Microbiomes of unreactive and pathologically altered ileocecal lymph nodes of slaughter pigs.

Microbe-laden dendritic cells are shifted to ileocecal lymph nodes (ICLNs), where microbes are concentrated and an adequate immune response is triggered. Hence, ICLNs are at a crucial position in immune anatomy and control processes of the local immune system. Pathological alterations in ICLNs, such as reactive hyperplasia, lymphadenitis purulenta, or granulomatosa, can harbor a multitude of pathogens and commensals, posing a potential zoonotic risk in animal production. The aim of this study was to characterize the microbial diversity of unreactive ICLNs of slaughter pigs and to investigate community shifts in reactive ICLNs altered by enlargement, purulence, or granulomatous formations. Pyrosequencing of 16S rRNA gene amplicons from 32 ICLNs yielded 175,313 sequences, clustering into 650 operational taxonomic units (OTUs). OTUs were assigned to 239 genera and 11 phyla. Besides a highly diverse bacterial community in ICLNs, we observed significant shifts in pathologically altered ICLNs. The relative abundances of Cloacibacterium- and Novosphingobium-associated OTUs and the genus Faecalibacterium were significantly higher in unreactive ICLNs than in pathologically altered ICLNs. Enlarged ICLNs harbored significantly more Lactobacillus- and Clostridium-associated sequences. Relative abundances of Mycoplasma, Bacteroides, Veillonella, and Variovorax OTUs were significantly increased in granulomatous ICLNs, whereas abundances of Pseudomonas, Escherichia, and Acinetobacter OTUs were significantly increased in purulent ICLNs (P < 0.05). Correlation-based networks revealed interactions among OTUs in all ICLN groups, and discriminant analyses depicted discrimination in response to pathological alterations. This study is the first community-based survey in ICLNs of livestock animals and will provide a basis to broaden the knowledge of microbe-host interactions in pigs.

Enumeration of clostridia in goat milk using an optimized membrane filtration technique.

A membrane filtration technique developed for counting butyric acid bacteria in cow milk was further developed for analysis of goat milk. Reduction of the sample volume, prolongation of incubation time after addition of proteolytic enzyme and detergent, and a novel step of ultrasonic treatment during incubation allowed filtration of goat milk even in the case of somatic cell counts (SCC) exceeding 10(6)/mL. However, filterability was impaired in milk from goats in late lactation. In total, spore counts were assessed in 329 farm bulk goat milk samples. Membrane filtration technique counts were lower than numbers revealed by the classic most probable number technique. Thus, method-specific thresholds for milk to evaluate the risk of late blowing have to be set. As expected, the spore counts of milk samples from suppliers not feeding silage were significantly lower than the spore counts of milk samples from suppliers using silage feeds. Not only were counts different, the clostridial spore population also varied significantly. By using 16S rRNA gene PCR and gene sequencing, 342 strains from 15 clostridial species were identified. The most common Clostridium species were Clostridium tyrobutyricum (40.4%), Clostridium sporogenes (38.3%), Clostridium bifermentans (7.6%), and Clostridium perfringens (5.3%). The 2 most frequently occurring species C. tyrobutyricum and C. sporogenes accounted for 84.7% of the isolates derived from samples of suppliers feeding silage (n=288). In contrast, in samples from suppliers without silage feeding (n=55), these species were detected in only 45.5% of the isolates.

Microbial community structure of plant-based meat alternatives.

A reduction in animal-based diets has driven market demand for alternative meat products, currently raising a new generation of plant-based meat alternatives (PBMAs). It remains unclear whether these substitutes are a short-lived trend or become established in the long term. Over the last few years, the trend of increasing sales and diversifying product range has continued, but publication activities in this field are currently limited mainly to market research and food technology topics. As their popularity increases, questions emerge about the safety and nutritional risks of these novel products. Even though all the examined products must be heated before consumption, consumers lack experience with this type of product and thus further research into product safety, is desirable. To consider these issues, we examined 32 PBMAs from Austrian supermarkets. Based on 16S rRNA gene amplicon sequencing, the majority of the products were dominated by lactic acid bacteria (either Leuconostoc or Latilactobacillus), and generally had low alpha diversity. Pseudomonadota (like Pseudomonas and Shewanella) dominated the other part of the products. In addition to LABs, a high diversity of different Bacillus, but also some Enterobacteriaceae and potentially pathogenic species were isolated with the culturing approach. We assume that especially the dominance of heterofermentative LABs has high relevance for the product stability and quality with the potential to increase shelf life of the products. The number of isolated Enterobacteriaceae and potential pathogens were low, but they still demonstrated that these products are suitable for their presence.

Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments.

Deep investigation of the microbiome of food-production and food-processing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.

Characterization of Leuconostoc carnosum and Latilactobacillus sakei during Cooked Pork Ham Processing.

Cooked ham is a popular, ready-to-eat product made of pork meat that is susceptible to microbial growth throughout its shelf life. In this study, we aimed to monitor the microbial growth and composition of nine vacuum-packed cooked ham lots using plate counting until the microbial limit of 7.4 log10 AMC/LAB CFU/g was exceeded. Eight out of nine lots exceeded the microbial limit after 20 days of storage. Lactic acid bacteria strains, particularly Leuconostoc carnosum and Latilactobacillus sakei, prevailed in vacuum-packed cooked ham. Leuconostoc carnosum 2 (Leuc 2) and Latilactobacillus sakei 4 (Sakei 4) were isolated from raw meat and the post-cooking area of the food processing facility. Carbohydrate utilization patterns of Leuc. carnosum PFGE types isolated from raw meat and the food processing environment differed from those isolated from cooked ham. These findings demonstrate how raw meat and its processing environment impact the quality and shelf life of cooked ham.

Metatranscriptomic Analyses Unravel Dynamic Changes in the Microbial and Metabolic Transcriptional Profiles in Artisanal Austrian Hard-Cheeses During Ripening.

Vorarlberger Bergkäse (VB) is an artisanal Austrian washed-rind hard cheese produced from alpine cows' raw milk without the addition of ripening cultures. Ripening time is a key factor in VB, as it strongly influences the microbial communities present in the cheeses and the organoleptic properties of the product. In this study, the microbial and metabolic transcriptional profiles in VB rinds at different ripening times were investigated. VB products before (30 days of ripening) and after (90 days of ripening) selling were selected, RNA was extracted and subjected to shotgun metatranscriptomic sequencing. The analysis revealed some of the previously described abundant bacterial taxa of Brevibacterium, Corynebacterium, Halomonas, Psychrobacter, and Staphylococcus to be highly active in VB rinds. Additionally, the investigation of most important metabolic pathways in cheese ripening clearly showed differences in the gene transcription profiles and the active microbiota between the two ripening points investigated. At 30 days of ripening, metabolic events related with the degradation of residual lactose, lactate, citrate, proteolysis, and lipolysis were significantly more transcribed and mainly associated with Staphylococcus. On the other hand, genes involved in the degradation of smaller compounds derived from previous metabolism (i.e., metabolism of free amino acids and fatty acids) were significantly more expressed in VB rinds with 90 of ripening, and mainly associated with Brevibacterium and Corynebacterium. These latter metabolic activities are responsible of the generation of compounds, such as methanethiol and 2,3-butanediol, that are very important for the flavor and aroma characteristics of cheeses. This study shows the dynamic changes in the gene transcriptional profiles associated with energy substrates metabolism and the generation of organoleptic compounds during VB ripening and uncovers bacterial taxa as key drivers of the ripening process. These taxa might be the target for future studies toward an accelerated cheese ripening and the enhancement of its organoleptic properties.

Co-Occurrence of Listeria spp. and Spoilage Associated Microbiota During Meat Processing Due to Cross-Contamination Events.

A large part of foodborne outbreaks related to Listeria monocytogenes are linked to meat and meat products. Especially, recontamination of meat products and deli-meat during slicing, packaging, and repackaging is in the focus of food authorities. In that regard, L. monocytogenes persistence in multi-species biofilms is one major issue, since they survive elaborate cleaning and disinfection measures. Here, we analyzed the microbial community structure throughout a meat processing facility using a combination of high-throughput full-length 16S ribosomal RNA (rRNA) gene sequencing and traditional microbiological methods. Samples were taken at different stages during meat cutting as well as from multiple sites throughout the facility environment to capture the product and the environmental associated microbiota co-occurring with Listeria spp. and L. monocytogenes. The listeria testing revealed a widely disseminated contamination (50%; 88 of 176 samples were positive for Listeria spp. and 13.6%; 24 of 176 samples were positive for L. monocytogenes). The pulsed-field gel electrophoresis (PFGE) typing evidenced 14 heterogeneous L. monocytogenes profiles with PCR-serogroup 1/2a, 3a as most dominant. PFGE type MA3-17 contributed to the resilient microbiota of the facility environment and was related to environmental persistence. The core in-house microbiota consisted mainly of the genera Acinetobacter, Pseudomonas, Psychrobacter (Proteobacteria), Anaerobacillus, Bacillus (Firmicutes), and Chryseobacterium (Bacteroidota). While the overall microbial community structure clearly differed between product and environmental samples, we were able to discern correlation patterns regarding the presence/absence of Listeria spp. in both sample groups. Specifically, our longitudinal analysis revealed association of Listeria spp. with known biofilm-producing Pseudomonas, Acinetobacter, and Janthinobacterium species on the meat samples. Similar patterns were also observed on the surface, indicating dispersal of microorganisms from this multispecies biofilm. Our data provided a better understanding of the built environment microbiome in the meat processing context and promoted more effective options for targeted disinfection in the analyzed facility.

PacBio and Illumina MiSeq Amplicon Sequencing Confirm Full Recovery of the Bacterial Community After Subacute Ruminal Acidosis Challenge in the RUSITEC System.

The impact of subacute rumen acidosis (SARA) on the rumen bacterial community has been frequently studied in in vivo trials. Here we investigated whether these alterations can be mirrored by using the rumen simulation technique (RUSITEC) as an in vitro model for this disease. We hypothezised that the bacterial community fully recovers after a subacute ruminal acidosis challenge. We combined a PacBio nearly full-length 16S rRNA gene analysis with 16S rRNA gene Illumina MiSeq sequencing of the V4 hypervariable region. With this hybrid approach, we aimed to get an increased taxonomic resolution of the most abundant bacterial groups and an overview of the total bacterial diversity. The experiment consisted of a control period I and a SARA challenge and ended after a control period II, of which each period lasted 5 d. Subacute acidosis was induced by applying two buffer solutions, which were reduced in their buffering capacity (SARA buffers) during the SARA challenge. Two control groups were constantly infused with the standard buffer solution. Furthermore, the two SARA buffers were combined with three different feeding variations, which differed in their concentrate-to-hay ratio. The induction of SARA led to a decrease in pH below 5.8, which then turned into a steady-state SARA. Decreasing pH values led to a reduction in bacterial diversity and richness. Moreover, the diversity of solid-associated bacteria was lower for high concentrate groups throughout all experimental periods. Generally, Firmicutes and Bacteroidetes were the predominant phyla in the solid and the liquid phase. During the SARA period, we observed a decrease in fibrolytic bacteria although lactate-producing and -utilizing families increased in certain treatment groups. The genera Lactobacillus and Prevotella dominated during the SARA period. With induction of the second control period, most bacterial groups regained their initial abundance. In conclusion, this in vitro model displayed typical bacterial alterations related to SARA and is capable of recovery from bouts of SARA. Therefore, this model can be used to mimic SARA under laboratory conditions and may contribute to a reduction in animal experiments.

The sources and transmission routes of microbial populations throughout a meat processing facility.

Microbial food spoilage is responsible for a considerable amount of waste and can cause food-borne diseases in humans, particularly in immunocompromised individuals and children. Therefore, preventing microbial food spoilage is a major concern for health authorities, regulators, consumers, and the food industry. However, the contamination of food products is difficult to control because there are several potential sources during production, processing, storage, distribution, and consumption, where microorganisms come in contact with the product. Here, we use high-throughput full-length 16S rRNA gene sequencing to provide insights into bacterial community structure throughout a pork-processing plant. Specifically, we investigated what proportion of bacteria on meat are presumptively not animal-associated and are therefore transferred during cutting via personnel, equipment, machines, or the slaughter environment. We then created a facility-specific transmission map of bacterial flow, which predicted previously unknown sources of bacterial contamination. This allowed us to pinpoint specific taxa to particular environmental sources and provide the facility with essential information for targeted disinfection. For example, Moraxella spp., a prominent meat spoilage organism, which was one of the most abundant amplicon sequence variants (ASVs) detected on the meat, was most likely transferred from the gloves of employees, a railing at the classification step, and the polishing tunnel whips. Our results suggest that high-throughput full-length 16S rRNA gene sequencing has great potential in food monitoring applications.

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.

Co-infection of Chicken Layers With Histomonas meleagridis and Avian Pathogenic Escherichia coli Is Associated With Dysbiosis, Cecal Colonization and Translocation of the Bacteria From the Gut Lumen.

Histomonosis in chickens often appears together with colibacillosis in the field. Thus, we have experimentally investigated consequences of the co-infection of birds with Histomonas meleagridis and avian pathogenic Escherichia coli (APEC) on the pathology, host microbiota and bacterial translocation from the gut. Commercial chicken layers were infected via oral and cloacal routes with lux-tagged APEC with or without H. meleagridis whereas negative controls were left uninfected. Except one bird, which died due to colibacillosis, no clinical signs were recorded in birds infected with bioluminescence lux gene tagged E. coli. In co-infected birds, depression and ruffled feathers were observed in 4 birds and average body weight gain significantly decreased. Typhlitis caused by H. meleagridis was present only in co-infected birds, which also had pronounced microscopic lesions in systemic organs such as liver, heart and spleen. The 16S rRNA gene amplicon sequencing showed that in co-infected birds, corresponding to the severity of cecal lesions, microbial species richness and diversity in caeca greatly decreased and the abundance of the Escherichia group, Helicobacter and Bacteroides was relatively higher with a reduction of commensals. Most of the shared Amplicon Sequencing Variants between cecum and blood in co-infected birds belonged to Pseudomonas, Staphylococcus, and members of Enterobacteriaceae while those assigned as Lactobacillus and members of Ruminococcaceae and Lachnospiraceae were found mainly in negative controls. In infected birds, E. coli in the cecal lumen penetrated into deeper layers, a phenomenon noticed with higher incidence in the dead and co-infected birds. Furthermore, numbers of lux-tagged E. coli in caeca were significantly higher at every sampling date in co-infected birds. Altogether, infection of layers with H. meleagridis and E. coli resulted in more severe pathological changes, dramatic shift in the cecal mucosa-associated microbiota, higher tissue colonization of pathogenic bacteria such as avian pathogenic E. coli in the gut and increased penetration of E. coli from the cecal lumen toward peritoneum. This study provides novel insights into the parasite-bacteria interaction in vivo highlighting the role of H. meleagridis to support E. coli in the pathogenesis of colibacillosis in chickens.

Stress- and growth rate-related differences between plate count and real-time PCR data during growth of Listeria monocytogenes.

To assess the overestimation of bacterial cell counts in real-time PCR in relation to stress and growth phase, four different strains of L. monocytogenes were exposed to combinations of osmotic stress (0.5 to 8% [vol/vol] NaCl) and acid stress (pH 5 to 7) in a culture model at a growth temperature of 10 degrees C or were grown under optimal conditions. Growth curves obtained from real-time PCR, optical density, and viable count data were compared. As expected, optical density data revealed entirely different growth curves. Good to moderate growth conditions yielded good correlation of real-time PCR data and plate count data (r(2) = 0.96 and 0.99) with similar cell counts. When growth conditions became worse, the numbers of CFU decreased during the stationary phase, whereas real-time-PCR-derived bacterial cell equivalents differed in this regard; the correlation worsened (r(2) = 0.84). However, fitted growth curves revealed that maximum growth rates calculated from real-time PCR data were not significantly different from those derived from plate count data. The overestimation of bacterial cell counts by real-time PCR observed in the stationary phase under higher-stress conditions might be explained by the accumulation of viable but nonculturable bacteria or dead bacteria and extracellular DNA. Considering these results, real-time PCR data collected from naturally contaminated samples should be viewed with caution.

Plasmids contribute to food processing environment-associated stress survival in three Listeria monocytogenes ST121, ST8, and ST5 strains.

Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis and is commonly isolated from food and food production facilities. Many L. monocytogenes strains contain plasmids, though the contributions of plasmids to survival in food production environments are unknown. Three L. monocytogenes ST5, ST8, and ST121 strains containing plasmids, which harbor putative stress response genes, were cured of their plasmids. Wildtype (WT) and plasmid-cured strains were exposed to disinfectant, oxidative, heat, acid, or salt stress. After stress exposure, cells were plated for colony forming unit (CFU) counts to determine survivors. L. monocytogenes WT strains exposed to 0.01% (vol/vol) H2O2, 1% (vol/vol) lactic acid, and 15% (wt/vol) NaCl, pH 5 showed significantly higher counts of survivors compared to the plasmid-cured strains. The number of survivors for the ST5 WT strain exposed to 10 µg/mL benzalkonium chloride (BC) was significantly higher than in the plasmid-cured strain. The ST8 and ST5 strains were exposed to elevated temperature (50° and 55 °C respectively); only the ST5 WT strain had significantly higher numbers of survivors than the plasmid-cured strains. Our data revealed that L. monocytogenes ST5, ST8, and ST121 plasmids contribute to tolerance against elevated temperature, salinity, acidic environments, oxidative stress and disinfectants.

Culture-Independent Evaluation of Bacterial Contamination Patterns on Pig Carcasses at a Commercial Slaughter Facility.

Traditionally, the microbiological status of meat is determined by culture-based techniques, although many bacteria are not able to grow on conventional media. The aim of this study was to obtain quantitative data on total bacterial cell equivalents, as well as taxa-specific abundances, on carcass surfaces during pig slaughter using quantitative real-time PCR. We evaluated microbial contamination patterns of total bacteria, Campylobacter, Escherichia coli, Lactobacillus group, Listeria monocytogenes, Salmonella, and Pseudomonas species throughout slaughtering and on different carcass areas. In addition, we compared contamination levels of breeding sow carcasses with fattening pig carcasses, and we assessed the efficacy of carcass polishing machines under two water amount conditions. Our results demonstrate that relevant meat-spoilage organisms show similar contamination patterns to total bacteria. The highest bacterial load was detected in the stunning chute (4.08 × 105 bacterial cell equivalents per cm2) but was reduced by 3 log levels after singeing and polishing (P < 0.001). It increased again significantly by a 4.73-fold change until the classification step. Levels of Campylobacter, Lactobacillus, and Pseudomonas species and of E. coli followed a similar trend but varied between 0 and 2.49 × 104 bacterial cell equivalents per cm2. Microbial levels did not vary significantly between sampled carcass areas for any analyzed taxa. Running the polishing machine with a low water amount proved to be less prone to microbial recontamination compared with a high water amount (17.07-fold change, P = 0.024). In the studied slaughterhouse, slaughter of breeding sows did not produce microbiologically safe meat products (>104 cells per cm2) and the implementation of specific hazard analysis critical control point systems for the slaughter of breeding sows should be considered. A larger cohort from different abattoirs is needed to confirm our results and determine whether this is universally valid.

Microbiota of newborn calves and their mothers reveals possible transfer routes for newborn calves' gastrointestinal microbiota.

The intestinal microbiota of newborns plays an important role in the development of immunity and metabolism. In livestock animals, knowledge of the intestinal microbiota is essential not only to prevent diseases but also to optimize weight gain and performance. The aim of our study was to examine faecal samples repeatedly within the first two days of life using 16S rRNA gene High Throughput Sequencing. Additionally, samples from the mouths of the calves and the vaginas, colostrum, and faeces of the dams were included to evaluate possible sources of the calf faecal microbiota. The calf faecal microbiota was highly variable during the first 48 hours post natum (p.n.). Significant changes were found in species diversity and richness, in copy numbers evaluated by qPCR and in predominant bacteria over time. The most pronounced changes occurred between 6 and 24 hours p.n. All calf faecal samples were dominated by Operational Taxonomic Units (OTUs) belonging to the family Enterobacteriaceae. Cow faecal samples showed significantly higher species richness, diversity, number of observed OTUs, and copy numbers compared to all other samples. OTUs belonging to the family Ruminococcaceae were most abundant in cow faecal and vaginal samples. Colostrum was dominated by Enhydrobacter affiliated OTUs. To identify possible inoculation routes for the calf microbiota, we analysed OTU sharing between samples. The calf microbiota during the first two days of life was clearly distinct from the dam's faecal microbiota. Furthermore, colostrum microbiota clearly differed from calf and cow faecal microbiota and thus most likely does not play an important role as inoculation source for calf microbiota during the first two days of life. In contrast, the cow vaginal and the calf faecal microbiota were more similar, suggesting that some of the calf faecal microbiota may derive from inoculation from the birth canal during birth.

Brevibacterium from Austrian hard cheese harbor a putative histamine catabolism pathway and a plasmid for adaptation to the cheese environment.

The genus Brevibacterium harbors many members important for cheese ripening. We performed real-time quantitative PCR (qPCR) to determine the abundance of Brevibacterium on rinds of Vorarlberger Bergkäse, an Austrian artisanal washed-rind hard cheese, over 160 days of ripening. Our results show that Brevibacterium are abundant on Vorarlberger Bergkäse rinds throughout the ripening time. To elucidate the impact of Brevibacterium on cheese production, we analysed the genomes of three cheese rind isolates, L261, S111, and S22. L261 belongs to Brevibacterium aurantiacum, whereas S111 and S22 represent novel species within the genus Brevibacterium based on 16S rRNA gene similarity and average nucleotide identity. Our comparative genomic analysis showed that important cheese ripening enzymes are conserved among the genus Brevibacterium. Strain S22 harbors a 22 kb circular plasmid which encodes putative iron and hydroxymethylpyrimidine/thiamine transporters. Histamine formation in fermented foods can cause histamine intoxication. We revealed the presence of a putative metabolic pathway for histamine degradation. Growth experiments showed that the three Brevibacterium strains can utilize histamine as the sole carbon source. The capability to utilize histamine, possibly encoded by the putative histamine degradation pathway, highlights the importance of Brevibacterium as key cheese ripening cultures beyond their contribution to cheese flavor production.

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.

Abundance and potential contribution of Gram-negative cheese rind bacteria from Austrian artisanal hard cheeses.

Many different Gram-negative bacteria have been shown to be present on cheese rinds. Their contribution to cheese ripening is however, only partially understood until now. Here, cheese rind samples were taken from Vorarlberger Bergkäse (VB), an artisanal hard washed-rind cheese from Austria. Ripening cellars of two cheese production facilities in Austria were sampled at the day of production and after 14, 30, 90 and 160days of ripening. To obtain insights into the possible contribution of Advenella, Psychrobacter, and Psychroflexus to cheese ripening, we sequenced and analyzed the genomes of one strain of each genus isolated from VB cheese rinds. Additionally, quantitative PCRs (qPCRs) were performed to follow the abundance of Advenella, Psychrobacter, and Psychroflexus on VB rinds during ripening in both facilities. qPCR results showed that Psychrobacter was most abundant on cheese rinds and the abundance of Advenella decreased throughout the first month of ripening and increased significantly after 30days of ripening (p<0.01). Psychrobacter and Psychroflexus increased significantly during the first 30 ripening days (p<0.01), and decreased to their initial abundance during the rest of the ripening time (p<0.05). Genome sequencing resulted in 17 to 27 contigs with assembly sizes of 2.7 Mbp for Psychroflexus, 3 Mbp for Psychrobacter, and 4.3 Mbp for Advenella. Our results reveal that each genome harbors enzymes shown to be important for cheese ripening in other bacteria such as: Cystathionine/Methionine beta or gamma-lyases, many proteases and peptidases (including proline iminopeptidases), aminotransferases, and lipases. Thus, all three isolates have the potential to contribute positively to cheese ripening. In conclusion, the three species quantified were stable community members throughout the ripening process and their abundance on cheese rinds together with the results from genome sequencing suggest an important contribution of these bacteria to cheese ripening.

Transcriptome Sequencing Reveals Novel Candidate Genes for Cardinium hertigii-Caused Cytoplasmic Incompatibility and Host-Cell Interaction.

Cytoplasmic incompatibility (CI) is an intriguing, widespread, symbiont-induced reproductive failure that decreases offspring production of arthropods through crossing incompatibility of infected males with uninfected females or with females infected with a distinct symbiont genotype. For years, the molecular mechanism of CI remained unknown. Recent genomic, proteomic, biochemical, and cell biological studies have contributed to understanding of CI in the alphaproteobacterium Wolbachia and implicate genes associated with the WO prophage. Besides a recently discovered additional lineage of alphaproteobacterial symbionts only moderately related to Wolbachia, Cardinium (Bacteroidetes) is the only other symbiont known to cause CI, and genomic evidence suggests that it has very little homology with Wolbachia and evolved this phenotype independently. Here, we present the first transcriptomic study of the CI Cardinium strain cEper1, in its natural host, Encarsia suzannae, to detect important CI candidates and genes involved in the insect-Cardinium symbiosis. Highly expressed transcripts included genes involved in manipulating ubiquitination, apoptosis, and host DNA. Female-biased genes encoding ribosomal proteins suggest an increase in general translational activity of Cardinium in female wasps. The results confirm previous genomic analyses that indicated that Wolbachia and Cardinium utilize different genes to induce CI, and transcriptome patterns further highlight expression of some common pathways that these bacteria use to interact with the host and potentially cause this enigmatic and fundamental manipulation of host reproduction. IMPORTANCE The majority of insects carry maternally inherited intracellular bacteria that are important in their hosts' biology, ecology, and evolution. Some of these bacterial symbionts cause a reproductive failure known as cytoplasmic incompatibility (CI). In CI, the mating of symbiont-infected males and uninfected females produces few or no daughters. The CI symbiont then spreads and can have a significant impact on the insect host population. Cardinium, a bacterial endosymbiont of the parasitoid wasp Encarsia in the Bacteroidetes, is the only bacterial lineage known to cause CI outside the Alphaproteobacteria, where Wolbachia and another recently discovered CI symbiont reside. Here, we sought insight into the gene expression of a CI-inducing Cardinium strain in its natural host, Encarsia suzannae. Our study provides the first insights into the Cardinium transcriptome and provides support for the hypothesis that Wolbachia and Cardinium target similar host pathways with distinct and largely unrelated sets of genes.

A systematic review of financial and economic assessments of bovine viral diarrhea virus (BVDV) prevention and mitigation activities worldwide.

Infection with bovine viral diarrhea virus (BVDV) results in major economic losses either directly through decreased productive performance in cattle herds or indirectly, such as through expenses for control programs. The aim of this systematic review was to review financial and/or economic assessment studies of prevention and/or mitigation activities of BVDV at national, regional and farm level worldwide. Once all predefined criteria had been met, 35 articles were included for this systematic review. Studies were analyzed with particular focus on the type of financially and/or economically-assessed prevention and/or mitigation activities. Due to the wide range of possible prevention and/or mitigation activities, these activities were grouped into five categories: i) control and/or eradication programs, ii) monitoring or surveillance, iii) prevention, iv) vaccination and v) individual culling, control and testing strategies. Additionally, the studies were analyzed according to economically-related variables such as efficiency, costs or benefits of prevention and/or mitigation activities, the applied financial and/or economic and statistical methods, the payers of prevention and/or mitigation activities, the assessed production systems, and the countries for which such evaluations are available. Financial and/or economic assessments performed in Europe were dominated by those from the United Kingdom, which assessed mostly vaccination strategies, and Norway which primarily carried out assessments in the area of control and eradication programs; whereas among non-European countries the United States carried out the majority of financial and/or economic assessments in the area of individual culling, control and testing. More than half of all studies provided an efficiency calculation of prevention and/or mitigation activities and demonstrated whether the inherent costs of implemented activities were or were not justified. The dairy sector was three times more likely to be assessed by the countries than beef production systems. In addition, the dairy sector was approximately eight times more likely to be assessed economically with respect to prevention and/or mitigation activities than calf and youngstock production systems. Furthermore, the private sector was identified as the primary payer of prevention and/or mitigation activities. This systematic review demonstrated a lack of studies relating to efficiency calculations, in particular at national and regional level, and the specific production systems. Thus, we confirmed the need for more well-designed studies in animal health economics in order to demonstrate that the implementation and inherent costs of BVDV prevention and/or mitigation activities are justified.