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.

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).

Relation to enterocins and herbal extracts of fecal hemolytic Escherichia coli from domestic ducks detected with MALDI-TOF mass spectrometry.

Surveillance studies have generally reported an increase in Escherichia coli strains resistant to major classes of antibiotics used for animals' treatment. The aim of this study was to test the susceptibility of 25 strains (isolated from 30 domestic Mallard ducks-Anas platyrhynchos, both sex, aged 8 to 14 wk, taxonomically alloted to the species E. coli using MALDI TOF mass spectrometry system) to antimicrobials (antibiotics, enterocins, and herbal extracts). Testing was performed using the agar disc method and the agar diffusion method. A total of 19 E. coli strains were multiresistant to antibiotics; but 10 of those strains were susceptible to enterocins with an inhibition activity of 100 AU/mL. All strains were susceptible to herbal extracts. These results indicate further benefit application of enterocins and herbal extracts to prevent/reduce problems caused with E. coli. Moreover, additional studies are in process.

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.

Assessment of biofilm formation by faecal strains of Enterococcus hirae from different species of animals.

Enterococcus hirae belongs in the Enterococcus faecium group within the genus Enterococcus. This species occurs naturally in the environment, commensally in the alimentary tracts of animals, and pathologically for example in humans with urinary infections. Some strains of E. hirae possess virulence factors, including biofilm formation. Biofilm growth protects bacteria against host de- fences; biofilm can be a source of persistent infection. Testing bacterial strains for their ability to form biofilm might therefore facilitate their treatment or prevention. This study focuses on bio- film formation by E. hirae strains derived from various animals. This kind of testing has never been done before. A total of 64 identified E. hirae from laying hens, ducks, pheasants, ostriches, rabbits, horses and a goat were tested by means of three methods; using Congo red agar, the tube method and microtiter plate agar. The majority of strains were found to form biofilm. 62.5% of strains were biofilm-forming, four categorized as highly positive (OD570 ≥1); most strains were low-grade biofilm positive (0.1 ≤ OD 570 ⟨ 1). Related to poultry, 55 E. hirae strains were tested and found to produce biofilm; 24 strains did not form biofilm, 31 strains were biofilm-forming; 27 strains showed low-grade biofilm formation, and four strains were highly biofilm-forming. Four strains from hens and ostriches reached the highest OD570 values, more than 0.500. Rabbit-de- rived E. hirae strains as well as strains isolated from horses and the goat were low-grade bio- film-forming. Microtiter plate assay proved to be the best tool for testing the in vitro biofilm for- mation capacity of E. hirae strains from different species of animals.

Characteristic and susceptibility to enterocins of enterococci in pheasants possessing virulence factor genes.

With an increasing number of pheasants as gamebirds being reared each year, these species are becoming a more prominent part of the workload of many veterinary practices. Only limited information can be found concerning the microflora of common pheasants. A significant part of the obligate microflora consists of lactic acid bacteria, including enterococci. In this study, faeces were sampled from 60 pheasants aged 16-17 weeks. Enterococcal counts reached 5.48±1.9 (log10) CFU/g. Strains (17) were taxonomically classified to the genus Enterococcus using the Maldi-Tof identification system; they were allotted to the species E. hirae (58.8%), E. faecium (23.5%) and E. faecalis (17.7%) by highly probable species identification or by secure genus identification/probable species identication. Species allocation was also confirmed using conventional biochemical tests. Most strains formed ß-hemolysis. Gelatinase active phenotype was found in three E. faecalis strains. Enterococci were ß-glucuronidase negative, mostly trypsin negative with slight or moderate production of α-chymotrypsin. EH52b and EF42 strains possessed the highest potential for pathogenicity. Average value of lactic acid was 1.78±0.33 mmo/L. Most strains were tetracycline resistant (82.4%). Polyresistant E. faecalis strains with positive gelatinase phenotype and possessing virulence factor genes confirmed using PCR (gelE, efaAfs, ccf cob, cpd) were sensitive to enterocins (activity 1600-25,600 AU/mL).

Use of bacteriocin-producing, probiotic strain Enterococcus faecium AL41 to control intestinal microbiota in farm ostriches.

Probiotic enterococci can produce bacteriocins. Enterococcus faecium AL41 is an Enterocin M-producing, probiotic strain which has previously shown beneficial effect in broiler chickens. In this study, it was used to control intestinal microbiota in farm ostriches in a 42-day experiment with an experimental group (EG, 40 ostriches) and a control group (CG, 46). In addition to feed mixture, the ostriches in EG received Ent. faecium AL41 (10(9) CFU ml(-1); by rifampicin-marked variant) 400 µl per animal per day in their drinking water for 21 days. Sampling was carried out at the start of the experiment (at day 0/1), at day 21 (after 21 days of AL41 application) and at day 42 (21 days after AL41 cessation). Faeces (mixture, n = 6) were treated using the standard microbiological dilution method and cultivated on selective media (ISO). The highest count of AL41 was found at day 42. Its identity was confirmed with PCR and Maldi-Tof. The ostriches were free of Salmonella and Campylobacter cells. At day 21, antimicrobial effect was demonstrated by significant reduction in coagulase-positive and negative staphylococci in EG compared to CG (P < 0·001) and coliforms, Enterobacteria and Pseudomonas-like bacteria (P < 0·001). We conclude that AL41 can be used to control intestinal microbiota in farm ostriches. Significance and impact of the study: Ostriches are excellent for high intensity farming in a wide range of climates, requiring only limited space and giving high yields per hectare. They are reared mainly for their meat. Although adult birds possess quite good immunity, young birds can be threatened by spoilage bacteria, especially when they are transferred from the nests to the farm area. Based on our previous results related to the beneficial effect of bacteriocin-producing, probiotic strain Enterococcus faecium AL41 in poultry or rabbits, we decided to test its ability to control intestinal microbiota in farming ostriches which has never been tested previously.