Strain isolation
To isolate bacterial species, skin and mucosal swabs were sampled (Amies agar gel without charcoal, Copan, Italy) from the buccal mucosa of 10 clinically healthy mares and geldings of breed Slovak warm-blood horse. Animals were kept 24/7 on the pasture in Northern Slovakia at the foot of the High Tatra Mountains. The swabs were placed into one mL of isotonic saline solution (pH 7.0) and treated according to standard microbiological method (ISO, 1:9). The aliquot (0.1 mL) from the appropriate dilutions was spread onto de Man-Rogosa-Sharpe agar (MRS, pH 6.4; Carl Roth GmbH + Co. KG, Karlsruhe, Germany) to select lactic acid bacteria. The agar plates were cultivated under anaerobic condition (Gas Pak Plus, BBL, Microbiology Systems, Cockeysville, USA) at 37°C for 48–72 h. The morphologically different colonies were reclaimed from the individual dilutions under the same growth conditions to obtain pure cultures. The isolates were selected based on Gram-positive staining and coccoid shape. For next testing, they were stored using the system Microbank™ (Pro-Lab Diagnostics, Ontario, Canada) at − 70°C.
Strain Identification: Dna Extraction, Pcr Amplification And Sequence Analysis
The genomic DNA was extracted from solitary pure colonies by using DNAzol direct (Molecular Research Center Inc., Cincinnati, USA) according to the manufacturer's instruction as previously reported Focková et al. (2022a). The 16S ribosomal RNA (rRNA) gene from isolate was amplified by PCR using the universal primers as follows: Bac27F(5-AGAGTTTGATCMTGGCTCAG-3) and 1492R (5-CGGYTACCTTGTTACGACTT-3 (Merck-Sigma Aldrich, Darmstadt, Germany). PCR reaction was carried out in a 50 µL PCR mixture containing 2 µL of DNA shield and 46 µL of a reaction mixture comprising One Taq 2x Master Mix with Standard Buffer (New England Biolabs, United Kingdom), diluted with water for molecular biology (PanReac AppliChem, Darmstadt, Germany) to 1x concentration and 1 µL of each primer (concentration 33 µM). The following PCR condition (thermocycler-TProfesional Basic, Biometra GmbH, Göttingen, Germany) was used: 94 ºC for 5 min followed by 30 cycles of denaturation at 94 ºC for 1 min, annealing at 55 ºC for 1 min, and primer extension at 72 ºC for 3 min, and finally at 72 ºC for 10 min. Aliquot PCR product was separated by horizontal 3% (w/v) agarose gel electrophoresis in Tris-acetate-EDTA buffer (pH 7.8) and visualized with GelRed (Biotium, Inc., Hayward, CA, USA). Amplified product was sent in low bind tube at minimal volume 15 µL for purification and sequencing in both directions using 1492R and Bac27F primer (Microsynth, Wien, Austria). The obtained 16S rRNA sequence was validated and assembled by Geneious 8.0.5 (Biomatters, Auckland, New Zealand) and subjected to BLASTn analysis (https://BLAST.ncbi.nlm.nih.gov/BLAST.cgi).
Growth Of Emo 1-1nik On Different Media
To check growing ability of the isolated strain, the following media were used: M-Enterococcus agar, Brain Heart infusion with agar (BHA), Slanetz-Bartley agar, and Trypticase-soy agar (Difco, Maryland, USA). The strain from blooded BHagar was inoculated on the selected agars formerly mentioned and the quality of grown colonies was checked after 48 h.
Biofilm-forming Ability Tests
The ability of the identified strain E. moraviensis EMo 1-1Nik to form biofilm was checked using the qualitative and quantitative methods. For the qualitative method Congo red agar was used and analysis described by Freemen et al. (1989). The Congo red agar plate was inoculated with the E. moraviensis EMo 1-1Nik strain. The plate was incubated at 37 ºC overnight. Biofilm formation was assessed through the presence of black colonies with dry crystalline consistency. The agar plate was then maintained at laboratory temperature and checked again at 48 and 72 h. Negative strain remained pink. Streptococcus equi subsp. zooepidemicus CCM 7316 was used as positive control (kindly provided by Dr. Eva Styková, University of Veterinary Medicine and Pharmacy in Košice, Slovakia).
The quantitative plate assay according to Chaieb et al. (2007) and Slížová et al. (2015) requested to pick up one colony of the tested EMo1-1Nik strain grown on BHAgar (Difco, USA) overnight at 37 ºC. This method was more detaily described in our previous study (Focková et al. 2022b). As a final step of analysis, a 150 µL volume was transferred from each microplate well into a new microplate well to measure absorbance (A570) in nm with the use of an Apollo 11 Absorbance Microplate reader LB 913 (Apollo, Berthold Technologies, Oak Ridge, Tennesee, USA). Testing of EMo 1-Nik1 strain was repeated in two independent runs with 12 replicates. Sterile BHI was used in analysis, serving as negative control. Streptococcus equi subsp. zooepidemicus CCM 7316 was used as positive control. Biofilm formation was classified as highly- positive (A570 ≥ 1), low-grade positive (0.1 ≤ A570 < 1) or negative (A570 < 0.1 (Chaieb et al. 2007; Slížová et al. 2015).
Virulence Factor Genes Control
The following genes for virulence factors were tested: gelE (gelatinase), esp (enterococcal surface protein), efaAfm (adhesin E. faecium), cylA (cytolysin A), hylEfm (hyaluronidase), agg (aggregation substance) and IS16 element (IS 16). The PCR product was separated using agarose gel electrophoresis (1.2% w/v, Sigma-Aldrich, Saint Louis, USA) with 1 µL/mL content of ethidium bromide (Sigma-Aldrich) using 0.5 x TAE buffer (Merck, Darmstadt, Germany). The PCR fragment was visualized with UV light. The strain E. faecalis 9Tr1 (our strain), E. faecium P36 (Dr. Semedo-Lemsaddek, University Lisbon, Portugal) was used as a positive control. The PCR was carried out in 25 µL volume, with a mixture consisting of 1x reaction buffer, 0.2 mmoL/L of deoxynucleoside triphosphate, 3 mmoL MgCl2, 1 µmoL/L of each primer, 1 U of Taq DNA polymerase, and 1.5 µL of DNA template with the cycling conditions as previously reported by Kubašová et al. (2017) and Lauková et al. (2014). The following PCR conditions (for gelE, agg, cylA, esp, efaAfm) were used: denaturation at 95°C for 3 min followed by 35 cycles for 30 s at 95°C, 30 s at 55°C, 30 s at 72°C and 5 min at 72°C. The PCR conditions for hyl and IS16 genes were as follows: denaturation at 94°C for 4 min, followed by 30 cycles for 30 s at 94°C, 30 s at 50°C, 30 s at 72°C and finally for 4 min at 72°C.
Hemolysis, Nuclease And Gelatinase Activity Testing
Hemolysis activity was evaluated using BH agar (Difco, USA) supplemented with 5% defibrinated sheep blood. Plate was incubated at 37 ºC for 48 h in an incubator. Presence/ absence of cleared zone around the colonies was interpreted as α/β-hemolysis, while those testing negative exhibited γ-haemolysis (Semedo-Lemsaddek et al. 2013).
Nuclease activity was analyzed as previously described by Lauková et al. (2022b). Tested strain was inoculated onto the surface of DNase agar (Oxoid, USA) and incubated for 24 h at 37 ºC. The production of deoxyribonuclease can be evaluated on this medium: colonies producing DNase hydrolyse the deoxyribonucleic acid (DNA) within the medium. After flooding and acidifying the medium with 1 N HCl (hydrochloric acid), the DNA precipitated, and the medium became turbid with cleared zones around DNase-positive colonies. As a positive control Staphylococcus pseudintermedius SPs 948 was used (our strain from ruminant).
Gelatinase test was performed by streaking single colonies onto Todd-Hewit agar (Difco, USA) supplemented with gelatine (Biomark, 30g/L) and incubating at 37°C for 48 h. After flooding the medium with 1.5% HgCl2 in 2.0% HCl, the medium became turbid with cleared zones around gelatinase-positive colonies. As a positive control Staphylococcus aureus ATCC 25923 was used.
Detection Of Bacteriocin - Producing Colonies Of Emo 1-1nik
EMo 1-1Nik strain was inoculated into 5 mL of BHI (pH, Difco) and incubated at 37 ˚C for 18 h. Then the appropriate amount (100 µL) was diluted in Ringer solution (Merck, Germany) and dilutions were plated on Brain heart agar (BHA, Difco) and incubated again at 37 ˚C for 24 h. Twenty-five colonies were grown on BHA from 10− 9 dilution. Plate was overlaid with 0.7% (v/w) BHI agar seeded with 0.2 mL of indicator strain Enterococcus avium EA5 (our strain) and incubated overnight 37 ˚C. Colonies with clear zones were assessed. Moreover, bacteriocin activity was checked using method according to Skalka et al. (1983), and inhibitory zone was expressed in mm.
Concentrated Substance Preparation
To prepare concentrated bacteriocin substance, 18 h culture of EMo1-1Nik strain (150 mL) in MRS broth (pH, Merck, Darmstadt, Germany) was centrifuged at 10,000 x g at 4 ˚C for half hour. Supernatant was divided into three parts (3 x 50 mL) and pH was adjusted to be pH 4.5, pH 6.3, and pH 7.3. Each volume (50 mL) of supernatant was then concentrated using Concentrator Plus (Eppendorf, Hamburg, Germany) to have concetrated substance in a final volume 4 mL. Then inhibitory activity was tested against indicator bacteria E. avium EA5 as well as against Listeria monocytogenes LM7223 (Olomouc, Czech Republic) according to De Vuyst et al. (1996) and expressed in arbitrary unit per mL.
Susceptibility Of Emo 1-1nik Strain To Mundticin Em 41/3
Susceptibility to Mundticin EM 41/3 was checked using agar diffusion test (De Vuyst et al. 1996) on BH agar plates. EMo 1-1Nik was incubated in BH broth. Inhibition activity was expressed in arbitrary units per mL meaning the highest dilution of Mundticin EM 41/3 which inhibited the growth of indicator strain (Focková et al. 2022).