Enterocins as Novel Feed Additives in Rabbit Diet: Enterocin Ent M and Durancin Ent ED26E/7, Their Combination, and Effects on Microbiota, Caecal Fermentation, and Enzymatic Activity.

The present study investigates the effects of enterocin Ent M and durancin Ent ED26E/7 applied separately and in combination on the intestinal microbiota, caecal enzymatic activity, and fermentaion of rabbits. Eighty rabbits (M91 meatline, aged 5 weeks, both sexes) were divided into groups E (Ent M; 50 µL/animal/day), D (Ent ED26E/7; 50 µL/animal/day), E + D (Ent M + Ent ED26E/7), and control (C). The additives were administered in drinking water for 21 days. Antimicrobial activity of Ent M and Ent ED26E/7 on coliforms (E, E + D: P < 0.001) and pseudomonads (D: P < 0.05) in feces was noted, compared to C. Ent M and Ent ED26E/7 application stimulated caecal enzymatic activity in rabbits. Pectinolytic (E vs. D, E + D: P < 0.01), inulolytic (E vs. E + D: P < 0.01; E vs. C: P < 0.05), and amylolytic (E vs. D, E + D. P < 0.001; E vs. C: P < 0.01) activities were influenced by Ent M, while cellulolytic (D vs. E + D: P < 0.01) and inulolytic (D vs. E + D, C: P < 0.01) activities by Ent ED26E/7 treatment. The cellulolytic and pectinolytic acitivities changed with time. Treatment × time interaction was detected for cellulose and xylan degradation. During Ent M and Ent ED26E/7 treatment, increased ammonia, lactic, butyric and iso-valeric acid, and lower acetic, propionic, iso-butyric, valeric, and caproic acid concentrations were noted. It can be concluded that Ent M and Ent ED26E/7 application can improve rabbit health due to reduced spoilage microbiota and enhanced caecal enzymatic activity.

Characterisation of Faecal Staphylococci from Roe Deer (Capreolus capreolus) and Red Deer (Cervus elaphus) and Their Susceptibility to Gallidermin.

Our current knowledge of microbiota in wild ruminants is limited. The goal of this study was to evaluate staphylococcal species in red and roe deer for various attributes (haemolysis, DNase, and urease activities; lactic acid and biofilm production; and antibiotic profile) and their susceptibility to gallidermin. Sixteen staphylococcal strains were identified from faeces of 21 free-living animals (9 adult female Cervus elaphus-red deer and 12 young female Capreolus capreolus-roe deer) sampled by the Polish colleagues in the Strzalowo Forest District, Piska Primaeval Forest. The variability in the species of staphylococci was determined. Seven species (Staphylococcus capitis, S. epidermidis, S. haemolyticus, S. hominis, S. pseudintermedius, S. vitulinus and S. warneri) and five clusters/groups of coagulase-negative staphylococci (CoNS) were identified. The strains were generally not haemolytic and Dnase negative; did not form biofilms or only produced low-grade biofilms; exhibited high levels of lactic acid; were urease positive; and were generally susceptible to antibiotics (only two strains were resistant to multiple antibiotics). However, all of the strains were susceptible to the lantibiotic bacteriocin gallidermin, with a minimal inhibitory concentration of 0.0156 µg (up to 6400 AU/ml in arbitrary units). This is the first study to perform a detailed study of the properties of CoNS from roe and red deer.

Enterocin M and Sage Supplementation in Post-weaning Rabbits: Effects on Growth Performance, Caecal Microbiota, Fermentation and Enzymatic Activity.

The effects of enterocin (Ent) M and sage extract applied separately and in combination were investigated. EntM (E 50 µL/animal/day in water) and sage extract (S 10 µL/animal/day in water) were applied individually and in combination (E+S) to rabbits during 21 days of treatment. The rabbits' growth was not significantly influenced by the additives. Lower feed conversion (FC) was noted in the experimental groups compared with controls, with the lowest data detected in E. The antimicrobial activity of EntM was noted (in E+S: lactic acid bacteria-P < 0.01; in E, E+S: enterococci, enterobacteria-P > 0.05; in E: clostridia-P > 0.05). The most significant changes in fermentation between weaned and older rabbits were noted in amylolytic activity at day 21 (E P < 0.05; E + S P < 0.05); prolonged reduction effect of sage extract on amylolytic activity was observed. The activity of cellulase, pectinase and xylanase was higher in older than in younger animals. Decrease in lactic acid and volatile fatty acids was noted during EntM administration, with significant effect on propionic acid concentration (E P < 0.05; E+S P < 0.001). The sage extract reduced propionic acid (S P < 0.001) and butyric acid levels (S P < 0.05) and increased the concentrations of butyric, iso-valeric, valeric, caproic acids and lactic acid (P < 0.001). It seems to be that EntM and sage supplementation may improve the economy of rabbit farms (increased FC) and the health status of rabbits (reduction of spoilage microbiota, enhanced enzymatic activities in caecum).

Fecal coagulase-negative staphylococci from horses, their species variability, and biofilm formation.

The intestinal microbiota has enormous impact on the health and performance of horses. Staphylococci belong in the phylum Firmicutes, and their occurrence, especially of methicillin-resistant strains and species, has been reported in horses previously. Moreover, biofilm formation is one of the virulence factors; it has been not completely studied in fecal coagulase-negative staphylococci (CoNS) from horses. Therefore, this study was focused on biofilm formation by various species of fecal CoNS from horses because it has been never reported before. In addition, their antibiotic profile was tested. Horses (42) of various breeds from Slovakia/Poland were sampled. Variability in the species of CoNS was detected in feces of horses. Thirty-two strains were identified by using the MALDI-TOF system and classified into nine species and three subspecies of CoNS: Staphylococcus capitis, S. cohnii subsp. cohnii, S. cohnii subsp. urealyticus, S. cohnii subsp. casei, S. epidermidis, S. haemolyticus, S. pasteuri, S. sciuri, S. vitulinus, S. warneri, and S. xylosus. The most frequent species was S. vitulinus. Twenty-two strains showed high biofilm production; 10 strains showed low-grade biofilm production. The highest biofilm formation was measured in the species S. xylosus. Eleven strains (of 32) were methicillin-resistant; the others were susceptible to methicillin.

Sensitivity to antimicrobials of faecal Buttiauxella spp. from roe and red deer (Capreolus capreolus, Cervus elaphus)detected with MALDI-TOF mass spectrometry.

Wild ruminants are an interesting topic for research because only limited information exists regarding their microbiota. They could also be an environmental reservoir of undesirable bacteria for other animals or humans. In this study faeces of the 21 free-living animals was sampled (9 Cervus elaphus-red deer, adult females, 12 Capreolus capreolus-roe deer, young females). They were culled by selective-reductive shooting during the winter season of 2014/2015 in the Strzalowo Forest District-Piska Primeval Forest (53° 36 min 43.56 sec N, 21° 30 min 58.68 sec E) in Poland. Buttiauxella sp. is a psychrotolerant, facultatively anaerobic, Gram-negative rod anaerobic bacte- rial species belonging to the Phylum Proteobacteria, Class Gammaproteobacteria, Order Entero- bacteriales, Family Enterobacteriacae and to Genus Buttiauxella. Buttiauxella sp. has never previ- ously been reported in wild ruminants. In this study, identification, antimicrobial profile and sensitivity to enterocins of Buttiauxella strains were studied as a contribution to the microbiota of wild animals, but also to extend knowledge regarding the antimicrobial spectrum of enterocins. Five strains were identified using the MALDI-TOF identification system (evaluation score value was up to 2.224) and allotted to the genus Buttiauxella including the species Buttiauxella gaviniae, B. ferragutiae, B. agrestis. Strains were DNase negative, and they hydrolysed esculin; fermentation of L-arabinose, D-mannitol and D-mannose was positive. Dulcitol, inositol reaction, urea and indol were negative. Buttiauxella strains did not form biofilm. They were resistant to at least one of the 13 antibiotics tested. B. agrestis 2/109/1 was resistant to amdinocillin, clindamycin and pen- icillin. However, Buttiauxella strains were sensitive to the enterocins used (inhibition activity ranged from 100 to 25 600 AU/ml).

The ability of rumen ciliates, Eudiplodinium maggii, Diploplastron affine, and Entodinium caudatum, to use the murein saccharides.

Murein polysaccharides may contribute to a considerable part of the dry matter of bacterial cells. Their utilization by protozoa inhabiting the rumen is, however, poorly recognized. The objective of this study was to examine the ability of three species of ciliates, i.e., Eudiplodinium maggii, Diploplastron affine, and Entodinium caudatum of digest, and ferment these saccharides. The cultivation experiments showed that the enrichment of growth medium with bacterial cell wall ß-glycans increased the ciliate number (p < 0.05). A statistically significant increase (p < 0.01) was followed by a continuous decrease (p < 0.01) in the percentage of individuals containing ß-glycans particles after 4- and 24-h incubation of ciliates with this substrate, respectively. The enzymatic experiments confirmed the ability of the examined protozoa to digest murein. E. caudatum exhibited the highest activity (8.2 unit (U)/mg protein per min), and E. maggii, the lowest (3.0 U/mg protein per min). The production rates of volatile fatty acids by starved and fed ciliate species were 0.7 and 1.6 (E. caudatum) pmol/ciliate cell per h, 30.5 and 42.5 (E. maggii) pmol/ciliate cell per h, and 8.3 and 19.2 (D. affine) pmol/ciliate cell per h (p < 0.05).

Chitinolytic enzymes of the rumen ciliate Eudiplodinium maggii.

The ability of rumen ciliates to digest chitin is clearly recognized. We investigated the chitinolytic system of the rumen ciliate Eudiplodinium maggii. The ciliates were grown in a selectively faunated sheep. They were isolated from the rumen and purified by sedimentation. A crude enzyme preparation was prepared following incubation of ciliates with antibiotics. This was done in order to reduce their contamination with intracellular bacteria. The activity of particular enzymes was examined by quantification of the products released from specific substrates. It was stated that the optimum conditions for the detected activities varied between 4.5 and 5.5 pH, and 45 and 55 °C. ß-N-Acetylglucosaminidase was found as an enzyme of the highest activity (4.2 µmol/l released product per mg protein per h). The activities of endochitinase and exochitinase were almost two times lower than that of ß-N-acetylglucosaminidase. Zymographic studies revealed the presence of two endochitinases, two exochitinases and two ß-N-acetylglucosaminidases in the examined preparation.

Mureinolytic ability of the rumen ciliate Diploplastron affine.

Rumen ciliate protozoa intensively engulf bacteria. However, their ability to utilize murein which is the main polysaccharide of bacterial cell wall has hardly been recognized. The present study concerns the ability of the rumen protozoa Diploplastron affine to digest and ferment murein. The ciliates were isolated from the rumen fluid and grown in vitro or inoculated into the rumen of defaunated sheep. The results of long-term cultivation of protozoa showed a positive correlation between their number and murein content in the culture medium. It was also found that bacteria-free D. affine ciliates incubated with or without murein produced volatile fatty acids at the rate of 12.3 and 8.7 pmol/h per protozoan, respectively, acetic, butyric and propionic acids being the three main acids released to the medium. Enzyme studies performed with the use of protozoan cell extract prepared from bacteria-free ciliates degraded murein at a rate of 25 U/mg protein per h; two mureinolytic enzymes were identified by zymographic technique in the examined preparation.

The ability of the rumen protozoan Eudiplodinium maggii to utilize chitin.

The ability was determined of the rumen ciliate Eudiplodinium maggii to utilize chitin from fungal cell wall. Cultivation experiments shoved that the population concentration (number of ciliates in vitro) was positively correlated with chitin doses. Cell extract prepared from the bacteria-free ciliates degraded colloidal chitin releasing 2.0 micromol reducing sugar per mg protein per h. End products of this reaction were chitotriose and N-acetylglucosamine. Incubation of the bacteria-free ciliates with chitin resulted in an increase in the concentration of acetic, propionic and butyric acids in the incubation medium. The production rate of total volatile fatty acids (VFA) by ciliates incubated with and without chitin was 45.0 and 30.5 pmol VFA per protozoan, respectively, the molar proportion of particular acids remaining unchanged.

The antimicrobial action of low-molar-mass chitosan, chitosan derivatives and chitooligosaccharides on bifidobacteria.

The crude fractions of chitooligosaccharides (COS) and low-molar-mass chitosans (LMWC) were prepared by enzyme hydrolysis of chitosan (CS). Specific growth rate of B. adolescentis, B. bifidum, B. breve, B. catenulatum, B. infantis and B. longum ssp. longum was determined in the presence of 0.025 and 0.5 % COS (<5 kDa), LMWC (5-10 kDa), and 0.025, 0.1 and 0.5% of CS, chitosan succinate and chitosan glutamate in vitro. Minimum inhibitory concentrations (MIC; assayed by colony counting on TPY agar plates) of COS-LMWC and CS ranged from 0.025% to 0.75% of CS-LMWC. The growth of all bifidobacterial strains in the presence of chitosan, its derivatives and LMWC decreased at a concentration of 0.025%; the bacterial growth was completely inhibited at a concentration of 0.5%. COS did not show any inhibitory effect, an increased growth rate was even observed in the case of B. bifidum, B. catenulatum and B. infantis.

Chitinolytic activity of the sheep rumen ciliate Diploplastron affine.

Supplementation of the rumen ciliate Diploplastron affine growth medium with commercial chitin stimulated growth of ciliates and the density of their population was positively correlated with chitin doses (r = 0.95; p < 0.01). The cell-free extracts prepared from bacteria-free ciliates degraded chitin to N-acetyl-D: -glucosamine and chitobiose. Three exochitinases, two endochitinases and two beta-N-acetylglucosaminidases were identified in the cell-free extract of protozoa. The molar mass of exochitinases was 80, 65 and 30 kDa, and endochitinases 75 and 50 kDa; the molar mass of one of the identified beta-N-acetylglucosaminidases was 45 kDa.

Why does the establishment of the starch preferring Entodinium caudatum in the rumen decrease the numbers of the fibrolytic ciliate Eudiplodinium maggii?

The effect of the establishment of Entodinium caudatum on the population of Eudiplodinium maggii was examined in the rumen of three sheep fed a hay/ground barley diet. The cell concentration of E. maggii were 15.9-38.5 and 11.7-12.4 x 10(3) cells per g of the rumen contents in the absence and presence of E. caudatum, respectively. Microscopic analysis showed that starch was the only material engulfed by eudiplodinia irrespective of the time after feeding and the presence or absence of E. caudatum. Up to 82-93% of individuals contained starch grains when E. maggii was the only ciliate species in the rumen; the proportion was 70-77% after entodinia had been established. The largest quantity of starch engulfed by E. maggii ciliates was 12.4-19.0 and 6.7-7.6 mg per 100 mg protozoal dry mass in the absence and presence of entodinia, respectively. No visible engulfment of hay was observed in vivo in spite of the fact that hay particles up to 42 microns in length were dominating in rumen fluid. Ingestion of fresh particles of hay separated from the rumen digesta was found when they were added in the proportion of 1 g per 40 mL suspension of ciliates. No preferential intake of starch was observed when E. maggii ciliates were incubated in vitro with a mixture of hay and barley starch. It is suggested that competition for starch between the two ciliate species was responsible for the drop in the numbers of E. maggii. This could result from a too low concentration of small particles of hay in the rumen fluid.