter spp., Enterobacter spp., Pseudomonas spp., and Brevundimonas spp. were the most abundant cultivable genera. In addition, the uncultivable part of the microbiome was dominated by Proteobacteria followed by Actinobacteria, Bacteroidota, Patescibacteria, and Verrucomicrobiota. Our findings indicate that MALDI-TOF MS offers a fast, reliable identification method and can act as an alternative to 16S rRNA gene Sanger sequencing of isolates. Moreover, the results suggest that MALDI-TOF MS paired with 16S rRNA gene amplicon sequencing have the potential to support the routine monitoring of the microbiological quality of irrigation water

Austrian Raw-Milk Hard-Cheese Ripening Involves Successional Dynamics of Non-Inoculated Bacteria and Fungi

Cheese ripening involves successional changes of the rind microbial composition that harbors a key role on the quality and safety of the final products. In this study, we analyzed the evolution of the rind microbiota (bacteria and fungi) throughout the ripening of Austrian Vorarlberger Bergkäse (VB), an artisanal surface-ripened cheese, by using quantitative and qualitative approaches. The real-time quantitative PCR results revealed that bacteria were more abundant than fungi in VB rinds throughout ripening, although both kingdoms were abundant along the process. The qualitative investigation was performed by high-throughput gene-targeted (amplicon) sequencing. The results showed dynamic changes of the rind microbiota throughout ripening. In the fresh products, VB rinds were dominated by Staphylococcus equorum and Candida. At early ripening times (14–30 days) Psychrobacter and Debaryomyces flourished, although their high abundance was limited to these time points. At the latest ripening times (90–160 days), VB rinds were dominated by S. equorum, Brevibacterium, Corynebacterium, and Scopulariopsis. Strong correlations were shown for specific bacteria and fungi linked to specific ripening periods. This study deepens our understanding of VB ripening and highlights different bacteria and fungi associated to specific ripening periods which may influence the organoleptic properties of the final products

Austrian Raw-Milk Hard-Cheese Ripening Involves Successional Dynamics of Non-Inoculated Bacteria and Fungi

Cheese ripening involves successional changes of the rind microbial composition that harbors a key role on the quality and safety of the final products. In this study, we analyzed the evolution of the rind microbiota (bacteria and fungi) throughout the ripening of Austrian Vorarlberger Bergkäse (VB), an artisanal surface-ripened cheese, by using quantitative and qualitative approaches. The real-time quantitative PCR results revealed that bacteria were more abundant than fungi in VB rinds throughout ripening, although both kingdoms were abundant along the process. The qualitative investigation was performed by high-throughput gene-targeted (amplicon) sequencing. The results showed dynamic changes of the rind microbiota throughout ripening. In the fresh products, VB rinds were dominated by Staphylococcus equorum and Candida. At early ripening times (14–30 days) Psychrobacter and Debaryomyces flourished, although their high abundance was limited to these time points. At the latest ripening times (90–160 days), VB rinds were dominated by S. equorum, Brevibacterium, Corynebacterium, and Scopulariopsis. Strong correlations were shown for specific bacteria and fungi linked to specific ripening periods. This study deepens our understanding of VB ripening and highlights different bacteria and fungi associated to specific ripening periods which may influence the organoleptic properties of the final products.This article is published as Quijada, Narciso M., Stephan Schmitz-Esser, Benjamin Zwirzitz, Christian Guse, Cameron R. Strachan, Martin Wagner, Stefanie U. Wetzels, Evelyne Selberherr, and Monika Dzieciol. "Austrian Raw-Milk Hard-Cheese Ripening Involves Successional Dynamics of Non-Inoculated Bacteria and Fungi." Foods 9, no. 12 (2020): 1851. doi: 10.3390/foods9121851.</p