The Strains Enterococcus faecalis as Contaminants of Raw Goat Milk and Their Treatment with Postbiotic Active Substances Produced by Autochthonous Lactococci

Abstract

Enterococci from raw goat milk were taxonomically allotted in the species Enterococcus faecalis using sequencing (16S rRNA and BLASTn analysis) with a percentage identity up to 99.91%. The virulence factor gene gelE was found in the strains EE/K3, EE/G3, and EE/G6. The agg gene was detected in the strain EE/G6, and the esp gene was detected in the strains EE/K5 and EE/G7. Each strain possessed at least one virulence factor gene. In the strain EE/G6, the gelE and esp genes were found. The strains EE/G6 and EE/G3 showed resistance to tetracycline and vancomycin. EE/G7 was resistant to vancomycin and gentamicin. All strains possessed low-grade biofilm-forming ability (0.1 < A570 ≤ 1.0). They possessed genes for biofilm formation (bopD, srt, and/or ace). They also produced esterase (20–40 nmo/L), esterase lipase, and α-chymotrypsin (10–40 nmoL). The values of acid phosphatase reached 20–40 nmoL. The strains EE/G3, EE/G6, and EE/G7 were observed to possess the most pathogenicity. However, all strains were susceptible to postbiotic active substances produced by two autochthonous lactococci, MK2/8 and MK1/3 (inhibitory activity up to 400 AU/mL). These postbiotic substances provide a new potential alternative to reducing contaminants in milk.

1. Introduction

Milk (including goat milk) represents an important nutritional source in the human diet [1]. It also plays a prominent role in physical and cognitive development [2,3]. Regarding goat milk, its specificity lies in its more abundant immunoglobulin content than human milk. As is well known, it is also higher in calcium compared with cow milk; it contains more trace elements than cow milk and a high content of some fatty acids as well [4,5]. Because milk is a highly nutritious product, it facilitates the growth and multiplication of a wide range of microbiota [6]. Among these can also appear various enterococcal species, including Enterococcus faecalis [7,8]. One of the reasons for their prevalence is their robust nature, since the majority of these species can grow at temperatures from 10° to 45 °C, in 6.5% NaCl, and in pH ranges from 4.0 to 9.6, and/or they can survive heat at 60 °C for 30 min [9]. Even the horizontal transfer of resistance genes from E. faecalis to methicillin-resistant Staphylococcus aureus has been reported previously [10]. Enterococci have often been associated with intrinsic or acquired antibiotic genes located on chromosomes, plasmids, transposons, and other virulence factor genes as well [11,12]. E. faecalis is a prevalent species among the dairy microbial community [13]. However, some authors have declared that enterococci contribute to the ripening of traditional fermented dairy products [14]. On the other hand, some authors have described virulence factor genes as possessing enterococci even in pasteurized milk [14]. They are also recognized for their ability to exchange genetic information through mobile elements and to spread antibiotic resistance among other bacteria [15].

However, the safety of food impacts consumers. Therefore, to prevent and/or reduce the effect of contaminants, especially those which possess virulence factor genes, including those harboring biofilm-forming or antibiotic resistance genes, novel challenges and tools are being experimented with. Among those which have seemed effective are allotted bacteriocins and/or postbiotic substances. Bacteriocins are antimicrobial-acting proteinaceous substances, which are more often purified to homogeneity with inhibitory effects against more or less relative bacteria. But those antimicrobial effective proteinaceous substances that are non-purified into homogeneity but confer host health benefits are now allotted into the group of postbiotics [16,17,18]. Even those substances (metabolites and secreted proteins) that are extracellularly released may be termed exopostbiotics [17]. Lauková et al. [19,20] reported raw goat-milk-derived strains, such as Lactococcus lactis MK1/3 (CCM 9209), as producing a postbiotic active substance with in vitro inhibitory activity against cheese and milk contaminant enterococci (inhibitory activity up to 3 200 AU/mL). Moreover, the strains Lactococcus lactis MK2/8 (AN PQ158272), MK2/7 (AN PQ158271), and MK2/2 (PQ158270) produced postbiotic active substances that inhibited 97.8% of indicator strains, including enterococci and staphylococci, reaching inhibitory activity in the range from 200 up to 800 AU/mL. Postbiotic substances are not only produced by probiotic bacteria [17]. Formerly mentioned, lactococci, as producers of postbiotic substances, show beneficial characteristics [19,20].

Therefore, the aim of this study was to detect the potential pathogenic risk of strains of the species Enterococcus faecalis isolated from raw goat milk and to show the inhibitory effect of postbiotic substances produced by autochthonous lactococci against these milk contaminants. The novelty of this study especially highlights the treatment effect of postbiotic substances secreted by autochthonous lactococci, from which E. faecalis contaminants were isolated from the same source, raw goat milk.

2. Materials and Methods

2.1. Enterococcus faecalis Strain Isolation and Identification

The strains were selected from the dilutions of 53 raw goat milk samples. They were supplied by breeders from farms located in the central Slovakia region. One milliliter from each milk sample was collected and then treated. There were standard hygienic conditions on the farms. Altogether, 132 goats were sampled throughout the year 2021. The standard microbiological dilution method was used to treat samples according to the ISO (International Organization for Standardization) as previously described by Lauková et al. [19]. The samples were diluted in Ringer solution (pH 7, Merck, Darmstadt, Germany, 1:9). Individual dilutions were plated on M-Enterococcus agar (Difco, Sparks, MD, USA). The pure selected colonies were stored for further testing by use of the Microbank system (Pro-Lab Diagnostics, Richmond Hill, ON, Canada).

The DNA extraction, PCR amplification, and sequencing were reported in detail in our previous study [19,20]. The isolation of DNA from enterococcal isolates was performed by DNAzol direct (Molecular Research Centre Inc. Cincinnati, OH, USA) according to the manufacturer’s instruction. The 16S ribosomal RNA (rRNA) genes from enterococci were amplified by a PCR cycler (TProfesional Basic, Biometra GmbH, Goettingen, Germany) using the universal primers [21,22] Bac27F (5-AGAGTTTGATCTTGGCTCAG-3) and 1492R (5-CGGTTACCTTACGACTT-3). The PCR reaction was carried out in a total master mix (One Taq 2X) with the standard buffer (New England Biolabs, Foster City, CA, USA). The PCR mixture was subjected to an initial denaturation cycle of 5 min at 94 °C followed by 30 cycles of denaturation at 94 °C for one min, annealing at 55 °C, and extension at 72 °C for 3 min followed by the final extension step at 72 °C for 10 min. The amplified PCR product was separated on 3% agarose gel electrophoresis in TRIS-acetate-EDTA buffer (pH 7.8) and visualized with GelRed (Biotium Inc. Hayword, CA, USA) under ultraviolet light. The PCR products were sent for Sanger sequencing (Microsynth, Austria GmbH, Wienna, Austria). All sequencing results (chromatogram) were analyzed using Geneious 8.0.5. (Biomatters, Auckland, New Zealand) and compared to the National Centre of Biotechnology Information GenBank database using the BLASTn program (http://www.ncbi.nlm.nih.gov/BLAST, accessed on 31 April 2022) for identification.

2.2. Hemolysis and Enzyme Analysis Using API ZYM Test

A hemolysis test was performed as reported by Semedo-Lemsaddek et al. [23]. Brain heart agar enriched with 5% defibrinated sheep blood (BHA, Difco, Sparks, MD, USA) was used for this test, performed in duplicate. Enterococci were inoculated on the BHA and cultivated at 37 °C overnight. The presence/absence of a cleared zone around the colonies that grew was assessed as α-, β-hemolysis and/or no hemolysis (ɤ-hemolysis), also including a positive control strain according to Semedo-Lemsaddek et al. [23]. The API ZYM panel (BioMerieux, Marcy L’ Etoile, France) was used to test enzyme production by selected strains to assess their character, as previously reported [19]. The panel includes the following enzymes: alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine 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. It corresponds with relevant values in nanomoles (5–40 nmoL). The test was performed in duplicate including the reference strain E. faecalis.

2.3. Virulence Factor Gene Analysis

The genes gelE (gelatinase), agg (aggregation substance), esp (enterococcal surface protein), hyl (hyaluronidase), cylA (cytolysin A), and IS16 (IS16 element) were tested. The procedure followed the previous description by Kubašová et al. [24] (

Table 1. Oligonucleotides used in this study to amplify virulence genes and biofilm in enterococci.

Genes	Primer Locus Sequence	Product Size
gelE	F: ACCCCGTATCATTGGTTT R: ACGCATTGCTTTTCCATC	419 bp
Element IS16	F: CATGTTCCACGAACCAGAG R: TCAAAAAGTGGGCTTGGC	547 bp
Cytolysin cylA	F: TAGCGAGTTATATCGTTCACTGTA R: CTCACCTCTTTGTATTTAAGCATG	1282 bp
Enterococcal surface protein esp	F: TTGCTAATGCTAGTCCACGACC R: GCGTCAACACTTGCATTGCCGAA	933 bp
Hyaluronidase	F: GAGTAGAGGAATATCTTAGC R: AGGCTCCAATTCTGT	661 bp
Aggregation substance agg	F: AAGAAAAAGAAGTAGACCAAC R: AAACGGCAAGACAAGTAAATA	1553 bp
ica	ica4F-TGGGATACTGATATGATTAC, ica4R-CCTCTGTCTGGGGCTTGACCATG	568 bp
bap	Sasp7c-TGTTGAAGTTAATACTGTACCTGC, Sasp6m-CCCTATATCGAAGGTGTAGAATTGCAC	970 bp
fsrA	F-5-GAGCCGTTATGCTCCTATGC-3 R-5-CTGCGGTAGTTGTTGGA-3	450 bp
ace	F-5-TTGATGCTGCTGCTGATGTG-3, R-5-ACGGATGAGCTTGTTGGGTA-3	400 bp
srt	F-5-TCTTGGTAGTGGGTCGTTGA-3 R-5-CGCAGTGTGTTTGATGTTGG-3	420 bp
bopD	F-5-GATCGTCTTCGCCATAGTAGG-3 R-5-RACACAACAGCCCTTGGCT-3	312 bp

). The PCR was carried out in a 25 µL volume with a mixture described previously by Focková et al. [25] under the following conditions (gelE, agg, cylA, and esp): denaturation at 95 °C for 3 min, 35 cycles for 30 s at 95 °C, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s and finally at 72° C for 5 min. The PCR conditions for the hyl and IS16 genes included denaturation at 94 °C for 4 min followed by 30 cycles at 94 °C for 30 s and at 50 °C for 30 s, then 30 s at 72 °C and extension at 72 °C for 4 min. The PCR products were separated using agarose electrophoresis (1.2%, v/w, Sigma Aldrich, Saint Louis, MO, USA) with a 1 µg/mL content of ethidium bromide (Sigma-Aldrich) using 0.5× TAE buffer (Merck, Darmstadt, Germany). The PCR fragments were visualized with ultraviolet light. Our strain E. faecalis 9Tr1 and E. faecium P36 (University in Lisbon, Faculty of Veterinary Medicine, Portugal) were positive controls.

2.4. Biofilm-Forming Ability and Biofilm Gene Testing

Two methods were applied for biofilm-forming ability testing: a quantitative plate assay [26] previously described by Lauková et al. [19,20] and the detection of genes responsible for biofilm formation using the appropriate primers and PCR. Following the quantitative method, a pure colony of the tested strain (grown on BH agar, Difco, overnight at 37 °C) was inoculated into 5 mL of Ringer solution (pH 7.0, 0.75% w/v) to have the suspension, which corresponded to 1 × 10⁸ cfu/mL. Then, 100 µL from the dilution was transferred into microtiter plate wells (Greiner Elisa 12 well strips, Frickenhausen GmbH, Frickenhausen, Germany). The next step was cultivation of the plate for 24 h at 37 °C. After that time, the biofilm formed in the plate well was washed twice with 200 µL of deionized water. The plate was dried at laboratory temperature for 40 min. This was followed by staining of the attached bacteria, performed with 200 µL of 0.1% crystal violet in deionized water at 25 °C for 30 min. After the dye solution aspirated away, the wells were washed twice with 200 µL of deionized water. Then, the water was removed, and the plate was dried for 30 min at laboratory temperature. The dye bound to the adhered biofilm was eluted using 200 µL of 95% ethanol. This was followed by the transfer of 150 µL from each well into a new microtiter plate to measure absorbance (A₅₇₀) using the absorbance reader Apollo 11 LB 913 (Apollo, Berthold Technologies, Oak Ridge, TN, USA). Two independent runs with 12 replicates were prepared for measurement, including a negative control (BHI broth). The positive control was the biofilm-forming strain Streptococcus equi subsp. zooepidemicus CCM 7316 (kindly provided by Dr. Eva Styková from the University of Veterinary Medicine and Pharmacy in Košice, Slovakia). The following classification was used to evaluate the biofilm-forming ability of the E. faecalis strains [26]: highly positive A570 ≥ 1.0; low-grade positive, 0.1 < A570 ≤ 1.0, and negative, A570 ≤ 0.1.

Several enterococcal virulence proteins have been studied that play an important role in biofilm-forming ability (ace, bopD, srtA, ica, Bap, and fsrA). Ace is a surface protein that is important in the initial adhesion of biofilm-forming bacteria [27]. The transcriptional regulator BopD is a protein that has a function in in vitro biofilm formation [28]. SrtA is an enzyme that is functional in surface protein processing in biofilm formation [29,30]. Fsr possesses a function in quorum sensing and may regulate many biofilm-related genes [30]. Ica and Bap are surface proteins participating in the cellular adhesion step in biofilm formation [31]. The primers used are included in Table 1. To detect the ica gene, the following mixture and cycles were used [28]: an initial denaturation cycle of 2 min at 94 °C, followed by denaturation at 94 °C for 20 s, annealing at 42 °C, 30 cycles for 20 s, extension at 72 °C for 50 s, and the final extension at 72 °C for 5 min. For the bap gene, it was an initial denaturation at 94 °C for 5 min, then 30 s at 94 °C, annealing at 55 °C, 30 cycles for 30 s, extension at 72 °C for 45 s, and the final extension at 72 °C for 7 min. To detect other genes, the PCR conditions followed those described by Hashem et al. [32]. The amplified PCR product was separated on 1.2% agarose gel electrophoresis (Sigma-Aldrich) with a 1.0 µg/mL content of ethidium bromide in 0.5× TAE buffer (Merck) and visualized under ultraviolet light. The positive control strains were those included in the references.

2.5. Relation of Enterococcus faecalis Strains to Antibiotics

Two assays were applied for antibiotic resistance phenotype testing. The agar disk diffusion method uses antibiotic resistance disks according to EUCAST, as reported by Focková et al. [25]. The following antibiotic disks were used: ampicillin (10 µg), penicillin (10 IU), erythromycin (15 µg), tetracycline (30 µg), vancomycin (30 µg), rifampicin (30 µg), chloramphenicol (30 µg), kanamycin (30 µg), streptomycin (30 µg), and gentamicin (120 µg). The overnight culture of the tested strains was spread (100 µL) on the surface of BHA plates with blood. Antibiotic disks were applied on the plate surface. The plates were incubated at 37 °C overnight. The zones around the disks were measured; this was tested in duplicate. Two zone sizes are included in millimeters.

Regarding the EUCAST (to assess the MIC, minimal inhibitory concentration), the E strip method was used, as previously described by Lauková et al. [20] and Focková et al. [25]. Instead of disks, E-strips were used as follows: ampicillin, erythromycin, tetracycline, vancomycin, kanamycin, and rifampicin (256-0.016 µg/mL); gentamicin (1024-0.064 µg/mL); and streptomycin (256-0.064 µg/mL, Oxoid, the United Kingdom). In both cases, the positive control was the strain E. faecalis ATCC 29212.

2.6. Treatment of Enterococcus faecalis Strains with Postbiotic Active Substances MK1/3 and MK2/8

The strains Lactococcus lactis MK2/8 and MK1/3 were both isolated from raw goat milk (Access Number in GenBank for MK2/8 PQ158272 for MK2/8) [19,20]. The concentrated (10-fold) postbiotic substance (CPS) was prepared from the supernatant as previously described in [20]. The inhibitory activity measured against the principal indicator Enterococcus avium EA5 was 800 AU/mL. The CPS from the strain MK1/3 was prepared as reported by Lauková et al. [19]. Inhibitory activity against Enterococcus avium EA5 (the principal indicator strain) reached 1600 AU/mL. The strain MK1/3 is included in patent no. 289266 by the Industrial Property Office of the Slovak Republic. The inhibitory activity of both postbiotic substances was analyzed by the agar spot test [33] and expressed in arbitrary unit per mL (AU/mL). It corresponds to the highest dilution of the CPS (ratio 1:1 in phosphate buffer, pH6.5), which inhibited the growth of the indicator strain. The CPS was stored at −20 °C.

3. Results

3.1. Species Strain Characterization

Among the isolated enterococci, strains of Enterococcus faecalis (5) species were identified (EE/K3, EE/K5, EE/G3, EE/G6, and EE/G7). They were identified as a result of sequencing (BLASTn 16S rRNA). A sequence identity percentage from 98.45% up to 99.91% (

Table 2. The species identification of isolated strains based on sequencing.

Strain	% Identity BLASTn 16S rRNA Sequence
Enterococcus faecalis EE/K3	98.45%
Enterococcus faecalis EE/K5	99.56%
Enterococcus faecalis EE/G3	98.65%
Enterococcus faecalis EE/G6	99.91%
Enterococcus faecalis EE/G7	99.50%

) was achieved compared with the strain E. faecalis ATCC19433 in GenBank. The strain EE/K3 reached a sequence similarity up to 98.45% (NR_115765) with the strain E. faecalis ATCC19433. The sequence similarity for the strain EE/K5 was identical (98.56%) (NR_ 115765.1) as the strain E. faecalis ATCC19433. The other strains also showed their sequence similarity/identity with the same strain, E. faecalis ATCC19433, in GenBank. A sequence similarity (NR_115765.1) of 98.65% was also allotted to the strain EE/G3 with the species E. faecalis. The highest sequence similarity (NR_115765.1) was noted for the strains EE/G6 (99.91%) and EE/G7 (99.50%).

3.2. Hemolysis and Enzyme Analysis Using the API ZYM Test and Virulence Factor Gene Analysis

The strains EE/K3 and EE/K5 showed α-hemolysis on blood agar. The other strains, E. faecalis EE/G3, EE/G6, and EE/G7, did not produce hemolysis (ɤ-hemolysis). Regarding the enzyme profile, the tested strains of E. faecalis did not produce α-mannosidase, α-fucosidase, N-acetyl-β-glucosaminidase, β-glucuronidase, or lipase. However, they produced high levels of esterase (C40;

Table 3. The enzyme production tested in E. faecalis strains using the API ZYM panel in nmoL.

	EE/K3	EE.EF/K5	EE/G3	EE/G6	EE/G7
Alkaline phosphatase	5	10	0	5	5
Esterase (C4)	20	40	30	30	20
Esterase lipase (C8)	20	30	20	20	30
Leucine arylamidase	10	10	10	10	10
Valine arylamidase	5	10	10	5	5
Cystin arylamidase	5	10	5	5	5
Trypsin	5	5	5	5	0
α-chymotrypsin	20	20	40	20	10
Acid phosphatase	40	40	20	30	20
Naphtol-AS-BI-phosphohydrolase	10	10	10	5	5
α-galactosidase	0	5	0	0	0
β-galactosidase	5	5	0	5	0
α-glucosidase	0	5	0	0	0
β-glucosidase	0	0	5	5	0

N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase, lipase, and β-glucuronidase were not produced by selected E. faecalis strains.

; from 20 nmoL to 40 nmoL). Levels of 20–30 nmoL were also measured for the enzyme esterase lipase. The strains showed a high value of α-chymotrypsin production (10–40 nmoL). The values of acid phosphatase were also high (20–40 nmoL). In the case of Naphtol-AS-BI-phosphohydrolase, a production of 5–10 nmoL was detected by the strains. The other enzymes were measured in low values (5–10 nmoL).

The genes for six virulence factors (hyl, IS16, cylA, gelE, agg, and esp) were evaluated. The E. faecalis were absent of the hyl, IS16, and cyl genes. The GelE gene was detected in three strains (EE/K3, EE/G3, and EE/G6,

Table 4. Virulence factor encoding genes detected in selected E. faecalis.

Strains	gelE Gene	agg Gene	esp Gene
EE/K3	+	−	−
EE/K5	−	−	+
EE/G3	+	−	−
EE/G6	+	+	−
EE/G7	−	−	+

The genes IS16, hyl, and cylA were not present in the E. faecalis strains.

). The Agg gene was detected only in the strain EE/G6, and the esp gene was detected in the strains EE/K5 and EE/G7. It can be summarized that each tested strain possessed one virulence factor gene. The Agg gene possessed the strain EE/G6, and the esp gene was found in two strains, EE/K5 and EE/G7. Two genes were found only in the strain EE/G6.

3.3. Biofilm-Forming Assay and Biofilm Gene Detection

Regarding the plate assay for biofilm-forming ability testing, all strains were measured with low-grade biofilm formation (0.1 < A570 ≤ 1.0, Table 4). The strain EE/G3 reached the highest value in biofilm formation (0.246 ± 0.171), followed by the strain EE/G6 (0.242 ± 0.166). The strains EE/K5 and EE/K3 were found to have similar values. Surprisingly, the strain with the most pathogenic character, EE/G7, showed the lowest value of biofilm formation (0.137 ± 0.062,

Table 5. Biofilm-formation-encoding genes detected in E. faecalis.

Strains	ace Gene	bopD Gene	srtA Gene	Biofilm
EE/K3	−	+	−	0.153 ± 0.077
EE/K5	+	+	−	0.157 ± 0.082
EE/G3	−	+	−	0.246 ± 0.171
EE/G6	−	+	+	0.242 ± 0.166
EE/G7	+	+	−	0.137 ± 0.062

The genes fsr, ica, and bap were not present in E. faecalis strains.

).

Six genes coding biofilm-forming ability were tested (ace, fsrA, bopD, srtA, ica, and bap). However, E. faecalis were absent of the ica, bap, and fsr genes. All strains had the bopD gene (Table 4). The gene srtA was detected in the strain EE/G6 only, and the ace gene was only detected in the strains EE/K5 and EE/G7 (Table 5). This means that the tested strains had at most two biofilm-forming genes, bopD and srtA (EE/G6) or ace and bopD (EE/K5 and EE/G7).

3.4. Relation of Enterococcus faecalis Strains to Antibiotics and Susceptibility to Postbiotic Substances Produced by the Strains Lactococcus lactis MK2/8 and MK1/3

All E. faecalis strains were kanamycin- and streptomycin-resistant (Table 5). They were susceptible to ampicillin, with a susceptibility zone of 15–23 mm. The zones for penicillin reached 16–20 mm and for erythromycin 15–26 mm. In the case of rifampicin, the zones measured 20–27 mm, and for chloramphenicol the susceptibility zones reached 12–21 mm. The strains EE/G3 and EE/G6 were resistant to tetracycline (R < 10). The strain EE/G7 was also resistant to gentamicin (

Table 6. Average size of inhibitory zone testing susceptibility to antibiotics of the strains E. faecalis using disk diffusion method expressed in mm from double testing.

	EE/K3	EE/K5	EE/G3	EE/G6	EE/G7
Ampicillin (10 µg)	15/22	23/15	20/10	20/10	22/10
Penicillin (10 IU)	20/20	20/20	20/20	16/16	19/19
Erythromycin (15 µg)	20/26	15/25	16/21	15/20	20/25
Tetracycline (30 µg)	20/25	15/23	R (<10)	R (<10)	20/23
Vancomycin (30 µg)	15/15	14/14	R (<10)	R (<10)	R (<10)
Rifampicin (30 µg)	25/25	20/23	22/25	21/27	20/22
Chloramphenicol (30 µg)	19/20	17/20	20/20	12/21	18/21
Gentamicin (120 µg)	12/11	12/12	15/11	17/17	R (<10)

). Three strains were resistant to three antibiotics (Table 6).

Using the E-strip method, the strains were susceptible to antibiotics (Table 6) with different minimal inhibitory concentrations (MICs). The resistance was determined in the case of streptomycin (Table 6). For ampicillin, the MIC ranged from 0.25 to 0.50 µg. For erythromycin, the MIC reached from 1.00 up to 2 µg (

Table 7. The minimal inhibitory concentration under which strains are inhibited and/or susceptible by antibiotic used (E-strip method).

	EE/K3	EE/K5	EE/G3	EE/G6	EE/G7
Ampicillin (256-0.016 µg/mL)	MIC: 0.25 µg (S)	0.50 µg (S)	0.50 µg (S)	0.50 µg (S)	0.50 µg (S)
Erythromycin (256-0.016 µg/mL)	1.00 µg (S)	1.00 µg (S)	2.00 µg (S)	2.00 µg (S)	1.5 µg (S)
Tetracycline (256-0.016 µg/mL	0.30 µg (S)	0.38 µg (S)	32.00 µg (S)	48.00 µg (S)	0.38 µg (S)
Vancomycin (256-0.016 µg/mL)	1.00 µg (S)	0.50 µg (S)	4.00 µg (S)	6.00 µg (S)	1.3 µg (S)
Streptomycin (256-0.064 µg/mL)	128.0 µg (R/S)	256 µg (R)	256 µg (R)	256 µg (R)	128.0 µg (R/S)
Chloramphenicol (256-0.016 µg/mL)	12.00 µg (S)	16.00 µg (S)	8.00 µg (S)	32.00 µg (S)	12.00 µg (S)
Gentamicin (1024-0.064 µg/mL)	16.00 µg (S)	24.00 µg (S)	32.00 µg (S)	24.00 µg (S)	32.00 µg (S)

S: susceptible; R: resistant; MIC: minimal inhibitory concentration.

). The MIC for tetracycline was in the range of 0.30 µg to 0.48 µg. For vancomycin, various MICs were achieved: 0.50 µg (EE/K5) to 6.00 µg (EE/G6). Differences were noted in the MIC for chloramphenicol at 12.0 µg–32.0 µg and for gentamicin as well (16,0 µg up to 32.0 µg).

However, the strains were susceptible to treatment with the postbiotic active substances MK2/8 (inhibitory effect from 100 to 400 AU/mL) and MK1/3 (from 100 to 400 AU/mL) as well. This susceptibility was well balanced (

Table 8. Susceptibility of E. faecalis to postbiotic substances produced by lactococci MK2/8 and MK1/3 (inhibitory activity in arbitrary unit per milliliter (AU/mL)).

Strains	Substance MK2/8	Substance MK1/3
EE/K3	400 AU/mL	100 AU/mL
EE/K5	100 AU/mL	400 AU/mL
EE/G3	400 AU/mL	100 AU/mL
EE/G6	400 AU/mL	400 AU/mL
EE/G7	400 AU/mL	400 AU/mL

The activity against E. avium (the principal indicator ranged from 800 up to 1600 AU/mL).

).

4. Discussion

4.1. Virulence Factor Genes

The species Enterococcus faecalis is a natural member of the intestinal microbiota of animals and humans, and it has also frequently been detected in various milks [5,7]. Especially, clinical strains of this species could threaten host health or cause problems during product processing. This impact has been focused on the presence of virulence factors in strains of this species. Moreover, the detection of virulence factor genes (which are responsible for their pathogenic phenotype) is of paramount interest. Regarding the biofilm formation, the presence of the following genes was tested in the strains E. faecalis: ace, bopD, fsr, srtA, bap, and ica. Among those tested genes, the genes bopD, srtA, and ace were detected. Ica and Bap are surface proteins participating in the cellular adhesion step in biofilm formation [31]. As previously mentioned [30], the fsr gene was allotted in the category regulatory system functioning in quorum sensing. The Ace gene has a function in initial adhesion in the biofilm-forming process [27]. López-Salas et al. [34] reported a significant correlation between the ace gene presence and tetracycline resistance, as also confirmed in our study in the strain EE/G6. Moreover, correlation between the agg gene with biofilm formation was found and also confirmed in the strain EE/G6, which showed low-grade biofilm formation. Ghaziasgar et al. [35] found that ace-positive isolates were significantly more potent for biofilm formation, and all ace-gene-containing enterococci had high biofilm development [36]. As formerly mentioned, the BopD protein has a function in vitro as the transcriptional regulator in biofilm formation [28]. The enzyme srtA functions in surface protein processing during biofilm formation [28]. Esp is surface protein—a specific adhesin that binds to collagen, which most frequently associated with the strains of the species E. faecalis [36], as also shown in our study. The contribution of aggregation substance (agg) in adhesion has been shown in many studies. Factor determinants like agg, esp, and ace impact biofilm formation on cultured human cells [36].

4.2. Antibiotic Resistance and Enzymes

Virulence factors have also shown antibiotic resistance. Some strains have been found to vancomycin-resistant, although this has not been detected Van genes. Vancomycin disturbs the development of the cell wall in growing bacteria, and resistance is based on changed targets for antibiotic–drug interactions [37].

Bacterial enzymes play their own role in the pathogenic characteristic of bacteria. Bacterial alkaline phosphatase (ALP) is phosphatase with the physiological role of dephosphorylating compounds. In humans, this is the enzyme associated with liver diseases or bone disorders. In the detected E. faecalis, the values of ALP were low (up to 10 nmoL). On the other side, ALP is commonly used in the dairy industry as an indicator of successful pasteurization. Acid phosphatase represents enzymes that are found in organisms ranging from bacteria to mammals, and they may play an integral role in microbial virulence or can be used for bacterial taxonomy and identification [38,39]. Microbial β-glucuronidase can play role in the development of different diseases, e.g., those related to estrogen metabolism [40]. A high value of chymotrypsin (20–40 nmoL) and/or acetyl-β-glucosaminidases may be related to the pathogenic tendency of the tested E. faecalis strains. However, N-acetyl-β-glucosaminidase was not produced by the E. faecalis strains. Also, β-glucuronidase was absent in the studied E. faecalis strains, which is an enzyme related to colon cancer.

4.3. Postbiotic Substances Against Contaminant Bacteria

There is extensive evidence regarding the use of postbiotic substances against contaminants. One important factor driving the interest in postbiotics is their inherent stability, both during industrial processes and storage [18,41]. Moreover, postbiotics could reasonably be expected to have a better safety profile than, e.g., probiotics. This is because the microbiota they produce are inanimate, and they have lost the capacity to replicate. This means they are without the same risks [18,41]. They have become very prominent in both nutrition and health sciences, as they are believed to confer various health benefits to consumers without the attendant concerns arising from the ingestion of, e.g., living forms of probiotics [18,41]. Postbiotics also can minimize the potential risk of antibiotic resistance, which is, however, still being discussed. They are also usually more stable during processing and/or storage [18,41].

Maintaining the safety of goat milk is important because it is a key functional food. This means it is a food with components partially or fully affecting consumer health [42]. The components in dairy that may purport and/or indicate milk as a functional food include above all calcium, vitamin D, probiotic and postbiotic active lactic acid bacteria, conjugated derivates of linoleic acid, and other bioactive peptides derived from milk proteins [42]. Venega-Ortega et al. [43] describe bioactive peptides from lactic acid bacteria as a sustainable approach for healthier foods. Pure bacteriocins from lactococci have been studied for decades; however, new methods to achieve pure and/or non-purified substances have been developed. Nisin (now called lantibiotic bacteriocin) was first described as a bacteriocin produced by some strains of the species Lactococcus lactis [44]. Then, lacticin 3147, produced by Lactococcus lactis subsp. lactis DPC3147 [45] isolated from Irish grain kefir used for making buttermilk, was described as a two-component bacteriocin with a hydrophobic nature. Its activity is acid-dependent. It is mostly used for anti-listeria purposes in yogurts or cheeses. Postbiotic substances secreted by raw-goat-milk-derived lactococci (MK1/3, MK2/8) have been mostly found to have an inhibitory effect against Gram-positive bacteria. These postbiotic-substance-producing strains are applied in yogurts [19,20]. This evidence indicates the need for further study of postbiotic active substances, especially from autochthonous lactococci, to understand their further use for the sustainable quality and safety of dairy products. The postbiotic substance Durancin ED26E/7 inhibited the growth of E. faecium and E. hirae from raw goat milk [5]. In addition, Luenglusontigit et al. [46] described a postbiotic preparation produced by E. faecalis PK1201 with an inhibitory effect against Clostridioides difficile.

5. Conclusions

Biofilm-forming strains of E. faecalis (from raw goat milk) with the presence of the ace, bopD, and/or srt genes encoding biofilm formation, with two strains possessing gelE, esp, and agg virulence factor genes, were susceptible to postbiotic active substances produced by the autochthonous strains Lactococcus lactis MK2/8 and MK1/3. Treatment of E. faecalis with postbiotic substances indicates an alternative eliminating strategy to fighting raw goat milk contaminants.