Mitsuokella Jalaludinii Research Articles

Fecal Microbiota Characteristics in Constipation-Predominant and Mixed-Type Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS) is a common condition that affects the lifestyle of patients. It is associated with significant changes in the composition of the gut microbiome, but the underlying microbial mechanisms remain to be fully understood. We study the fecal microbiome of patients with constipation-predominant IBS (IBS-C) and mixed-type IBS (IBS-M). We sequenced the V3 region of the 16S rRNA on the Ion Torrent PGM sequencing platform to study the microbiome. In the patients with IBS-C and IBS-M, an increase in alpha diversity was found, compared to the healthy group, and differences in beta diversity were also noted. At the phylum level, both IBS subtypes showed an increase in the Firmicutes/Bacteroidetes ratio, as well as an increase in the abundance of Actinobacteria and Verrucomicrobiota. Changes in some types of bacteria were characteristic of only one of the IBS subtypes, while no statistically significant differences in the composition of the microbiome were detected between IBS-C and IBS-M. This study was the first to demonstrate the association of Turicibacter sanguinis, Mitsuokella jalaludinii, Erysipelotrichaceae UCG-003, Senegalimassilia anaerobia, Corynebacterium jeikeium, Bacteroides faecichinchillae, Leuconostoc carnosum, and Parabacteroides merdae with IBS subtypes.

177 Modulation of fecal microbiome by phytogenics, a postbiotic, and mix of organic acids with essential oils in weaned pigs subjected to F18 Escherichia coli challenge

Abstract This study evaluated the effects of bioactive feed ingredients on fecal microbiome of weaned pigs subjected to F18 enterotoxigenic Escherichia coli (ETEC) challenge. Two blocks of weaned pigs [n = 72; Yorkshire/Landrace/Duroc; initial body weight (BW) = 6.62 ± 1.37 kg] were balanced by gender and litter and randomly allocated to one of the six treatments with 12 pens/block and 6 pigs/pen. Treatments included: 1) basal diet (NC), 2) NC + 0.1% phytogenic (Fresta Protect, Delacon, Austria; PFP), 3) NC + 0.2% postbiotic (Dia-V Nursery, Diamond V, IA; PB), 4) NC + 0.2% new phytogenic prototype (Delacon, Austria; NPP), 5) NC + 0.2% organic acid and essential oil blend (OA+EO), and 6) NC + 0.1% phytogenic (Fresta Protect) + 0.2% postbiotic (Dia-V Nursery; PFP+PB). The trial lasted 15 d, with a 7-d pre-challenge and 8-d post-challenge periods. Pigs were orally inoculated with 5 x 1010 CFU/mL of F18 ETEC (O147:H2, LT:STb:STa:Stx2:F18) on d 8 and 9 of study after 5-h fasting. Fecal swabs were collected before challenge (d 8) and 3- and 8-d post-challenge (d 10 and 15 of study) and subjected to shotgun metagenomics. Diarrhea score and E. coli shedding (CFU/mL) were monitored daily and analyzed using a multi-level mixed effect ordinal logistic regression with block and pen as random effects. Microbiome data were center-log-ratio transformed and analyzed for diversity, abundance, and interaction networks. At 3-d post-challenge, the OA+EO, PFP+PB and NPP supplemented pigs had reduced (P < 0.001) E. coli shedding compared with NC. Diarrhea score tended to be less (P = 0.054) in PFP+PB fed pigs 5-d post-challenge and was less (P = 0.02) in PFP+PB, OA+EO, and NPP 6-d post-challenge than NC. Alpha-diversity declined (P < 0.01) 3-d post-challenge with most reduction in PFP and OA+EO but recovered 8-d post-challenge except for PFP and OA+EO (P < 0.01). At the baseline and 8-d post-challenge, the largest number of differentially abundant taxa (P < 0.05) were observed in PFP+PB compared with NC. The PFP+PB reduced the abundances of Escherichia.sp000208585 and Campylobacter.P585 which were negatively correlated (P < 0.01) with average daily gain (ADG) and BW, and positively correlated (P < 0.01) with diarrhea score and E. coli shedding. The PFP+PB increased beneficial species including Prevotella.sp900543585, Mitsuokella jalaludinii, and Faecalibacterium prausnitzii with the first two positively correlated (P < 0.01) with ADG and BW, and negatively correlated (P < 0.01) with diarrhea score and E. coli shedding. By d-8 post-challenge, PFP+PB microbial network had the highest degree of negative connections suggestive of a more competitive microbial ecosystem against opportunistic microorganisms compared with NC. In conclusion, co-supplementation of Fresta Protect phytogenic and postbiotic promoted a microbiome with greater beneficial and reduced detrimental bacteria resulting in low diarrhea score post-challenge.

254 Intestinal Oxidative Damages By F18+Escherichia Coli and Its Amelioration with an Antibacterial Bacitracin Fed to Nursery Pigs

Abstract This study investigated the intestinal oxidative damages caused by F18+Escherichia coli and its amelioration with an antibacterial bacitracin fed to nursery pigs. Thirty-six weaned pigs (6.31 ± 0.08 kg BW) were allotted in RCBD. Treatments were: NC, not challenged/not treated; PC, challenged (F18+E. coli at 5.2 × 109 CFU)/not treated; AGP, challenged (F18+E. coli at 5.2 × 109 CFU)/treated with bacitracin (30 g/t). The F18+E. coli was orally inoculated to pigs on challenged treatments at d 7. The unchallenged pigs received an oral dose of sterile saline solution. The fecal score was recorded daily. Body weight and feed intake were recorded weekly. On d 14 and 28, fecal and blood samples were collected. On d 28, all pigs were euthanized to collect intestinal samples. F18+E. coli counting, microbiota composition, and intestinal health variables were analyzed. Data were analyzed using the mixed procedure on SAS 9.4. Overall, PC reduced (P < 0.05) ADG (353 to 282 g), G:F (0.71 to 0.55), villus height (527 to 394 µm), and VH:CD (2.22 to 1.58), whereas AGP increased (P < 0.05) ADG (298 to 344 g), and G:F (0.55 to 0.61). PC increased (P < 0.05) fecal score from d 7 to 14 (3.0 to 4.6), from 14 to 21 (2.8 to 3.9), and from d 21 to 28 (2.5 to 3.2); increased F18+E. coli (5.9 to 7.5 log DNA) in feces at d 14, and protein carbonyl (2.15 to 3.61 nmol/mg protein) in jejunal mucosa. AGP reduced (P < 0.05) fecal score from d 7 to 14 (4.6 to 4.0) and from d 14 to 21 (3.9 to 3.4) and reduced F18+E. coli (4.3 to 3.9 log DNA) in jejunal mucosa. PC reduced (P < 0.05) Prevotella stercorea (5.1 to 1.5%) population in jejunal mucosa, whereas AGP increased (P < 0.05) Phascolarctobacterium succinatutens (3.0 to 10.0%) and reduced (P < 0.05) Mitsuokella jalaludinii (8.7 to 2.0%) populations in feces. Collectively, F18+E. coli challenge increased fecal score and disrupted the microbiota composition, harming intestinal health by increasing oxidative stress and damaging the intestinal epithelium, ultimately impairing growth performance. Dietary bacitracin reduced oxidative damages caused by F18+ E. coli thereby protecting intestinal health by reducing F18+E. coli population in jejunal mucosa and fecal score, consequently improving the growth performance of nursery pigs.

Intestinal Damages by F18+Escherichia coli and Its Amelioration with an Antibacterial Bacitracin Fed to Nursery Pigs.

This study investigated intestinal oxidative damage caused by F18+Escherichia coli and its amelioration with antibacterial bacitracin fed to nursery pigs. Thirty-six weaned pigs (6.31 ± 0.08 kg BW) were allotted in a randomized complete block design. Treatments were: NC, not challenged/not treated; PC, challenged (F18+E. coli at 5.2 × 109 CFU)/not treated; AGP challenged (F18+E. coli at 5.2 × 109 CFU)/treated with bacitracin (30 g/t). Overall, PC reduced (p < 0.05) average daily gain (ADG), gain to feed ratio (G:F), villus height, and villus height to crypt depth ratio (VH:CD), whereas AGP increased (p < 0.05) ADG, and G:F. PC increased (p < 0.05) fecal score, F18+E. coli in feces, and protein carbonyl in jejunal mucosa. AGP reduced (p < 0.05) fecal score and F18+E. coli in jejunal mucosa. PC reduced (p < 0.05) Prevotella stercorea populations in jejunal mucosa, whereas AGP increased (p < 0.05) Phascolarctobacterium succinatutens and reduced (p < 0.05) Mitsuokella jalaludinii populations in feces. Collectively, F18+E. coli challenge increased fecal score and disrupted the microbiota composition, harming intestinal health by increasing oxidative stress, and damaging the intestinal epithelium, ultimately impairing growth performance. Dietary bacitracin reduced reduced F18+E. coli populations and the oxidative damages they cause, thereby improving intestinal health and the growth performance of nursery pigs.

Dietary wheat and reduced methane yield are linked to rumen microbiome changes in dairy cows.

Fermentation of pasture grasses and grains in the rumen of dairy cows and other ruminants produces methane as a by-product, wasting energy and contributing to the atmospheric load of greenhouse gasses. Many feeding trials in farmed ruminants have tested the impact of dietary components on feed efficiency, productivity and methane yield (MeY). Such diets remodel the rumen microbiome, altering bacterial, archaeal, fungal and protozoan populations, with an altered fermentation outcome. In dairy cows, some dietary grains can reduce enteric methane production. This is especially true of wheat, in comparison to corn or barley. Using a feeding trial of cows fed rolled wheat, corn or barley grain, in combination with hay and canola, we identified wheat-associated changes in the ruminal microbiome. Ruminal methane production, pH and VFA concentration data together with 16S rRNA gene amplicon sequences were used to compare ruminal bacterial and archaeal populations across diets. Differential abundance analysis of clustered sequences (OTU) identified members of the bacterial families Lachnospiraceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Selenomonadaceae, Anaerovoracaceae and Fibrobacteraceae having a strong preference for growth in wheat-fed cows. Within the methanogenic archaea, (at >99% 16S rRNA sequence identity) the growth of Methanobrevibacter millerae was favoured by the non-wheat diets, while Methanobrevibacter olleyae was unaffected. From the wheat-preferring bacteria, correlation analysis found OTU strongly linked to reduced MeY, reduced pH and raised propionic acid levels. OTU from the genera Shuttleworthia and Prevotella_7 and especially Selenomonadaceae had high anti-methane correlations. An OTU likely representing (100% sequence identity) the fumarate-reducing, hydrogen-utilising, rumen bacterium Mitsuokella jalaludinii, had an especially high negative correlation coefficient (-0.83) versus MeY and moderate correlation (-0.6) with rumen pH, strongly suggesting much of the MeY suppression is due to reduced hydrogen availablity. Other OTU, representing as yet unknown species from the Selenomonadaceae family and the genera Prevotella_7, Fibrobacter and Syntrophococcus also had high to moderate negative MeY correlations, but low correlation with pH. These latter likely represent bacterial species able to reduce MeY without causing greater ruminal acidity, making them excellent candidates, provided they can be isolated, for development as anti-methane probiotics.

Development of Electrochemical DNA Biosensor for Equine Hindgut Acidosis Detection.

The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse’s feeding regime is excessively carbohydrate rich. This drop in pH below six causes hindgut acidosis and may lead to laminitis. Lactic acid-producing bacteria Streptococcus equinus and Mitsuokella jalaludinii have been found to produce high amounts of L-lactate and D-lactate, respectively. Early detection of increased levels of these bacteria could allow the horse owner to tailor the horse’s diet to avoid hindgut acidosis and subsequent laminitis. Therefore, 16s ribosomal ribonucleic acid (rRNA) sequences were identified and modified to obtain target single stranded deoxyribonucleic acid (DNA) from these bacteria. Complementary single stranded DNAs were designed from the modified target sequences to form capture probes. Binding between capture probe and target single stranded deoxyribonucleic acid (ssDNA) in solution has been studied by gel electrophoresis. Among pairs of different capture probes and target single stranded DNA, hybridization of Streptococcus equinus capture probe 1 (SECP1) and Streptococcus equinus target 1 (SET1) was portrayed as gel electrophoresis. Adsorptive stripping voltammetry was utilized to study the binding of thiol modified SECP1 over gold on glass substrates and these studies showed a consistent binding signal of thiol modified SECP1 and their hybridization with SET1 over the gold working electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to examine the binding of thiol modified SECP1 on the gold working electrode and hybridization of thiol modified SECP1 with the target single stranded DNA. Both demonstrated the gold working electrode surface was modified with a capture probe layer and hybridization of the thiol bound ssDNA probe with target DNA was indicated. Therefore, the proposed electrochemical biosensor has the potential to be used for the detection of the non-synthetic bacterial DNA target responsible for equine hindgut acidosis.

Mitsuokella Jalaludinii Supplementation Improved Nutrient Utilization of Broilers Fed Low-Available Phosphorus Diet

ABSTRACT Phytase enzyme is supplemented to poultry feed to improve phosphorus (P) availability. Mitsuokella jalaludinii, bacteria isolated from the rumen of cattle, has been reported as a cheaper alternative source of phytase. As much nutrients are trapped within the phytate complex, we hypothesized that the supplementation of M. jalaludinii phytase to poultry feed would enhance nutrient utilization by poultry. In the current study, the efficacy of freeze-dried M. jalaludinii cells (Mj) as feed supplement for broilers fed low-available phosphorus (low-aP) diet was evaluated. Day-old male Cobb raised in battery cages were assigned to three treatment groups [normal-available phosphorus diet with heat-deactivated Mj (DMj); low-aP diet with DMj; and low-aP diet with Mj], each consisting of four replicates (10 birds per replicate) for a 3-weeks feeding period. Feed intake was recorded daily from day 1-21, whereas broilers were weighted at day 1, 7, 14, and 21. Total excreta were collected at day 11-13 and 18-20. At day 21, twelve broilers from each treatment group were slaughtered to collect plasma and tibia. The results showed that Mj significantly enhanced broilers’ live weight and feed conversion ratio compared to the control groups (p<0.05). Supplementation with Mj have also enhanced the level of P, Ca, Mn, Cu, and Zn in the sera; and Ca and Mn in the tibia at day 18-20 sampling period (p<0.05). As Mj supplementation can enhance nutrient utilization particularly in broilers fed with low-aP diet, it could provide the market with another option in improving broilers’ growth rate at a lower cost.

Effects of supplementing freeze-dried Mitsuokella jalaludinii phytase on the growth performance and gut microbial diversity of broiler chickens.

Inclusion of phytase in animal feedstuff is a common practice to enhance nutrients availability. However, little is known about the effects of phytase supplementation on the microbial ecology of the gastrointestinal tract. In this study, freeze-dried Mitsuokella jalaludinii phytase (MJ) was evaluated in a feeding trial with broilers fed a low available phosphorus (aP) diet. A total of 180 male broiler chicks (day-old Cobb) were assigned into three dietary treatments: Control fed with 0.4% (w/w) of available phosphorus (aP); Group T1 fed low aP [0.2% (w/w)] supplemented with MJ; and T2 fed low aP and deactivated MJ. The source of readily available P, dicalcium phosphate (DCP), was removed from low aP diet, whereby additional limestone was provided to replace the amount of Ca normally found in DCP. For each treatment, 4 replicate penswere used, where each pen consisted of 15 animals. The animals' energy intake and caecal bacterial community were monitored weekly for up to 3weeks. The apparent metabolizable energy (AME) and apparent digestibility of dry matter (ADDM) of broilers fed with different diets were determined. In addition, the caecal microbial diversities of broilers were assessed using high-throughput next-generation sequencing targeting the V3-V4 region of bacterial 16S rRNA. The results showed that broilers fed with T1 diet have better feed conversion ratio (FCR) when compared to the Control (p<.05) and T2 diets (p<.05), demonstrating the efficiency of MJ as a supplement to low aP diet. Nevertheless, MJ did not significantly affect the microbial population and diversity in broilers' caeca, which mainly consists of members from Bacteroidetes, Firmicutes, and Proteobacteria. Regardless, significant variations in the caecal bacterial composition were observed over time, probably due to succession as the broilers aged. This is the first reported study on the effect of MJ on the microbial diversity of broiler's caeca.

Preservation of phytase enzyme produced by anaerobic rumen bacteria, Mitsuokella jalaludinii

Poultry feed consists of feed ingredients like soybean meal and corn, which contain high levels of phytate that is poorly utilised especially by the monogastric animals that lack of phytase. Hence, phytase has been extensively applied as a feed supplement in poultry production due to the efficiency of this enzyme in improving phosphorous (P) availability, thus reducing P excretion to the environment as well as reducing the feed cost by reducing inorganic P supplementation. Mitsuokella jalaludinii, an obligate anaerobe, Gram-negative rumen bacterium, produces high phytase activity. Birds supplemented with bacterial preparation of M. jalaludinii showed comparable performance to that of commercial phytase. However, the anaerobic nature of this bacterium renders difficulty in the use of live cells as feed supplement in commercial poultry production. Therefore, this study was conducted to determine a suitable method to preserve phytase activity of M. jalaludinii regardless of cells viability. Mitsuokella jalaludinii was grown in MF medium under anaerobic condition and the cells were subjected to various treatments to preserve the enzyme, including bead beating, compressed air, moist heat, dry heat and freeze-drying under aerobic condition. The results showed that the total number of viable cells were significantly (p&lt;0.05) reduced when the cells were subjected to bead beating, whereas no viable cells were detected for compressed air, moist heat, dry heat and freeze-drying. Bead beating, compressed air, moist heat and dry heat treatments resulted in the reduction of phytase activity. However, only freeze-drying method was able to preserve high level of phytase activity significantly (p&lt;0.05).

Cloning of a novel phytase from an anaerobic rumen bacterium, Mitsuokella jalaludinii, and its expression in Escherichia coli

The full length phytase gene of Mitsuokella jalaludinii was successfully cloned and was found to be 1 047 bp in length, with 348 amino acids, and was designated as PHY7 phytase gene. A comparison of the sequence of PHY7 phytase gene of M. jalaludinii with various microbial phytase gene sequences showed that it was not similar to those from other bacteria except Selenomonas ruminatium, thus suggesting that they may both express a new class of phytase. The PHY7 phytase gene was subsequently subcloned into bacterial expression vector, pET32a, for expression in Escherichia coli strain Rosetta-gami. Expression of the recombinant phytase gene was optimised and characterised. The recombinant phytase was estimated to be approximately 55 kDa by SDS-PAGE analysis. The recombinant phytase exhibited optimum activity at 55°C, pH 4.5 and showed good pH stability from pH 3.5 to 5.5 (>78% relative activity). Metal ions such as Ca2+, Mg2+, and K+ were found to exert significant stimulatory effect on the recombinant phytase activity while Cu2+, Fe3+, and Zn2+ greatly inhibited the enzyme activity. The recombinant phytase showed moderate resistance to trypsin proteolysis, but susceptible to pepsin proteolysis. The results of the study showed that several characteristics of recombinant phytase were slightly different from the native enzyme. Unfavourable characteristics such as reduced pH stability and metal ion effects should be taken into consideration during feed enzyme formulation.

Shifts in microbiota species and fermentation products in a dietary model enriched in fat and sucrose

The gastrointestinal tract harbours a 'superorganism' called the gut microbiota, which is known to play a crucial role in the onset and development of diverse diseases. This internal ecosystem, far from being a static environment, can be manipulated by diet and dietary components. Feeding animals with high-fat sucrose (HFS) diets entails diet-induced obesity, a model which is usually used in research to mimic the obese phenotype of Western societies. The aim of the present study was to identify gut microbiota dysbiosis and associated metabolic changes produced in male Wistar rats fed a HFS diet for 6 weeks and compare it with the basal microbial composition. For this purpose, DNA extracted from faeces at baseline and after treatment was analysed by amplification of the V4-V6 region of the 16S ribosomal DNA (rDNA) gene using 454 pyrosequencing. Short-chain fatty acids, i.e. acetate, propionate and butyrate, were also evaluated by gas chromatography-mass spectrometry. At the end of the treatment, gut microbiota composition significantly differed at phylum level (Firmicutes, Bacteroidetes and Proteobacteria) and class level (Erisypelotrichi, Deltaproteobacteria, Bacteroidia and Bacilli). Interestingly, the class Clostridia showed a significant decrease after HFS diet treatment, which correlated with visceral adipose tissue, and is likely mediated by dietary carbohydrates. Of particular interest, Clostridium cluster XIVa species were significantly reduced and changes were identified in the relative abundance of other specific bacterial species (Mitsuokella jalaludinii, Eubacterium ventriosum, Clostridium sp. FCB90-3, Prevotella nanceiensis, Clostridium fusiformis, Clostridium sp. BNL1100 and Eubacterium cylindroides) that, in some cases, showed opposite trends to their relative families. These results highlight the relevance of characterising gut microbial population differences at species level and contribute to understand the plausible link between diet and specific gut bacterial species that are able to influence the inflammatory status, intestinal barrier function and obesity development.

Characterization of rumen bacterial strains isolated from enrichments of rumen content in the presence of propolis.

Propolis presents many biological properties, including antibacterial activities, and has been proposed as an additive in ruminant nutrition. Twenty bacterial strains, previously isolated from enrichments of Brazilian cow rumen contents in the presence of different propolis extracts (LLOS), were characterized using phenotyping and 16S rRNA identification. Seven strains were assigned to Streptococcus sp., most likely S. bovis, and were all degrading starch. One amylolytic lactate-utilizing strain of Selenomonas ruminantium was also found. Two strains of Clostridium bifermentans were identified and showed proteolytic activity. Two strains were assigned to Mitsuokella jalaludinii and were saccharolytic. One strain belonged to a Bacillus species and seven strains were affiliated with Escherichia coli. All of the 20 strains were able to use many sugars, but none of them were able to degrade the polysaccharides carboxymethylcellulose and xylans. The effect of three propolis extracts (LLOS B1, C1 and C3) was tested on the in vitro growth of four representative isolates of S. bovis, E. coli, M. jalaludinii and C. bifermentans. The growth of S. bovis, E. coli and M. jalaludinii was not affected by the three propolis extracts at 1 mg ml(-1). C. bifermentans growth was completely inhibited at this LLOS concentration, but this bacterium was partially resistant at lower concentrations. LLOS C3, with the lower concentration of phenolic compounds, was a little less inhibitory than B1 and C1 on this strain.

Effect of fumarate reducing bacteria on in vitro rumen fermentation, methane mitigation and microbial diversity

The metabolic pathways involved in hydrogen (H(2)) production, utilization and the activity of methanogens are the important factors that should be considered in controlling methane (CH(4)) emissions by ruminants. H(2) as one of the major substrate for CH(4) production is therefore should be controlled. One of the strategies on reducing CH(4) is through the use of hydrogenotrophic microorganisms such as fumarate reducing bacteria. This study determined the effect of fumarate reducing bacteria, Mitsuokella jalaludinii, supplementation on in vitro rumen fermentation, CH(4) production, diversity and quantity. M. jalaludinii significantly reduced CH(4) at 48 and 72 h of incubation and significantly increased succinate at 24 h. Although not significantly different, propionate was found to be highest in treatment containing M. jalaludinii at 12 and 48 h of incubation. These results suggest that supplementation of fumarate reducing bacteria to ruminal fermentation reduces CH(4) production and quantity, increases succinate and changes the rumen microbial diversity.

Characterization, metabolites and gas formation of fumarate reducing bacteria isolated from Korean native goat (Capra hircus coreanae)

Fumarate reducing bacteria, able to convert fumarate to succinate, are possible to use for the methane reduction in rumen because they can compete for H(2) with methanogens. In this, we isolated fumarate reducing bacteria from a rumen of Korean native goat and characterized their molecular properties [fumarate reductase A gene (frdA)], fumarate reductase activities, and productions of volatile fatty acids and gas. Eight fumarate reducing bacteria belonging to Firmicutes were isolated from rumen fluid samples of slaughtered Korean black goats and characterized their phylogenetic positions based on 16S rRNA gene sequences. PCR based analyses showed that only one strain, closely related to Mitsuokella jalaludinii, harbored frdA. The growths of M. jalaludinii and Veillonella parvula strains were tested for different media. Interestingly, M. jalaludinii grew very well in the presence of hydrogen alone, while V. parvula grew well in response of fumarate and fumarate plus hydrogen. M. jalaludinii produced higher levels of lactate (P≤0.05) than did V. parvula. Additionally, M. jalaludinii produced acetate, but not butyrate, whereas V. parvula produced butyrate, not acetate. The fumarate reductase activities of M. jalaludinii and V. parvula were 16.8 ± 0.34 and 16.9 ± 1.21 mmol NADH oxidized/min/mg of cellular N, respectively. In conclusion, this showed that M. jalaludinii can be used as an efficient methane reducing agent in rumen.

Mitsuokella jalaludinii inhibits growth of Salmonella enterica serovar Typhimurium

Salmonella continues to be a significant human health threat, and the objective of this study was to identify microorganisms with the potential to improve porcine food-safety through their antagonism of Salmonella. Anaerobic culture supernatants of 973 bacterial isolates from the gastrointestinal tract and feces of swine were screened for their capacity to inhibit the growth of Salmonella enterica serovar Typhimurium. Growth inhibition of 1000-fold or greater was observed from 16 isolates, and 16S rRNA sequencing identified the isolates as members of the genera Mitsuokella, Escherichia/Shigella, Anaerovibrio, Selenomonas, and Streptococcus. Four isolates were identified as Mitsuokella jalaludinii, and the mechanism of Salmonella Typhimurium growth inhibition by M. jalaludinii was further investigated. M. jalaludinii stationary phase culture supernatants were observed to significantly inhibit growth, and featured the production of lactic, succinic, and acetic acids. Aerobic and anaerobic S. Typhimurium growth was restored when the pH of the culture supernatants (pH 4.6) was increased to pH 6.8. However, S. Typhimurium growth in fermentation acid-free media was the same at pH 4.6 and pH 6.8 – indicating a synergistic effect between fermentation acid production and low pH as the cause of S. Typhimurium growth inhibition. Furthermore, exposure of S. Typhimurium to M. jalaludinii culture supernatants inhibited Salmonella invasion of HEp-2 cells by 10-fold. The results identify M. jalaludinii as a possible inhibitor of Salmonella growth and invasion in swine, and thus a potential probiotic capable of improving food safety.

English

The phytase from&nbsp;Mitsuokella jalaludinii, a novel phytase-producing rumen bacterium, was purified 120-fold to near homogeneity and characterized. The phytase was completely cell-associated and about half of the enzyme activity was released when the bacterial cells were incubated with 1.5 mol/l KCl solution for 8 h. The optimum pH for phytase activity was in the range of 4.0 to 5.0 and the optimum temperature was 55 to 60&deg;C. The phytase was stable at pH 4.0 to 7.0. It was highly specific to sodium phytate as the substrate, strongly inhibited by Cu2+, Zn2+, Fe2+and Fe3+, significantly stimulated by Ba2+&nbsp;and slightly stimulated by Mn2+&nbsp;and Ca2+. The metal ions chelating agents, namely trisodium citrate, potassium sodium tartrate and EDTA, did not show any inhibitory effect on the phytase activity of&nbsp;M. jalaludinii.&nbsp;The phytase was also not inhibited by sulfhydryl inhibitor, 2-mercaptoethanol, and a carboxyl inhibitor, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). &nbsp; Key words:&nbsp;Mitsuokella jalaludinii, bacterial phytase, rumen bacteria.

Effects of freeze‐dried Mitsuokella jalaludinii culture and Natuphos® phytase supplementation on the performance and nutrient utilisation of broiler chickens

Phytate-bound phosphorus (P) in poultry diets is poorly available to chickens. Hence exogenous phytase is often added to their diets. Mitsuokella jalaludinii is a rumen bacterial species that produces high phytase activity. In this study the effects of freeze-dried active M. jalaludinii culture (FD-AMJC) and Natuphos(®) phytase (phytase N) supplementations on the growth performance and nutrient utilisation of broiler chickens fed a low-available P (aP) diet were evaluated. Supplementation of FD-AMJC or phytase N to the low-aP diet improved the feed intake, feed conversion rate, body weight gain, dry matter (DM) digestibility and P, Ca and Mn retention, increased the tibia bone ash content, Ca and P concentrations in tibia DM and P and Zn concentrations in plasma and reduced the P excretion of broiler chickens. However, the feed conversion rate, P and Ca retention, DM digestibility and reduction of P excretion were better with FD-AMJC than phytase N supplementation. Supplementation of FD-AMJC to the low-aP diet also improved the apparent metabolisable energy value of the diet, Cu and Zn retention and crude protein digestibility, but phytase N supplementation did not. FD-AMJC supplementation was more efficient in improving nutrient utilisation and reducing P excretion in chickens than phytase N supplementation.

Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis

Carbohydrate-induced laminitis in horses is characterized by marked changes in the composition of the hindgut microbiota, from a predominantly Gram-negative population to one dominated by Gram-positive bacteria. The objective of this study was to monitor changes in the relative abundance of selected hindgut bacteria that have previously been implicated in the pathophysiology of equine laminitis using fluorescence in situ hybridization (FISH). Caecal cannulae were surgically implanted in five Standardbred horses and laminitis induced by oral administration of a bolus dose of oligofructose. Caecal fluid and faecal specimens were collected over a 48 h period at 2 to 4 h intervals post-oligofructose administration and subjected to FISH using probes specific for nine bacterial groups to determine changes in their relative abundance compared with total bacteria hybridizing to the generic EUBMIX probe. Additionally, hoof biopsies were taken over the course of the experiment at 6 h intervals and evaluated for histopathological changes consistent with laminitis, allowing changes in hindgut microbiota to be correlated with the onset of lesions in the foot. Of the microorganisms specifically targeted, streptococci of the Streptococcus bovis/equinus complex were the only bacteria that consistently proliferated in both caecal fluid and faeces immediately before the onset of histological signs of laminitis. Furthermore, lactobacilli, Enterobacteriaceae, Allisonella histaminiformans, enterococci, Bacteroides fragilis, Mitsuokella jalaludinii and Clostridium difficile did not establish significant populations in the hindgut before the onset of equine laminitis.

The genetic diversity of lactic acid producing bacteria in the equine gastrointestinal tract

Seventy-two lactic acid producing bacterial isolates (excluding streptococci) were cultured from the gastrointestinal tract of six horses. Two of the horses were orally dosed with raftilose to induce lactic acidosis and laminitis while the remaining four were maintained on a roughage diet. Near complete 16S rDNA was amplified by PCR from the genomic DNA of each isolate. Following RFLP analysis with the restriction enzymes MboI, HhaI and HinfI, the PCR products from the 18 isolates that produced l- and/or d-lactate were subsequently cloned and sequenced. DNA sequence analysis indicated that the majority of the isolates were closely related to species within the genus Lactobacillus, including Lactobacillus salivarius, Lactobacillus mucosae and Lactobacillus delbrueckii. Four isolates were closely related to Mitsuokella jalaludinii. Lactic acid producing bacteria (LAB) from the equine gastrointestinal tract was dominated by representatives from the genus Lactobacillus, but also included d-lactate-producing bacteria closely related to M. jalaludinii. Identification and characterization of LAB from the equine gastrointestinal tract should contribute to our understanding and management of fermentative acidosis, ulceration of the stomach and laminitis.

Efficacy of supplementation of a phytase-producing bacterial culture on the performance and nutrient use of broiler chickens fed corn-soybean meal diets

We evaluated the efficacy of supplementation of active Mitsuokella jalaludinii culture (AMJC) on the growth performance, nutrient use, and mineral concentrations in tibia bone and plasma of broiler chickens fed corn-soybean meal diets. Dietary treatments included low-nonphytate P (NPP) feed (containing 0.24% and 0.232% NPP for chicks from 1 to 21 and 22 to 42 d of age, respectively), low-NPP feed added with different levels of AMJC (equivalent to 250, 500, 750, and 1,000 U phytase/kg of feed), and normal-NPP feed (containing 0.46 and 0.354% NPP for chicks from 1 to 21 and 22 to 42 d of age, respectively). Supplementation of AMJC to low-NPP feed increased (P < 0.05) weight gain and feed intake and decreased (P < 0.05) feed:gain ratio of chickens during the whole experiment (Days 1 to 42). Supplementation of AMJC increased (P < 0.05) the AME value, digestibility of DM and CP, and retention of P, Ca, and Cu. Mn retention in broilers was only increased (P < 0.05) by AMJC supplementation from 18 to 20 d of age, and Zn retention was improved (P < 0.05) only at a high level of AMJC (equivalent to 1,000 U phytase/kg of feed) supplementation. Chicks fed low-NPP feed added with AMJC had similar tibia ash percentages as those fed the normal-NPP diet. Generally, supplementing AMJC to low-NPP feed increased (P < 0.05) Ca, decreased significantly (P < 0.05) Mn and Cu, but did not affect Zn and P concentrations in tibia ash. Supplementing AMJC also increased (P < 0.05) plasma P but had no effect on plasma Ca or Mn. Plasma Zn concentration was increased only when a high level of AMJC (equivalent to 1,000 U phytase/kg of feed) was used. In conclusion, AMJC supplementation to low-NPP feed improved growth performance; AME value; digestibility of CP and DM; use of Ca, P, and Cu; and bone mineralization.