Capture and transport of white rhinoceroses (Ceratotherium simum) cause shifts in their fecal microbiota composition towards dysbiosis.

Translocations of Rhinocerotidae are commonly performed for conservation purposes but expose the animals to a variety of stressors (e.g. prolonged fasting, confinement, novel environment, etc.). Stress may change the composition of gut microbiota, which can impact animal health and welfare. White rhinoceroses in particular can develop anorexia, diarrhea and enterocolitis after translocation. The aim of this study was to investigate the associations of age, sex and translocation on the rhinoceros' fecal bacterial microbiota composition. fecal samples were collected from rhinoceroses at capture (n = 16) and after a >30-hour road transport (n = 7). DNA was isolated from these samples and submitted for 16S rRNA V3-V4 phylotyping. Alpha diversity indices of the rhinoceros' fecal microbiota composition of different age, sex and before and after transport were compared using non-parametric statistical tests and beta diversity indices using Permutational Multivariate Analysis Of Variance (PERMANOVA). Resulting P-values were alpha-corrected (Padj.). Alpha and beta diversity did not differ between rhinoceroses of different age and sex. However, there was a significant difference in beta diversity between fecal samples collected from adult animals at capture and after transport. The most abundant bacterial phyla in samples collected at capture were Firmicutes and Bacteroidetes (85.76%), represented by Lachnospiraceae, Ruminococcaceae and Prevotellaceae families. The phyla Proteobacteria (Padj. = 0.009) and Actinobacteria (Padj. = 0.012), amongst others, increased in relative abundance from capture to after transport encompassing potentially pathogenic bacterial families such as Enterobacteriaceae (Padj. = 0.018) and Pseudomonadaceae (Padj. = 0.022). Important commensals such as Spirochaetes (Padj. = 0.009), Fibrobacteres (Padj. = 0.018) and Lachnospiraceae (Padj. = 0.021) decreased in relative abundance. These results indicate that the stressors associated with capture and transport cause an imbalanced fecal microbiota composition in white rhinoceroses that may lead to potentially infectious intestinal disorders. This imbalance may result from recrudescence of normally innocuous pathogens, increased shedding of pathogens or increased vulnerability to new pathogens.

Autochthonous fungi are central components in microbial community structure in raw fermented sausages.

Raw meat sausage represents a unique ecological niche rich in nutrients for microbial consumption, making it particularly vulnerable to microbial spoilage. Starter cultures are applied to improve product stability and safety as well as flavour characteristics. However, the influence of starter cultures on microbial community assembly and succession throughout the fermentation process is largely unknown. In particular the effect on the fungal community has not yet been explored. We evaluate the microbiological status of four different raw meat sausages using high-throughput 16S rRNA gene and ITS2 gene sequencing. The objective was to study temporal changes of microbial composition during the fermentation process and to identify potential keystone species that play an important role within the microbial community. Our results suggest that fungi assigned to the species Debaryomyces hansenii and Alternaria alternata play a key role in microbial community dynamics during fermentation. In addition, bacteria related to the starter culture Lactobacillus sakei and the spoilage-associated genera Acinetobacter, Pseudomonas and Psychrobacter are central components of the microbial ecosystem in raw fermented sausages. Elucidating the exact role and interactions of these microorganisms has the potential to have direct impacts on the quality and safety of fermented foods.

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.

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.

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.

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 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.

The application of rumen simulation technique (RUSITEC) for studying dynamics of the bacterial community and metabolome in rumen fluid and the effects of a challenge with Clostridium perfringens.

The rumen simulation technique (RUSITEC) is a well-established semicontinuous in vitro model for investigating ruminal fermentation; however, information on the stability of the ruminal bacterial microbiota and metabolome in the RUSITEC system is rarely available. The availability of high resolution methods, such as high-throughput sequencing and metabolomics improve our knowledge about the rumen microbial ecosystem and its fermentation processes. Thus, we used Illumina MiSeq 16S rRNA amplicon sequencing and a combination of direct injection mass spectrometry with a reverse-phase LC-MS/MS to evaluate the dynamics of the bacterial community and the concentration of several metabolites in a RUSITEC experiment as a function of time and in response to a challenge with a pathogenic Clostridium perfringens (C. perfringens) strain. After four days of equilibration, samples were collected on days 5, 6, 7, 10, 12 and 15 of the steady-state and experimental period. From a total of six fermenters, three non-infected fermenters were used for investigating time-dependent alterations; three fermenters were incubated with C. perfringens and compared with the non-infected vessels at days 10, 12 and 15. Along the time-line, there was no statistically significant change of the overall bacterial community, however, some phylotypes were enriched at certain time points. A decrease in Fibrobacter and Elusimicrobia over time was followed by an increase in Firmicutes and Actinobacteria. In contrast, classical fermentation measurements such as pH, redox potential, NH3-N, short chain fatty acids and the concentrations of metabolites determined by metabolomics (biogenic amines, hexoses and amino acids) remained stable throughout the experiment. In response to C. perfringens addition the concentrations of several amino acids increased. Although the overall bacterial community was not altered here either, some minor changes such as an enrichment of Synergistetes and Bacteroidetes were detectable over time. In conclusion, both, the bacterial community composition and the metabolome in the RUSITEC system were relatively stable during the experiment.

Metatranscriptome Sequencing Reveals Insights into the Gene Expression and Functional Potential of Rumen Wall Bacteria.

Microbiota of the rumen wall constitute an important niche of rumen microbial ecology and their composition has been elucidated in different ruminants during the last years. However, the knowledge about the function of rumen wall microbes is still limited. Rumen wall biopsies were taken from three fistulated dairy cows under a standard forage-based diet and after 4 weeks of high concentrate feeding inducing a subacute rumen acidosis (SARA). Extracted RNA was used for metatranscriptome sequencing using Illumina HiSeq sequencing technology. The gene expression of the rumen wall microbial community was analyzed by mapping 35 million sequences against the Kyoto Encyclopedia for Genes and Genomes (KEGG) database and determining differentially expressed genes. A total of 1,607 functional features were assigned with high expression of genes involved in central metabolism, galactose, starch and sucrose metabolism. The glycogen phosphorylase (EC:2.4.1.1) which degrades (1->4)-alpha-D-glucans was among the highest expressed genes being transcribed by 115 bacterial genera. Energy metabolism genes were also highly expressed, including the pyruvate orthophosphate dikinase (EC:2.7.9.1) involved in pyruvate metabolism, which was covered by 177 genera. Nitrogen metabolism genes, in particular glutamate dehydrogenase (EC:1.4.1.4), glutamine synthetase (EC:6.3.1.2) and glutamate synthase (EC:1.4.1.13, EC:1.4.1.14) were also found to be highly expressed and prove rumen wall microbiota to be actively involved in providing host-relevant metabolites for exchange across the rumen wall. In addition, we found all four urease subunits (EC:3.5.1.5) transcribed by members of the genera Flavobacterium, Corynebacterium, Helicobacter, Clostridium, and Bacillus, and the dissimilatory sulfate reductase (EC 1.8.99.5) dsrABC, which is responsible for the reduction of sulfite to sulfide. We also provide in situ evidence for cellulose and cellobiose degradation, a key step in fiber-rich feed digestion, as well as oxidative stress response and oxygen scavenging at the rumen wall. Archaea, mainly Methanocaldococcus and Methanobrevibacter, were found to be metabolically active with a high number of transcripts matching to methane and carbohydrate metabolism. These findings enhance our understanding of the metabolic function of the bovine rumen wall microbiota.

Evidence of In Vivo Absorption of Lactate and Modulation of Short Chain Fatty Acid Absorption from the Reticulorumen of Non-Lactating Cattle Fed High Concentrate Diets.

Short-chain fatty acids (SCFAs) and lactate are endproducts of rumen fermentation and important energy sources for the host ruminant. Because their rapid accumulation results in ruminal acidosis, enhancement of the absorption of SCFA and lactate across reticuloruminal wall is instrumental in increasing energy supply and preventing ruminal acidosis in cattle. This study investigated whether the reticuloruminal absorption of SCFAs and lactate was altered by different strategies of high concentrate feeding. Eight rumen-cannulated, non-lactating Holstein cows were fed a forage-only diet (baseline) and then gradually adapted over 6 d to a 60% concentrate level. Thereafter, this concentrate-rich diet was fed for 4 wk either continuously (Con; n = 8) or interruptedly (Int; n = 8). Absorption of SCFAs and lactate was determined in vivo from the experimental buffer introduced into the washed reticulorumen. The buffer contained acetate, propionate, butyrate and lactate at a concentration of 60, 30, 10 and 5 mmol/L, respectively and Cr-EDTA as a marker for correcting ruminal water fluxes. The reticuloruminal absorption after 35 and 65 min of buffer incubation was measured at the baseline, after 1 wk of 60% concentrate feeding in the interrupted model (Int-1) and after 4 wk of concentrate feeding in both feeding models (Int-4 and Con-4). Data showed that the absorption rates of individual and total SCFAs during the first 35 min of incubation of Con-4 were highest (~1.7 times compared to baseline), while Int-1 and Int-4 were similar to respective baseline. Lactate was not absorbed during forage-only baseline and 1-wk concentrate feeding, but after 4-wk feeding of concentrates in both models. In conclusion, SCFAs absorption across the reticulorumen of non-lactating cattle was enhanced by the 4-wk continuous concentrate feeding, which seems to be more advantageous in terms of rumen acidosis prevention compared to the interrupted feeding model. The study provides evidence of lactate absorption across the reticulorumen of non-lactating cattle after both continuous and interrupted 4-wk concentrate feeding.

Epimural Indicator Phylotypes of Transiently-Induced Subacute Ruminal Acidosis in Dairy Cattle.

The impact of a long-term subacute rumen acidosis (SARA) on the bovine epimural bacterial microbiome (BEBM) and its consequences for rumen health is poorly understood. This study aimed to investigate shifts in the BEBM during a long-term transient SARA model consisting of two concentrate-diet-induced SARA challenges separated by a 1-week challenge break. Eight cows were fed forage and varying concentrate amounts throughout the experiment. In total, 32 rumen papilla biopsies were taken for DNA isolation (4 sampling time points per cow: at the baseline before concentrate was fed, after the first SARA challenge, after the challenge break, and after the second SARA challenge). Ruminal pH was continuously monitored. The microbiome was determined using Illumina MiSeq sequencing of the 16S rRNA gene (V345 region). In total 1,215,618 sequences were obtained and clustered into 6833 operational taxonomic units (OTUs). Campylobacter and Kingella were the most abundant OTUs (16.5 and 7.1%). According to ruminal pH dynamics, the second challenge was more severe than the first challenge. Species diversity estimates and evenness increased during the challenge break compared to all other sampling time points (P < 0.05). During both SARA challenges, Kingella- and Azoarcus-OTUs decreased (0.5 and 0.4 fold-change) and a dominant Ruminobacter-OTU increased during the challenge break (18.9 fold-change; P < 0.05). qPCR confirmed SARA-related shifts. During the challenge break noticeably more OTUs increased compared to other sampling time points. Our results show that the BEBM re-establishes the baseline conditions slower after a SARA challenge than ruminal pH. Key phylotypes that were reduced during both challenges may help to establish a bacterial fingerprint to facilitate understanding effects of SARA conditions on the BEBM and their consequences for the ruminant host.

Psychrophile spoilers dominate the bacterial microbiome in musculature samples of slaughter pigs.

The aim of this study was to disentangle the microbial diversity on porcine musculature. The hypervariable V1-V2 region of the 16S rRNA gene was amplified from DNA samples of clinically healthy slaughter pigs (n=8). Pyrosequencing yielded 37,000 quality-controlled reads and a diverse microbiome with 54-159 OTUs per sample was detected. Interestingly, 6 out of 8 samples were strongly dominated by 1-2 highly abundant OTUs (best hits of highly abundant OTUs: Serratia proteamaculans, Pseudomonas syringae, Aeromonas allosaccharophila, Brochothrix thermosphacta, Acidiphilium cryptum and Escherichia coli). In 1g musculature scraping, 3.20E+06 16S rRNA gene copies and 4.45E+01 Enterobacteriaceae rRNA gene copies were detected with qPCR. We conclude that i.) next-generation sequencing technologies help encompass the full content of complex, bacterial contamination, ii.) psychrophile spoilers dominated the microbiota and iii.) E. coli is an effective marker species for pork contamination, as it was one of very few abundant species being present in all samples.

The Metabolically Active Bacterial Microbiome of Tonsils and Mandibular Lymph Nodes of Slaughter Pigs.

The exploration of microbiomes in lymphatic organs is relevant for basic and applied research into explaining microbial translocation processes and understanding cross-contamination during slaughter. This study aimed to investigate whether metabolically active bacteria (MAB) could be detected within tonsils and mandibular lymph nodes (MLNs) of pigs. The hypervariable V1-V2 region of the bacterial 16S rRNA genes was amplified from cDNA from tonsils and MLNs of eight clinically healthy slaughter pigs. Pyrosequencing yielded 82,857 quality-controlled sequences, clustering into 576 operational taxonomic units (OTUs), which were assigned to 230 genera and 16 phyla. The actual number of detected OTUs per sample varied highly (23-171 OTUs). Prevotella zoogleoformans and Serratia proteamaculans (best type strain hits) were most abundant (10.6 and 41.8%, respectively) in tonsils and MLNs, respectively. To explore bacterial correlation patterns between samples of each tissue, pairwise Spearman correlations (r s) were calculated. In total, 194 strong positive and negative correlations |r s| ≥ 0.6 were found. We conclude that (i) lymphatic organs harbor a high diversity of MAB, (ii) the occurrence of viable bacteria in lymph nodes is not restricted to pathological processes and (iii) lymphatic tissues may serve as a contamination source in pig slaughterhouses. This study confirms the necessity of the EFSA regulation with regard to a meat inspection based on visual examinations to foster a minimization of microbial contamination.