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.

Chlamydiae are obligate intracellular bacteria comprising well-known human pathogens and ubiquitous symbionts of protists, which are characterized by a unique developmental cycle. Here we comprehensively analyzed gene expression dynamics of Protochlamydia amoebophila during infection of its Acanthamoeba host by RNA sequencing. This revealed a highly dynamic transcriptional landscape, where major transcriptional shifts are conserved among chlamydial symbionts and pathogens. Our data served to propose a time-resolved model for type III protein secretion during the developmental cycle, and we provide evidence for a biphasic metabolism of P. amoebophila during infection, which involves energy parasitism and amino acids as the carbon source during initial stages and a postreplicative switch to endogenous glucose-based ATP production. This fits well with major transcriptional changes in the amoeba host, where upregulation of complex sugar breakdown precedes the P. amoebophila metabolic switch. The biphasic chlamydial metabolism represents a unique adaptation to exploit eukaryotic host cells, which likely contributed to the evolutionary success of this group of microbes. IMPORTANCE Chlamydiae are known as major bacterial pathogens of humans, causing the ancient disease trachoma, but they are also frequently found in the environment where they infect ubiquitous protists such as amoebae. All known chlamydiae require a eukaryotic host cell to thrive. Using the environmental chlamydia Protochlamydia amoebophila within its natural host, Acanthamoeba castellanii, we investigated gene expression dynamics in vivo and throughout the complete chlamydial developmental cycle for the first time. This allowed us to infer how a major virulence mechanism, the type III secretion system, is regulated and employed, and we show that the physiology of chlamydiae undergoes a complete shift regarding carbon metabolism and energy generation. This study provides comprehensive insights into the infection strategy of chlamydiae and reveals a unique adaptation to life within a eukaryotic host cell.

Despite the importance of gut microbiota for broiler performance and health little is known about the composition of this ecosystem, its development and response towards bacterial infections. Therefore, the current study was conducted to address the composition and structure of the microbial community in broiler chickens in a longitudinal study from day 1 to day 28 of age in the gut content and on the mucosa. Additionally, the consequences of a Campylobacter (C.) jejuni infection on the microbial community were assessed. The composition of the gut microbiota was analyzed with 16S rRNA gene targeted Illumina MiSeq sequencing. Sequencing of 130 samples yielded 51,825,306 quality-controlled sequences, which clustered into 8285 operational taxonomic units (OTUs; 0.03 distance level) representing 24 phyla. Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Tenericutes were the main components of the gut microbiota, with Proteobacteria and Firmicutes being the most abundant phyla (between 95.0 and 99.7% of all sequences) at all gut sites. Microbial communities changed in an age-dependent manner. Whereas, young birds had more Proteobacteria, Firmicutes, and Tenericutes dominated in older birds (>14 days old). In addition, 28 day old birds had more diverse bacterial communities than young birds. Furthermore, numerous significant differences in microbial profiles between the mucosa and luminal content of the small and large intestine were detected, with some species being strongly associated with the mucosa whereas others remained within the luminal content of the gut. Following oral infection of 14 day old broiler chickens with 1 × 108 CFU of C. jejuni NCTC 12744, it was found that C. jejuni heavily colonized throughout the small and large intestine. Moreover, C. jejuni colonization was associated with an alteration of the gut microbiota with infected birds having a significantly lower abundance of Escherichia (E.) coli at different gut sites. On the contrary, the level of Clostridium spp. was higher in infected birds compared with birds from the negative controls. In conclusion, the obtained results demonstrate how the bacterial microbiome composition changed within the early life of broiler chickens in the gut lumen and on the mucosal surface. Furthermore, our findings confirmed that the Campylobacter carrier state in chicken is characterized by multiple changes in the intestinal ecology within the host.

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.

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.

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.

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.

Cardinium is an intracellular bacterial symbiont in the phylum Bacteroidetes that is found in many different species of arthropods and some nematodes. This symbiont is known to be able to induce three reproductive manipulation phenotypes, including cytoplasmic incompatibility. Placing individual strains of Cardinium within a larger evolutionary context has been challenging because only two, relatively slowly evolving genes, 16S rRNA gene and Gyrase B, have been used to generate phylogenetic trees, and consequently, the relationship of different strains has been elucidated in only its roughest form.

The increased concentrate amounts in cow diets may initiate changes in both particle-associated (PaM) and epimural microbiota (EpM) with the potential for promoting the establishment of pathogens. Clay minerals have shown promising potentials in binding harmful microorganisms and metabolites due to their high adsorption capacity. This study evaluated the effects of a clay-mineral based product (CM) on PaM, EpM, fermentation parameters, and epithelial gene expression in cows fed a high-concentrate diet. Eight rumen-cannulated non-lactating Holstein cows received a concentrate mix supplemented with CM or not (CON) in a change-over design with an initial 100% roughage diet phase (RD, 1 week), followed by intermittent 65%-high-concentrate-diet phases (HC1, HC2; 1 and 2 week duration, respectively), interrupted by 1 week roughage only. Rumen samples for short-chain fatty acids, ammonia, and lactate quantification, as well as PaM, and epithelial biopsies for EpM examination and epithelial gene expression were collected via the cannula once during each feeding phase. Phylogenetic distance analysis of Illumina MiSeq sequencing of the 16S rRNA gene region V345 showed a clear clustering of RD microbiota compared to HC in PaM, showing the impact of the high-concentrate diet on the bacterial community. In the EpM this effect was less pronounced, due to higher variability in RD. In the PaM, a decrease (P < 0.01) of community diversity occurred with the onset of HC feeding, while in the EpM there was an increase in diversity (P < 0.05). In the PaM, CM increased the relative abundance of genus Butyrivibrio (P < 0.01), a commensal bacterium of the rumen, which was, with 6.4%, the second most abundant genus. There, the CM supplementation decreased the genera Lactobacillus, Fusobacterium, and Treponema (P = 0.05), which are potentially either lactate producing or opportunistic pathogens. In the EpM, CM decreased the relative abundance of Succiniclasticum genus (P < 0.01), a possible endotoxin producer, and increased bacteria that are associated with a normobiotic rumen, such as Campylobacter (P = 0.06). Barrier function genes were upregulated in HC2 and nutrient transport genes downregulated in HC1 (P < 0.05); however, there was little effect on pro-inflammatory genes at the epithelium. The CM showed a significant decreasing effect on the cellular metabolism genes HMGCS1 (P = 0.04). Our results suggest that CM supplementation can increase the relative abundance of commensal microbiota and decrease bacteria that could negatively impact the rumen milieu and health during high-concentrate feeding.

From birth to the time after weaning the gastrointestinal microbiota of calves must develop into a stable, autochthonous community accompanied by pivotal changes of anatomy and physiology of the gastrointestinal tract. The aim of this pilot study was to examine the fecal microbiota of six Simmental dairy calves to investigate time-dependent dynamics of the microbial community. Calves were followed up from birth until after weaning according to characteristic timepoints during physiological development of the gastrointestinal tract. Pyrosequencing of 16S rRNA gene amplicons from 35 samples yielded 253,528 reads clustering into 5410 operational taxonomic units based on 0.03 16S rRNA distance. Operational taxonomic units were assigned to 296 genera and 17 phyla with Bacteroidetes, Firmicutes, and Proteobacteria being most abundant. An age-dependent increasing diversity and species richness was observed. Highest similarities between fecal microbial communities were found around weaning compared with timepoints from birth to the middle of the milk feeding period. Principal coordinate analysis revealed a high variance particularly in samples taken at the middle of the milk feeding period (at the age of approximately 40 days) compared to earlier timepoints, confirming a unique individual development of the fecal microbiota of each calf. This study provides first deep insights into the composition of the fecal microbiota of Simmental dairy calves and might be a basis for future more detailed studies.

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.

Dietary composition largely influences pig's gastrointestinal microbiota and represents a useful prophylactic tool against enteric disturbances in young pigs. Despite the importance for host-microbe interactions and bacterial colonization, dietary responses of the mucosa-associated bacterial communities are less well investigated. In the present study, we characterized the mucosa-associated bacterial communities at the Pars non-glandularis of the stomach, ileum and colon, and identified shifts in these communities in response to different dietary calcium-phosphorus (Ca-P) contents (100% versus 190% of the Ca and P requirements) in combination with two basal diets (wheat-barley- or corn-based) in weaned pigs. Pyrosequencing of 16S rRNA genes from 93 mucosal samples yielded 447,849 sequences, clustering into 997 operational taxonomic units (OTUs) at 97% similarity level. OTUs were assigned to 198 genera belonging to 14 different phyla. Correlation-based networks revealed strong interactions among OTUs at the various gastrointestinal sites. Our data describe a previously not reported high diversity and species richness at the Pars non-glandularis of the stomach in weaned pigs. Moreover, high versus adequate Ca-P content significantly promoted Lactobacillus by 14.9% units (1.4 fold change) at the gastric Pars non-glandularis (P = 0.035). Discriminant analysis revealed dynamic changes in OTU composition in response to dietary cereals and Ca-P contents at all gastrointestinal sites which were less distinguishable at higher taxonomic levels. Overall, this study revealed a distinct mucosa-associated bacterial community at the different gut sites, and a strong effect of high Ca-P diets on the gastric community, thereby markedly expanding our comprehension on mucosa-associated microbiota and their diet-related dynamics in weaned pigs.

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.

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.

Using ceftiofur during the first days of life is a common preventative strategy against several bacterial diseases in pig production. This study aimed to evaluate short- and long-term effects of early use of ceftiofur on the fecal microbiota development in suckling and growing pigs. Sixty-four piglets from eight litters were assigned to the antibiotic (AB; n = 32) or control group (control; n = 32). Twelve hours postpartum (day 0) AB piglets received an intramuscular injection of ceftiofur (5.0 mg/kg body weight) or a placebo. DNA was extracted from fecal samples collected on days 0, 12, 28, and 97 for deep-sequencing of the 16S rRNA gene. The AB administration disturbed the maturational changes in the fecal microbiome, whereby effects were sex-specific. Sex-related differences in AB metabolism in females and males may have caused these diverging AB-effects on the fecal microbiota. Especially the loss of bacterial diversity and of certain taxa in female AB pigs may have contributed to the decreased body weight of these females on day 97 of life. Taken together, this study showed that an AB injection with ceftiofur 12 h postpartum markedly affected the successional changes in the fecal microbiota composition in male and female pigs, with long-term consequences for host performance.