Impact of early inoculation of probiotics to suckling piglets on postweaning diarrhoea - A challenge study with Enterotoxigenic E. Coli F18.

Postweaning diarrhoea caused by Enterotoxigenic Escherichia coli (ETEC) is a threat to the pig industry. With an intensified focus on finding alternatives to the use of medical zinc oxide and antibiotics in newly weaned pigs, the objective of this study was to investigate the effect of early inoculation of probiotics to suckling piglets on subsequently ETEC faecal shedding and immune parameters in ETEC F18-challenged weaned piglets. Sixty pigs weaned on day 28 of age were assigned to three treatment groups: (i) Negative Control (non-challenged), (ii) Positive Control (challenged) and (iii) Probiotic (challenged and inoculated with a multi-species probiotic product during suckling). On days 1 and 2 postweaning, pigs in the Positive Control and Probiotic groups were challenged with 5 × 108 colony-forming unit ETEC F18/pig/day, whereas pigs in the Negative Control group were provided with NaCl. Growth and diarrhoea incidence were not significantly affected by ETEC challenge or probiotic administration. ETEC F18 shedding and C-reactive protein concentration in plasma were significantly lower in the Negative Control group, confirming a successful challenge model. Pigs in the Probiotic group had a significantly reduced number of pigs shedding ETEC F18 and STb toxin in faeces compared with the Positive Control group. Probiotic application did not significantly impact the concentration of C-reactive protein, haptoglobin and cytokines in plasma nor haematology numbers. In conclusion, weaned pigs administered with a multi-species probiotic product early in life had a more rapid response towards the pathogen challenge and a faster clearance of ETEC compared with the Positive Control group. Administration of a multi-species probiotic to newborn piglets may thus promote resilience in the newly weaned pig. However, further studies with pigs subjected to a more severe pathogen challenge are needed to confirm these results and to investigate the mechanism of action of the probiotic intervention.

Screening of probiotic candidates in a simulated piglet small intestine in vitro model.

The CoMiniGut in vitro model mimicking the small intestine of piglets was used to evaluate four probiotic strains for their potential as a preventive measure against development of diarrhea in weaned pigs. In the in vitro system, piglet digesta was inoculated with pathogenic enterotoxigenic Escherichia coli F4 (ETEC F4), and the short-chain fatty acid profile and the gut microbiota composition were assessed. A total of four probiotic strains were evaluated: Enterococcus faecium (CHCC 10669), Lactobacillus rhamnosus (CHCC 11994), Bifidobacterium breve (CHCC 15268) and Faecalibacterium prausnitzii (CHCC 28556). The significant differences observed in metabolite concetration and bacterial enumeration were attributed to variation in inoculating material or pathogen challenge rather than probiotic treatment. Probiotic administration influenced the microbiota composition to a small extend. Learnings from the present study indicate that the experimental setup, including incubation time and choice of inoculating material, should be chosen with care.

Pre-clinical evaluation of the effect of co-medication with antibiotics and oral steroids in Göttingen Minipigs on the biological activity of the probiotic medicinal product TSO (Trichuris suis ova).

The probiotic medicinal product TSO (Trichuris suis ova) is administered to patients with active ulcerative colitis in an ongoing clinical phase IIb trial where the typical co-medications are steroids (prednisolone or budesonide) and antibiotics (e.g., phenoxymethylpenicillin). The present pre-clinical study evaluates the effects of these co-medications on the biological activity of TSO in Göttingen Minipigs. This translationally relevant pre-clinical model allows administration of TSO with and without oral steroids or antibiotics in a manner similar to the administration to patients, followed by quantification of the biological activity of TSO. The biological activity of TSO was not affected by oral steroids but was reduced by oral antibiotics. Fecal calprotectin, the common marker of intestinal inflammation in patients with UC, did not differ between groups.

Functional in vitro screening of probiotic strains for inoculation of piglets as a prophylactic measure towards Enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC), being the major cause of post-weaning diarrhoea (PWD) in newly weaned piglets, induces poor performance and economic losses in pig production. This functional in vitro screening study investigated probiotic strains for use in suckling piglets as a prophylactic strategy towards PWD. Nine strains were evaluated based on their ability to: enhance intestinal epithelial barrier function, reduce adherence of ETEC F18 to intestinal cells, inhibit growth of ETEC F18, and grow on porcine milk oligosaccharides. Strains included in the screening were of the species Lactobacillus, Enterococcus, Bifidobacterium and Bacillus. Our in vitro screening demonstrated genus-, species and strain-specific differences in the mode of action of the tested probiotic strains. Some of the tested bifidobacteria were able to grow on the two porcine milk oligosaccharides, 3'-sialyllactose sodium salt (3'SL) and Lacto-N-neotetraose (LNnT), whereas most lactic acid bacteria strains and both Bacillus subtilis strains failed to do so. All probiotic strains inhibited growth of ETEC F18 on agar plates. All but the bifidobacteria reduced binding of ETEC F18 to Caco-2 cell monolayers, with the Enterococcus faecium strain having the most profound effect. All three lactic acid bacteria and Bifidobacterium animalis subsp. lactis counteracted the ETEC F18-induced permeability across Caco-2 cell monolayers with the E. faecium strain exhibiting the most pronounced protective effect. The findings from this in vitro screening study indicate that, when selecting probiotic strains for suckling piglets as a prophylactic strategy towards PWD, it would be advantageous to choose a multi-species product including strains with different modes of action in order to increase the likelihood of achieving beneficial effects in vivo.

Global transcriptome analysis of Rhizobium favelukesii LPU83 in response to acid stress.

Acidic environments naturally occur worldwide and inappropriate agricultural management may also cause acidification of soils. Low soil pH values are an important barrier in the plant-rhizobia interaction. Acidic conditions disturb the establishment of the efficient rhizobia usually used as biofertilizer. This negative effect on the rhizobia-legume symbiosis is mainly due to the low acid tolerance of the bacteria. Here, we describe the identification of relevant factors in the acid tolerance of Rhizobium favelukesii using transcriptome sequencing. A total of 1924 genes were differentially expressed under acidic conditions, with ∼60% underexpressed. Rhizobium favelukesii acid response mainly includes changes in the energy metabolism and protein turnover, as well as a combination of mechanisms that may contribute to this phenotype, including GABA and histidine metabolism, cell envelope modifications and reverse proton efflux. We confirmed the acid-sensitive phenotype of a mutant in the braD gene, which showed higher expression under acid stress. Remarkably, 60% of the coding sequences encoded in the symbiotic plasmid were underexpressed and we evidenced that a strain cured for this plasmid featured an improved performance under acidic conditions. Hence, this work provides relevant information in the characterization of genes associated with tolerance or adaptation to acidic stress of R. favelukesii.

Multidonor FMT capsules improve symptoms and decrease fecal calprotectin in ulcerative colitis patients while treated - an open-label pilot study.

Background: Growing evidence indicates that gut dysbiosis is a factor in the pathogenesis of ulcerative colitis (UC). Fecal microbiota transplantation (FMT) appears to be promising in inducing UC remission, but there are no reports regarding administration using capsules. Methods: Seven patients with active UC, aged 27-50 years, were treated with 25 multidonor FMT capsules daily for 50 days as a supplement to their standard treatment in an open-label pilot study. The primary objective was to follow symptoms through the Simple Clinical Colitis Activity Index (SCCAI). Secondary objectives were to follow changes in fecal calprotectin and microbial diversity through fecal samples and quality of life through the Inflammatory Bowel Disease Questionnaire (IBDQ). Participants were followed through regular visits for six months. Results: From a median of 6 at baseline, the SCCAI of all participants decreased, with median decreases of 5 (p = .001) and 6 (p = .001) after 4 and 8 weeks, respectively. Three of the seven patients had flare-up/relapse of symptoms after the active treatment period. The median F-calprotectin of ≥1800 mg/kg at baseline decreased significantly during the treatment period, but increased again in the follow-up period. The median IBDQ improved at all visits compared to baseline. The fecal microbiota α-diversity did not increase in the study period compared to baseline. All participants completed the treatment and no serious adverse events were reported. Conclusion: Fifty days of daily multidonor FMT capsules temporarily improved symptoms and health-related life quality and decreased F-calprotectin in patients with active UC.

Mesorhizobium sanjuanii sp. nov., isolated from nodules of Lotus tenuis in the saline-alkaline lowlands of Flooding Pampa, Argentina.

Two rhizobial strains, BSA136T and BSA150, related to the genus Mesorhizobium were isolated from root nodules of Lotus tenuis grown in saline-alkaline lowlands soil from Argentina. These strains showed different repetitive element palindromic PCR fingerprinting patterns but shared more than 99 % sequence similarity for both 16S rRNA and recA genes. Despite the symbiotic nodC gene sequences of our strains being related to the canonical Lotus biovar species comprising Mesorhizobium loti and Mesorhizobium japonicum, the 16S rRNA phylogenetic marker suggests that their taxonomical identities are closely related to Mesorhizobium helmanticense, Mesorhizobium metallidurans, Mesorhizobium thianshanense, Mesorhizobium gobiense and Mesorhizobium tarimense. Multilocus sequence analysis performed with seven housekeeping genes confirmed that BSA136T belongs to a separate clade within the genus Mesorhizobium. The results of comparisons for in silico DNA-DNA hybridization and average nucleotide identity indexes between the genomes of BSA136T and closest-related Mesorhizobium species were below the threshold for species delineation. Phenotypic features differentiated BSA136T from its closest-related species. On the basis of our results, BSA136T and BSA150 can be considered to represent a novel species of the genus Mesorhizobium, for which the name Mesorhizobium sanjuanii sp. nov. is hereby proposed. The type strain of this species is BSA136T (=CECT 9305T=LMG 30060T), for which the draft genome sequence is available.

A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static and flow conditions.

Pseudomonas aeruginosa is an opportunistic human pathogen that forms highly stable communities - biofilms, which contribute to the establishment and maintenance of infections. The biofilm state and intrinsic/acquired bacterial resistance mechanisms contribute to resistance/tolerance to antibiotics that is frequently observed in P. aeruginosa isolates. Here we describe the isolation and characterization of six novel lytic bacteriophages: viruses that infect bacteria, which together efficiently infect and kill a wide range of P. aeruginosa clinical isolates. The phages were used to formulate a cocktail with the potential to eliminate P. aeruginosa PAO1 planktonic cultures. Two biofilm models were studied, one static and one dynamic, and the phage cocktail was assessed for its ability to reduce and disperse the biofilm biomass. For the static model, after 4 h of contact with the phage suspension (MOI 10) more than 95% of biofilm biomass was eliminated. In the flow biofilm model, a slower rate of activity by the phage was observed, but 48 h after addition of the phage cocktail the biofilm was dispersed, with most cells eliminated (> 4 logs) comparing with the control. This cocktail has the potential for development as a therapeutic to control P. aeruginosa infections, which are predominantly biofilm centred.

The effect of Lactobacillus paracasei subsp. paracasei L. casei W8® on blood levels of triacylglycerol is independent of colonisation.

Gut microbiota (GM) dysbiosis has been linked to obesity and its metabolic complications such as cardiovascular disease (CVD). The risk of developing CVD increases with elevated concentration of serum triacylglycerol (TAG). In a blinded, randomised two-arm parallel human intervention study we have previously found that four weeks of supplementation with Lactobacillus paracasei subsp. paracasei L. casei W8® (L. casei W8) compared to placebo reduced the concentration of TAG in 64 young healthy adults, an effect, likely mediated by a decreased stearoyl- CoA desaturase-1 (SCD1) activity. In the present study we analysed faecal samples obtained during the intervention study to investigate whether this effect was related to the ability of L. casei W8 to colonise the human gut after supplementation of L. casei W8 (1010 cfu daily) as determined by qPCR specific for L. paracasei and L. casei (L. casei group); whether L. casei W8 consumption affected GM composition as determined by 16S rRNA gene targeted 454/FLX amplicon sequencing; and whether these changes were associated with changes in TAG concentration and SCD1 activity. Faecal samples were collected at baseline, after four weeks supplementation and two weeks after the supplementation was ended, and fasting blood samples were collected at baseline and after 4 weeks. Four weeks supplementation with L. casei W8 did not affect the overall composition of the GM; however, an increase in the relative abundance of the L. casei group from 8.48×10-6% of the total GM compared to 2.83×10-3% at baseline (P<0.001) was observed. Two weeks after supplementation ended, the relative abundance of the L. casei group was still increased 14 times compared to before the intervention (P<0.01). However, neither the increase in the abundance of the L. casei group nor overall GM composition correlated with changes in blood lipids or SCD1 activity.

Combined use of bacteriophage K and a novel bacteriophage to reduce Staphylococcus aureus biofilm formation.

Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-resistant S. aureus (MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a broad host range among S. aureus bacteria. Morphologically, the phage belongs to the Myoviridae family and comprises a large double-stranded DNA (dsDNA) genome of 141,907 bp. DRA88 was mixed with phage K to produce a high-titer mixture that showed strong lytic activity against a wide range of S. aureus isolates, including representatives of the major international MRSA clones and coagulase-negative Staphylococcus. Its efficacy was assessed both in planktonic cultures and when treating established biofilms produced by three different biofilm-producing S. aureus isolates. A significant reduction of biofilm biomass over 48 h of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus biofilms.

Genome Sequences of Two Leuconostoc pseudomesenteroides Strains Isolated from Danish Dairy Starter Cultures.

The lactic acid bacterium Leuconostoc pseudomesenteroides can be found in mesophilic cheese starters, where it produces aromatic compounds from, e.g., citrate. Here, we present the draft genome sequences of two L. pseudomesenteroides strains isolated from traditional Danish cheese starters.

Genome Sequence of Leuconostoc mesenteroides subsp. cremoris Strain T26, Isolated from Mesophilic Undefined Cheese Starter.

Leuconostoc is the main group of heterofermentative bacteria found in mesophilic dairy starters. They grow in close symbiosis with the Lactococcus population and are able to degrade citrate. Here we present a draft genome sequence of Leuconostoc mesenteroides subsp. cremoris strain T26.

Characterization of the gut microbiota in leptin deficient obese mice - Correlation to inflammatory and diabetic parameters.

Gut microbiota have been implicated as a relevant factor in the development of type 2 diabetes mellitus (T2DM), and its diversity might be a cause of variation in animal models of T2DM. In this study, we aimed to characterise the gut microbiota of a T2DM mouse model with a long term vision of being able to target the gut microbiota to reduce the number of animals used in experiments. Male B6.V-Lep(ob)/J mice were characterized according to a number of characteristics related to T2DM, inflammation and gut microbiota. All findings were thereafter correlated to one another in a linear regression model. The total gut microbiota profile correlated to glycated haemoglobin, and high proportions of Prevotellaceae and Lachnospiraceae correlated to impaired or improved glucose intolerance, respectively. In addition, Akkermansia muciniphila disappeared with age as glucose intolerance worsened. A high proportion of regulatory T cells correlated to the gut microbiota and improved glucose tolerance. Furthermore, high levels of IL-10, IL-12 and TNF-α correlated to impaired glucose tolerance, blood glucose or glycated haemoglobin. The findings indicate that gut microbiota may contribute to variation in various disease read-outs in the B6.V-Lep(ob)/J model and considering them in both quality assurance and data evaluation for the B6.V-Lep(ob)/J model may have a reducing impact on the inter-individual variation.

Specificity and stability of the Acromyrmex-Pseudonocardia symbiosis.

The stability of mutualistic interactions is likely to be affected by the genetic diversity of symbionts that compete for the same functional niche. Fungus-growing (attine) ants have multiple complex symbioses and thus provide ample opportunities to address questions of symbiont specificity and diversity. Among the partners are Actinobacteria of the genus Pseudonocardia that are maintained on the ant cuticle to produce antibiotics, primarily against a fungal parasite of the mutualistic gardens. The symbiosis has been assumed to be a hallmark of evolutionary stability, but this notion has been challenged by culturing and sequencing data indicating an unpredictably high diversity. We used 454 pyrosequencing of 16S rRNA to estimate the diversity of the cuticular bacterial community of the leaf-cutting ant Acromyrmex echinatior and other fungus-growing ants from Gamboa, Panama. Both field and laboratory samples of the same colonies were collected, the latter after colonies had been kept under laboratory conditions for up to 10 years. We show that bacterial communities are highly colony-specific and stable over time. The majority of colonies (25/26) had a single dominant Pseudonocardia strain, and only two strains were found in the Gamboa population across 17 years, confirming an earlier study. The microbial community on newly hatched ants consisted almost exclusively of a single strain of Pseudonocardia while other Actinobacteria were identified on older, foraging ants in varying but usually much lower abundances. These findings are consistent with recent theory predicting that mixtures of antibiotic-producing bacteria can remain mutualistic when dominated by a single vertically transmitted and resource-demanding strain.

Metamobilomics--expanding our knowledge on the pool of plasmid encoded traits in natural environments using high-throughput sequencing.

A metamobilome is defined as a metagenome of circular genetic elements within a certain community. Metagenomic analyses of plasmids provide insights into the composition and structure of environmental plasmid communities. It is a promising method that will provide information about the types of plasmids that are present within environmental samples, and will give overviews about occurrences of plasmids as well as accessory genetic elements carried on these plasmids. A metamobilome library was constructed by combining multiple displacement amplification with pyrosequencing. This method provided a fast, efficient and unbiased strategy to investigate the communal gene pool of circular genetic elements (the metamobilome). We compared our wastewater metamobilome library with a wastewater metagenome library, against chromosomes, plasmids, phages and IS element databases, respectively. This showed that very few strictly chromosomal reads were present in our metamobilome library. Furthermore, data analysis showed that our library was strongly enriched for genes encoding plasmid-selfish traits, such as stability and conjugation, and most strikingly several hundred new putative plasmid replicases have been recovered.

Early life treatment with vancomycin propagates Akkermansia muciniphila and reduces diabetes incidence in the NOD mouse.

AIMS/HYPOTHESIS: Increasing evidence suggests that environmental factors changing the normal colonisation pattern in the gut strongly influence the risk of developing autoimmune diabetes. The aim of this study was to investigate, both during infancy and adulthood, whether treatment with vancomycin, a glycopeptide antibiotic specifically directed against Gram-positive bacteria, could influence immune homeostasis and the development of diabetic symptoms in the NOD mouse model for diabetes. METHODS: Accordingly, one group of mice received vancomycin from birth until weaning (day 28), while another group received vancomycin from 8 weeks of age until onset of diabetes. Pyrosequencing of the gut microbiota and flow cytometry of intestinal immune cells was used to investigate the effect of vancomycin treatment. RESULTS: At the end of the study, the cumulative diabetes incidence was found to be significantly lower for the neonatally treated group compared with the untreated group, whereas the insulitis score and blood glucose levels were significantly lower for the mice treated as adults compared with the other groups. Mucosal inflammation was investigated by intracellular cytokine staining of the small intestinal lymphocytes, which displayed an increase in cluster of differentiation (CD)4(+) T cells producing pro-inflammatory cytokines in the neonatally treated mice. Furthermore, bacteriological examination of the gut microbiota composition by pyrosequencing revealed that vancomycin depleted many major genera of Gram-positive and Gram-negative microbes while, interestingly, one single species, Akkermansia muciniphila, became dominant. CONCLUSIONS/INTERPRETATION: The early postnatal period is a critical time for microbial protection from type 1 diabetes and it is suggested that the mucolytic bacterium A. muciniphila plays a protective role in autoimmune diabetes development, particularly during infancy.

Genetic labelling and application of the isoproturon-mineralizing Sphingomonas sp. strain SRS2 in soil and rhizosphere.

AIMS: To construct a luxAB-labelled Sphingomonas sp. strain SRS2 maintaining the ability to mineralize the herbicide isoproturon and usable for monitoring the survival and distribution of strain SRS2 on plant roots in laboratory systems. METHODS AND RESULTS: We inserted the mini-Tn5-luxAB marker into strain SRS2 using conjugational mating. In the transconjugant mutants luciferase was produced in varying levels. The mutants showed significant differences in their ability to degrade isoproturon. One luxAB-labelled mutant maintained the ability to mineralize isoproturon and was therefore selected for monitoring colonization of barley roots. CONCLUSIONS: We successfully constructed a genetically labelled isoproturon-mineralizing-strain SRS2 and demonstrated its ability to survive in soil and its colonization of rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: The construction of a luxAB-labelled strain SRS2 maintaining the degradative ability, provides a powerful tool for ecological studies serving as the basis for evaluating SRS2 as a bioremediation agent.

Presence of N-acyl homoserine lactones in soil detected by a whole-cell biosensor and flow cytometry.

Quorum sensing enables bacteria to regulate expression of certain genes according to population density. N-acyl homoserine lactone (AHL)-based quorum sensing is known to be widespread among gram-negative bacteria. Several bacterial whole-cell biosensors for AHL detection have been developed and some were used in in situ studies of AHL production. From these studies our knowledge of the significance of quorum sensing in various environments has been improved. However, very little is known about production of AHLs in soil environments. In the present study, an approach for detecting AHL production in bulk soil was developed. A whole-cell biosensor based on the regulatory region of the lux-operon from Vibrio fischeri fused to gfp was constructed, resulting in a luxR-PluxI-gfpmut3*-fusion in the high copy plasmid, pAHL-GFP. Escherichia coli MC4100 harboring pAHL-GFP responded to the AHL-compound N-octanoyl homoserine lactone (OHL) by expressing green fluorescence. In situ application of E. coli MC4100/pAHL-GFP was tested by adding OHL in different concentrations to sterile soil microcosms. E. coli MC4100/pAHL-GFP were incubated in the soil microcosms and extracted by an improved Nycodenz-extraction method optimized for flow cytometry. The presence of induced cells was then verified by single-cell analysis by flow cytometry. OHL concentrations between 0.5 and 50 nmol per g soil were detected. When introducing the AHL-producing Serratia liquefaciens to soil microcosms, expression of green fluorescent protein was induced in E. coli MC4100/pAHL-GFP. Thereby, the ability of this strain to detect excretion of AHLs by S. liquefaciens in sterile soil was shown. The use of an improved extraction method and a whole-cell biosensor combined with flow cytometry analysis proved to be promising tools in future studies of AHL production by microbial populations in soil environments.

Recognition of nucleotide G745 in 23 S ribosomal RNA by the rrmA methyltransferase.

Methylation of the N1 position of nucleotide G745 in hairpin 35 of Escherichia coli 23 S ribosomal RNA (rRNA) is mediated by the methyltransferase enzyme RrmA. Lack of G745 methylation results in reduced rates of protein synthesis and growth. Addition of recombinant plasmid-encoded rrmA to an rrmA-deficient strain remedies these defects. Recombinant RrmA was purified and shown to retain its activity and specificity for 23 S rRNA in vitro. The recombinant enzyme was used to define the structures in the rRNA that are necessary for the methyltransferase reaction. Progressive truncation of the rRNA substrate shows that structures in stem-loops 33, 34 and 35 are required for methylation by RrmA. Multiple contacts between nucleotides in these stem-loops and RrmA were confirmed in footprinting experiments. No other RrmA contact was evident elsewhere in the rRNA. The RrmA contact sites on the rRNA are inaccessible in ribosomal particles and, consistent with this, 50 S subunits or 70 S ribosomes are not substrates for RrmA methylation. RrmA resembles the homologous methyltransferase TlrB (specific for nucleotide G748) as well as the Erm methyltransferases (nucleotide A2058), in that all these enzymes methylate their target nucleotides only in the free RNA. After assembly of the 50 S subunit, nucleotides G745, G748 and A2058 come to lie in close proximity lining the peptide exit channel at the site where macrolide, lincosamide and streptogramin B antibiotics bind.