Composition and diversity of 16S rRNA based skin bacterial microbiome in healthy horses.

Characterization of microbiota structure on the skin of healthy horses is important for further development of modulation strategies to ensure optimal bacterial composition for physiological processes. This requirement is also supported by the relatively high incidence of dermatological diseases in horses and thus the need to manage them therapeutically. The taxonomic analysis of skin samples (n = 30) from five different body parts of clinically healthy Shetlands ponies females (neck, back, abdomen, pastern, muzzle) kept under homogeneous conditions (in open stalls with paddock, feed with dry hay, green grass ad libitum and granulated feed) was performed using amplification of V3-V4 region of the 16S rRNA gene. Results indicate that bacteria associated with healthy equine skin represent 18 phyla, 29 classes and 119 families. The most abundant phyla were Proteobacteria (30.8 ± 9.1%) followed by Actinobacteriota (20.4 ± 7.6%), Firmicutes (19.5 ± 10.1%), Bacteroidota (8.5 ± 5.0%) and Deinococcota (7.2 ± 14.8%). Among 229 genera identified, Corynebacterium (7.4 ± 6.5%) was the most abundant genus in skin sites of horses, followed by Deinococcus (7.1 ± 14.9%) and Macrococcus (5.0 ± 8.2%). Indices for the richness and diversity of species within bacterial populations for five regions of horses skin revealed no significant variations observed for species richness (Chao1, p-value 0.2001) but significant result for species evenness (Shannon, p-value 0.0049) with maximum on the neck and minimum on the back skin site. The clustering was seen across samples from different skin sites but also across samples collected from individual horses.

Virulence determinants and antibiotic resistance in staphylococci isolated from the skin of captive bred reptiles.

Knowledge of the composition and properties of skin microbiota in healthy reptiles is essential for preservation strategies and thus the prevention of skin dysbiosis leading to dermatological diseases. Despite the greatly increasing popularity of reptiles as pets, only a few studies have dealt with this topic. Therefore, the aim of this work was to analyse species composition of bacteria isolated from skin swabs of 40 reptiles (17 species) using MALDI-TOF spectrometry and to characterise the virulence properties of identified staphylococci (n = 51). The most common species were Staphylococcus xylosus and S. sciuri. Bacilli, enterococci, Escherichia coli, Salmonella sp. and Acinetobacter sp. were also common. The most frequent antimicrobial resistance in staphylococcal isolates was observed for ampicillin (100.0%) and cefoxitin (98.0%) with the blaZ gene being most prevalent (58.8%). In contrast, all staphylococci were susceptible to gentamicin, kanamycin and imipenem. Slime and biofilm production was observed in 86.3% and 76.5% of isolates, respectively. Gelatinase, DNase, protease and lipase activity was found more rarely (41.2%; 25.5%; 27.5% and 21.6%). Since reptiles are a reservoir of bacteria for their owners, common multi-drug resistance (84.3%, MAR index average 0.29 ± 0.09) and biofilm formation must be kept in mind, especially in the case of injury when handling reptiles.

Antibacterial activity of plant-derived compounds and cream formulations against canine skin bacteria.

An urgent need to find alternative antimicrobial compounds effective in the prevention and treatment of skin infections led us to study the inhibitory activity of eight plant-derived bioactive compounds (betulin, curcumin, glycyrrhizic acid, guaiazulene, piperine, quercetin, quinine, tannic acid) against 14 canine skin isolates (11 Gram-positive and three Gram-negative bacteria) selected based on antibiotic resistance and virulence features. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined using the broth microdilution method. In detail, the results for the eight different plant compounds showed their inhibitory activity in the concentration range from 0.04 to more than 16 mg/ml (MIC) and from 0.25 to more than 16 mg/ml (MBC). The most potent compounds appear to be tannic acid, followed by quinine and curcumin (MIC 0.04-16.0 mg/ml). The most susceptible strain to the tested agents in general was Bacillus cereus AE13, while Enterococcus faecium AA14 was the most resistant strain (the highest MICs) among the tested bacteria. The two most potent plant-derived compounds (tannic acid and quinine) were tested in mixture in different ratios (1:1, 1:2, 2:1). The lowest MIC and MBC values were observed for the 1:2 ratio, which was used for preparation of creams with different cream bases. One of the cream formulations (cream F) was effective up to 63.0 mg/ml (MIC) with a microbial inactivation time of 1-6 h according to the tested strain. This study provides evidence that some plant-derived compounds could have an antimicrobial effect against canine skin bacteria, the strength of which is bacterial strain dependent.

Antimicrobial activity of organic acids against canine skin bacteria.

Canine skin is often a source of bacterial strains that are characterized by the presence of important virulence factors and a high antimicrobial resistance. These bacteria are involved in the pathogenesis of infectious skin diseases, which are very frequent in dogs. Moreover, canine skin isolates are easily spread to other animals and humans. The aim of this study was to evaluate the inhibitory and bactericidal activity of eight organic acids (L-lactic, acetic, propionic, butyric, citric, succinic, glycolic, L-ascorbic acid) against 14 canine skin isolates (11 Gram-positive and three Gram-negative species). The advantages of the tested organic acids are their gentleness to the skin and their affordability. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the broth microdilution method. All tested acids showed a bactericidal effect against the selected bacteria, with the exception of their bacteriostatic effect against the Bacillus cereus strain. The lowest MIC showed acetic acid (MIC between 0.5 and 2.0 mg/mL) and propionic acid (MIC 0.8 - 3.3 mg/mL), whereas L-ascorbic acid (MIC 4.0 - 16.0 mg/mL) seems to be weaker among the tested acids. Two Staphylococcus aureus strains and a strain of Escherichia coli were observed to be more resistant compared to coagulase-negative staphylococci.

Distribution and Characterization of Staphylococci Isolated From Healthy Canine Skin.

Despite increasing interest to study skin microbiota with progressive methods, there are almost no data on staphylococcal species distribution on skin of healthy dogs available. Therefore we decide to characterize staphylococci isolated from 8 different body sites (inner pinna, chin, nasal skin, back, axilla, abdomen, interdigital area and perianal region) of healthy canine skin. A total of 91 staphylococci were isolated from 30 dogs living in East Slovakia. Swabs of each dog were cultivated and colonies analysed using MALDI-TOF spectrometry. The vast majority of isolated staphylococci belonged to S pseudintermedius species (48%) followed by S hominis (15%) and S aureus (10%). S haemolyticus, S warneri, S epidermidis, S capitis, S xylosus, S pasteuri, S intermedius and S succinus were also isolated (<10%). The most frequent resistance in staphylococcal isolates was observed for chloramphenicol (73%) and penicillin (67%) followed by erythromycin (42%), tetracycline (26%), and oxacillin (20%). Multi-drug resistance was found in 50% of isolates. All strains were gentamicin and vancomycin sensitive and were strong or moderate biofilm producers with high acid and alkaline phosphatase activities. Over half of strains were haemolytic (57%) and produced gelatinase (54%), DNAse (84%) and lipase (64%). It seems, multiresistant biofilm forming staphylococci could be commonly detected also in healthy dogs and could probably serve as reservoir for other dogs or owners because of constant exchange of their microbiota.

Effect of enterocin M and durancin ED26E/7 supplementation on blood parameters, immune response and jejunal morphometry in rabbits.

Natural feed additives application in rabbit nutrition can help to control and prevent digestive disturbances and improve gut health and immunity around the critical weaning period. While probiotics are frequently used in rabbits, in vivo administration of bacteriocins is often limited. Therefore, the present study evaluates the effect of enterocin EntM, durancin EntED26E/7 and their combination on serum biochemistry, phagocytic activity and jejunal morphometry of rabbits. Eighty rabbits (aged 35 days, meat line M91, both sexes) were divided into experimental groups E (EntM; dose 50 µl/animal/day, activity 25,600 AU/ml), D (EntED26E/7; 50 µl/animal/day, 12,800 AU/ml), E + D (50 µl EntM + 50 µl EntED26E/7 /animal/day) and control group (C). Additives were administrated in drinking water for 21 days. Both enterocins positively influenced tested serum parameters, with emphasis on durancin EntED26E/7 administration, alone and/or in combination with EntM. Increased total proteins (E, D: p < 0.001), urea (D: p < 0.001), albumin (D: p < 0.05) and triglycerids (E, D, E+D: p < 0.001) were found. Hypocholesterolaemic effect of both additives was recorded (p < 0.001), with the lowest HDL concentration in E + D. The most of tested hepatic enzymes were positively influenced by enterocins combination (E + D; p < 0.001). The lowest AST was noted in group D (p < 0.001). Mineral profile was also improved (p < 0.001), with the highest values in D. Oxidative stress, was not evoked during enterocins application. Both additives showed a tendency to improve phagocytic activity (prolonged effect of EntED26D/7; D, E+D: p < 0.05) and jejunal morphometry parameters (increased villus cut surface; E, D, E+D; p < 0.001). Diet supplementation with EntM and mostly with EntED26E/7 can improve serum biochemistry, phagocytic activity and jejunal morphometry.

Slovak Local Ewe's Milk Lump Cheese, a Source of Beneficial Enterococcus durans Strain.

Slovak ewe's milk lump cheese is produced from unpasteurized ewe's milk without any added culture. Because of the traditional processing and shaping by hand into a lump, this cheese was given the traditional specialty guaranteed (TSG) label. Up till now, there have existed only limited detailed studies of individual microbiota and their benefits in ewe's milk lump cheese. Therefore, this study has been focused on the beneficial properties and safety of Enterococcus durans strains with the aim to contribute to basic dairy microbiology but also for further application potential and strategy. The total enterococcal count in cheeses reached 3.93 CFU/g (log 10) ± 1.98 on average. Based on a MALDI-TOF mass spectrometry evaluation, the strains were allotted to the species E. durans (score, 1.781-2.245). The strains were gelatinase and hemolysis-negative (γ-hemolysis) and were mostly susceptible to commercial antibiotics. Among the strains, E. durans ED26E/7 produced the highest value of lactase enzyme ß-galactosidase (10 nmoL). ED26E/7 was absent of virulence factor genes such as Hyl (hyaluronidase), IS 16 element and gelatinase (GelE). To test safety, ED26E/7 did not cause mortality in Balb/c mice. Its partially purified bacteriocin substance showed the highest inhibition activity/bioactivity against Gram-positive indicator bacteria: the principal indicator Enterococcus avium EA5 (102,400 AU/mL), Staphylococcus aureus SA5 and listeriae (25,600 AU/mL). Moreover, 16 staphylococci (out of 22) were inhibited (100 AU/mL), and the growth of 36 (out of 51) enterococcal indicators was as well. After further technological tests, E. durans ED26E/7, with its bacteriocin substance, can be supposed as a promising additive to dairy products.

Effects of silybin supplementation on nutrient digestibility, hematological parameters, liver function indices, and liver-specific mi-RNA concentration in dogs.

BACKGROUND: Hepatopathies are an important group of disorders in dogs where proper nutritional care is crucial. Supplementation with a hepatoprotectant like silybin can improve liver function and should not interfere with nutrient digestibility. The purpose of this study was to investigate the effect of both pure silybin and commercial hepatoprotectant on nutrients digestibility, liver function indices and health status in healthy dogs (EXP1). Moreover, the second experiment (EXP2) investigated the effect of commercial hepatoprotectant on liver function tests and liver-associated miRNAs concentration in dogs with idiopathic liver disorder. RESULTS: Nutrient digestibility was not affected by treatment in EXP1. Supplementation did alter the serum fatty acid profile, with no clinical relevance. The levels of liver markers such as ALT, AST and GGT significantly decreased. In EXP2, supplementation with commercial hepatoprotectant containing silybin improved liver function tests. A decrease was observed in liver serum markers such as ALT, AST and miR122 concentration. CONCLUSIONS: EXP1 confirmed that silybin (whether pure or as a commercial hepatoprotectant) does not interfere with digestion which subsequently exerts no detrimental effect on dogs' health and metabolism. In EXP2, dietary supplementation with commercial hepatoprotectant containing silybin resulted in a decreased activity of serum liver markers, accompanied by a decrease in the concentration of liver-specific miRNA molecules. Liver function indices were consequently improved. Silybin supplementation can thus serve as an effective therapeutical tool in dogs with hepatopathies.

Effect of Hydrolyzed Yeast Administration on Faecal Microbiota, Haematology, Serum Biochemistry and Cellular Immunity in Healthy Dogs.

Fungal microorganisms are regularly found in the gastrointestinal tract of healthy and diseased dogs especially from the phyla Ascomycota and Basidiomycota; however, it is necessary to better understand their role in host health. One of the most commonly studied yeast species in humans or animals is Saccharomyces cerevisiae especially in its live cell form. Scarce knowledge on its hydrolysate product effects in dogs forced us to test diet supplemented with hydrolyzed brewery S. cerevisiae (at a dose 0.3% of the diet) for 14 days to healthy adult dogs. Twenty German Shepherds were randomly divided into 2 groups: control and experimental, ten dogs in each. The experiment lasted 42 days (blood and faeces sample collection at days 0, 14, 28 and 42). The results of this straighforward experiment showed significant increase in the abundance of bifidobacteria (day 14), lactic acid bacteria (day 42) and clostridia (day 42). The faecal pH was significantly increased at day 28. In blood serum, the concentration of triglyceride and cholesterol decreased (day 42) while activities of alanine aminotransferase (at day 14) and aspartate aminotransferase significantly increased (at days 28 and 42). Activities of these enzymes were above reference range top in 7 dogs (ALT) and 4 dogs (AST). Haematological paramaters and activity of phagocytes as well as on percentage of lymphocyte subsets CD4+, CD8+, CD4+CD8+ and CD21+ were not changed during the experiment. The important point of these results is their onset mostly in the post-supplementation period. The observation of some unexpected effects emphasizes the need for reassessment to use yeasts products for dogs but further studies using different doses are necessary.

Enterocin M-Producing Enterococcus faecium CCM 8558 Demonstrating Probiotic Properties in Horses.

The effects of non-authochtonous Enterococcus faecium AL41 = CCM 8558, enterocin M-producing and probiotic strain were tested on the microbiota, phagocytic activity, hydrolytic enzymes, biochemical parameters and dry matter in horses based on its previous benefits demonstrated in other animals. E. faecium CCM 8558 sufficiently colonized the digestive tract of horses. At day 14, its counts reached 2.35 ± 0.70 CFU/g (log 10) on average. The identity of CCM 8558 was confirmed by means of PCR after its re-isolation from horse faeces. The inhibition activity of CCM 8558 was demonstrated against Gram-negative aeromonads, counts of which were significantly reduced (P < 0.001). After 14 days application of CCM 8558, a tendency towards increased phagocytic activity (PA) was measured; PA value was 73.13% ± 8.55 on average at day 0/1; at day 14, it was 75.11 ± 8.66%. Cellulolytic, xylanolytic and pectinolytic activity in horse faeces was significantly increased (P < 0.001) at day 14 (after CCM 8558 application) and amylolytic activity as well (P < 0.01) compared to day 0/1. Inulolytic activity increased with mathematical difference 1.378. Dry matter value reached 20.81 ± 2.29% on average at day 0/1; at day 14, it was 20.77 ± 2.59% (P = 0.9725). Biochemical parameters were influenced mostly in the physiological range. These results achieved after application of CCM 8558 in horses are original, giving us further opportunity to continue these studies, to measure additional parameters and to show the benefits of CCM 8558 application in horses.

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.

Can Enterocin M in Combination with Sage Extract Have Beneficial Effect on Microbiota, Blood Biochemistry, Phagocytic Activity and Jejunal Morphometry in Broiler Rabbits?

The present study investigated the effects of enterocin (Ent) M and sage extract alone and also in combination on the gut microflora, phagocytic activity, blood biochemistry, and morphometry of rabbits. Sixty-four rabbits (aged five weeks, M91 meatline, both sexes) were divided into three experimental groups: E (EntM; 50 µL/animal/day), S (sage; 10 µL/animal/day), and E + S (EntM + sage) groups and control group (C). The additives were administered in drinking water for a period of 21 days. Dietary supplementation of EntM and sage significantly reduced the coliforms (E: p < 0.001; S: p < 0.001; E + S: p < 0.001) in feces, while simultaneous addition of EntM and sage decreased enterococci (E + S: p < 0.0001), lactic acid bacteria (E + S: p < 0.01), and coagulase-positive staphylococci (E + S: p < 0.0001) in the appendix. Sage addition reduced HDL (S: p < 0.001) and LDL cholesterol (S: p < 0.001; E + S: p < 0.001), LDL/HDL ratio (S: p < 0.001; E + S: p < 0.01), and increased urea (S: p < 0.01; E + S: p < 0.001) and creatinine (S: p < 0.001; E + S: p < 0.001) in serum. EntM and sage application, alone or in combination, improve the jejunal morphometry (p < 0.0001) in rabbits.

Some safety aspects of enterococci isolated from Slovak lactic acid dairy product "zincica".

In Slovakia, dairy products made from ewes' milk have a long tradition. These products include the lactic acid product called "zincica" which is a by-product occurring during the preparation of ewes' lump cheese. There is no information in the literature regarding the special properties of the microbiota, especially lactic acid Firmicutes, which can survive in "zincica." From the safety aspect, enterococci are a controversial group of bacteria, and those from "zincica" have never been tested for their properties. The "zincica" used in our study was supplied by several different agrofarms producing ewes' lump cheese in central Slovakia. The species Enterococcus faecium (strains EF30E1, EF32E1, EF34E1, EF34E5) and Enterococcus faecalis (strains EE30E4, EE35E1, E31E2, altogether 7) were detected in samples from "zincica" identified using MALDI-TOF spectrometry with secure genus identification/probable species identification and then confirmed by means of PCR. Enterococci were hemolysis-negative and the genes of the typical enterococcal virulence factors were mostly absent; the gelE gene was found in two E. faecium strains (EF30E1 and EF32E1), the agg gene was detected in E. faecalis EE35E1, and the esp gene was found in two E. faecalis strains (EE30E4 and EE31E2). No strains harbored the cytolysin A gene. Biofilm formation was detected in four strains (EF30E1, EF32E1, EF34E1, and EF34E5), indicating highly positive and low-grade positive biofilm formation. Enterococci were mostly susceptible to antibiotics tested for their phenotype. This is the first study to analyze enterococci in "zincica."

Bacteriocin production and distribution of bacteriocin-encoding genes in enterococci from dogs.

Many enterococcal strains produce bacteriocins, which could be useful as natural food preservatives through inhibition of pathogenic and spoilage microorganisms. There is little knowledge of the distribution and spectrum of bacteriocin activity and the distribution of bacteriocin-encoding genes in enterococci isolated from dogs. Therefore, we subjected 160 enterococcal isolates (E. faecium n=92, E. faecalis n=35, E. hirae n=28, E. casseliflavus n=3, E. mundtii n=2) from 105 samples of dog faeces to polymerase chain reaction (PCR) detection of genes for enterocin A, P, B, L50A, L50B, AS-48, and bac31 and to screening for bacteriocin activity. The results showed the presence of at least one of the tested genes in 54/160 isolates, with E. faecium the most common gene-possessing species. The most frequently occurring gene for production of enterocin A was observed in combination with enterocin P and B. Bacteriocin activity was observed in 76/160 isolates against at least one of 5 indicator bacteria from the genus Listeria, Enterococcus, Streptococcus and Staphylococcus. Four selected strains (IK25, Bri, I/Dz, P10) were active mostly against different species of Enterococcus (in the range 400-25 600 AU/mL) and Listeria sp. (800-12 800 AU/mL) but no Gram-negative bacteria were inhibited. Protein character, thermostability (up to 121°C) and stability at different pH values (3.0-10.0) were confirmed for crude bacteriocins of these four strains. The antimicrobial substance of E. faecium IK25 strain was identified as enterocin B using molecular weight detection and the presence of genes.

Enterococci Isolated from Trout in the Bukovec Water Reservoir and Cierny Váh River in Slovakia and Their Safety Aspect.

The aim of this study was to investigate enterococci as lactic acid bacteria and as part of Firmicutes phylum. We focused on the virulence factor, biofilm formation, and antibiotic resistance and also on lactic acid production and enterocin gene detection. Intestinal samples were taken from 50 healthy trout (3 Salmo trutta and 47 Salmo gairdneri) collected in April 2007, 2010, and 2015 from different locations at the Bukovec water reservoir and the Cierny Váh River in Slovakia. Twenty pure colonies were identified using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification system based on protein fingerprints, and then seven identified strains were also phenotyped. Based on the identification methods used, the identified enterococci (7) belong taxonomically to four different enterococcal species: Enterococcus durans, E. faecium, E. mundtii, and E. thailandicus. They were hemolysis, DNase, and gelatinase negative with acceptable enzymatic activity. They did not form biofilm and were mostly susceptible to antibiotics. All strains produced lactic acid amounting to 1.78 ± 0.33 mmol/l on average and possessed the gene for enterocin A production. This is the first study reporting more detailed properties of enterococci from trout in Slovakian wild water sources, and it produces new possibilities for studying microbiota in trout.

Virulence Factor Genes Incidence among Enterococci from Sewage Sludge in Eastern Slovakia following Safety Aspect.

The sewage sludges represent a potential health hazard because of the quantity of different microbiota detected in sewages. Among microbiota detected in sewages, also belong representatives of the phylum Firmicutes. In the past, environmental enterococci in addition to coliforms were widely used as indicators of faecal contamination. Regarding the enterococcal strains as potential pathogenic bacteria, their pathogenicity is mainly caused by production of virulence factors. Therefore, the aim of the study was to analyse incidence of virulence factors in enterococci from cows' dung water. Species identification of 24 enterococci using MALDI-TOF MS system allotted 23 strains to the species Enterococcus faecium with highly probable species identification and E. faecalis EEV20 with a score value meaning secure genus identification/probable species identification. Enterococci were absent of cytolysin A gene, hyaluronidase gene, and element IS gene. It can be concluded that they are not invasive which is very important from safety aspect. The most frequently detected gene was adhesin E. faecium (efaAfm, in 22 E. faecium strains and in one E. faecalis). Adhesin efaAfs gene was detected in E. faecalis EEV20 and in two E. faecium. GelE gene was present in three strains. E. faecium EF/EC31 was absent of virulence factor genes.

Oral administration of bacteriocin-producing and non-producing strains of Enterococcus faecium in dogs.

The current effort to incorporate microbial cultures in canine nutrition and thus intake of them on daily base increases our interest in careful and more complex study of their effects in dogs. Many of the commercially used strains have not been tested in dogs and are incorporated only on the base of beneficial effects observed in humans with specific disorders. Moreover, no information on the effects of bacteriocin-producing strains in dogs is available. Therefore, we decided to test and to compare overall effect of bacteriocin non-producing Enterococcus faecium DSM 32820 and enterocin B-producing E. faecium LMG 30881 strain (both of canine origin). Dogs were divided into three treatment groups of ten dogs each: control; DSM 32820 group; and LMG 30881 group, dosing 109 CFU/day/dog. The experiment lasted 35 days with a 14-day treatment period (sample collection at days 0, 7, 14, 35). Despite bacteriocin production is believed that may provide a competitive advantage over neighbouring sensitive strains within shared environment, results indicated somewhat better survival for the DSM 32820 compared to the LMG 30881 group. Furthermore, dogs of DSM 32820 group had optimal faecal consistency throughout the experiment, significantly stimulated phagocytic activity (days 7 and 14) and metabolic burst activity of leukocytes (days 14 and 35) and lower serum glucose concentration (day 35). In contrast, dogs of LMG 30881 group showed higher faecal count of Gram-negative bacteria (day 35), lower haemoglobin and glucose concentration (day 35), and higher metabolic burst activity (days 14 and 35). These results are further evidence of the existence of inter-strain differences in efficacy despite the same origin.

Evaluation of enterococci for potential probiotic utilization in dogs.

Some strains of the genus Enterococcus are effective probiotic bacteria if they meet safety and probiotic criteria. In our study, 17 canine enterococci previously selected from a group of 160 isolates based on safety criteria were screened for some functional properties relevant to their use as probiotics. The results of antimicrobial resistance testing showed sensitivity of eleven strains to EFSA recommended antimicrobials. In contrast, the most frequent resistance was observed for cefotaxim (15/17) and oxacillin (13/17). PCR detection of resistance genes (vanA, vanB, vanC, tetM, tetL, ermB, and mefA) revealed the presence of mefA gene in five Enterococcus faecium strains and vanA gene in one strain. The production of enzymes commonly associated with intestinal diseases was in general rare (ß-glucosidase 2/17, α-chymotrypsin 1/17, N-acetyl-ß-glucosaminidase 0/17, and ß-glucuronidase 0/17). The measurement of strain survival rate (%) under the conditions simulating gastric (pH 2.5) and bile juices (0.3% bile) showed considerable differences between strains (< 0.01 to 4.7% after 90 min for gastric juices, 48.0 to 254.0% after 180 min for bile). The concentration of produced L-lactic acid ranged between 83.1 to 119.3 mmol/L after 48 h cultivation depending on the strain. All strains fermented 16 out of 49 different carbohydrates (range from 17 to 23/49). Antimicrobial activity was recorded for two strains against some species of Listeria sp. and Enterococcus sp. Finally, two E. faecium candidates (IK25 and D7) were selected for testing in dogs, and hereafter they could possibly extend the currently limited range of beneficial bacteria of canine origin used as a dietary supplement for dogs.

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.

Enterocin M and its Beneficial Effects in Horses-a Pilot Experiment.

Probiotic bacteria or their antimicrobial proteinaceous substances called bacteriocins (enterocins) hold promising prophylactic potential for animal breeding. This study present the results achieved after application of Enterocin M in horses. Enterocin M has never been applied to horses before. Clinically healthy horses (10) were involved in this pilot experiment. They were placed in the stables of the University of Veterinary Medicine and Pharmacy, Kosice, Slovakia, with the approval of the University Ethics Committee. The animals were fed twice a day with hay and oats, or alternatively grazed with access to water ad libitum. The experiment lasted 6 weeks. Sampling was performed at the start of the experiment, at day 0-1, at day 21 (3 weeks of Enterocin M application), and at day 42 (3 weeks of cessation). Feces were sampled directly from the rectum and blood from the vena jugularis; the samples were immediately treated and/or stored for analyses. Each horse itself represented a control animal (compared to its status at the start of the experiment, day 0-1). After initial sampling, the horses were administered 100 µl of Ent M (precipitate, 12,800 AU/ml) in a small feed bolus to ensure it was consumed; Ent M was applied for 3 weeks (21 days). Fecal samples were treated using the standard microbial dilution method; phagocytic activity was assessed with standard and flow cytometry; biochemistry and metabolic profiles were tested using commercial kits and standard methods. Administration of Ent M led to mathematical reduction of coliforms, campylobacters (abP < 0.05), and significant reduction of Clostridium spp. (abP < 0.001, bcP < 0.001); increase of PA values was noted (P < 0.05, P < 0.0001); no negative influence on hydrolytic enzyme profile or biochemical blood parameters was noted.