Microbiota, Phagocytic Activity, Biochemical Parameters and Parasite Control in Horses with Application of Autochthonous, Bacteriocin-Producing, Probiotic Strain Enterococcus faecium EF 412.

The beneficial influence of bacteriocin-producing, probiotic, mostly non-autochthonous bacteria has already been reported in various animals. However, their use in horses provides limited information, and results with autochthonous bacteria have not been reported. Therefore, the main objective of this model study was to test the effect of autochthonous, bacteriocin-producing faecal strain Enterococcus faecium EF 412 application in horses. One gram of freeze-dried EF 412 strain (109 CFU/mL for 21 days) was applied to horses in a small feed ball. Clinically healthy horses (12), Slovak warm-blood breed of various ages (5-13 years), were involved in a 35-day-long experiment, also functioning as control for themselves. They were stabled in separate boxes (university property), fed twice a day (hay, whole oats or grazed) with water access ad libitum. Sampling was performed at the start of the experiment, i.e. at days 0/1, 21 (3 weeks of EF 412 application) and at day 35 (2 weeks of EF 412 cessation). EF 412 colonized GIT of horses was 3.54 ± 0.75 CFU/g (log 10) at day 21. The eggs of the nematode Strongylus spp. were not found in horses after EF 412 application, and Eimeria spp. oocysts were similarly not found. The other microbiota were not reduced as evaluated by the use of standard method. Using next-generation sequencing, at phylum level, phyla Bacteroidetes and Firmicutes dominated and at family level, they were Bacteroidales BS11 and S24-7 gut goups and Lentisphaerae. In horses, the increasing tendency in phagocytic activity was noted after EF 412 application. Biochemical parameters were in the physiological range. Total protein value was significantly decreased at day 21 compared with day 0/1 as well as with day 35 (P < 0.05). Cholesterol and triglycerides were influenced (decreased) at day 21 compared with day 0/1 and day 35. Neither nematode eggs Strongylus spp. nor Eimeria spp. oocysts were found in faeces after EF 412 application. Autochthonous, faecal strain E. faecium EF 412 showed promising application potential.

Microbiome Associated with Slovak Raw Goat Milk, Trace Minerals, and Vitamin E Content.

In Slovakia, goat milk production for direct consumption and cheese processing has attracted growing interest. However, there is a lack of information regarding the microbial consortium in Slovak raw goat milk analyzed by next-generation sequencing and trace elements and vitamin E as well. A randomly selected samples (G24-G50) of raw goat milk from different animals at farms in Slovakia were analyzed. The phylum Actinobacteria dominated (62.8%), followed by the phyla Firmicutes (20.5%), Proteobacteria (7.4%), and Bacteroidetes (6.4%). The family Microbacteriaceae was detected in the highest percentage (60.2%) followed by Staphylococcaceae, Bacteroidaceae, Streptococcaceae, Lactobacillaceae, Enterobacteriaceae, and others. Regarding the genera, the most prevalent was genus Curtobacterium (47.4%) followed by the genera such as Staphylococcus (8.3%) and Bifidobacterium (4%). The genera Streptococcus, Lactococcus, Enterococcus, Lactobacillus, and Lacticaseibacillus were evaluated in abundance percentage in range 1%-3.2%. The genus Veillonella reached abundance 3.2%. The genera Enterobacter, Pseudomonas (1.3% and 0.5%), and Bacteroides (6.4%) were evaluated in small percentage abundance too. Zinc was detected with the highest mean value (2.561 ± 0.6823 mg/L) in raw goat milk, followed by iron (1.383 ± 0.5087 mg/L). The mean value of copper and manganese was 0.1746 ± 0.0463 mg/L and 0.051 ± 0.0238 mg/L. The vitamin E reached the mean value 0.3783 ± 0.1976 mg/L. This study is an original contribution showing microbial consortium in raw goat milk from Slovak farms. It also contributes to trace elements and vitamin E status in raw goat milk showing it as a nutritionally healthy food.

Enterocin M in Interaction in Broiler Rabbits with Autochthonous, Biofilm-Forming Enterococcus hirae Kr8 Strain.

Young rabbits are susceptible to gastrointestinal diseases caused by bacteria. Enterococcus hirae can be associated with diseases. But enterocins produced by some enterococcal species can prevent/reduce this problem. Therefore, the interaction of enterocin M with a biofilm-forming, autochthonous E. hirae Kr8+ strain was tested in rabbits to assess enterocin potential in vivo. Rabbits (96), aged 35 days, both sexes, meat line M91 breed were divided into four groups, control C and three experimental groups. The rabbits in C received the standard diet, rabbits in experimental group 1 (E1) received 108 CFU/mL of Kr8+, a dose 500 µL/animal/day, E2 received Ent M (50 µL/animal/day), and E3 received both Kr8+ and Ent M in their drinking water over 21 days. The experiment lasted 42 days. Feces and blood were sampled at day 0/1 (at the start of the experiment, fecal mixture of 96 animals, n = 10), at day 21 (five fecal mixtures per group, n = 5), and at day 42 (21 days after additives cessation, the same). At days 21 and 42, four rabbits from each group were slaughtered, and cecum and appendix were sampled for standard microbial analysis. Ent M showed decreased tendency of Kr8+. Using next-generation sequencing, the phyla detected with the highest abundance were Firmicutes, Verrucomicrobia, Bacteroidetes, Tenericutes, Proteobacteria, Cyanobacteria, Saccharibacteria, and Actinobacteria. Interaction of Ent M with some phyla resulted in reduced abundance percentage. At day 21, significantly increased phagocytic activity (PA) was found in E1 and E2 (p < 0.001). Kr8+ did not attack PA and did not stimulate oxidative stress. But Ent M supported PA. The prospective importance of this study lies in beneficial interaction of enterocin in host body.

Effect of enterocins against methicillin-resistant animal-derived staphylococci.

The occurence and spread of animal-derived methicillin resistant staphylococci (MRS) worldwide is a current problem, especially due to their increasing incidence in food animals and their products, with possible contamination of food consumers and handlers. Staphylococci isolated from animals (n = 123) were identified with MALDI-TOF mass spectrometry and screened for methicillin/oxacillin/cefoxitin resistance (MR) using the disk diffusion method. Twenty-three phenotypically MRS strains were analysed using PBP2' Latex Agglutination Test Kit to confirm the phenotypic MR and PCR was performed for mecA gene detection; mecA gene positive strains were furtherly confirmed by means of sequencing. The susceptibility of MRS to 11 partially-purified enterocins (Ent) produced by E. faecium, E. durans and E. mundtii strains of animal, feed/food and environmental origin was checked using agar spot tests. Out of 23 MRS, PBP testing confirmed MR in 17 strains. Three Staphylococcus epidermidis and one S. vitulinus were mecA positive. The majority of MRS, including two mecA gene-positive strains S. epidermidis R44/1 and P3/Tr2a, were susceptible to the tested enterocins, mainly to Ent7420, EntA(P)/EK13, Ent412, Ent55 and Ent9296 (in the range 100 - 12,800 AU/mL). The most susceptible strains appeared to be the mecA gene-positive S. epidermidis SE R44/1 and SE P3/Tr2a strains, inhibited by eight enterocins out of 11 tested (100-200 AU/mL). Only four strains (including mecA gene positive S. epidermidis SE P3/Tr1 and S. vitulinus SV K12PL/1) were resistant to the tested antimicrobial substances. These results indicate that the enterocins used offer a promising option for prevention and treatment of bacterial infection caused by MRS in animals.

Effect of Sustained Administration of Thymol on Its Bioaccessibility and Bioavailability in Rabbits.

The objective of this study was the detection of thymol in rabbit plasma, tissues, large intestinal content, and faeces. Forty-eight rabbits were divided into control and experimental groups (thymol 250 mg/kg feed). Thymol was administered for 21 days and then withdrawn for 7 days. Concentration of thymol in the intestinal wall (IW) was significantly higher than in plasma (p < 0.05) and liver (p < 0.05); in the kidneys it was significantly higher than in plasma (p < 0.05) and liver (p < 0.05) during thymol addition. Thymol in IW was significantly higher than in plasma also after withdrawal (p < 0.01). Significant correlation (rs = -1.000, p < 0.01) between IW and plasma points to the intensive absorption of thymol from the intestine, while the correlation between plasma and liver (rs = 0.786, p < 0.05) indicates intensive biotransformation and excretion processes in liver. Significant correlation between liver and kidney (rs = 0.738, p < 0.05) confirms the intensive metabolism of thymol in the kidney. During the withdrawal period, thymol was detected above trace amounts only in faeces, and was significantly higher than in the colon during both periods (p < 0.01). Results show intensive biotransformation of thymol in the rabbit organism.

Enterocin Structural Gene Screening in Enterococci from Pannon White Breed Rabbits.

This study was focused on screening the most frequently occurring enterocin (Ents) genes in faecal enterococci from the Pannon White breed of rabbits, in which this parameter never has been tested. Enterococci tested were Ent genes absent, except Enterococcus faecium EF9a, which possessed genes for four Ents. Strains produced a high amount of lactic acid (1.25 ± 0.055 mmol/L, on average) and sufficiently tolerate bile. Inhibition activity of crude bacteriocin substance-partially purified (Ent, CE EF9a)-was tested against altogether 53 indicator strains. The growth of 38 strains of different staphylococcal species was not inhibited. However, the growth of fifteen other Gram-positive indicators was inhibited with inhibition activity ranged from 100 to 12,800 AU/mL. Listeria monocytogenes LM 7562 was the most susceptible to CE EF9a treatment (12,800 AU/mL). Molecular mass of CE EF9a ranged between 3000 and 10,000 Da. After heat treatment at different temperatures and time, CE EF9a was active with the inhibition activity 12,800 AU/mL. After storage of CE EF9a at different conditions, it was active even after 2-month storage at room temperature (200 AU/mL against LM 7562 strain). Inhibition activity of CE EF9a after 2-month storage at 4 °C was 1600 AU/mL and the most stable was CE EF9a at - 20 °C; the activity 12,800 AU/mL was kept still after 2 months. It indicates a small, thermostable substance; however, in further detail, purification analysis is necessary.

Benefits of Enterocin M and Sage Combination on the Physico-chemical Traits, Fatty Acid, Amino Acid, and Mineral Content of Rabbit Meat.

Higher rabbit meat consumption can be ensured by increasing of its quality due to its improved nutritional properties. The effect of enterocin M (EntM) and sage on growth performance, physico-chemical properties, fatty acid (FA), amino acid (AA), and mineral concentrations of rabbit meat was evaluated. Sixty-four rabbits (M91 meatline, both sexes) were divided into three experimental: E (EntM), S (sage), E + S (EntM + sage) groups and control group (C). The additives were administrated in drinking water during 21 days. Lower pH (E, S: P < 0.05; E + S: P < 0.001) and decrease in water content were noted in all experimental groups compared with controls (C). Higher values of lightness (L*), yellowness (b*), and redness (a*; except group E) were measured. The sage administration increased the fat and protein contents (P < 0.05), the meat energy value (S vs. C: P < 0.01; S vs. E and E + S: P < 0.05), the concentrations of arachidonic (P < 0.05), eicosapentaeonic (P < 0.05), and oleic acids (P < 0.01), magnesium (P < 0.05), and potassium (P < 0.01) content. The additives did not influence the rabbit meat AA composition. The sage diet inclusion could improve the quality of rabbit meat due to its higher protein, fat, and energy contents and enhance the PUFA and mineral content of rabbit meat. Moreover, the effect of EntM on meat color parameters, FA and AA composition, has not been tested in rabbits previously.

Relation to enterocins of variable Aeromonas species isolated from trouts of Slovakian aquatic sources and detected by MALDI-TOF mass spectrometry.

Aeromonads represent bacteria thought to be primarily mostly autochthonous to aquatic environments. This study was focused on the relation with antibiotics and enterocins of identified Aeromonas species isolated from the intestine of trouts living in Slovakian aquatic sources. Intestinal samples from 50 trouts (3 Salmo trutta and 47 Salmo gairdnerii) were collected in April of years 2007, 2010, and 2015 from trouts of different water sources in Slovakia (pond Bukovec near Kosice, river Cierny Váh). Due to the MALDI-TOF mass spectrometry evaluation, 25 strains were proposed to the genus Aeromonas involving nine different species (Aeromonas bestiarum-nine strains, Aer. salmonicida-four strains, Aer. encheleia, Aer. eucrenophila, Aer. molluscorum, Aer. media, Aer. sobria, Aer. popoffii, Aer. veronii). Phenotypic evaluation of individual strains confirmed their species identification. Twenty-five strains of different Aeromonas species were sensitive to azithromycin, amikacin, mecillinam, mezlocillin, piperacillin, gentamicin, chloramphenicol, and tetracycline. On the other side, they were resistant to carbenicillin and ticarcillin. The growth of Aer. bestiarum R41/1 was inhibited by treatment with Ent M and Ent 2019 (inhibition activity 100 AU/mL). Aer. bestiarum R47/3 was inhibited by eight enterocins (100 AU/mL). It is the first study testing enterocins to inhibit the growth of Aeromonas species from trouts.

Benefits of combinative application of probiotic, enterocin M-producing strain Enterococcus faecium AL41 and Eleutherococcus senticosus in rabbits.

This study presents the effects of the probiotic and enterocin M-producing strain Enterococcus faecium AL41 on microbiota, phagocytic activity (PA), oxidative stress, performance and biochemical parameters when applied individually or in combination with Eleutherococcus senticosus in rabbits. The novelty of the study lies in the use of our non-rabbit-derived strain (AL41 = CCM8558) which produces new enterocin M. Ninety-six post-weaned rabbits (Hyplus breed) aged 5 weeks were divided into three experimental groups, 24 in each: E. senticosus (ES, 30 g/100 kg) in feed, E. faecium AL41 (10(9) CFU/mL marked by rifampicin to differentiate it from other enterococci) in water, and ES + AL. AL41 colonized sufficiently in rabbits to reduce coliforms, staphylococci, pseudomonads and clostridia. Slight decrease in bacteria was also found in the caecum and appendix. Phagocytic activity was significantly increased in the experimental groups compared to the control group (CG) (p < 0.001; p < 0.05). Applications did not evoke oxidative stress. Biochemical parameters in blood and caecal organic acids were slightly influenced. Average daily weight gain was slightly higher in ES and AL + ES. Combinative application of E. faecium with E. senticosus can be beneficial in rabbits. AL41 strain alone and in combination with ES produced reduction in spoilage bacteria; the highest stimulation of PA was in the AL41 + ES group.