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.

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.

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.

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 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.

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.

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.

Evaluation of Probiotic Lactobacillus fermentum CCM 7421 Administration with Alginite in Dogs.

There are growing efforts to find applications for various naturally occurring organo-mineral rocks. They have so far been preferentially used in agriculture and forestry; however, medicine and nutrition may also be interesting areas for their application. This study investigates the effects of dietary supplementation with canine-derived probiotic strain Lactobacillus fermentum CCM 7421 in combination with alginite in dogs. Alginite is a loam-like material of volcanic origin composed of clay minerals and fossilised unicellular algae. The effects of these additives on faecal microbiota, faecal characteristics, short-chain fatty acid profile, haematology, serum biochemistry and cellular immunity parameters were monitored. Forty dogs were randomly divided into four treatment groups: control group (C), alginite-supplemented group (A; 1% diet), probiotic group (LF; L. fermentum CCM 7421 at a dose of 109 cfu/day/dog) and combined group (A + LF group); 10 dogs in each group. The experiment lasted for 35 days with a 14-day treatment period (sample collection at days 0, 7, 14 and 35). The results of this straightforward experiment showed beneficial effects in the combined A + LF group. In detail, a decrease in faecal coliforms and clostridia and an increase in lactic acid bacteria, haemoglobin and serum magnesium levels compared to baseline were observed in the A + LF group (P < 0.05). In contrast, sole application of alginite (A group) led to several unexpected effects such as an increase in clostridial population and serum alanine aminotrasferase and a decrease in haemoglobin concentration (P < 0.05). The addition of alginite prevented a decrease in faecal pH and serum mineral content observed in the LF group. This indicates the possibility of applying alginite also in the nutrition of dogs as a combinative additive with probiotic bacteria for restoring optimal acid-alkali balance without affecting positive probiotic effects.

Health benefits observed after probiotic Lactobacillus fermentum CCM 7421 application in dogs.

The importance of the intestinal microbiota has attracted much interest in recent years particularly with respect to ways in which the microbiota can be manipulated in order to improve health. Improving gut health through the use of probiotic microorganisms has become an area of research activity in both human and animal nutrition. However, the amount of research using companion animals is insufficient. The present review evaluates and compares the effects achieved after application of canine-derived probiotic strain Lactobacillus fermentum CCM 7421 to healthy dogs as well as to dogs suffering from gastrointestinal disorders. The experiments involved varying duration of application (4 days-14 days), dosage (107-109 CFU), form of application (fresh culture or lyophilized from) or combination with natural substances. Results from nine independent studies show the ability of probiotic strains to establish themselves in the canine gastrointestinal tract, alter the composition of intestinal microbiota and metabolites (organic acids), and modulate the physiology (serum biochemical parameters) and immunity parameters in dogs. Almost all observed effects were irrespective of dose or duration of probiotic administration.

Safety assessment of commensal enterococci from dogs.

Enterococci form a complex, diverse, and very important group of bacteria from the technological and food safety aspect, or from the health-improving aspect as probiotics. Generally, enterococci are considered to be of low pathogenic potential, which is associated mostly with clinical strains. In these strains, production of virulence factors as well as resistance to many antimicrobial drugs could complicate treatment of nosocomial infections. Because there is a lack of information on incidence of these attributes in animal commensal enterococci, we screened 160 strains originating from feces of clinically healthy dogs in Eastern Slovakia (n = 105). The predominant species were Enterococcus faecium (57.5%) followed by Enterococcus faecalis (21.9%), and Enterococcus hirae (17.5%), while Enterococcus casseliflavus (1.9%) and Enterococcus mundtii (1.2%) rarely occurred. Among the tested antibiotics, gentamicin (high level) was the most effective drug against canine enterococci (95% of isolates were sensitive). In contrast, the highest resistance recorded (71.9%) was to teicoplanin. PCR screening showed the highest incidence of virulence genes in E. faecalis species. The most frequently detected were genes encoding adhesins efa Afm and efa Afs and sex pheromone cpd. IS16 gene, a marker specific for hospital strains, appeared in nine E. faecium strains. No strain was positive for DNase activity, 8.8% of the isolated strains showed gelatinase activity, and almost 100% strains produced tyramine. It seems commensal-derived enterococci from dogs could also to some extent be potential reservoir of risk factors for other microbiota or organisms.

Experimental application of Lactobacillus fermentum CCM 7421 in combination with chlorophyllin in dogs.

Chlorophyll belongs in a larger class of phytochemical plant pigments currently receiving more attention as a physiologically active dietary component. Although most research has focused on its biological activities such as its antioxidant, antimutagenic, anti-inflammatory or apoptotic effects in humans or rodents, there is limited knowledge at this time about the combinative possibilities of chlorophyll with probiotic bacteria. Our aim was to test the growth characteristics of canine-derived probiotic strain Lactobacillus fermentum CCM 7421 in the presence of different concentrations of chlorophyllin in vitro. Antimicrobial activity of chlorophyllin against canine indicator bacteria was also detected. In the in vivo study, chlorophyllin, L. fermentum CCM 7421 and the combination of both additives on faecal microbiota, faecal organic acid concentrations, haematological and immunological parameters in dogs were tested. Forty dogs were divided into 4 treatment groups; control (C); receiving chlorophyllin (60 mg/day/dog, CH group); L. fermentum CCM 7421 (10(8) CFU/day/dog, LF group); and both additives (CH + LF group), 10 dogs in each group. The experiment lasted for 28 days with a 14-day treatment period (sample collection at days 0, 7, 14 and 28). Results showed no growth inhibition of strain CCM 7421 by 0.05-0.25 % of chlorophyllin in broth after 24 h. Reduced growth of staphylococci, Listeria monocytogenes and Citrobacter freundii was observed at 1 % chlorophyllin (P < 0.05). In dogs, lower coliform bacteria numbers and higher concentration of propionic acid in faeces of the CH group during the treatment compared to baseline were detected (P < 0.01). Phagocytic activity of leukocytes was stimulated in all three treated groups of dogs (P < 0.05).