Development of a multi-locus sequence typing system helps reveal the evolution of Cardinium hertigii, a reproductive manipulator symbiont of insects.

BACKGROUND: 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. RESULTS: We developed a Multi Locus Sequence Typing (MLST) system that provides researchers with three new genes in addition to Gyrase B for inferring phylogenies and delineating Cardinium strains. From our Cardinium phylogeny, we confirmed the presence of a new group D, a Cardinium clade that resides in the arachnid order harvestmen (Opiliones). Many Cardinium clades appear to display a high degree of host affinity, while some show evidence of host shifts to phylogenetically distant hosts, likely associated with ecological opportunity. Like the unrelated reproductive manipulator Wolbachia, the Cardinium phylogeny also shows no clear phylogenetic signal associated with particular reproductive manipulations. CONCLUSIONS: The Cardinium phylogeny shows evidence of diversification within particular host lineages, and also of host shifts among trophic levels within parasitoid-host communities. Like Wolbachia, the relatedness of Cardinium strains does not necessarily predict their reproductive phenotypes. Lastly, the genetic tools proposed in this study may help future authors to characterize new strains and add to our understanding of Cardinium evolution.

Microbial shifts in Minas artisanal cheeses from the Serra do Salitre region of Minas Gerais, Brazil throughout ripening time.

The Minas artisanal cheese is a traditional product in its way of producing. Produced in the Minas Gerais state, Brazil, this cheese is made using raw cow's milk with the addition of an endogenous starter culture called "pingo", responsible for inoculating specific microorganisms that could enhance flavor and sensorial aspects. There are seven regions able to produce and commercialize this product - Araxá, Campo das Vertentes, Canastra Cerrado, Serra do Salitre, Serro and Triângulo Mineiro. This study aimed to assess the bacterial community of raw milk, endogenous starter culture and to uncover possible shifts in the bacterial community of the rind and core of cheeses at sixty days of ripening located in the Serra do Salitre region by Illumina MiSeq 16S rRNA gene amplicon sequencing. Raw milk and starter culture are responsible for inoculating specific bacteria into the cheese, with Planococcaceae and Streptococcaceae being prevalent throughout ripening time. The Planococcaceae family seems to develop strong interactions with the Leuconostocaceae family on the surface of these cheeses, and is associated with environmental aspects of the region, probably leading to a microbial signature of these products. Additionally, abiotic factors such as geographical location, moisture and acidity are major drivers in the microbial shift.

Effects of clay mineral supplementation on particle-associated and epimural microbiota, and gene expression in the rumen of cows fed high-concentrate diet.

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.

Adaptation of the cecal bacterial microbiome of growing pigs in response to resistant starch type 4.

Resistant starch (RS) exacerbates health benefits on the host via modulation of the gut bacterial community. By far, these effects have been less well explored for RS of type 4. This study aimed at gaining a community-wide insight into the impact of enzymatically modified starch (EMS) on the cecal microbiota and hindgut fermentation in growing pigs. Castrated male pigs (n = 12/diet; 29-kg body weight) were fed diets with either 70% EMS or control starch for 10 days. The bacterial profile of each cecal sample was determined by sequencing of the V345 region of the 16S rRNA gene using the Illumina MiSeq platform. EMS diet reduced short-chain fatty acid concentrations in cecum and proximal colon compared to the control diet. Linear discriminant analyses and K means clustering indicated diet-specific cecal community profiles, whereby diversity and species richness were not different among diets. Pigs showed host-specific variation in their most abundant phyla, Firmicutes (55%), Proteobacteria (35%), and Bacteroidetes (10%). The EMS diet decreased abundance of Ruminococcus, Parasutterella, Bilophila, Enterococcus, and Lactobacillus operational taxonomic units (OTU), whereas Meniscus and Actinobacillus OTU were increased compared to those with the control diet (P < 0.05). Quantitative PCR confirmed results for host effect on Enterobacteriaceae and diet effect on members of the Lactobacillus group. The presence of less cecal short-chain fatty acids and the imputed metabolic functions of the cecal microbiome suggested that EMS was less degradable for cecal bacteria than the control starch. The present EMS effects on the bacterial community profiles were different than the previously reported RS effects and can be linked to the chemical structure of EMS.

Dietary calcium concentration and cereals differentially affect mineral balance and tight junction proteins expression in jejunum of weaned pigs.

Ca plays an essential role in bone development; however, little is known about its effect on intestinal gene expression in juvenile animals. In the present study, thirty-two weaned pigs (9·5 (SEM 0·11) kg) were assigned to four diets that differed in Ca concentration (adequate v. high) and cereal composition (wheat-barley v. maize) to assess the jejunal and colonic gene expression of nutrient transporters, tight junction proteins, cytokines and pathogen-associated molecular patterns, nutrient digestibility, Ca balance and serum acute-phase response. To estimate the impact of mucosal bacteria on colonic gene expression, Spearman's correlations between colonic gene expression and bacterial abundance were computed. Faecal Ca excretion indicated that more Ca was available along the intestinal tract of the pigs fed high Ca diets as compared to the pigs fed adequate Ca diets (P> 0.05). High Ca diets decreased jejunal zonula occludens 1 (ZO1) and occludin (OCLN) expression, up-regulated jejunal expression of toll-like receptor 2 (TLR2) and down-regulated colonic GLUT2 expression as compared to the adequate Ca diets (P< 0.05). Dietary cereal composition up-regulated jejunal TLR2 expression and interacted (P= 0.021) with dietary Ca on colonic IL1B expression; high Ca concentration up-regulated IL1B expression with wheat-barley diets and down-regulated it with maize diets. Spearman's correlations (r> 0·35; P< 0·05) indicated an association between operational taxonomic units assigned to the phyla Bacteroidetes, Firmicutes and Proteobacteria and bacterial metabolites and mucosal gene expression in the colon. The present results indicate that high Ca diets have the potential to modify the jejunal and colonic mucosal gene expression response which, in turn, interacts with the composition of the basal diet and mucosa-associated bacteria in weaned pigs.

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.

Quantification of Gram-positive bacteria: adaptation and evaluation of a preparation strategy using high amounts of clinical tissue.

BACKGROUND: A preparation method for quantification of bacteria in tissues is obligatory to reduce tissue mass, concentrate the target, purify, remove inhibitory substances and to achieve constant target recovery rates. No preparation method has been available until now for a high mass of tissue applicable for routine use and analytical veterinary diagnostics. RESULTS: This study describes an easy-to-use tissue preparation protocol to quantify Gram-positive bacteria from a large volume of tissue matrix. A previously published sample preparation method (Matrix-Lysis) from food science was successfully adapted for clinical use on tissues from pigs, including cerebrum, spinal cord, lung, liver, ileum, colon, caecum, kidney and muscle tissue. This tissue preparation method now permits quantification of pathogens from 5 g of organic matrix, which is a 20-200 fold increase by weight compared to other methods. It is based on solubilization of the sample matrix with either a chaotrope plus detergent or divalent salts as solubilization agents. The method was designed as a modular system, offering the possibility to change lysis buffers, according to tissue solubilization characteristics and the intended detection method (molecular or culture). Using Listeria monocytogenes as model organism, viable cell quantification or DNA extraction and quantitative real-time PCR were performed after Matrix-Lysis to determine recovery rates and detection limit (LOD). The adapted Matrix-Lysis protocol resulted in high recovery rates (mean value: 76% ± 39%) for all tested organs, except kidney, and recovery was constant over 5 log scales for all tested buffer systems. The LOD for Matrix-Lysis with subsequent plate count method (PCM) was as low as 1 CFU/5 g, while for qPCR based detection the LOD was 102 bacterial cell equivalents (BCE)/5 g for two buffer systems. CONCLUSIONS: This tissue preparation is inexpensive and can be easily used for routine and analytical veterinary diagnostics. Inoculation studies or hazard assessments can profit from this tissue preparation method and it is anticipated that this study will be a valuable source for further research on tissue preparation strategies.

Changing dietary calcium-phosphorus level and cereal source selectively alters abundance of bacteria and metabolites in the upper gastrointestinal tracts of weaned pigs.

Several dietary ingredients may affect the bacterial community structure and metabolism in the porcine gut and may therefore influence animals' health and performance. This study investigated the effects of cereal source and calcium-phosphorus (CaP) level in the diet on bacterial microbiota and metabolites, nutrient intake, and gut environment in weaned pigs. Pigs (n=8/treatment) were fed wheat-barley- or corn-based diets with an adequate or high CaP level for 14 days. Effects on microbiota in the stomach, ileum, and midcolon were assessed using quantitative PCR. Data showed that Enterobacteriaceae, Campylobacter spp., and Helicobacter spp., which all contain highly immune reactive lipopolysaccharide (LPS), were abundant at all gut sites. Diet effects on bacteria and metabolites were moderate and occurred mainly in the upper gut, whereas no effects on bacteria, fermentation products, and LPS could be observed in the colon. Differences in carbohydrate intake with corn versus wheat-barley diets selectively stimulated Bifidobacterium in the stomach and ileum. There was a growth advantage for a few bacterial groups in the stomach and ileum of pigs fed the high versus adequate CaP level (i.e., gastric Enterobacteriaceae and ileal Enterococcus, Bacteroides-Prevotella-Porphyromonas, and Campylobacter). Interestingly, gastrointestinal pH was not affected by dietary CaP level. The present findings demonstrate the stability of the bacterial community and gut environment toward dietary changes even in young pigs. The results on stimulation of gastric and ileal Bifidobacterium by corn diets may be employed in nutritional strategies to support gut health after weaning.

Early Parenteral Administration of Ceftiofur has Gender-Specific Short- and Long-Term Effects on the Fecal Microbiota and Growth in Pigs from the Suckling to Growing Phase.

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.

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.

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.

Autochthonous facility-specific microbiota dominates washed-rind Austrian hard cheese surfaces and its production environment.

Cheese ripening involves the succession of complex microbial communities that are responsible for the organoleptic properties of the final products. The food processing environment can act as a source of natural microbial inoculation, especially in traditionally manufactured products. Austrian Vorarlberger Bergkäse (VB) is an artisanal washed-rind hard cheese produced in the western part of Austria without the addition of external ripening cultures. Here, the composition of the bacterial communities present on VB rinds and on different processing surfaces from two ripening cellars was assessed by near full length 16S rRNA gene amplification, cloning and sequencing. Non-inoculated aerobic bacteria dominated all surfaces in this study. VB production conditions (long ripening time, high salt concentration and low temperatures) favor the growth of psychro- and halotolerant bacteria. Several bacterial groups, such as coryneforms, Staphylococcus equorum and Halomonas dominated VB and were also found on most environmental surfaces. Analysis of OTUs shared between different surfaces suggests that VB rind bacteria are inoculated naturally during the ripening from the processing environment and that cheese surfaces exert selective pressure on these communities, as only those bacteria better adapted flourished on VB rinds. This study analyzed VB processing environment microbiota and its relationship with VB rinds for the first time, elucidating that the processing environment and the cheese microbiota should be considered as microbiologically linked ecosystems with the goal of better defining the events that take place during cheese maturation.

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.

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.

Biphasic Metabolism and Host Interaction of a Chlamydial Symbiont.

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.

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.