Enterococcus moraviensis EMo 1-1Nik of horse origin:characteristics and potential bacteriocin-producing strain.

Nowadays, developed more precisious identification techniques have allowed to validate newer enterococcal species. Among them, the species Enterococcus moraviensis was also validated, at first from surface waters. However, in this study, characteristics and potential to bacteriocin production by the strain E. moraviensis EMo 1-1Nik isolated from buccal mucosa of Slovak warm-blood horse breed has been studied. BLASTn analysis allotted this strain to the species E. moraviensis with percentage identity BLASTn 16S rRNA sequence in the strain up to 100% (99.93% similarity with E. moraviensis NR113937.1). The strain EMo 1-1Nik has been provided with GenBank accession number MW326085. It is hemolysis-negative (γ-hemolysis), deoxyribonuclease-negative and gelatinase-negative; absent of virulence factor genes, low-grade biofilm-positive (0.133 ± 0.36), mostly susceptible to tested antibiotics. Moreover, 60% of EMo1-1Nik colonies were found as bacteriocin-producing against the principal indicator Enterococcus avium EA5. The concentrated substance (CS, pH 4.5) of EMo1-1Nik showed the inhibitory activity against EA5 strain (800 AU/mL); CSs with pH 6.3 and 7.3 reached inhibitory activity 100 AU/mL against EA5 strain. CS was thermo-stable and it does not lost activity after enzymes treatment. Oppositelly, EMo 1-1Nik was susceptible to Mundticin EM 41/3 (800 AU/mL) produced by horse fecal strain E. mundtii EM 41/3 and enterocins (up to 51 200 AU/mL). In spite of the preliminary results, it has been shown a potential to produce bacteriocin substance of the safe strain E. moraviensis EMo1-1Nik. The additional studies are in processing.

Antibiofilm Activity of Weissella spp. and Bacillus coagulans Isolated from Equine Skin against Staphylococcus aureus.

The aim of this study was to evaluate the antimicrobial and antibiofilm activity of Weissella cibaria, Weissella hellenica and Bacillus coagulans, isolated from equine skin, against biofilm-forming Staphylococcus aureus CCM 4223 and clinical isolate methicillin-resistant S. aureus (MRSA). Non-neutralized cell-free supernatants (nnCFS) of tested skin isolates completely inhibited the growth and biofilm formation of S. aureus strains and caused dispersion of the 24 h preformed biofilm in the range of 21-90%. The majority of the pH-neutralized cell-free supernatants (nCFS) of skin isolates inhibited the biofilm formation of both S. aureus strains in the range of 20-100%. The dispersion activity of B. coagulans nCFS ranged from 17 to 77% and was significantly lower than that of nnCFS, except for B. coagulans 3T27 against S. aureus CCM 4223. Changes in the growth of S. aureus CCM 4223 in the presence of catalase- or trypsin-treated W. hellenica 4/2D23 and W. cibaria 4/8D37 nCFS indicated the role of peroxides and/or bacteriocin in their antimicrobial activities. For the first time, the presence of the fenD gene, associated with biosurfactants production, was detected in B. coagulans. The results of this study showed that selected isolates may have the potential for the prevention and treatment of biofilm-forming S. aureus infections.

Detection of Periodontal Pathogens from Dental Plaques of Dogs with and without Periodontal Disease.

Dental plaque bacteria are one of the main factors responsible for the development of a periodontal disease, which is the most common infectious disease in dogs. The aim of this study was to identify the presence of periodontal disease-related bacteria in the dental plaque of dogs. Plaque samples were taken from dogs with and without periodontal disease. Samples were analyzed for the presence of Porphyromonas gulae, Tannerella forsythia and Treponema denticola using a PCR technique amplifying 16S rRNA genes of P. gulae and T. forsythia and flaB2 genes of Treponema species, including T. denticola. The presence of T. forsythia was confirmed in all samples. P. gulae was detected in all dogs with periodontal disease and in 71.43% of dogs without periodontal disease. Treponema spp. were detected in 64.29% of the samples. Based on Sanger sequencing and Basic Local Alignment Search Tool algorithm, Treponema spp. were identified as T. denticola and Treponema putidum. T. denticola was present in 28.57% of dogs with periodontal disease, while T. putidum was present in 42.86% of dogs with periodontal disease and in 57.14% of dogs without periodontal disease. T. putidum was positively correlated with both P. gulae and T. forsythia, suggesting that it may be involved in the development of periodontal disease.

Antiadhesive, antibiofilm and dispersion activity of biosurfactants isolated from Bacillus amyloliquefaciens 3/22.

The aim of this work was to monitor the potential antibiofilm properties of biosurfactants (BS) isolated from Bacillus amyloliquefaciens 3/22 against biofilm formation of the indicator strain Staphylococcus aureus CCM 4223. In this work, the effect of BS 3/22 on biofilm growth during co-incubation, inhibition of biofilm-forming cell adhesion and biofilm dispersion was studied. BS 3/22 inhibited biofilm formation, with its formation decreasing significantly (p < 0.05; p < 0.01; p < 0.001) with increasing BS 3/22 concentration. BS 3/22 also showed antiadhesive activity, which correlated with the concentration used. The dispersing effect of isolated BS 3/22 on a 24-hour biofilm was also detected. BS 3/22 were effective in biofilm dispersion even at lower concentrations compared to antiadhesive activity and inhibition of biofilm formation.

The Presence of Treponema spp. in Equine Hoof Canker Biopsies and Skin Samples from Bovine Digital Dermatitis Lesions.

Equine hoof canker and bovine digital dermatitis are infectious inflammatory diseases of the hooves with an unknown etiology. However, anaerobic spirochetes of the genus Treponema are considered to be potential etiological agents. The aim of this study was to find a suitable way to isolate DNA and to detect the presence of treponemal DNA in samples of equine hoof canker and bovine digital dermatitis. DNAzol®® Direct and column kits were used to isolate DNA from samples of equine hoof canker and bovine digital dermatitis. The presence of Treponema spp. was detected using PCR and Sanger sequencing. DNAzol®® Direct is suitable for isolating DNA from these types of samples. Treponemal DNA was detected in equine hoof samples as well as in bovine digital dermatitis skin samples. In equine hoof biopsies, the most frequently detected was Treponema pedis (8/13). Treponema brennaborense (2/13) and Treponema denticola (2/13) were also found. In the case of bovine digital dermatitis, Treponema medium ssp. bovis was confirmed in 14 of 36 skin samples. Treponema pedis (9/36), Treponema vincentii (1/36), Treponema phagedenis (1/36), and Treponema brennaborense (1/36) were detected as well. DNAzol®® Direct was more appropriate for isolation of treponemal DNA because the columns isolation method was more equipment and time-consuming. The presence of several Treponema spp. was determined in the samples. In horses, the most commonly detected species was a T. pedis, while in cattle it was T. medium ssp. bovis.

Bacillus amyloliquefaciens-Derived Lipopeptide Biosurfactants Inhibit Biofilm Formation and Expression of Biofilm-Related Genes of Staphylococcus aureus.

Biosurfactants (BSs) are surface-active compounds produced by diverse microorganisms, including the genus Bacillus. These bioactive compounds possess biological activities such as antiadhesive, antimicrobial and antibiofilm effects that can lead to important applications in combating many infections. Based on these findings, we decided to investigate the antibiofilm activity of BSs from the marine Bacillus amyloliquefaciens against Staphylococcus aureus CCM 4223. Expression of biofilm-related genes was also evaluated using qRT-PCR. Isolated and partially purified BSs were identified and characterized by molecular tools and by UHPLC-DAD and MALDI-TOF/MS. Bacillus amyloliquefaciens 3/22, that exhibited surfactant activity evaluated by oil spreading assay, was characterized using the 16S rRNA sequencing method. Screening by PCR detected the presence of the sfp, srfAA, fenD and ituD genes, suggesting production of the lipopeptides (LPs) surfactin, fengycin and iturin. The above findings were further supported by the results of UHPLC-DAD and MALDI-TOF/MS. As quantified by the crystal violet method, the LPs significantly (p < 0.001) reduced biofilm formation of S. aureus in a dose-dependent manner and decreased expression of biofilm-related genes fnbA, fnbB, sortaseA and icaADBC operon. Data from our investigation indicate a promising therapeutic application for LPs isolated from B. amyloliquefaciens toward prevention of S. aureus biofilm infections.

Antiadhesive, antibiofilm and dispersion activity of biosurfactants isolated from Bacillus amyloliquefaciens 3/22.

The aim of this work was to monitor the potential antibiofilm properties of biosurfactants (BS) isolated from Bacillus amyloliquefaciens 3/22 against biofilm formation of the indicator strain Staphylococcus aureus CCM 4223. In this work, the effect of BS 3/22 on biofilm growth during co-incubation, inhibition of biofilm-forming cell adhesion and biofilm dispersion was studied. BS 3/22 inhibited biofilm formation, with its formation decreasing significantly (p < 0.05; p < 0.01; p < 0.001) with increasing BS 3/22 concentration. BS 3/22 also showed antiadhesive activity, which correlated with the concentration used. The dispersing effect of isolated BS 3/22 on a 24-hour biofilm was also detected. BS 3/22 were effective in biofilm dispersion even at lower concentrations compared to antiadhesive activity and inhibition of biofilm formation.

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 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.

Biosurfactants and their role in the inhibition of the biofilmforming pathogens.

Resistance of pathogenic bacteria is currently one of the major medical problems. Most microbial infections are based on the formation of biofilms, which are a significant reservoir of pathogens. The aim of this study is to determine the antibiofilm and antimicrobial activity of biosurfactants isolated from intestinal lactobacilli and marine bacteria. Biosurfactants (BS) isolated from the strains L. fermentum 2I3, L. fermentum B2/6, L. reuteri SL16, L. reuteri B6/1, S. luteola 3/22, Brevibacillus sp. 4/9, Brevibacillus sp. 2/30 and B. amyloliquefaciens 1/6K significantly (p < 0.001) inhibited the biofilm formation of S. aureus CCM 3953 and P. mirabilis CCM 7188, with higher inhibition detected in BS of marine bacteria when compared to BS isolated from lactobacilli. The results suggest that the mechanism of the antibiofilm effect of BS isolated from lactobacilli against both the reference strains is the same and it is not the result of their antimicrobial action. In contrast, the mechanism of the antibiotic effect of BS isolated from marine bacteria probably depends on the properties of the indicator strain. Key words: biosurfactants biofilm pathogens inhibition.

Bovine vaginal strain Kocuria kristinae and its characterization.

Kocuria spp. are widely distributed in nature. They are Gram-positive, coagulase-negative, coccoid bacteria belonging to the family Micrococcaceae, suborder Micrococcineae, order Actinomycetales, class Actinobacteria. In general, limited knowledge exists concerning the properties associated with the representants of the genus Kocuria, Kocuria kristinae as well. Following our previous results, K. kristinae Kk2014 Biocenol(™) (CCM 8628) was isolated from vagina of a healthy cow. Its taxonomical allottation was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identification system and phenotypic characteristics. Kk2014 strain showed strong adherence capability to the vaginal mucus, produced organic acids which can play a role in prevention of unsuitable contamination, and showed in vitro antagonistic/antimicrobial activity against strains Arcanobacterium pyogenes CCM 5753, Fusobacterium necrophorum CCM 5982, Streptococcus equi subsp. zooepidemicus CCM 7316, and Gardnerella vaginalis CCM 6221. Antimicrobial activity ranged from 100 to 200 AU/mL, up to 32 mm in size, respectively.