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21 April 2026

10 Pages

Postbiotic Substance Durancin-like ED26E/7 as a Tool for In Vitro Treatment of Water-Derived Strains Enterococcus haemoperoxidus and Enterococcus moraviensis

,
,
and
Centre of Biosciences of the Slovak Academy of Sciences, Institute of Animal Physiology, Šoltésovej 4-6, 04 001 Košice, Slovakia
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Author to whom correspondence should be addressed.
Hygiene2026, 6(2), 22;https://doi.org/10.3390/hygiene6020022
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Abstract

Enterococci are found not only in warm-blooded animals but also as a resident population of water and an indicator of fecal pollution. The species Enterococcus haemoperoxidus and E. moraviensis are relatively new, having been detected in water. Strains with genes for biofilm formation can serve as reservoirs for gene transfer. The aim and novelty of this study were to determine whether the postbiotic substance (PS) of proteinaceous character (Durancin-like) produced by our strain Enterococcus durans ED26E/7 can inhibit in vitro the growth of biofilm-forming E. haemoperoxidus and E. moraviensis isolated from water sources. E. haemoperoxidus and E. moraviensis lacked the biofilm-forming genes ica, bap, ace, and fsrA. The bopD gene was found only in three strains of E. haemoperoxidus. The srtA gene was present in two strains of E. haemoperoxidus and E. moraviensis. Using the quantitative test, three strains showed low-grade biofilm-forming ability. They exhibited ɤ-hemolysis, and they were mostly susceptible to antibiotics. However, they were susceptible to PS Durancin-like ED26E/7. E. haemoperoxidus, the strains EHae466 and EMo494, showed the highest susceptibility to Durancin-like ED26E/7.

1. Introduction

In general, water is one of the essential resources for sustaining life. However, surface water is especially prone to bacterial contamination. This is because water receives wastes and pollutants from human and animal sources. The contaminated water may pose a health risk to the population [1,2]. To reduce public health risks, monitoring the water for any microbial pollutants is essential. Enterococci are bacteria not only associated with warm-blooded animals but also have been detected in extra-intestinal habitats [3]. They are ubiquitous bacteria distributed in natural environments. Even the incidence of enterococci in water (primarily Enterococcus faecalis and E. faecium) has been considered as an indicator of fecal pollution [4]. There, enterococci were found to be the resident population in water [4]. Besides E. faecalis and E. faecium, the species E. durans and E. hirae have especially been considered to be of fecal origin [5]. However, non-fecal-origin enterococci can also be found in surface waters, e.g., the species E. casseliflavus. Among “newly” discovered species of enterococci frequently indicated in drinking and surface waters were the species E. haemoperoxidus and E. moraviensis [3,5,6]. Differences in enterococcal species composition across various water sources may be influenced by environmental factors such as warm temperatures, which favor bacterial growth. In the case of surface water, rainfall can also contribute to these factors, transporting bacteria into water along with runoff from surrounding vegetation and soil, animal feces, and/or sewage leakage [2]. Fecal contamination from animal and human sources can pose health risks to local populations exposed to environmental water through municipal, agricultural, and recreational uses [2]. Taučer-Kapteijn et al. [3] found the species E. haemoperoxidus and E. moraviensis in the feces of geese, and E. moraviensis in the feces of rabbits and foxes. On the other hand, in our previous study [7], E. haemoperoxidus and E. moraviensis from waters possessed genes for the production of proteinaceous antimicrobial compounds- bacteriocins-Enterocins, which are postbiotic substances purified into homogeneity. Substances of proteinaceous character that are not purified to homogeneity are indicated as postbiotic substances [8]. The postbiotic substance Durancin-like ED26E/7 is produced by the strain Enterococcus durans ED26E/7 isolated from the ewe’s milk lump cheese [9]. It is a safe strain; it does not cause mortality in BALB/c model mice. Postbiotic substances produced by this strain inhibited the principal indicator Enterococcus avium EA5 with an inhibitory activity of 102,400 AU/mL. Moreover, the growth of Staphylococcus aureus SA5—the other staphylococci—and Listeriae was inhibited [9]. The aim of this study was to determine whether the postbiotic substance (PS) can successfully inhibit the growth of the strains E. haemoperoxidus and E. moraviensis from water sources in vitro, indicating PS as a tool for maintaining sustainable hygiene conditions following further in situ studies. The reason is that contaminated water, mainly drinking and/or swimming pool waters, can cause waterborne disease. According to the literature, there are some bacteriocins that have been successfully used in vitro models to fight skin infections, but also oral, respiratory, gastrointestinal, and urogenital tract infections [10], e.g., lantibiotic nisin in bovine mastitis [11] and even Enterocin 96 produced by the strain E. faecalis WHE96 isolated from Munster cheese used against cutaneous infections [12]. In this study, besides the basic properties, E. haemoperoxidus and E. moraviensis were tested to have biofilm genes (ica, ace, bap, bopD, fsrA, and srtA), and for their biofilm-forming ability, antibiotic profile using E-strips, enzyme production, and susceptibility to the postbiotic substance Durancin-like ED26E/7 to indicate it as an original and novel tool to inhibit in vitro water-derived contaminants.

2. Materials and Methods

2.1. Source of Strains

The studied strains were derived from drinking, swimming pool water, and a spring in the region of North Moravia (Czech Republic) [7]. They were kindly supplied by Pavel Švec from the Czech Collection of Microorganisms at Masaryk University in Brno (Brno, Czech Republic) for their next studies. The strains’ identification method was previously reported by Švec et al. [6,7] and Pantuček et al. [13]. They were identified using ribotyping with EcoRI and HindIII. Moreover, tRNA intergenic length polymorphism analysis (tDNA-PCR) was applied to identify these species strains, followed by 16S rDNA sequence analysis, DNA base composition analysis, and DNA-DNA hybridization [5,6]. The following strains were studied: Enterococcus haemoperoxidus EHae382 isolated from drinking water in location Čeladná, EHae562 (drinking water in Lhotka), EHae 466 (swimming pool in Třinec), EHae 473 (drinking water in Třinec), Enterococcus moraviensis CCM 4860 isolated from drinking water in location Fryčovice, EMo494 (drinking water in Frýdek-Místek), EMo CCM4859 and EMo CCM4885 (drinking water in Frýdek-Místek), and EMo330T CCM4856T=LMG19487T (sample from spring in location Hájek) [5].

2.2. Detection of Biofilm-Associated Genes and Quantification of Biofilm-Forming Ability

The detection of the following genes responsible for biofilm formation was analyzed: ace, bap, bopD, fsrA, ica, and srtA. They are proteins responsible for different phases of biofilm formation [14,15,16,17,18]. The primers used for this test are summarized in Table 1. Processing was performed using the thermocycler BJ Mini (Bio-Rad Laboratories, San Francisco, CA, USA). The following process was carried out to detect the bap gene: initial denaturation at 94 °C (5 min), followed by 30 s at 94 °C, annealing at 55 °C, 30 cycles for 30 s, extension at 72 °C for 45 s, and final extension at 72 °C for 7 min. The ica gene was amplified with an initial denaturation cycle of 2 min at 94 °C, followed by denaturation at 94 °C for 20 s, annealing at 42 °C for 30 cycles of 20 s, extension at 72 °C for 50 s, and final extension at 72 °C for 5 min. The other genes were detected according to the protocol reported by Hashem et al. [19]. The amplified PCR product was separated by 1.2% agarose gel electrophoresis (Sigma-Aldrich, St. Louis, MO, USA) using 1.0 μg/mL content of ethidium bromide in 0.5 × TAE buffer (Merck KGaA, Darmstadt, Germany). The product was visualized under ultraviolet light. The positive control strains were included in the references as previously reported by Lauková et al. [20].
Table 1. Oligonucleotides used in the study to amplify biofilm-forming ability in Enterococcus haemoperoxidus and E. moraviensis.
The biofilm-forming ability of the E. haemoperoxidus and E. moraviensis strains was tested using the quantitative method [21]. A pure colony of the strain grown on BHA (Difco, Tucker, GA, USA) at 37 °C for 18–24 h was picked up and transferred into 5 mL of Ringer solution (pH 7.0; 0.75% w/v; Merck, Darmstadt, Germany), to have a suspension which corresponded to 1.0 × 108 CFU/mL. The dilution (100 μL) was transferred into the microtiter plate wells (Greiner, ELISA 12-well strips, Frickenhausen GmBH, Frickenhausen, Germany). The plates were cultivated for 24 h at 37 °C. The biofilm formed in the plate well was washed twice with 200 μL of deionized water. The plates were dried at the laboratory’s temperature for 40 min. The attached bacteria were stained with 200 μL of 0.1% crystal violet in deionized water at 25 °C for 1 h. After the dye solution was aspirated, the wells were washed twice with 200 μL of deionized water, and the plates were dried for 30 min at room temperature. The dye bound to the adhered biofilm was eluted with 200 μL of 95% ethanol. To measure absorbance at A570, a volume of 150 μL was transferred to each well of a new microtiter plate. The reader Apollo 11 LB913 (Apollo, Berthold technologies, Oak Ridge, TN, USA) was used. Two independent runs with 12 replicates were prepared for measurement, including a negative control (BHI) and the positive Streptococcus equi subsp. zooepidemicus CCM7316 control (kindly supplied by Dr. Eva Styková from the University of Veterinary Medicine and Pharmacy in Košice, Slovakia). The following classification, according to Chaieb et al. [21], was used: highly positive strain A570 ≥ 1.0, low-grade positive, 0.1 < A570 ≤ 1.0, and negative A570 ≤ 0.1

2.3. Hemolysis, Enzyme Production Testing by API ZYM Panel

To evaluate hemolysis, a method previously reported by Semedo-Lemsaddek et al. [22] was used. The strains were inoculated onto Brain Heart agar (BHA, Difco, Sparks, NV, USA) plates enriched with 5% defibrinated sheep blood and cultivated at 37 °C for 18 h. The presence or absence of a cleared zone around grown colonies was assessed as α, β, and ɤ-hemolysis (negative). A positive control strain was also included [22].
Enzyme production was tested using the commercial API ZYM panel (BioMérieux, Marcy L’Étoile, France) as previously reported [20]. The production of the following enzymes was tested: alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystin arylamidase, trypsin, α-chymotrypsin, acid phosphatase, Naphtol-AS-Bi-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase. The color intensity was assessed from 0 to 5, which corresponds with relevant values in nanomoles (5–40 nmol). Testing was performed in duplicate, including a reference strain.

2.4. Antibiotic Profile

Antibiotic susceptibility was tested according to EUCAST guidelines, assessing the minimal inhibitory concentration (MIC). The E-strip method previously described by Lauková et al. [20] was used. The tested strain in BHI broth (Difco, Sparks, NV, USA) was incubated in the incubator at 37 °C overnight. Then, 100 μL of broth culture was spread onto BHA (Difco, Sparks, NV, USA). The individual E-strips were placed on the surface of the BHA, and the plates were incubated at 37 °C overnight. The MICs were read according to the manufacturer’s instructions. The following E-strips were used: ampicillin (256–0.016 µg/mL); erythromycin (256–0.016 µg/mL); tetracycline (256–0.016 µg/mL); vancomycin (256–0.016 µg/mL); streptomycin (256–0.064 µg/mL); chloramphenicol (256–0.016 µg/mL; and gentamicin (1024–0.064 µg/mL. The E-strips were supplied by Oxoid (Basingstoke, Hampshire, United Kingdom). Kanamycin was tested by the disc method (Lachema, Brno, Czech Republic) at a concentration of 30 µg. The positive control was Enterococcus faecalis ATCC 29212, recommended by the manufacturer.

2.5. Postbiotic Substance Preparation and Treatment of Strains

A partially purified postbiotic substance ED26E/7 was prepared according to Lauková et al. [9]. Briefly, E. durans ED26E/ was grown in 500 mL of De Man–Rogose–Sharpe broth (MRS, Merck, Darmstadt, Germany) at 37 °C for 18 h. The broth culture was centrifuged (10,000× g) for half an hour at 4 °C. The pH of the supernatant was adjusted to 4.2, and the supernatant was filtered through a 0.45 µm filter (Millipore Corporation, Bedford, MA, USA), followed by precipitation of the supernatant with ammonium sulfate (80% saturation) at 4 °C for 18 h. The precipitate was re-suspended in the minimum volume of phosphate buffer (pH 6.5). Inhibitory activity was measured using the agar spot test [23] against Enterococcus avium EA5 (the principal indicator, our strain) and expressed in arbitrary units per mL. AU/mL is defined as a two-fold dilution of the prepared postbiotic substance in phosphate buffer, the highest dilution of which still causes inhibition of the indicator (tested) strain. Inhibitory activity against Enterococcus avium EA5 reached 102,400 AU/mL. The broth cultures of the tested (indicator) strains of E. haemoperoxidus and E. moraviensis, grown overnight in BH broth (Difco, USA), were checked at 600 nm (Spectrometer Specol 11, Zeiss Jena, Germany) to an absorbance of 0.8–1.0. The BHA plates (1.5%, w/v) were overlaid with 4 mL of soft BH agar (0.7%, w/v) inoculated with 200 µL of the indicator strain. The PS (Durancin-like ED26E/7) was diluted 2-fold in phosphate buffer (pH 6.5, 1:1), and 10 μL of the appropriate dilution was spotted on the surface of BHA. The plates were kept in the fridge for 10 min to improve diffusion, then they were incubated at 37 °C. The first check was after 4 h, then after 18 h.

3. Results

3.1. Biofilm Genes Detection and Biofilm-Forming Ability in Strains

The tested strains of E. haemoperoxidus and E. moraviensis lacked ica, bap, ace, and fsrA genes. The bopD gene was found only in three strains of E. haemoperoxidus (EHae382, EHae473, and EHae562; Table 2). The srtA gene was present in two strains of E. haemoperoxidus (EHae382, EHae562), and in two strains of E. moraviensis (EMo494 and EMo CCM4856T). This means that EHae 466, EMo CCM4856T, EMo CCM4859, EMo CCM4860, and EMo CCM4885 did not contain any of the six tested genes responsible for biofilm formation (Table 2). Five strains out of nine (three of E. haemoperoxidus and two strains of E. moraviensis) contain at least one gene. EHae382 and EHae562 contain both bopD and srtA genes (Table 2). While the bopD gene was detected only in the E. haemoperoxidus strains, the srtA gene was detected in the strains of both species (Table 2).
Table 2. Detection of biofilm-forming ability and biofilm genes (±SD).
Using the quantitative method to test biofilm-forming ability, three out of nine tested strains were found to have low-grade biofilm-forming ability (0.1 < A570 ≤ 1.0): one strain of E. haemoperoxidus EHae473 and two strains of E. moraviensis (CCM4859 and CCM4860, Table 2). In general, the biofilm-forming ability of the tested strains ranged from 0.109 ± 0.02 to 0.178 ± 0.00, with the highest value observed for the strain EMoCCM4885 (0.178 ± 0.00). Six strains (three of each species) did not show biofilm-forming ability (measured values from 0.061 ± 0.02 to 0.097 ± 0.03, Table 2).

3.2. Test of Hemolysis, Enzyme Production

The strains of E. haemoperoxidus and E. moraviensis lacked hemolysis. They were assessed as having a negative reaction, meaning ɤ-hemolysis.
The enzymes alkaline phosphatase, lipase (C14), α-galactosidase, β-glucuronidase, α-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase were not produced by the tested strains. The values of the produced enzyme esterase (C4) in the tested strains ranged from 5 to 20 nmol. For esterase-lipase (C8), measured values ranged from 5 to 10 nmol. The values of leucine arylamidase ranged from 5 to 10 nmol for both species (Table 3). The enzyme valine arylamidase was produced by only two strains (5 nmol; Table 3): one E. haemoperoxidus (EHae466) and one E. moraviensis (CCM4860). Similarly, the enzyme cystin arylamidase was produced by three strains: EHae562, EHae466, and the strain of E. moraviensis CCM4860. The strains of E. haemoperoxidus did not produce trypsin; among the strains of E. moraviensis, the production of trypsin was measured only in EMo CCM4860 (5 nmol). The enzyme α-chymotrypsin was produced by all E. haemoperoxidus but only at a low value (5 nmol). Two strains of E. moraviensis, EMo CCM4860 and EMo494, produced a low (5 nmol) amount of α-chymotrypsin. Acid phosphatase was measured only in strain CCM4885 of E. moraviensis. The E. hemoperoxidus strains did not produce acid phosphatase. Naphtol-AS-BI-phosphohydrolase was not produced only by the strain EHae466, but the values for the other strains ranged from 5 to 10 nmol. In total, the highest amounts were measured for the enzyme β-glucosidase (10–40 nmol). The value of 40 nmol reached the strain EHae466. The same value, 40 nmol, was measured in EHae466 for the enzyme β-galactosidase. In the other strains, the enzyme β-galactosidase was measured at levels of 5–20 nmol. The strain E. moraviensis CCM4860 produced 10 of 19 tested enzymes, the highest number of any strain tested. The other strains produced 6–8 enzymes in total (Table 3).
Table 3. The production of enzymes by the tested enterococci.

3.3. Susceptibility to Antibiotics and Postbiotic Substance ED26E/7

The strains were resistant to kanamycin (30 μg/mL). This means the strains were not inhibited by kanamycin, but were resistant to kanamycin. However, E. haemoperoxidus and E. moraviensis were the most susceptible to antibiotics (Table 4). The minimal inhibitory concentrations (MICs) for ampicillin among the strains ranged from 0.023 µg/mL to 0.125 µg/mL (Table 4). The MICs for erythromycin ranged from 1.00 µg/mL to 1.75 µg/mL. The strains were susceptible to tetracycline, with MICs ranging from 0.125 µg/mL to 0.19 µg/mL. The MICs for vancomycin ranged from 0.50 µg/mL to 1.25 µg/mL. The MICs for streptomycin ranged from 4.16 µg/mL to 32.00 µg/mL, for chloramphenicol from 1.50 µg/mL to 8.00 µg/mL, and for gentamicin from 5.00 µg/mL to 19.00 µg/mL. The strain EMo494 was the most susceptible to antibiotics; its MICs regarding individual antibiotics were the lowest, except tetracycline (Table 4). All MICs fall within the susceptible range according to the EUCAST assessment. The values for gentamicin and streptomycin are below the resistance breakpoints, also confirming their susceptibility.
Table 4. Susceptibility to antibiotics (minimal inhibitory concentration, MIC), and to postbiotic substance ED26E/7 of the strains of Enterococcus haemoperoxidus and E. moraviensis.
In addition, the strains were susceptible to postbiotic substance (PS) ED26E/7 treatment with inhibitory activity ranging from 200,0 ± 0.0 to 12,800 ± 0.0 AU/mL. E. moraviensis EMo 494 was the most susceptible not only to antibiotics as formerly reported, but it is also the most susceptible to PS ED26E/7 (an inhibitory activity of 12,800 AU/mL. The strain of E. haemoperoxidus EHae466 was the most susceptible among this species; the other strains showed inhibitory activity ranging from 200 to 6400 AU/mL. However, the E. moraviensis strains were more susceptible to PS, with inhibitory scores ranging from 2000 to 12,800 AU/mL (Table 4).

4. Discussion

The strains of the species E. haemoperoxidus and E. moraviensis possess, like the other enterococci, the innate ability to form biofilms (surface-attached bacterial communities), which enhance survival, persistence, and resistance to environmental stress. Biofilm growth enables bacteria to persist, making their eradication difficult. Biofilms develop in multiple stages to form highly ordered communities [24]. At first, bacteria adhere to a surface or host tissue (primary attachment phase), followed by the accumulation phase, when they proliferate to form multicellular aggregates. During the maturation phase, structures that allow nutrient penetration occur [25,26]. At the end, bacteria detach from the biofilm and disseminate to new sites (dispersal phase) [26]. There are genes responsible for individual phases/stages of biofilm formation. Therefore, to assess biofilm formation in the strains, the detection of those genes has been requested. In this study, six genes were tested: ace, ica, bap, bopD, fsrA, and srtA. Ace is a surface protein important in the initial adhesion of biofilm-forming bacteria [14]. The transcriptional regulator BopD protein has been shown to function in vitro for biofilm formation [15]. SrtA is an enzyme that functions in surface protein processing in biofilm formation [16,17]. FsrA possesses a function in quorum sensing and may regulate many biofilm-related genes [17]. Ica and bap are surface proteins that participate in the cellular adhesion step in biofilm formation [18]. E. haemoperoxidus and E. moraviensis studied were absent of ica, bap, ace, and fsrA genes. The bopD gene was detected only in the strains of E. haemoperoxidus, and the srtA gene was detected more frequently in E. moraviensis strains. Two E. heamoperoxidus contained both genes. Although both genes were detected in the strains EHae382 and EHae562, quantifying their biofilm-forming ability using the plate well method was negative. On the other hand, E. moraviensis EMo CCM4859 and EMo CCM4860 were absent of genes, but with biofilm-forming ability tested by the plate well assay at low-grade intensity. However, the strains were susceptible to most antibiotics as assessed according to the EUCAST guide. Moreover, they were susceptible to PS ED26E/7. Algburi et al. [27] reported that the lantibiotic bacteriocin-subtilosin prevents biofilm formation by inhibiting bacterial quorum sensing. In our previous study, the susceptibility to Enterocin M (produced by the environmental strain E. faecium AL41-CCM8558) and to EntA/P (produced by another environmental strain E. faecium EK13=CCM7419) was described [7]. As reported [7], the strains of E. moraviensis were susceptible to both substances. The most susceptible strains were EMo CCM4857 and EMo CCM4860 (51,200 AU/mL). The strains of E. haemoperoxidus were less susceptible to PS ED26E/7. EMo494 was the most susceptible to Ent M [7]. Similarly, in this study, EMo494 was the most susceptible to PS ED26E/7 (12,800 AU/mL). EHae 382 was resistant to both Ent M and Ent A/P. However, it was susceptible to PS ED26E/7 (400 AU/mL). Enterococcal indicator bacteria, in general, have been susceptible to PS produced by enterococci, e.g., the strains of E. thailandicus from beavers are susceptible to PS ED26E/7 (inhibitory activity 6400 AU/mL) [28]. Moreover, lantibiotic nisin, alone and also in combination with antibiotics, was effective against Staphylococcus biofilms [29]. On the other hand, Lauková et al. [7] reported previously that EHae466 alone produces a thermo-stable antimicrobial (postbiotic) substance, which is active against 21 Gram-positive indicator strains, including enterococci, staphylococci, and listeriae (inhibitory activity up to 12,800 AU/mL) [7]. Moreover, the strain E. moraviensis EMo1-1Nik of horse origin produces a thermo-stable PS that inhibits Gram-positive species [30]. In general, PS/bacteriocins can act in different ways, e.g., by inhibiting septum formation in susceptible bacteria, or by depolarizing the membrane [31]. In particular, class II bacteriocins act this way, and PS/EntED26/7 seems to belong to this class II, a group of small antimicrobial peptides.
In this study, the strains of both species showed ɤ-hemolysis, which corresponds with their general characterization. They do not produce harmful enzymes and are susceptible to most antibiotics (except chromosomally encoded kanamycin). They are mostly non-biofilm producers. The strain EHae466 even produces the useful enzyme β-galactosidase (a key enzyme in energy production and as a source of carbon through the breakdown of lactose to galactose and glucose). It is also susceptible to antibiotics, lacks genes for biofilm formation, and produces a heat-stable active PS [7], all of beneficial properties. The fact that tested strains mostly do not produce biofilm can be associated with their susceptibility to antimicrobials. Silva et al. [32] reported that, e.g., ampicillin and/or vancomycin-resistant enterococci produce more biofilm mass than those susceptible, suggesting a link between resistance and biofilm formation [32]. Results indicate that PS shows in vitro efficacy of 12,800 AU/mL against low-biofilm-forming water enterococci, warranting pilot-scale water-treatment trials. As we know, the future is within the framework of the food–human–animal–environment strategy.

5. Conclusions

E. haemoperoxidus and E. moraviensis studied lacked ica, bap, ace, and fsrA genes. The bopD gene was found only in the strains of E. haemoperoxidus (EHae382; EHae473, and EHae562). The srtA gene was present in the strains of E. haemoperoxidus (EHae382; EHae562), and also in two strains of E. moraviensis (EHae382 and EHae562). The strains (one EHae and two EMo) were low-grade biofilm-forming. All strains were hemolysis-negative (ɤ-hemolysis), mostly susceptible to antibiotics, and susceptible to the postbiotic substance ED26E/7. Among the species E. heamoperoxidus, the strain EHae466 was the most susceptible (6400 AU/mL) to PS ED26E/7. Regarding the species E. moraviensis, the strain EMo494 was the most susceptible, as well as in general (12,800 AU/mL). Despite the preliminary results, it is suggested to assume PS as a potential tool to reduce water-derived contaminant bacteria. Of course, additional continuing studies are requested.

Author Contributions

Conceptualization, A.L.; methodology, A.L., E.B. and J.Š.; validation, A.L.; investigation, A.L., E.B. and J.Š.; data curation, A.L.; writing—original draft preparation, A.L.; writing—review and editing, A.L.; supervision, A.L.; project administration, A.L. and M.P.S.; funding acquisition, M.P.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Slovak Scientific Agency VEGA (Vedecká grantová agentúra (Slovak Scientific Agency)), the project no. 2/0006/2017, and 2/0009/25.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

We are grateful to Ivo Sedláček and Pavel Švec from the Masaryk University in Brno (Czech Republic), Czech Culture Collection, for their kindness in supplying us with E. haemoperoxidus and E. moraviensis strains for their next studies. Hemolysis results were previously included in the article by Lauková et al. [7]. Properties of the strains Enterococcus haemoperoxidus and E. moraviensis, new species among enterococci. In Folia Microbiologica 2007, 53, 273–279. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Conflicts of Interest

The authors declare no conflicts of interest.

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