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Article

Treatment of Fecal Enterococci from European Brown Hares (Lepus europaeus) with Postbiotic Substances

by
Andrea Lauková
1,*,
Jana Ščerbová
1,
Ľubica Chrastinová
2,† and
Monika Pogány Simonová
1
1
Center of Biosciences, Slovak Academy of Sciences, Šoltésovej 4-6, 040 01 Košice, Slovakia
2
National Agricultural and Food Center, Hlohovecká 2, 951 41 Nitra-Lužianky, Slovakia
*
Author to whom correspondence should be addressed.
†
Deceased author.
Processes 2026, 14(10), 1587; https://doi.org/10.3390/pr14101587
Submission received: 18 February 2026 / Revised: 4 May 2026 / Accepted: 12 May 2026 / Published: 14 May 2026
(This article belongs to the Section Biological Processes and Systems)
Versions Notes

Abstract

The occurrence of the European brown hare (Lepus europaeus Pallas 1778) has declined throughout Europe in recent years. However, it remains economically valuable as an important game species. To date, information on the individual microbiota of the European hare has been limited. The phylum Firmicutes (Bacillota) was dominant, and enterococci belong to this phylum. However, they can carry virulence factor genes. Therefore, this study aimed to address two aspects: the health of hares due to their recent decline, and, as a game animal, the protection of consumers’ health. Based on MALDI-TOF mass spectrometry, five strains were identified as Enterococcus faecium and two as E. faecalis; these findings were confirmed by genotyping using PCR and phenotypic analysis. The average value of lactic acid production was 0.680 ± 0.005 mmol/L. The strains lacked the virulence factor genes esp, agg, and gelE. However, they showed susceptibility to antibiotics and to postbiotic substances, even to 13 of 14 tested. PS/Ent M appears to be the most active PS against tested strains, with inhibitory activity of 25,600 AU/mL. Postbiotic substances represent a new tool for preventing unwanted microbiota in game animals.

1. Introduction

Populations of European brown hares (Lepus europaeus Pallas 1778) are widespread across Europe. However, they have been found to have declined dramatically throughout Europe in recent years [1]. As a consequence, the European brown hare has been listed in Appendix III of the Bern Convention on the Conservation of European Wildlife and Natural Habitats [2]. It is classified as near-threatened or threatened on several Red Lists of threatened species, e.g., in Austria, Norway, Germany, and elsewhere [3]. In Slovakia, the European hare has also become a rare species. The European hare is important not only for its economic and hunting value as a game species, but also for its role in the natural biodiversity of each country, including Slovakia [4]. In Slovakia, this species mostly occurs in the southwestern part of the country, where flat, low-relief land prevails. Leporids are small mammalian herbivores that select food to obtain the necessary nutrients and energy for their bodies [5]. They feed mostly on a herbivorous diet [6]. The population density of the European hare can be affected by climate, food availability, disease, predators, reproductive rate, adaptability, and anthropogenic factors [1,6]. In general, wild animals are rarely studied from a microbial perspective because they can be hunted only at certain times of the year. Therefore, to date, information on the European hare’s microbiota has been limited. Recently, several articles have reported on the gut microbiota of the European brown hare, assessed using next-generation sequencing [7,8,9,10]. Stalder et al. [7] reported that the dominant phyla were Firmicutes (now known as Bacillota) and Bacteroidetes, with relative abundances of 45.51% and 19.30%. However, a high abundance was observed among representatives of the family Enterobacteriaceae, a group of potentially pathogenic bacteria commonly associated with intestinal dysbiosis in hares. They also reported the effects of environmental and regional factors on microbiota composition and detailed the microbial community of European hares using next-generation sequencing [7]. Padula et al. [8] found that the most abundant phyla in the gut and intestines of European hares in Italy were Bacteroidetes and Firmicutes. In that study, the amplicon sequence variants were classified into 735 bacterial genera, belonging to 285 families and 36 phyla. Similarly, using the standard microbiological method, representatives of the genus Enterococcus dominated [11]. The genus Enterococcus belongs to the phylum Firmicutes (Bacillota). Another association is that enterococci have been detected in various herbal plant sources [12], which are a common part of the hare’s diet. Although enterococci are mostly associated with warm-blooded animals, they have been detected in extra-intestinal habitats [13] as previously mentioned. These strains can carry virulence factor genes, which, because hares are game animals, can then also threaten human health. Moreover, European hares can be infected with coccidia (Eimeria spp.) oocysts, and this protozoan infection can cause hares’ mortality [14]. Therefore, the aim of this study was to address two aspects: the health of hares due to their recent decline, and, on the other hand, as a game animal, to protect consumers’ health. In this study, enterococci from European hares were identified and tested for antibiotic profiles, lactic acid production, detection of virulence factor genes, and their susceptibility to postbiotic substances as a tool to prevent unwanted microbiota. The postbiotic treatment in hares as game animals has not yet been studied.

2. Materials and Methods

2.1. Total Counts, Strains Identification, and Lactic Acid Production

Twenty-six Lepus europaeus were hunted in six districts of southwestern Slovakia (Trnava, Čataj, Lehnice, Žiharec, Vlčany, and Čilížska Radvaň) during the 2007–2008 winter season by our colleagues at the National Agricultural and Food Center in Nitra-Lužianky (Slovakia) [14]. Hare handling was approved in accordance with Slovak law and the guidelines of the Institutional Ethic Committee and the Institutional Scientific Practice of the National Agricultural and Food Center in Nitra-Lužianky (SK CH 17016 and SK U 18016). Fecal samples were transported in a box to our laboratory and processed using the standard microbiological dilution method (ISO 6887-1:2017, International Organization for Standardization) [15]. Samples were diluted in Ringer solution (pH 7.0, Merck, Darmstadt, Germany, 1:9). Appropriate dilutions were spread onto M-Enterococcus agar plates (pH = 7.2, Difco, Sparks, MD, USA) and incubated at 37 °C for 24–48 h. Total counts were calculated and expressed as colony-forming units per gram (CFU/g) ± SD. Individual pure colonies were picked and stored for further analyses using the Microbank system (Pro-Lab Diagnosis, Richmond Hill, ON, Canada).
Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, Bruker Daltonics, Billerica, MA, USA), seven strains were selected based on bacterial protein “fingerprints” [16]. Bacterial cell lysates were prepared according to the manufacturer’s instructions. The evaluation score was determined using the MALDI Biotyper 3.0 identification database (Bruker Daltonics). The MALDI-TOF MS score is interpreted as indicating highly probable species identification (2.300–3.000), secure genus identification/probable species identification (2.000–2.299), and probable genus identification (1.700–1.999) as previously reported by Lauková et al. [17]. The positive control was the reference strain included in the Bruker Daltonics database.
For PCR identification, DNA was isolated from a single loopful of a bacterial colony. The suspension (10 µL) was resuspended in 30 µL of water and vortexed for 10 min. Supernatant aliquots served as template DNA and were added to the 39.5 µL reagent mixture. The mixture contained 0.5 μM of each primer, 0.2 mM of each deoxynucleotide (dATP, dTTP, dCTP, dNTPs, Invitrogen, Waltham, MA, USA), 2.5 mM MgCl2 (Invitrogen), 10× PCR buffer (Invitrogen), and 1.25 μL of Taq polymerase (Invitrogen), with water added to a total volume of 50 μL as previously described by Simonová et al. [18]. The primer sequences for PCR amplification of enterococci were as follows: for Enterococcus faecium F: 5′-GCAAGGCTTCTTAGAGA-3′, R: 5′-CATCGTGTAAGCTAACTTC-3′; for E. faecalis F: 5′-ATCAAGTACAGTTAGTCTT-3′, R: 5′-ACGATTCAAAGCTAACTG-3′ [19,20]. The amplification protocol included: an initial denaturation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, and a final extension at 72 °C for 10 min. A thermocycler Techgene KRD (Techne, London, UK) was used. The PCR products (10 µL) were separated by electrophoresis on a 0.8% agarose gel (Sigma-Aldrich, Taufkirchen, Germany) buffered with 1× TAE (Merck, Germany) and containing 1.0 µg/mL ethidium bromide (Sigma-Aldrich). The molecular mass standard (100 bp DNA ladder, Invitrogen) was used according to the manufacturer’s instructions. Phenotypic characterization of selected enterococci was performed using the BBL Crystal Gram-positive ID panel (Becton Dickinson, Sparks, MD, USA) and the Remel Rapid Rap ID test (USA) with control strains (ATCC 2192129 and ATCC 11576).
As indicated, seven enterococcal strains were finally studied for additional properties. Even with limited enterococcal detection, they can serve as a bacterial model for bacterial susceptibility to the postbiotic substances (PSs).
A validated quantitative method using a spectrophotometer (Specol 11, Zeiss, Jena, Germany) was used to assess lactic acid production by the identified strains. This property was analyzed to confirm that the strains belong to the group of lactic acid-producing bacteria (LAB). The method is based on the conversion of lactic acid to acetaldehyde by sulfuric acid. Acetaldehyde from this reaction reacts with p-hydroxybifenyl to form a colored complex. Testing was performed in triplicate. The LA value was measured at 565 nm (A565) and reported in mmol/L [21].

2.2. Determination of Virulence Factor Genes

PCR screening for three virulence factor genes (the most frequently detected in enterococci) was performed as previously reported by Kubašová et al. [22]. DNA from the identified enterococci was extracted using the rapid alkaline lysis method [23]. The genes for aggregation substance (agg), enterococcal surface protein (esp), and gelatinase (gelE) were tested using the appropriate primers and a C1000TM thermocycler (BioRad Laboratories, Hercules, CA, USA). The oligonucleotides used to amplify the genes (5′ to 3′ sequences) are as follows: for agg, F:5′-AAGAAAAAGAAGTAGACCAAC-3′, R:5′-AAACGGCAAGCAAAGTAAATA-3′ (1553 bp product size) [24]; for esp, F:5′-TTGCTAATGCTAGTCCACGACC-3′, R:5′-GCGTCAACACTTGCATTGCCGAA-3′, 933 bp [24]; and for gelE, F:5′-ACCCCGTATCATTGGTTT-3′, R:5′-ACGCATTGCTTTTCCATC-3′, 419 bp [24]. The PCRs were carried out in a 25 μL volume; the mix consisted of 1 × reaction buffer, 0.2 mmol/L deoxynucleoside triphosphates, 3 mmol/L MgCl2, 1 μmol/L of each primer, 1 U of Taq DNA polymerase, and 1.5 μL of DNA template. The cycling conditions for gene amplification included an initial step at 95 °C for 3 min, 354 cycles of 30 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C, and a final step at 72 °C for 5 min. The PCR products were separated by agarose electrophoresis (1.2% w/v agarose, Sigma-Aldrich, Saint Louis, MO, USA) containing 1 μL/mL ethidium bromide (Sigma-Aldrich) in 0.5× TAE buffer (Merck, Germany). PCR products were visualized under UV light. E. faecalis P36 and E. faecium F10 (kindly provided by Teresa Semedo-Lemsaddek of the University of Lisbon, Faculty of Veterinary Medicine, Portugal) served as positive controls.

2.3. Antibiotic Phenotype Test

Antibiotic susceptibility of enterococci from European hares was tested using the agar disk diffusion method [25]. Briefly, strains were grown overnight in Brain Heart Infusion/broth (Difco). A volume of 100 μL was spread on Brain Heart agar (Difco). Antibiotic discs were placed on the inoculated agar plate. Plates were then refrigerated for 10 min to improve diffusion, then incubated at 37 °C for 18 h. Antibiotic discs were supplied by Oxoid (Basingstoke, UK) and Difco (Tucker, GA, USA). The following antibiotics were tested: clindamycin (Cc-2 µg), ampicillin (Amp-10 μg), gentamicin (Gn-10 μg), penicillin (Pnc-10IU), azithromycin (Azm-15 μg), erythromycin (Ery-15 μg), tetracycline (Tc-30 μg), kanamycin (K-3 μg), vancomycin (Va-30 μg), and chloramphenicol (C-30 μg). Susceptibility and/or resistance of enterococci were expressed as the mean diameter of inhibitory zones (mm) from duplicate tests. The positive control was the strain ATCC 2192129. Evaluation was performed according to the manufacturer’s instructions.

2.4. Susceptibility to Postbiotic Substances

Fourteen postbiotic substances were used to treat enterococci from European hares. Their sources are listed in Table 1. Eleven PSs were produced by various enterococcal strains, and three were produced by lactococci. PS MK2/8, MK1/3, and MK2/2 are produced by Lactococcus lactis strains MK2/8, MK1/3, and MK2/2 isolated from raw goat milk [26]; the GenBank accession numbers for the MK2/8 strain are PQ158272, and for MK1/3 ON114093. PS/Ent M and PS/Ent A/P were previously purified to homogeneity [27,28]. According to the definition of Chikindas et al. [29], they are bacteriocins. However, in this study, they were used in a non-purified homogeneous form, i.e., as postbiotic substances. They are produced by the environmental strains E. faecium EK13 (CCM7419) and E. faecium AL41 (CCM8558) Mareková et al. [28,30]. PS 55 is produced by the strain E. faecium EF55 isolated from chicken [31]. PS 412 and PS EM41/3 are produced by the horse strains E. faecium EF 412 and E. mundtii EM41/3 [32,33]. Moreover, PS Sac and PS As are also produced by horse strains E. saccharolyticus Es3/11 D27 and E. asini EAs 1/11D27 [34,35]. PS 2019 is produced by the strain E. faecium EF2019 (CCM7420) from rabbits [36]. The strain E. faecium CCM4231 of ruminal origin is a producer of PS 4231 [37]. PS/ED26E/7 is produced by E. durans ED26E/7, isolated from ewe’s milk lump cheese [17], and PS EM41 is produced by ostrich-derived E. faecium EF EM41 [38]. Based on basic characterization and properties, postbiotic substances produced by enterococcal strains are likely classified as class II bacteriocins. They are thermostable and retain inhibitory activity even after long-term storage at −20 °C. These proteinaceous substances are produced in the early growth phase of their producer strains and usually exhibit a broad antimicrobial spectrum. Postbiotic substances produced by lactococci retain their activity after long-term storage at −20 °C and are thermostable. PSs were prepared according to the protocols reported in previous studies (Table 1). Briefly, PS-producing strains were grown in MRS/M17 broth (Difco) overnight at 37 °C. The cultures were centrifuged at 10,000× g, and the supernatants were adjusted to the pH specified in individual PS protocols (as previously indicated). The supernatants were precipitated with sodium sulfate (40% saturation for enterococcal PS, and 80% saturation for lactococcal PS) at different temperatures for 4 h or 18 h as per the individual protocols. Then, centrifugation at 10,000× g at 4 °C was performed, and the precipitates were resuspended in the minimum volume of phosphate buffer (pH 6.5). Susceptibility to PSs was tested using the agar spot test [39]. Brain Heart agar plates (1.5% w/v; Becton Dickinson, Cockeysville, MD, USA) served as the bottom layer. Soft BH agar (0.7 w/v, 4 mL) inoculated with 200 μL of an overnight culture (A600 up to 1.0) of the tested/indicator strain grown in BHI (Becton Dickinson) served as the overlay. Then, 10 μL of each PS, diluted 1:1 in phosphate buffer (pH 6.5), was spotted onto the agar surface. Plates were placed in the fridge for 10 min to enhance diffusion and incubated at 37 °C overnight. The first check was performed after 4 h of incubation, followed by another after 24 h. The inhibitory activity/susceptibility of the tested strains was expressed in arbitrary units per mL (AU/mL), defined as the highest dilution of a PS that inhibits the tested strain.

3. Results

3.1. Strain Identifications; Lactic Acid Production

Although the total enterococci count in fecal samples from European brown hares was low (1.24 ± 0.11 CFU/g on average), seven strains were finally selected based on MALDI-TOF mass spectrometry and identified as Enterococcus faecium (five strains) and E. faecalis (two strains, Table 2). The evaluation scores for two strains reached 2.322 (EFTr/11b) and 2.360 (EEZih/8a), indicating highly probable species identification. Four strains were identified with secure genus/probable species identification (Table 2), showing scores of 2.137 (EFTr/1a), 2.094 for the strain EFZih/2b, 2.277/EF/L25/24, and 2.015 for strain EEZih2a. Strain EFZih/8b was assigned a score of 1.901, indicating probable genus identification (Table 2). In addition, PCR product visualization confirmed the species as E. faecium and E. faecalis. For phenotypic tests, the tested strains showed positive reactions for aesculin, mannitol, and glucose. The sorbitol reaction for E. faecalis was positive, and for E. faecium negative. Similarly, arginine showed a positive reaction in E. faecalis, but was negative in E. faecium. Additionally, the other parameters measured by the kits were associated with the reference strains for both species (ATCC 2192129 and CCM 2123). LA production by enterococci was well balanced, ranging from 0.625 ± 0.009 mmol/L (EFZih/8b) to 0.725 ± 0.006 mmol/L (EF/L25/24 (Table 2). The average value reached 0.681 ± 0.005 mmol/L.

3.2. Virulence Factor Genes and Antibiotic Phenotype Test

The strains lacked tested virulence factor genes (esp, agg, gelE). The tested enterococci were mostly susceptible to antibiotics (Table 3). They were 100% susceptible to ampicillin (average inhibitory zone 17–25 mm, MIC 10IU), chloramphenicol (inhibitory zones 19–25 mm, MIC 30 µg), azithromycin (12–21 mm, MIC 15 µg), erythromycin (18–22 mm, MIC 15 µg), tetracycline (20–29 mm, MIC 30 µg), and vancomycin (11–17 mm, MIC 30 µg). Enterococci were resistant to kanamycin. The largest average inhibitory zone of 20–29 mm was measured for tetracycline. Enterococci were the least susceptible to vancomycin (average inhibitory zone size, 11–17 mm). However, penicillin-susceptible strains had an average inhibitory zone of 11–20 mm (Table 3). Two strains, EFZih/8b and EEZih/2a, were Pnc-resistant. Regarding gentamicin (Gn), only strain EFTr11/b was susceptible; the other four strains showed an intermediate reaction (zone size of 10 mm). Similarly, an average inhibitory zone of 10 mm was observed for strain EEZih/2a with clindamycin. The other strains were susceptible to clindamycin (12–26 mm, MIC 2 μg). Only E. faecium EFTr11/b was susceptible to all antibiotics except kanamycin. The other strains showed an intermediate reaction to one antibiotic, except for kanamycin, to which they were resistant.

3.3. Susceptibility to Postbiotic Substances

In general, the strains were susceptible to PSs. Four strains (3 E. faecium and 1 E. faecalis, Table 4) were susceptible to 13 of 14 PSs tested. The strains EFL25/24 and EEZih/8a were treated with only two PSs (PS EntM and PSEM41), to which they were susceptible (Table 4). Later, they did not grow, so they were not tested with other PSs. Inhibitory activity varied with the PS used, ranging from 100 AU/mL to 25.600 AU/mL (Table 4). Enterococci were susceptible to PSs produced by lactococci (PS MK2/8 and MK1/3), with inhibitory activity of 1 600 AU/mL–6 400 AU/mL, respectively, and 400–3.200 AU/mL (PS MK1/3). Using PS MK2/2, the inhibitory activity against three strains was 12.800 AU/mL (EFZih2/b). The strains were most susceptible to PS/EntM, with inhibitory activity mostly at 25.600 AU/mL. Susceptibility to PS ED26E/7 was found in five strains (up to 3 200 AU/mL); two strains were not treated. EEZih/2a was the most susceptible to PS EM41 (25.600 AU/mL). Susceptibility to PS EM41/3 was well balanced across all strains, with inhibitory activity of 3 200 AU/mL. PSs produced by Enterococcus asini EAs 1/11D27 and E. saccharolyticus Es 3/11 D27 inhibited indicator strains, with inhibitory activity up to 400 AU/mL. PS Ent A/P exhibited well-balanced inhibition against the tested enterococci, although with low inhibitory activity (100 AU/mL). On the other hand, PS 4231 inhibited only three of five tested strains, but with an inhibitory activity of 12.800 AU/mL. EFTr11/b and EFZih/2b treated with PS 55 were susceptible with activities of 6 400 AU/mL. The highest activity (3 200 AU/mL) was measured with PS EM41 and PS 2019. The strain EFTr/1a was susceptible to 13 of 14 postbiotic substances, with inhibitory activity ranging from 100 AU/mL for PS ESac to 12.800 for PS 4231. EFTr11/b was also susceptible to 13 of 14 postbiotic substances, with activity ranging from 100 (PS 2019) to 25.600 AU/mL for PS-EntM. EFZih/8b was susceptible to 14 postbiotic substances (100 AU/mL-PS 4231) up to 25.600 in the case of PS EntM. EFZih/2b was inhibited by 12 PSs; PSEM41 was not used against it; and treatment with PS 4231 was negative, the strain was not inhibited (100-EntA/P to 25.600 for EntM). Although EF/L25/24 and EEZih/8a were treated only with PS EntM and PS EM41, they were susceptible, meaning their growth was inhibited by both PSs, with high inhibitory activity up to 25.600 AU/mL for both PSs (Table 4). Finally, EEZih/2a was susceptible to 13 of 14 PSs, with inhibitory activities of 100 AU/mL for PS ESac, and 25.600 AU/mL for PS EntM and PS EM41. In summary, 13 of 14 PSs were effective against enterococcal indicators. However, each one of the Ps inhibited at least the growth of two indicator strains. The most effective was PS EntM.

4. Discussion

As previously indicated, the European brown hare is a mammal well-adapted to its environment. It is also a widely distributed and important game species throughout Europe [7]. Its taxonomy places it in the class Mammalia, order Lagomorpha, family Leporidae, and genus Lepus. Its population has declined, and the cause has not yet been identified, although disease has been considered a primary factor [4]. Recent research has highlighted the importance of the gut microbiota’s impact on host physiology [7]. In the European hare, the detailed microbial composition was characterized only recently using next-generation sequencing [7], which showed dominance of the phyla Firmicutes (Bacillota) and Bacteroidetes. Moreover, Shanmuganandam et al. [10] reported the same dominant phyla, Firmicutes and Bacteroidetes, in the fecal microbiomes of wild hares and rabbits. In this study, enterococci belonging to the genus Enterococcus and the phylum Firmicutes (Bacillota) were identified using genotypic and phenotypic analyses, and fingerprinting (MALDI-TOF MS). Two main species, E. faecium and E. faecalis, were confirmed, although only a limited number of strains were selected; however, they were sourced from six different regions. These species are dominant across various animal sources. They were detected in, e.g., broiler rabbits [7], horses [32], sheep [37], mouflons and bison [40], goats, cattle, pigs [41], poultry [31], and/or dogs [22]. The methods of their identification are validated and reliable for taxonomy allocation [7,42]. Genotypic analysis and MALDI-TOF MS (fingerprinting) are rapid, accurate methods for the identification of bacteria. Phenotypic analysis serves as an additional contribution.
It appears that the detected enterococci did not constitute a reservoir of antibiotic resistance, as they were mostly susceptible to antibiotics, based on phenotypic testing of antibiotics. Enterococci, especially E. faecium and E. faecalis, are naturally chromosomally resistant to kanamycin [43]. Thal et al. [43] reported ampicillin susceptibility in enterococci, as in our study, and this susceptibility also included gentamicin susceptibility, as noted in this study. A total of 97 enterococci from various animal sources were analyzed therein. Antibiotic susceptibility also confirms that the European hare, as a wild animal, was not exposed to antibiotics. If resistance is observed, it may be attributed to the conjugal transfer of resistance plasmids [43]. E. faecium EFTr11/b was susceptible to all antibiotics except kanamycin and produced a postbiotic (antimicrobial) substance [11], indicating its selection for further studies associated with PSs. Kanamycin resistance in enterococci is chromosomally encoded. As presented in this study, LA production by enterococci was high and well balanced. This confirms their classification within the lactic acid bacteria group. Previously, high LA values were also observed in strains of this species from other sources [21].
Postbiotics, as previously reported [29,44], are preparations of inanimate microorganisms and their components that have emerged as a promising functional ingredient in animal health and nutrition. This category also includes proteinaceous substances purified to homogeneity, such as bacteriocins, as well as proteinaceous substances not purified to homogeneity [29], which exhibit antimicrobial/inhibitory effects against more or less related bacteria. They exhibit not only in vitro antimicrobial effects but also in vivo effects across different animals. They inhibit pro-inflammatory molecules such as TNF-α and IL-6; enhance the anti-inflammatory cytokine IL-10, promoting the maturation and function of immune cells; and increase IgA production [45]. Fecal coliforms (p < 0.001) and pseudomonads (p < 0.05) were reduced in broiler rabbits after the application of the postbiotics PS EntM and PS Durancin ED26E/7 as reported by Pogány Simonová et al. [46]. Moreover, postbiotic substances produced by lactococci were successfully used in situ to enrich animal-derived foods, thereby serving as functional foods [26,27]. PS MK2/8 and MK1/3 were reported to inhibit E. faecalis strains isolated from raw goat milk [47]. However, activity ranged from 100 to 400 AU/mL. Enterococci from hares were most susceptible to PSs produced by lactococci. Here, enterococci lacked virulence factor genes (gelE, agg, esp) in contrast to E. faecalis from raw goat milk, where those genes were detected in some strains [47]. Szabóová et al. [48] reported the anti-Eimeria oocysts effect of PS Ent 4231 produced by E. faecium CCM4231 of ruminant origin. A significant reduction (p < 0.001) was observed after its application. In addition, Petrová et al. [49] reported the direct effect of PS Ent M and Durancin-like ED26E/7 on Trichinella spiralis fecundity in an in vitro test.
Given the European hare as a game animal, it is necessary to protect it against eimeriosis and/or other related infections, and this service could provide previously documented postbiotic substances. In this study, enterococci were tested in limited amounts from hares, and the detected enterococci did not show pathogenic characteristics. However, their inhibition and susceptibility to PSs broadened the spectrum of PS inhibitory activity and suggested their possible use in treating game meat. In each case, the multifunctional benefits of postbiotics make them a valuable tool for maintaining healthy game animals and for producing game products that support welfare and productivity, thereby eliminating the need for antibiotics [45].

5. Conclusions

Seven fecal lactic acid-producing strains of Enterococcus faecium (five strains) and E. faecalis (two strains) from European hares were studied. Their taxonomic assignment to the genus Enterococcus and the phylum Firmicutes (Bacillota) was based on MALDI-TOF mass spectrometry and on genotypic and phenotypic analyses. The enterococci studied lacked the virulence factor genes esp, agg, and gelE. They were mostly susceptible to antibiotics and postbiotic substances. Five strains were susceptible to 13 of 14 postbiotic substances tested. The most effective was PS EntM. Results demonstrate the benefits of PSs against contaminant strains in the European hare. These contaminant strains pose a risk to consumer health because the European hare is a game animal. Although additional studies are underway, postbiotic substances offer a new tool for preventing spoilage microbiota in game animals.

Author Contributions

Conceptualization, A.L.; methodology, A.L., J.Š. and M.P.S.; validation, A.L.; investigation, A.L., J.Š. and M.P.S.; resources, Ľ.C.; data curation, A.L.; writing—original draft preparation, A.L.; writing—review and editing, A.L.; supervision, A.L.; project administration, A.L.; funding acquisition, A.L. and M.P.S. Author Ľubica Chrastinová passed away prior to the publication of this manuscript. All other authors have read and agreed to the published version of this manuscript.

Funding

The research was partially funded by the Slovak Scientific Agency VEGA (project nos. 2/0006/17 and 2/0009/25), and by COST action CA22166 (Safety in the Game Meat Chain, acronym SafeGameMeat).

Data Availability Statement

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

Acknowledgments

We are grateful to our colleagues from the National Agricultural and Food Center in Nitra-Lužianky, Slovakia, Jaroslav Slamečka and Rastislav Jurčík, for providing us with the European hare sample. We also thank Margita Bodnárová for her skillful laboratory work. During the preparation of this manuscript, the language was checked using the Grammarly tool (Grammarly Versions and Features, 2026). 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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Table 1. The list of postbiotic substances (PSs) used against enterococci from the European hares.
Table 1. The list of postbiotic substances (PSs) used against enterococci from the European hares.
Postbiotic SubstancesProducer StrainPS Preparation
PS MK2/8Lactococcus lactis MK2/8Lauková et al. (2024) [26]
PS MK1/3Lactococcus lactis MK1/3Lauková et al. (2025) [27]
PS MK2/2Lactococcus lactis MK2/2Lauková et al. (2024) [26,34,35]
PS Ent MEnterococcus faecium AL41 = CCM8558Mareková et al. (2007) [30]
PS 55Enterococcus faecium EF55Strompfová and Lauková (2007) [31]
PS 412 Enterococcus faecium EF 412Lauková et al. (2008) [32]
PS Ent A/PEnterococcus faecium EK13 = CCM7419Mareková et al. (2003) [28]
PS 4231Enterococcus faecium CCM4231Lauková et al. (1993) [37]
PS EM41Enterococcus faecium EM41Lauková et al. (2017) [38]
PS 2019E. faecium EF2019 = CCM7420Pogány Simonová et al. (2020) [36]
PS EM41/3Enterococcus mundtii EM41/3Focková et al. (2022) [33]
PS ED26E/7Enterococcus durans ED26E/7Lauková et al. (2021) [17]
PS EAsEnterococcus asini EAs 1/11D27 Lauková et al. (2024a) [34]
PS SacEnterococcus saccharolyticus Es3/11 D27Lauková et al. (2024b) [35]
PS MK2/8, a postbiotic substance produced by the strain Lactococcus lactis subsp. lactis MK2/8; PS MK1/3 is produced by Lactococcus lactis subsp. lactis MK1/3; PS MK2/2 is produced by Lactococcus lactis subsp. lactis MK2/2; PS Ent M (here indicated as a PS because a non-purified substance was used) is produced by Enterococcus faecium AL41 = CCM8558; PS 55 is produced by E. faecium EF55; PS 412 is produced by E. faecium EF 412; PS Ent A/P is produced by E. faecium EK13 = CCM7419 (here indicated as a PS because a non-purified substance was used); PS 4231 is produced by E. faecium CCM4231; PS EM41 is produced by E. faecium EM41; PS 2019 is produced by E. faecium EF2019 = CCM7420; PS/EM41/3 is produced by E. mundtii EM41/3; PS ED26E/7 is produced by E. durans ED26E/7; PS/EAs is produced by E. asini EAs 1/11D27; PS/Sac, E. saccharolyticus is produced by E. saccharolyticus Es 3/11 D27.
Table 2. Identification score of isolated enterococci, positive PCR reaction, and lactic acid production in mmol/L.
Table 2. Identification score of isolated enterococci, positive PCR reaction, and lactic acid production in mmol/L.
StrainsMALDI-TOF ScorePCRLactic Acid
EF Tr/1a2.137+0.655 ± 0.007
EF Tr/11b2.322+0.715 ± 0.004
EFZih/8b1.901+0.625 ± 0.009
EFZih/2b2.094+0.675 ± 0.007
EF/L25/242.277+0.725 ± 0.006
EEZih/8a2.360+0.695 ± 0.005
EEZih2a2.015+0.680 ± 0.000
+ PCR reaction was positive for the species identification: EF, Enterococcus faecium, EE, E. faecalis. The MALDI-TOF MS score is evaluated as a highly probable species identification (2.300–3.000), secure genus identification and/or probable species identification (2.000–2.299), and probable genus identification (1.700–1.999), Lauková et al. [17].
Table 3. Antibiotic profile of identified enterococci using the phenotype test.
Table 3. Antibiotic profile of identified enterococci using the phenotype test.
StrainsCcAmp GnPncEryAzmCVaTc
EFTr/1a+24 (S)+25 (S)+10 (I)+17 (S)+22 (S)+17 (S)+23 (S)+16 (S)+26 (S)
EETr11/b+26 (S)+21 (S)+12 (S)+20 (S)+22 (S)+21 (S)+25 (S)+17 (S)+29 (S)
EFZih/8b+15 (S)+21 (S)+10 (I)+9 (R) +21 (S)+15 (S)+20 (S)+13 (S)+24 (S)
EFZih/2b+22 (S)+20 (S)+7 (R)+17 (S)+17 (S)+12 (S)+22 (S)+14 (S)+25 (S)
EF/L25/24+19 (S)+17 (S)+10 (I)+17 (S)+18 (S)+17 (S)+19 (S)+17 (S)+20 (S)
EEZih/8a+12 (S)+21 (S)+10 (I)+11 (S)+22 (S)+19 (S)+21 (S)+14 (S)+21 (S)
EEZih/2a+10 (I)+21 (S)+9 (R)+9 (R)+20 (S)+15 (S)+20 (S)+11 (S)+27 (S)
EF, Enterococcus faecium, EE, E. faecalis; clindamycin (Cc-2 µg), ampicillin (Amp-10 μg), gentamicin (Gn-10 μg), penicillin (Pnc-10IU), erythromycin (Ery-15 μg), azithromycin (Azm-15 μg), chloramphenicol (C-30 μg), vancomycin (Va-30 μg), and tetracycline (Tc-30 μg). The strains were resistant to kanamycin (K-30 µg). S: susceptibility, R: resistance, I: dubious (intermediate) reaction, inhibitory zone of small size according to CLSI. Antibiotic susceptibility was expressed as the mean diameter of inhibitory zones (mm). The positive control was the strain ATCC 2192129.
Table 4. Susceptibility to postbiotic substances of tested enterococci.
Table 4. Susceptibility to postbiotic substances of tested enterococci.
PSEFTr/1aEF Tr11/bEFZih/8bEFZih/2bEF/L25/24EEZih/8aEEZih/2a
PS MK2/83 200 ± 0.003 200 ± 0.001 600 ± 0.006 400 ± 0.00ntnt3 200 ± 0.00
PS MK1/3400 ± 0.00800 ± 0.003 200 ± 0.00800 ± 0.00ntnt400 ± 0.00
PS EntA/P100 ± 0.00100 ± 0.00100 ± 0.00100 ± 0.00ntnt100 ± 0.00
PS EntM6 400 ± 0.0025.600 ± 0.0025.600 ± 0.0025.600 ± 0.0012.800 ± 0.0025.600 ± 0.0025.600 ± 0.00
PS 412200 ± 0.003 200 ± 0.003 200 ± 0.003 200 ± 0.00ntnt200 ± 0.00
PS 55100 ± 0.006 400 ± 0.00400 ± 0.006 400 ± 0.00ntnt100 ± 0.00
PS 423112.800 ± 0.00ng100 ± 0.00ngntnt12.800 ± 0.00
PS EAs400 ± 0.00400 ± 0.00400 ± 0.00400 ± 0.00ntnt200 ± 0.00
PS ESac100 ± 0.00200 ± 0.00200 ± 0.00200 ± 0.00 ntnt100 ± 0.00
PS20191 600 ± 0.00100 ± 0.003 200 ± 0.001 600 ± 0.00ntnt1 600 ± 0.00
PS EM41/33 200 ± 0.003 200 ± 0.003 200 ± 0.003 200 ± 0.00ntnt3 200 ± 0.00
PS MK2/2ng400 ± 0.003 200 ± 0.0012.800 ± 0.00ntntng
PSEM416 400 ± 0.006 400 ± 0.0012.800 ± 0.00nt6 400 ± 0.0012.800 ± 0.0025.600 ± 0.00
PS ED26E/73 200 ± 0.001 600 ± 0.003 200 ± 0.00800 ± 0.00ntnt400 ± 0.00
Inhibitory activity of postbiotic substances (susceptibility to PSs) is expressed in arbitrary units per mL (AU/mL). nt: not tested (they were not treated with those substances); ng: negative, PS did no inhibit strain; PS: postbiotic substance; EF: Enterococcus faecium; EE: E. faecalis.
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Lauková, A.; Ščerbová, J.; Chrastinová, Ľ.; Pogány Simonová, M. Treatment of Fecal Enterococci from European Brown Hares (Lepus europaeus) with Postbiotic Substances. Processes 2026, 14, 1587. https://doi.org/10.3390/pr14101587

AMA Style

Lauková A, Ščerbová J, Chrastinová Ľ, Pogány Simonová M. Treatment of Fecal Enterococci from European Brown Hares (Lepus europaeus) with Postbiotic Substances. Processes. 2026; 14(10):1587. https://doi.org/10.3390/pr14101587

Chicago/Turabian Style

Lauková, Andrea, Jana Ščerbová, Ľubica Chrastinová, and Monika Pogány Simonová. 2026. "Treatment of Fecal Enterococci from European Brown Hares (Lepus europaeus) with Postbiotic Substances" Processes 14, no. 10: 1587. https://doi.org/10.3390/pr14101587

APA Style

Lauková, A., Ščerbová, J., Chrastinová, Ľ., & Pogány Simonová, M. (2026). Treatment of Fecal Enterococci from European Brown Hares (Lepus europaeus) with Postbiotic Substances. Processes, 14(10), 1587. https://doi.org/10.3390/pr14101587

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