Oral vaccination of young broilers with a live Salmonella Typhimurium vaccine reduces caecal and internal organ colonization following a Salmonella Infantis challenge in a seeder-bird model.

Poultry products are an important source of foodborne Salmonella infections in humans. Amongst these, the prevalence of S. Infantis is rising. In this study, the protection efficacy of an authorized live-attenuated S. Typhimurium vaccine against S. Infantis, was examined using a seeder-bird model in broilers. Vaccinated birds displayed a significantly lower colonization of S. Infantis bacteria in the caeca compared to the non-vaccinated counterparts (P = 0.017), with no significant differences observed in the spleen among the groups, three days post-infection. Thirty-two days post-infection, the disparity in average S. Infantis concentration between all-vaccinated and non-vaccinated birds was significant in both caeca (P = 0.0003) and spleen (P = 0.0002). Interestingly, a third group, consisting of seeder birds that were not vaccinated but housed with vaccinated penmates, exhibited significantly lower S. Infantis levels in both caeca (P = 0.0014) and spleen (P < 0.0001) compared to the non-vaccinated group. These findings underscore the potential of a live-attenuated S. Typhimurium vaccine administered to 2-day-old chicks in conferring protection against S. Infantis in broilers up to slaughter age.

Bacterial chondronecrosis with osteomyelitis related Enterococcus cecorum isolates are genetically distinct from the commensal population and are more virulent in an embryo mortality model.

Bacterial chondronecrosis with osteomyelitis (BCO) is a common cause of broiler lameness. Bacteria that are found in BCO lesions are intestinal bacteria that are proposed to have translocated through the intestinal epithelium and have spread systemically. One of the specific bacterial species frequently isolated in BCO cases is Enterococcus cecorum. In the current study, caecal isolates were obtained from birds derived from healthy flocks (12 isolates from 6 flocks), while isolates derived from caeca, colon, pericardium, caudal thoracic vertebrae, coxo-femoral joint, knee joint and intertarsal joint (hock) were obtained from broilers derived from BCO outbreaks (111 isolates from 10 flocks). Pulsed field gel electrophoresis was performed to determine similarity. Clonal E. cecorum populations were isolated from different bones/joints and pericardium from animals within the same flock, with intestinal strains carrying the same pulsotype, pointing to the intestinal origin of the systemically present bacteria. Isolates from the intestinal tract of birds from healthy flocks clustered away from the BCO strains. Isolates from the gut, bones/joints and pericardium of affected animals contained a set of genes that were absent in isolates from the gut of healthy animals, such as genes encoding for enterococcal polysaccharide antigens (epa genes), cell wall structural components and nutrient transporters. Isolates derived from the affected birds induced a significant higher mortality in the embryo mortality model as compared to the isolates from the gut of healthy birds, pointing to an increased virulence.

Broiler performance correlates with gut morphology and intestinal inflammation under field conditions.

Maintaining optimal gut health is a key driver for a well-performing broiler flock. Histology of intestinal sections and quantification of villus structure can be used to evaluate gut health. While these measurements have been used in experimental models to evaluate gut health, less is known about the associations of these parameters with performance in commercial broiler farms. The objective of the present study was to evaluate possible associations of intestinal villus structure and the inflammatory condition of the gut with Ross 308 broiler performance in 50 commercial farms. On day 28 of the production round, 20 randomly selected broilers per farm were weighed, euthanized, and a duodenal section was collected to determine villus length, crypt depth and the CD3+ T-lymphocytes area percentage (CD3+ %). We found a relatively low coefficient of variance (CV) for the villus length (between farms; 9.67%, within farms; 15.97%), while the CD3+ (%) had a high CV (between farms; 29.78%, within farms; 25.55%). At flock level, the CD3+ (%) was significantly correlated with the villus length (r = -0.334), crypt depth (r = 0.523) and the villus-to-crypt ratio (r = -0.480). The crypt depth was significantly correlated with the European production index (EPI) (r = -0.450) and feed conversion ratio (FCR) (r = 0.389). At broiler level, a significant association was found between the individual body weight (day 28), CD3+ (%) and villus-to-crypt ratio. These data thus show that gut villus structure is significantly associated with bird performance under commercial conditions. RESEARCH HIGHLIGHTSGut histology parameters vary between and within farms.Broiler performance is associated with gut morphology.

Bacteria-derived long chain fatty acid exhibits anti-inflammatory properties in colitis.

OBJECTIVE: Data from clinical research suggest that certain probiotic bacterial strains have the potential to modulate colonic inflammation. Nonetheless, these data differ between studies due to the probiotic bacterial strains used and the poor knowledge of their mechanisms of action. DESIGN: By mass-spectrometry, we identified and quantified free long chain fatty acids (LCFAs) in probiotics and assessed the effect of one of them in mouse colitis. RESULTS: Among all the LCFAs quantified by mass spectrometry in Escherichia coli Nissle 1917 (EcN), a probiotic used for the treatment of multiple intestinal disorders, the concentration of 3-hydroxyoctadecaenoic acid (C18-3OH) was increased in EcN compared with other E. coli strains tested. Oral administration of C18-3OH decreased colitis induced by dextran sulfate sodium in mice. To determine whether other bacteria composing the microbiota are able to produce C18-3OH, we targeted the gut microbiota of mice with prebiotic fructooligosaccharides (FOS). The anti-inflammatory properties of FOS were associated with an increase in colonic C18-3OH concentration. Microbiota analyses revealed that the concentration of C18-3OH was correlated with an increase in the abundance in Allobaculum, Holdemanella and Parabacteroides. In culture, Holdemanella biformis produced high concentration of C18-3OH. Finally, using TR-FRET binding assay and gene expression analysis, we demonstrated that the C18-3OH is an agonist of peroxisome proliferator activated receptor gamma. CONCLUSION: The production of C18-3OH by bacteria could be one of the mechanisms implicated in the anti-inflammatory properties of probiotics. The production of LCFA-3OH by bacteria could be implicated in the microbiota/host interactions.

Dietary muramidase degrades bacterial peptidoglycan to NOD-activating muramyl dipeptides and reduces duodenal inflammation in broiler chickens.

Muramidases constitute a superfamily of enzymes that hydrolyse peptidoglycan (PGN) from bacterial cell walls. Recently, a fungal muramidase derived from Acremonium alcalophilum has been shown to increase broiler performance when added as a feed additive. However, the underlying mechanisms of action are not yet identified. Here, we investigated the hypothesis that this muramidase can cleave PGN to muramyl dipeptide (MDP), activating nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) receptors in eukaryotic cells, potentially inducing anti-inflammatory host responses. Using Micrococcus luteus as a test bacterium, it was shown that muramidase from A. alcalophilum did not display antimicrobial activity, while it could cleave fluorescently labelled PGN. It was shown that the muramidase could degrade PGN down to its minimal bioactive structure MDP by using UPLC-MS/MS. Using HEK-Blue™-hNOD2 reporter cells, it was shown that the muramidase-treated PGN degradation mixture could activate NOD2. Muramidase supplementation to broiler feed increased the duodenal goblet cell and intraepithelial lymphocyte abundance while reducing duodenal wall CD3+ T lymphocyte levels. Muramidase supplementation to broiler feed only had moderate effects on the duodenal, ileal and caecal microbiome. It was shown that the newly discovered muramidase hydrolysed PGN, resulting in MDP that activates NOD2, potentially steering the host response for improved intestinal health.

Valid publication of the names Caecibacterium and Caecibacterium sporoformans.

Descriptions of the genus Caecibacterium and its proposed type species Caecibacterium sporoformans were published in the IJSEM by Onrust et al. (Int J Syst Evol Microbiol 2017;67:4589-4594). The type strain was deposited as LMG 27730 and DSM 26959. DSM 26959 is a patent strain, and therefore the names were effectively, but not validly, published based on Rule 30(4) of the International Code of Nomenclature of Prokaryotes. The type strain of C. sporoformans is now available from the Deutsche Sammlung von Mikroorganismen und Zellkulturen as DSM 103070 and no restrictions have been placed on its distribution. We here present new descriptions of the genus and its type species so that the names can be validly published.

Host intestinal biomarker identification in a gut leakage model in broilers.

Intestinal health problems are a major issue in the poultry industry. Quantifiable easy-to-measure biomarkers for intestinal health would be of great value to monitor subclinical intestinal entities that cause performance problems and to evaluate control methods for intestinal health. The aim of the study was to identify host protein biomarkers for intestinal inflammation and intestinal barrier damage. Proteomic analysis was conducted on ileal and colonic content samples of broilers under an experimental gut damage and inflammation model. Effects of the challenge treatment resulted in a worse gut condition based on macroscopic gut appearance (p < 0.0001). Also microscopic changes such as shortening of the villi and increased crypt depth (p < 0.0001) as well as higher infiltration of T-lymphocytes (p < 0.0001) were seen in the duodenal tissue of challenged animals. Several candidate proteins associated with inflammation, serum leakage and/or tissue damage were identified with an increased abundance in intestinal content of challenged animals (p < 0.05). Conversely, brush border enzymes were less abundant in intestinal content of challenged animals (p < 0.05). These candidate biomarkers have potential to be used in the field for detection of gut barrier failure in broilers.

Evidence that the stress hormone cortisol regulates biofilm formation differently among Flavobacterium columnare isolates.

The impact of cortisol on Flavobacterium columnare biofilm formation was explored. Firstly, the dynamics of biofilm formation by one highly (HV) and one low virulent (LV) F. columnare isolate with and without the stress hormone cortisol under microfluidic flow conditions was characterized. This to confirm that F. columnare cells could form biofilm under cortisol supplementation, and to compare the temporal and structural differences between different treatment groups. One trial revealed that in both isolates cell aggregates resembling biofilms occurred within 7-h post-inoculation. Consequently, cell clusters were sloughed away, followed by a rebuilding of bacterial cell aggregates, suggestive for a high spreading capacity. While the HV isolate revealed cell aggregates formed upstream at all time-points, for the LV isolate this was only seen upon cortisol supplementation. Secondly, the transcriptional effect of genes (gldK, gldL, gldM, gldN, sprA, sprE, sprT, and porV) belonging to the Type IX secretion system involved in gliding motility was investigated in planktonic and biofilm cells of a HV and LV isolate to which no, a low (LD) or high (HD) dose of cortisol was added. Significantly lower expression of gliding genes gldK, gldL, gldM and gldN, and of protein secretion regulator porV was seen in the LV isolate planktonic cells supplemented with a HD-cortisol. The LV isolate biofilm cells treated with the HD-cortisol showed a significant upregulation of sprT, encoding mobile surface adhesion important in bacterial colonization. This is the first evidence for the co-regulatory effect of cortisol on biofilm formation and F. columnare gliding gene expression.

Biomarkers for monitoring intestinal health in poultry: present status and future perspectives.

Intestinal health is determined by host (immunity, mucosal barrier), nutritional, microbial and environmental factors. Deficiencies in intestinal health are associated with shifts in the composition of the intestinal microbiome (dysbiosis), leakage of the mucosal barrier and/or inflammation. Since the ban on growth promoting antimicrobials in animal feed, these dysbiosis-related problems have become a major issue, especially in intensive animal farming. The economical and animal welfare consequences are considerable. Consequently, there is a need for continuous monitoring of the intestinal health status, particularly in intensively reared animals, where the intestinal function is often pushed to the limit. In the current review, the recent advances in the field of intestinal health biomarkers, both in human and veterinary medicine are discussed, trying to identify present and future markers of intestinal health in poultry. The most promising new biomarkers will be stable molecules ending up in the feces and litter that can be quantified, preferably using rapid and simple pen-side tests. It is unlikely, however, that a single biomarker will be sufficient to follow up all aspects of intestinal health. Combinations of multiple biomarkers and/or metabarcoding, metagenomic, metatranscriptomic, metaproteomic and metabolomic approaches will be the way to go in the future. Candidate biomarkers currently are being investigated by many research groups, but the validation will be a major challenge, due to the complexity of intestinal health in the field.

Specific members of the predominant gut microbiota predict pouchitis following colectomy and IPAA in UC.

OBJECTIVE: Pouchitis is the most common complication after colectomy with ileal pouch-anal anastomosis (IPAA) for UC and the risk is the highest within the 1st year after surgery. The pathogenesis is not completely understood but clinical response to antibiotics suggests a role for gut microbiota. We hypothesised that the risk for pouchitis can be predicted based on the faecal microbial composition before colectomy. DESIGN: Faecal samples from 21 patients with UC undergoing IPAA were prospectively collected before colectomy and at predefined clinical visits at 1 month, 3 months, 6 months and 12 months after IPAA. The predominant microbiota was analysed using community profiling with denaturing gradient gel electrophoresis followed by quantitative real-time PCR validation. RESULTS: Cluster analysis before colectomy distinguished patients with pouchitis from those with normal pouch during the 1st year of follow-up. In patients developing pouchitis, an increase of Ruminococcus gnavus (p<0.001), Bacteroides vulgatus (p=0.043), Clostridium perfringens (p=0.011) and a reduction of two Lachnospiraceae genera (Blautia (p=0.04), Roseburia (p=0.008)) was observed. A score combining these five bacterial risk factors was calculated and presence of at least two risk factors showed a sensitivity and specificity of 100% and 63.6%, respectively. CONCLUSIONS: Presence of R. gnavus, B. vulgatus and C. perfringens and absence of Blautia and Roseburia in faecal samples of patients with UC before surgery is associated with a higher risk of pouchitis after IPAA. Our findings suggest new predictive and therapeutic strategies in patients undergoing colectomy with IPAA.

Caecibacterium sporoformans gen. nov., sp. nov., an anaerobic, butyrate-producing, spore-forming bacterium isolated from chicken caecum.

Strains of a Gram-stain-negative, rod-shaped and immotile bacterium were isolated from broiler chicken caecal content. The isolates required strict anaerobic conditions for growth, formed spores, were catalase-positive and oxidase-negative. They produced butyrate as the major metabolic end product in reinforced clostridial medium broth. The genomic DNA G+C content of the isolated strains was 32.5-34.6 mol%. The major cellular fatty acids were C16 : 0 FAME, C14 : 0 FAME, C19 : 0CYC 9,10DMA and C16 : 0DMA. The fatty acid composition of the cell wall showed no similarity to any strain in the midi database. 16S rRNA gene sequence analysis showed that the nearest phylogenetic neighbours were Anaerostipes hadrus and Clostridium populeti (92 % sequence similarity) within Clostridium cluster XIVa of the phylum Firmicutes. Therefore, a novel genus is proposed, with the name Caecibacterium sporoformans gen. nov., sp. nov. The type strain of Caecibacterium sporoformans is LMG 27730T=DSM 26959T.

The response of canine faecal microbiota to increased dietary protein is influenced by body condition.

BACKGROUND: High protein diets shift the faecal microbiota into a more unfavourable composition in obese humans. In lean dogs, higher protein consumption is accompanied with increased production of putrefactive fermentation products, whereas obese dogs have a different gut microbiota compared to lean dogs. Still, the impact of high dietary protein on gut microbiota in obese dogs remains unclear. The aim of this study was to investigate faecal microbial changes in lean and obese dogs in response to two different levels of dietary protein. Six healthy lean and six obese Beagles were fed a high protein diet (HP) and a low protein diet (LP) for 28 days each in a crossover design. Denaturing gradient gel electrophoresis and quantitative PCR were performed on faecal samples for microbial profiling. Plasma acylcarnitine and fermentation metabolites were measured. RESULTS: Dogs fed HP had higher concentrations of protein fermentation metabolites including faecal ammonia, isovalerate, isobutyrate, phenol, indole, serum indoxyl sulphate and plasma 3-OH isovalerylcarnitine compared to dogs fed LP, whereas no changes in faecal concentrations of acetate and butyrate were observed. The abundances of clostridial clusters IV and XIVa, covering the majority of butyrate-producing bacteria, and of the butyrate kinase gene, one of the terminal genes of the butyrate synthesis pathway were higher in dogs on HP compared to LP. Significant interactions between diet and body condition were found for the abundance of Firmicutes, Lactobacillus and clostridial cluster I. The similarity coefficient of faecal microbiota between the two diets was smaller in obese dogs than in lean dogs. CONCLUSIONS: High protein diet increased the abundance and activity of butyrate-producing bacteria in Beagles independent of the body condition. In addition, increasing dietary protein content had a greater overall impact on faecal microbiota in obese compared to lean dogs.

Microbial shifts associated with necrotic enteritis.

An outbreak of necrotic enteritis (NE) is a complex process requiring one or a number of predisposing factors rather than just the presence of pathogenic Clostridium perfringens. Examples are dietary influences, such as high levels of non-starch polysaccharides and fishmeal, and factors that evoke epithelial cell damage, such as Fusarium mycotoxins in feed and Eimeria infections. Recent studies have shown that different predisposing factors induce similar shifts in the intestinal microbiota composition. Butyrate-producing-strains of the Ruminococcaceae family are decreased in abundance by both fishmeal and Eimeria. Similarly, a decreased abundance of butyrate-producing-strains belonging to the Lachnospiraceae family has been induced by fishmeal. Also shifts are observed in the lactic acid-producing bacteria, such as decreased abundance of Lactobacillus johnsonii or Weissella confusa, when broilers were fed a fishmeal-based diet or a Fusarium mycotoxin contaminated diet. Finally, the abundance of Candidatus Savagella was decreased in broilers following Eimeria challenge or feeding a fumonisins contaminated diet. The nature of the microbiota shifts indicate that immune modulatory actions of the intestinal microbiota may play a critical role in the effect on the necrosis inducing activity of C. perfringens. Indeed, colonization with butyrate-producing bacteria plays a key role in counteracting inflammation in the gut and preserving intestinal integrity, while Candidatus Savagella is involved in stimulating Th17 and immunoglobulin A responses. Lactic acid bacteria stimulate colonization of lactate-utilizing and butyrate-producing Lachnospiraceae. Future research needs to clarify the role of the microbiota changes in the pathogenesis of NE.

Does canine inflammatory bowel disease influence gut microbial profile and host metabolism?

BACKGROUND: Inflammatory bowel disease (IBD) refers to a diverse group of chronic gastrointestinal diseases, and gut microbial dysbiosis has been proposed as a modulating factor in its pathogenesis. Several studies have investigated the gut microbial ecology of dogs with IBD but it is yet unclear if this microbial profile can alter the nutrient metabolism of the host. The aim of the present study was to characterize the faecal bacterial profile and functionality as well as to determine host metabolic changes in IBD dogs. Twenty-three dogs diagnosed with IBD and ten healthy control dogs were included. Dogs with IBD were given a clinical score using the canine chronic enteropathy clinical activity index (CCECAI). Faecal short-chain fatty acids (SCFA) and ammonia concentrations were measured and quantitative PCR was performed. The concentration of plasma amino acids, acylcarnitines, serum folate, cobalamin, and indoxyl sulfate was determined. RESULTS: No significant differences in the abundance of a selection of bacterial groups and fermentation metabolites were observed between the IBD and control groups. However, significant negative correlations were found between CCECAI and the faecal proportion of Lactobacillus as well as between CCECAI and total SCFA concentration. Serum folate and plasma citrulline were decreased and plasma valine was increased in IBD compared to control dogs. Increased plasma free carnitine and total acylcarnitines were observed in IBD compared with control dogs, whereas short-chain acylcarnitines (butyrylcarnitine + isobutyrylcarnitine and, methylmalonylcarnitine) to free carnitine ratios decreased. Dogs with IBD had a higher 3-hydroxyisovalerylcarnitine + isovalerylcarnitine to leucine ratio compared to control dogs. CONCLUSIONS: Canine IBD induced a wide range of changes in metabolic profile, especially for the plasma concentrations of short-chain acylcarnitines and amino acids, which could have evolved from tissue damage and alteration in host metabolism. In addition, dogs with more severe IBD were characterised by a decrease in faecal proportion of Lactobacillus.

Interactions of highly and low virulent Flavobacterium columnare isolates with gill tissue in carp and rainbow trout.

The interactions of Flavobacterium columnare isolates of different virulence with the gills of carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum) were investigated. Both fish species were exposed to different high (HV) or low virulence (LV) isolates and sacrificed at seven predetermined times post-challenge. Histopathological and ultrastructural examination of carp and rainbow trout inoculated with the HV-isolate disclosed bacterial invasion and concomitant destruction of the gill tissue, gradually spreading from the filament tips towards the base, with outer membrane vesicles surrounding most bacterial cells. In carp, 5-10% of the fish inoculated with the LV-isolate became moribund and their gill tissue displayed the same features as described for the HV-isolate, albeit to a lesser degree. The bacterial numbers retrieved from the gill tissue were significantly higher for HV- compared to LV-isolate challenged carp and rainbow trout. TUNEL-stained and caspase-3-immunostained gill sections demonstrated significantly higher apoptotic cell counts in carp and rainbow trout challenged with the HV-isolate compared to control animals. Periodic acid-Schiff/alcian blue staining demonstrated a significantly higher total gill goblet cell count for HV- and LV-isolate challenged compared to control carp. Moreover, bacterial clusters were embedded in a neutral matrix while being encased by acid mucins, resembling biofilm formation. Eosinophilic granular cell counts were significantly higher in the HV-isolate compared to LV-isolate inoculated and control carp. The present data indicate a high colonization capacity, and the destructive and apoptotic-promoting features of the HV-isolate, and point towards important dynamic host mucin-F. columnare interactions warranting further research.

Fumonisins affect the intestinal microbial homeostasis in broiler chickens, predisposing to necrotic enteritis.

Fumonisins (FBs) are mycotoxins produced by Fusarium fungi. This study aimed to investigate the effect of these feed contaminants on the intestinal morphology and microbiota composition, and to evaluate whether FBs predispose broilers to necrotic enteritis. One-day-old broiler chicks were divided into a group fed a control diet, and a group fed a FBs contaminated diet (18.6 mg FB1+FB2/kg feed). A significant increase in the plasma sphinganine/sphingosine ratio in the FBs-treated group (0.21 ± 0.016) compared to the control (0.14 ± 0.014) indicated disturbance of the sphingolipid biosynthesis. Furthermore, villus height and crypt depth of the ileum was significantly reduced by FBs. Denaturing gradient gel electrophoresis showed a shift in the microbiota composition in the ileum in the FBs group compared to the control. A reduced presence of low-GC containing operational taxonomic units in ileal digesta of birds exposed to FBs was demonstrated, and identified as a reduced abundance of Candidatus Savagella and Lactobaccilus spp. Quantification of total Clostridium perfringens in these ileal samples, previous to experimental infection, using cpa gene (alpha toxin) quantification by qPCR showed an increase in C. perfringens in chickens fed a FBs contaminated diet compared to control (7.5 ± 0.30 versus 6.3 ± 0.24 log10 copies/g intestinal content). After C. perfringens challenge, a higher percentage of birds developed subclinical necrotic enteritis in the group fed a FBs contaminated diet as compared to the control (44.9 ± 2.22% versus 29.8 ± 5.46%).

A decrease of the butyrate-producing species Roseburia hominis and Faecalibacterium prausnitzii defines dysbiosis in patients with ulcerative colitis.

OBJECTIVE: Bacteria play an important role in the onset and perpetuation of intestinal inflammation in inflammatory bowel disease (IBD). Unlike in Crohn's disease (CD), in which dysbiosis has been better characterised, in ulcerative colitis (UC), only small cohorts have been studied and showed conflicting data. Therefore, we evaluated in a large cohort if the microbial signature described in CD is also present in UC, and if we could characterise predominant dysbiosis in UC. To assess the functional impact of dysbiosis, we quantified the bacterial metabolites. DESIGN: The predominant microbiota from 127 UC patients and 87 age and sex-matched controls was analysed using denaturing gradient gel electrophoresis (DGGE) analysis. Differences were quantitatively validated using real-time PCR. Metabolites were quantified using gas chromatography-mass spectrometry. RESULTS: Based on DGGE analysis, the microbial signature previously described in CD was not present in UC. Real-time PCR analysis revealed a lower abundance of Roseburia hominis (p<0.0001) and Faecalibacterium prausnitzii (p<0.0001) in UC patients compared to controls. Both species showed an inverse correlation with disease activity. Short-chain fatty acids (SCFA) were reduced in UC patients (p=0.014), but no direct correlation between SCFA and the identified bacteria was found. CONCLUSIONS: The composition of the fecal microbiota of UC patients differs from that of healthy individuals: we found a reduction in R hominis and F prausnitzii, both well-known butyrate-producing bacteria of the Firmicutes phylum. These results underscore the importance of dysbiosis in IBD but suggest that different bacterial species contribute to the pathogenesis of UC and CD.

Faecalicoccus acidiformans gen. nov., sp. nov., isolated from the chicken caecum, and reclassification of Streptococcus pleomorphus (Barnes et al. 1977), Eubacterium biforme (Eggerth 1935) and Eubacterium cylindroides (Cato et al. 1974) as Faecalicoccus pleomorphus comb. nov., Holdemanella biformis gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov., respectively, within the family Erysipelotrichaceae.

Strains LMG 27428(T) and LMG 27427 were isolated from the caecal content of a chicken and produced butyric, lactic and formic acids as major metabolic end products. The genomic DNA G+C contents of strains LMG 27428(T) and LMG 27427 were 40.4 and 38.8 mol%. On the basis of 16S rRNA gene sequence similarity, both strains were most closely related to the generically misclassified Streptococcus pleomorphus ATCC 29734(T). Strain LMG 27428(T) could be distinguished from S. pleomorphus ATCC 29734(T) based on production of more lactic acid and less formic acid in M2GSC medium, a higher DNA G+C content and the absence of activities of acid phosphatase and leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and histidine arylamidases, while strain LMG 27428 was biochemically indistinguishable from S. pleomorphus ATCC 29734(T). The novel genus Faecalicoccus gen. nov. within the family Erysipelotrichaceae is proposed to accommodate strains LMG 27428(T) and LMG 27427. Strain LMG 27428(T) ( =DSM 26963(T)) is the type strain of Faecalicoccus acidiformans sp. nov., and strain LMG 27427 ( =DSM 26962) is a strain of Faecalicoccus pleomorphus comb. nov. (type strain LMG 17756(T) =ATCC 29734(T) =DSM 20574(T)). Furthermore, the nearest phylogenetic neighbours of the genus Faecalicoccus are the generically misclassified Eubacterium cylindroides DSM 3983(T) (94.4% 16S rRNA gene sequence similarity to strain LMG 27428(T)) and Eubacterium biforme DSM 3989(T) (92.7% 16S rRNA gene sequence similarity to strain LMG 27428(T)). We present genotypic and phenotypic data that allow the differentiation of each of these taxa and propose to reclassify these generically misnamed species of the genus Eubacterium formally as Faecalitalea cylindroides gen. nov., comb. nov. and Holdemanella biformis gen. nov., comb. nov., respectively. The type strain of Faecalitalea cylindroides is DSM 3983(T) =ATCC 27803(T) =JCM 10261(T) and that of Holdemanella biformis is DSM 3989(T) =ATCC 27806(T) =CCUG 28091(T).

Butyricicoccus pullicaecorum, a butyrate producer with probiotic potential, is intrinsically tolerant to stomach and small intestine conditions.

Butyrate has several beneficial properties that are essential to maintain gastrointestinal health. Therefore butyrate-producing bacteria are seen as the next generation of probiotics. The butyrate-producing bacterium Butyricicoccus pullicaecorum (a clostridial cluster IV strain) is such a promising probiotic candidate for people suffering from inflammatory bowel disease. To exert its beneficial properties, it is crucial that B. pullicaecorum survives the harsh conditions of the upper gastrointestinal tract to arrive in the colon in a viable and metabolically active state. Before developing a stable formulation of B. pullicaecorum for oral administration, it is important to know its intrinsic acid and bile tolerance. We monitored the survival during and short chain fatty acid production after incubation in conditions simulating the stomach and small intestine using in vitro batch experiments. In case of acid conditions (pH 2 and pH 3), B. pullicaecorum was viable and active but not cultivable. Cultivability was restored during subsequent small intestine conditions. Importantly, bile and pancreatic juice had no lethal effect. Milk, as a suspension medium, only had a protective effect on the cultivability during the first hour at pH 2. B. pullicaecorum was still metabolically active after upper gastrointestinal conditions and produced short chain fatty acids, but a shift from butyrate to acetate production was observed. Although the butyrate-producing anaerobe B. pullicaecorum showed good intrinsic acid and bile tolerance in terms of viability and metabolic activity, colonization efficiency and butyrate production under colon conditions is needed to further evaluate its probiotic potential.

Butyricicoccus pullicaecorum in inflammatory bowel disease.

OBJECTIVE: Many species within the phylum Firmicutes are thought to exert anti-inflammatory effects. We quantified bacteria belonging to the genus Butyricicoccus in stools of patients with ulcerative colitis (UC) and Crohn's disease (CD). We evaluated the effect of Butyricicoccus pullicaecorum in a rat colitis model and analysed the ability to prevent cytokine-induced increases in epithelial permeability. DESIGN: A genus-specific quantitative PCR was used for quantification of Butyricicoccus in stools from patients with UC or CD and healthy subjects. The effect of B pullicaecorum on trinitrobenzenesulfonic (TNBS)-induced colitis was assessed and the effect of B pullicaecorum culture supernatant on epithelial barrier function was investigated in vitro. RESULTS: The average number of Butyricicoccus in stools from patients with UC and CD in active (UC: 8.61 log10/g stool; CD: 6.58 log10/g stool) and remission phase (UC: 8.69 log10/g stool; CD: 8.38 log10/g stool) was significantly lower compared with healthy subjects (9.32 log10/g stool) and correlated with disease activity in CD. Oral administration of B pullicaecorum resulted in a significant protective effect based on macroscopic and histological criteria and decreased intestinal myeloperoxidase (MPO), tumour necrosis factor α (TNFα) and interleukin (IL)-12 levels. Supernatant of B pullicaecorum prevented the loss of transepithelial resistance (TER) and the increase in IL-8 secretion induced by TNFα and interferon γ (IFN gamma) in a Caco-2 cell model. CONCLUSIONS: Patients with inflammatory bowel disease have lower numbers of Butyricicoccus bacteria in their stools. Administration of B pullicaecorum attenuates TNBS-induced colitis in rats and supernatant of B pullicaecorum cultures strengthens the epithelial barrier function by increasing the TER.