In ongoing feed-trial studies, we screened several lactic acid bacteria from pigs fed different sources and levels of dietary fiber and undertook morphological, biochemical, and molecular characterization of strains with potential probiotic properties [910]. The first aim of the current study was to determine the probiotic properties of a new strain characterized as Enterococcus (E.) faecalis and designated as E. faecalis PSCT3-7. Probiotics administered to animals are usually incorporated to carrier medium. Vermiculite, an important support and carrier medium for a range of nutrients for animals, is also reported to create a protective envelope around microorganisms [514]. Therefore, our second aim was to examine the properties of E. faecalis PSCT3-7 adsorbed onto vermiculite.

The strain investigated in this study was taken from 23 lactobacilli strains we recently screened from the intestines of pigs fed dietary fiber sources containing 50% sawdust for 4 weeks (Unpublished). The strain was selected as it exhibited the greatest activity of digestive enzymes, including protease, cellulase, phytase, and α-amylase, based on API ZYM system results. Identification of E. faecalis PSCT3-7 was based on morphological and biochemical characterization using a negative catalase reaction, a positive PYR test (Murex Diagnostika, Germany), and the API 20 Strep system (bioMérieux, Germany) as described previously [16]. Its identity was confirmed by MALDI-TOF mass spectrometry and 16S rRNA sequence analysis and comparison with sequences available in the GenBank database using the BLAST algorithm (National Center for Biothechnology Information, USA). Sequence comparisons done by the Korean Culture Center of Microorganisms (Korea) showed 100% similarity of the strain to E. faecalis with the best sequence match with E. faecalis EU708623 (Fig. 1).

Fermentation experiments were carried out in lactobacilli MRS medium as described previously [9]. In a 48 h study, pH and dissolved oxygen (DO) were automatically monitored, and the growth of cells was monitored by measuring the optical density at 600 nm (OD₆₀₀). Bacterial counts were performed by using one milliliter samples taken throughout the incubation period. During the 48 h incubation period, the pH of the culture gradually decreased (panel A in Fig. 2) and the level of DO sharply fell from 17 mg/L to 5.3 mg/L during the first 12 h, increased thereafter, and reached 18 mg/mL by 48 h (panel B in Fig. 2). The OD₆₀₀ results showed a sharp increase, peaked at 12 h, and remained stable and high until 48 h (panel C in Fig. 2). Similar to the OD₆₀₀ changes, E. faecalis PSCT3-7 showed an initial exponential growth followed by a stationary phase (panel D in Fig. 2).

Acid and bile tolerance tests were performed as described previously [9], in pH-adjusted medium (pH 2–8) or by adding 0.3% bile salts (Sigma, USA) in the medium. E. faecalis PSCT3-7 tolerated a pH of 3 and grew well in a pH range of 3 to 8 for 2 h (panel E in Fig. 2). Intestinal contents or feed matrix may prevent exposure of probiotic bacteria to bile thereby increasing the survival and functioning of the organism in the gastrointestinal tract [3]. Hence, we tested whether adsorption to vermiculite may improve the acid and bile tolerance of E. faecalis PSCT3-7 by adding 1% (w/v) vermiculite in the media and found no enhancement in acid tolerance of the strain (panel F in Fig. 2). However, while E. faecalis PSCT3-7 showed viability for 3 h in medium containing 0.3% bile salts, a longer survival (up to 6 h) of the strain was observed when 1% vermiculite was added in the bile-containing medium (data not shown). The survival and growth of E. faecalis PSCT3-7 over a wide range of pH and in media containing bile salts suggest that the strain can survive and grow both in the acidic environment of the stomach and in the presence of intestinal bile salts.

The inhibitory activity of E. faecalis PSCT3-7 (starting inoculum: 10⁵, 10⁷ or 10⁹ colony-forming unit [CFU]/mL) against Salmonella enterica serotype Typhimurium (Salmonella Typhimurium, KCTC 2515, starting inoculum: 10³ or 10⁶ CFU/mL to model early and late infections in pigs) was determined by performing co-culture experiments in triplicate. A one-way ANOVA with Dunnett's post hoc analysis was performed for Salmonella Typhimurium CFU/mL obtained after incubation without or with 3 levels of E. faecalis PSCT3-7. Significantly lower Salmonella Typhimurium CFU/mL (p < 0.05) was observed between the highest E. faecalis PSCT3-7 levels (10⁹ CFU/mL) and all other treatments, both at 6 and 12 h post-incubation. While Salmonella Typhimurium CFU/mL continuously decreased in co-cultures with 10⁹ CFU/mL E. faecalis PSCT3-7, Salmonella Typhimurium cell counts reached more than 10¹⁰ CFU/mL by 12 h in experiments with out or 10⁴ or 10⁷ CFU/mL E. faecalis PSCT3-7 (panels A and B in Fig. 3). The CFU/mL of Salmonella Typhimurium in the absence or presence of different levels of E. faecalis PSCT3-7 was fitted with the inhibitor-versus response model built in GraphPad Prism (GraphPad Software, USA), with the equation: Y = Bottom + (Top − Bottom)/(1 + (X/IC₅₀)), where Top and Bottom are Salmonella Typhimurium CFU/mL in the absence of E. faecalis PSCT3-7 and at maximum growth inhibition in the presence of E. faecalis PSCT3-7, and IC₅₀ is the minimum level of E. faecalis PSCT3-7 required to inhibit the growth of Salmonella Typhimurium by 50% in the co-culture experiments. The IC₅₀ values were determined to be 1.39 × 10⁸ and 2.39 × 10⁸ CFU/mL for Salmonella Typhimurium inocula of 10³ and 10⁶ CFU/mL, respectively (panels C and D in Fig. 3). Mechanisms for inhibition of Salmonella Typhimurium growth by E. faecalis PSCT3-7 remain to be described. However, it has been reported that E. faecalis strains adhere to intestinal cells and produce antimicrobial substances [713], suggesting that E. faecalis PSCT3-7 may remain the dominant microflora in intestines; thereby, preventing invasion by Salmonella Typhimurium strains of infected animals. Furthermore, addition of vermiculite in the culture medium did not affect the antibacterial activity of E. faecalis PSCT3-7 against Salmonella Typhimurium (data not shown), suggesting that vermiculite could be used as a potential carrier for administration of E. faecalis PSCT3-7 to pigs.

Minimum inhibitory concentrations (MIC) of several antibacterial agents against E. faecalis PSCT3-7 were determined according to Clinical and Laboratory Standards Institute guidelines and by using Staphylococcus aureus and Escherichia coli quality control strains [4]. E. faecalis PSCT3-7 showed resistance to several antibacterial agents with MIC (µg/mL) values of greater than 512 (colistin, spectinomycin, streptomycin), 256 (chloramphenicol, florfenicol, norfloxacin, novobiocin), 128 (cephalexin), 64 (bacitracin, marbofloxacin), and 32 (gentamycin), while it was susceptible to amoxicillin at a MIC value of 1 µg/mL. This resistance pattern may be considered advantageous because probiotic strains with antibiotic resistance could be useful for restoring gut microbiota when administered to animals that are undergoing antibiotic treatment [8].

The ultrastructural morphology of E. faecalis PSCT3-7 was studied by using a scanning electronic microscope (SEM; models S-4300 and EDX-350; Hitachi, Japan). Sample preparations were essentially as described previously [616]. The SEM images revealed that E. faecalis PSCT3-7 is spherical or oval and is divided with perfect symmetry (panel A in Fig. 4). In addition, binary fission of the bacteria was evident in the SEM images.

Probiotics prepared as feed additives should have stability and longevity to ensure extended physiological activity. This can be achieved by encapsulation or by using carrier medium, such as vermiculite [2]. Adsorption of E. faecalis PSCT3-7 onto vermiculite was studied by using a SEM. Vermiculite-adsorbed E. faecalis PSCT3-7 was prepared according to a previous method [16]. Panel B in Fig. 4 shows the typical silicate clay with porous and lamellar structures of expanded vermiculite. SEM images show good adsorption capacity of E. faecalis PSCT3-7 onto 1% vermiculite with several cells adsorbed on the surface of the plate and inside the porous structures (panel C in Fig. 4).

In summary, E. faecalis PSCT3-7 possesses the essential characteristics of a potential probiotic bacterial strain, including the ability to survive in bile salts and at a low pH. Moreover, it has antibacterial activity against Salmonella Typhimurium and good adsorption onto vermiculite. Future studies will further establish the clinical functionality of the strain in piglets challenged with enteric bacteria.