Exploring Enterococcus Species for their Next-Generation Probiotics Potential

2.1 Prospects of next-generation probiotics as biotherapeutics

Probiotics have been used as fermented foods by humans for a long time, but the beneficial effects of these bacteria were not well understood until the development of new and emerging techniques. Probiotics of the current generation are demonstrated to have a role in the maintenance of the gut microbiota and can reduce inflammatory responses, allergic illnesses, and autoimmune diseases [51]. New NGPs with potential beneficial characteristics for human health expand this traditional probiotic spectrum and contribute to the development and elaboration of new food products that respond to the population’s growing interest in health and quality of life, again making the NGPs an important area in the food industry. The biotherapeutics potential of NGPs should be stated after passing the three phases of clinical trials, that is, preclinical, toxicological studies, and pharmacodynamics [46, 52, 53, 54].

The availability of microbe-free animal models will aid in the identification of safe NGPs that meet regulatory parameters such as the US-LBP FDA’s program (US Live Biotherapeutic Products, Food and Drug Administration). The efficacy and safety of the next generation of probiotics and the technological elements of using these microbes in food preparation are considered the most important concerns in processing. The majority of these microorganisms have metabolic properties that make them challenging to utilize as sustainable methods in the development of novel products, particularly in large-scale food production [53]. However, limited studies have been conducted to establish the effectiveness and safety of these microorganisms, necessitating more research in in vivo systems and clinical trials. In the food industry, there must be studies that analyze the potential applications of next-generation probiotics in food matrices and their impacts on the intrinsic technical and sensory properties of food. In the future, these microbes may be employed as bio-therapeutic items and sold mostly as nutritional supplements [46].

2.2 The next-generation probiotics microorganisms

As described, the NGPs have advanced functions over traditional probiotics and are used in a specific application. A number of species, mostly from the gut microbiota, are targeted to be screened for their next-generation probiotic potential. These include Akkermansia muciniphila, Faecalibacterium prausnitzii, Bacteroides fragilis, Eubacterium hallii, Bifidobacterium spp., Prevotella copri, Christensenella minuta, and Roseburia spp. [48, 55, 56]. It is important to mention here that the NGPs include commensal species that have no previous history of safe use. Likewise, the conventional probiotics, with the beneficial effects of NGP, are also strain-dependent [47]. The NGPs of various bacterial species are previously documented [46, 57, 58].

3.1 Enterococcus faecium

E. faecium is one of the most prevalent enterococcal species in the environment and has received exceptional attention for its probiotic potential. It is generally considered a safe species and is hence widely used in different industries. It has a small genome size (2.23 to 3.72 Mbp) and a short generation time, which make it suitable for its NGP potential. The production of multiple enterocins also aids in their NGP potential [1, 39]. E. faecium also showed greater environmental persistence and became dormant under stress conditions [39]. Currently, a good number of probiotic strains from E. faecium have been identified and developed commercially, indicating its excellent NGP potential. These developed probiotics have good potential to treat different diseases and aid in intestinal microflora restoration probably due to their short lag phase and generation time. Its strains can hinder the growth of E. coli, Salmonella Serovars, Shigella spp., and Enterobacter spp. in vitro, while also showing resistance to low pH and bile. Some strains showed anti-inflammatory and immunomodulatory potential, which is because of their inhibition of nuclear factor-κB [59, 60, 61].

There are vast areas in which E. faecium probiotics are used. For instance, they are used in the treatment of diseases, the restoration of gut flora, and as food and feed supplements for pets [29]. Some strains also decrease cholesterol and help in the production of cheese as starter culture [62]. E. faecium probiotics are taken via different routes, like oral and inhalation, depending on the specific applications [63]. The commercially available E. faecium probiotics, such as E. faecium SF-68 and ECOFLOR, have proven benefits and are not associated with any potential side effects [62]. E. faecium M74 strains in boilers increase serum calcium contents and antioxidant status [64]. Likewise, the heat-killed E. faecium NHRD IHARA is suggested to be used as feed additives [65]. Its potential in veterinary medicine is also elucidated and found to have promising results [66]. Thus, highlighting these probiotics, it is concluded that E. faecium has excellent NGP potential and can be explored for more applications.

3.2 Enterococcus faecalis

E. faecalis has the highest prevalence in hospital settings and is considered one of the virulent enterococcal species. Generally, E. faecalis causes 75–80% of the enterococcal infections. Due to its widespread distribution and the ability to tolerate harsh conditions, it is often isolated from medical settings, while a low proportion is also collected from other environmental niches. It has a larger genome size (2.7 Mb) than E. faecium and generally shows more virulence characters instead of resistance [1, 37].

Unlike E. faecium, the NGP potential of E. faecalis is less investigated due to its apparent virulence profile, but still, few strains of it are commercialized as probiotics. For example, Symbioflor® 1 is a leading E. faecalis probiotic that has the properties of immune regulation and combats chronic sinusitis and bronchitis [40]. In combinatorial treatment, the effectiveness of Symbioflor® 1 was found to enhance the treatment potential of respiratory diseases [67, 68]. Upon extensive investigation, the E. faecalis strains can be used in different applications, including medicinal aspects and food supplements. E. faecalis strains are generally considered in that category, which requires extensive characterization, particularly their genomic analysis.

3.3 Enterococcus hirae

E. hirae is a non-motile enterococcal species that was identified in 1985 and has lower environmental prevalence. It is frequently isolated from the intestinal flora of different domestic animals [69]. It is associated with different diseases, like septicemia and focal necrosis. E. hirae has the potential to produce acid from melibiose and sucrose and shows close resemblance to E. durans [69]. Its NGP potential is explored due to its anti-inflammatory and antibacterial potential. E. hirae strains also have tolerance potential, good hydrophobicity, and no antibiotic resistance. The ability to produce enterocins and their antibacterial activity against Micrococcus luteus, Listeria monocytogenes, Salmonella Typhi, Staphylococcus aureus, Shigella flexneri, Pseudomonas fluorescens, E. coli O157:H7, etc., can aid in their NGP potential [70]. Its major enterocin is Hiracin JM79, which belongs to Class IIa antilisteria-pediocin-like bacteriocins [71].

The currently available E. hirae probiotics not only confirmed their NGP potential but can also indicate their widespread applications. These NGPs show the therapeutic potential and have immunomodulatory effects without any documented side effects [72]. Adnan et al. documented the NGP potential of E. hirae F2 strain and identified its antimicrobial and antioxidant activities, while suggesting its promising probiotic potential [73].

3.4 Enterococcus durans

E. durans has close similarities with E. hirae and has a low environmental prevalence [74]. It is found in the gut in rare cases and is responsible for different animals’ diseases like enteritis [69]. Characteristically, it is positive for the arginine test and shows negative tests against arabinose, raffinose, pyruvate, and sorbose [74]. Like other enterococcal probiotics, E. durans also has the tolerance potential, adherence capacity, and ability to colonize mucosal surfaces, have no virulence traits, show antibacterial potential, and have immune modulatory effects. It also has an effect on cytokine production and enhances IgA secretion [75]. These properties enable E. durans strains to be targeted as NGP and, hence, play a role in wide applications.

Different E. durans strains are identified as having good NGP potential. For instance, E. durans M4 was found to have anti-inflammatory effects and preserve intestinal epithelial integrity, indicating its prophylactic potential [76]. Another strain, E. durans VJI19, was found to have antifungal and pro-apoptotic activities based on its palmitic acid and 2,3-dihydro-3,5-dihydroxy-6-methyl 4H pyran-4-one. Additional properties such as the presence of hydrophobicity, tolerance, and colonization potential suggest its NGP potential [77]. Likewise, the potential of E. durans 6H and E. durans LAB18s was also screened and identified its NGP potential [60, 78]. Human serum cholesterol lowering strain E. durans KLDS 6.0930 is also documented as a potential probiotic strain [8].

3.5 Enterococcus lactis

E. lactis has a close resemblance to E. faecium and was first isolated in 2012 from milk. It has distinct genomic and carbohydrate metabolism properties and is thus considered a separate species [79, 80]. The absence of antibiotic resistance genes and virulence traits makes the species more suitable for NGP potential even than E. faecium with applications in the food and health industries [80]. The disease-causing potential of E. lactis is much less than that of E. faecium. Due to their close resemblance with E. faecium some probiotics that are claimed to contain E. faecium actually contain E. lactis [81]. E. lactis shows gelatinase negative potential and carries enterocin-producing genes [79]. The existing E. lactis probiotic strains mostly have the cholesterol-lowering potential and susceptibility to antibiotics, besides other probiotic properties [79].

Once the E. lactis is recognized as a separate species, it attracts researchers to identify its NGP potential because of its apparent safe profile and enterocins production capacity [79]. The NGP potential of E. lactis makes it a suitable candidate to prevent and help in the treatment of different human diseases, like malignant tumors. It prevents the tumor in different ways, such as reducing the expression of pro-carcinogenic enzymes, improving normal cell proliferation, and hindering inflammation-associated cell apoptosis. For instance, E. lactis BT 16 and E. lactis MTCC 25438 strains, which are isolated from cheese and goat milk, respectively, have the cholesterol-lowering potential and thus indicate their NGP potential [68, 82]. Other strains, such as E. lactis YHC20, E. lactis FM11-1, and E. lactis IW5, showed good cellular hydrophobicity and carbohydrate and bile salt hydrolase (BSH) activity [83, 84, 85]. The NGP potential of strain E. lactis PMD74 was investigated, and it was observed that, besides other probiotic properties, this strain shows greater survivability in fermented milk [86].

3.6 Enterococcus gallinarum

E. gallinarum, a motile microbe, besides other enterococcal species, is part of the normal stool flora of chickens and cattle but was also isolated from clinical samples. It is considered less pathogenic than other clinically isolated species [87, 88, 89, 90]. It shows 99.8% homology in 16S rDNA similarities with E. casseliflavus, making it difficult to distinguish [91]. It causes common enterococcal infections such as bacteremia, endocarditis, and meningitis. A rare effect on the central nervous system is also identified [92, 93]. It is separated from its close partner, E. casseliflavus, as it does not produce the yellow pigment [89].

The next-generation probiotic potential of E. gallinarum, though it is not authorized for probiotics, is still established and some strains have been found that have the NGP potential [94]. The NGP potential is mostly dependent on the strains antimicrobial potential; for example, E. gallinarum T71 and W21 were found to have broad-spectrum activity and hence can be investigated for use as starter cultures in foods [95]. Another strain, E. gallinarum L-1, was evaluated on the cellular immune systems of four different fish species and found to stimulate the leucocytes of the tested fishes [96].

3.7 Enterococcus casseliflavus

E. casseliflavus is the second motile specie of the genus Enterococcus that has the capacity to produce a yellow pigment, and hence, it is separated from other motile specie, that is, E. gallinarum [89, 97]. This species shows an environmental adaptation to aquatic vegetation [97]. Its inulin fermentation property makes it a distinguishing species and has the potential of environmental persistence, facilitated by cell-associated carotenoid pigments that also protect it against photooxidation [89]. Another unique feature of this specie is the presence of acetoin dehydrogenase genes (ECAG_02019 to ECAG_02022), which helps in the conversion of acetoin to acetaldehyde and acetyl coenzyme A [89]. Despite enterococcal species, it is considered a less virulent species [94].

Unlike other enterococcal species, E. casseliflavus and E. gallinarum are not authorized for probiotic potential, but recently they were found in two probiotic products indicating their NGP potential [94]. This potential is mostly because of its antimicrobial potential, bacteriocins production, improving growth performance, immune modulation, and resistance against pathogens [98]. Studies also showed the tolerance potential of E. casseliflavus, particularly in its fermentation profile [99]. Deng et al. [94], identifying the new insight of antibiotic resistance in enterococcal probiotics, declared that both E. casseliflavus and E. gallinarum are free from antibiotic resistance genes but can carry low-level vancomycin resistance genes (i.e., vanC), thus influencing the host immunity [23, 94, 100]. Recently, a study was conducted to identify the response of cancer patients to chemotherapy treatment, and it was identified that E. casseliflavus is more abundant in responding compared to non-responding patients, thus indicating its potential to be used as a biological biomarker for chemotherapy response in lung cancer patients [101].

3.8 Enterococcus avium

E. avium is a member of the genus Enterococcus that is commonly isolated from bird droppings [87] and was previously included in group Q Streptococcus [74]. It is also isolated from hospital settings and identified as showing similar resistance to E. raffinosus [74], but being negative to raffinose metabolism, it is considered a separate species [102]. The pathogenic nature of this specie is less understood but still involved in different enterococcal infections, including peritonitis [74]. E. avium also has the potential to produce enterocins like Avicin A, a double-glycine lead bacteriocin belonging to the class IIa anti-listeria-pediocin-like bacteriocins [71].

The next-generation probiotic potential of E. avium is rare, but there are some positive attributes that might be helpful in its NGP status. For instance, the production of Avicin A, which is isolated from babies’ feces, indicates its distribution in the GIT and helps in the maintenance of normal commensal flora [71]. The results of another study showed that E. avium has no virulence factors, antibiotic resistance, inhibits the growth of L. monocytogenes, shows adhesion to epithelial cells, and has greater cholesterol assimilation potential. These properties indicate the NGPs of E. avium and could be used in functional foods, particularly in broiler chicks [103, 104]. In contrast, a sample isolated from animals in Tunisia indicated the presence of vancomycin resistance in E. avium isolate [105].

3.9 Enterococcus mundtii

E. mundtii belongs to the group E. faecium, has ubiquitous distribution, and is found in plants, the gut of animals, and raw milk with low GC contents of 38–39% [106]. Functionally, E. mundtii lacks catalase and cytochrome C oxidase enzymes but can perform carbohydrate fermentations. It has the potential to produce enterocins, that is, Bacteriocin ST15, and thus has antibacterial activity against Pseudomonas, Clostridium, Klebsiella, Lactobacillus, Acinetobacter [106].

The NGP potential of E. mundtii is greatly investigated and identified, as many strains have the NGP potential. Tolerance, hydrophobicity, higher auto-aggregation, and no hemolytic activity, besides no antibiotic resistance genes, are the major probiotic attributes [107]. The NGPs potential is mostly because of its bacteriocin and enzyme production abilities, anti-pathogenic capacity, and tolerance potential. Nawaz et al. documented the different enzymatic activities (i.e., proteolytic, lipolytic, amylolytic, and cellulolytic) of E. mundtii QAUEM2808, suggesting its role in aroma and flavor development in food [106]. Likewise, another strain, E. mundtii ST4SA, is also documented as a potential probiotic strain [8]. Grau et al. documented that administration of the E. mundtii strain in Tribolium castaneum (the red flour beetle) indicated the desired probiotic properties, increasing the researcher’s interest in using this microbe for in vitro probiotic characterization [107]. Similarly, experiments conducted to check the strain’s survivability in the presence of antibiotics indicated that E. mundtii ST4V has the potential to survive in the presence of most of the administered antibiotics, thus, suggesting its potential to be used as a probiotic in those individuals who are receiving medical treatment [108].

3.10 Enterococcus raffinosus

E. raffinosus is a non-motile, enterococcal species, first identified in 1989, and shows negative tests for tellurite and arginine but positive tests for mannitol, sorbose, arabinose, raffinose, and pyruvate [74, 109, 110, 111]. In recent years, the pathogenesis in humans has been identified but it is considered a less pathogenic species, causing infections with heterogenicity like sinusitis, urinary tract infection, vaginal infection, and decubital ulcer [74, 102, 109]. This bacterium also harbors the VanA genes, thus creating the glycopeptide resistance [74]. The complete genome of E. raffinosus CX012922 was conducted by Zhao et al., which showed a close relationship with E. gilvus and E. avium, and had pathogenicity-encoding genes in its genome. The presence of giant mega-plasmids (~1 Mb) in its genome is the key feature of this species, which provides it with different functions including host adaptation [110]. The E. raffinosus less pathogenic nature and good sugar metabolic potential can be targeted for its next-generation probiotic potential. However, to the best of our knowledge, there are no current reports showing the NGP of this species.

3.11 Enterococcus villorum

E. villorum belongs to the E. faecium group and is mostly isolated from domestic animals [69]. It is separated from other enterococcal species using methyl-β-D-glucopyranoside and sucrose tests, while a positive D-xylose test is used occasionally [69]. Due to the limited information, it is difficult to determine the exact nature, different potentials, and applications of this species. The sequence of E. villorum strain LMG 12287 T was found identical to E. porcinus strain ATCC 700913 T, hence creating confusion but the name E. villorum was used earlier than E. porcinus, and hence, the latter is considered a junior synonym of E. villorum [112]. A study conducted on the comparative genomics of enterococcal species showed tetracycline resistance genes in E. villorum [113]. Likewise, prophages from the Phycodnaviridae family and type II CRISPR-Cas system genes were also identified in E. villorum [113].

Due to the scarcity of available data on the different aspects of E. villorum, it is difficult to explore the NGP potential of this species. To the best of our knowledge, there are no data touching its NGP potential. But we anticipate that the presence of CRISPR-Cas genes and sugar metabolism can be used to identify its next-generation probiotic potential.

3.12 Enterococcus viikkiensis

E. viikkiensis is a member of the genus Enterococcus, which shows light gray translucent colonies on its growth media. It was isolated from broiler and broiler production lines [114, 115]. Characteristically, this species grows on bile-esculin agar and temperatures ranging from 10 to 37°C but cannot grow at 4 and 45°C, but shows tolerance to 6.5% NaCl [116]. Li et al. isolated a new enterococcal species, that is, Enterococcus xiangfangensis that has similarities with the E. viikkiensis strain [117, 118]. In the study by Glaeser et al., it was also identified that E. viikkiensis strains harbor the VanA gene besides other E. lemanii clusters [115].

The health profiles, such as pathogenesis, enterocin production, probiotic potential, are still undefined for this specie; hence, it is difficult to elaborate on its next-generation probiotic potential. In a broad sense, one study has shown that it harbors the vancomycin resistance genes, hence making it unfit for next-generation probiotic potential. But as its brighter side is not yet conclusively established, further studies are required that target its NGP potential.

3.13 Enterococcus italicus

E. italicus is a newly identified enterococcal species that belongs to group V and has now been isolated from a variety of cheeses around the globe [119, 120, 121, 122, 123]. Besides other sources, it is also isolated from dairy products [124]. It shows a close relationship with E. sulfureus and E. saccharolyticus, which have 96 and 97% 16S rDNA sequence similarity, respectively [114]. Its unique properties include no growth at 6.5% NaCl and the lack of acid production from L-arabinose, melibiose, raffinose, and ribose [114, 125].

The safety profile of E. lactis is good as there are no potential virulence factors or antibiotic resistance genes identified, thus representing its promising role in adjunct culture and use in different industries [121, 123, 126]. For instance, a study was conducted to evaluate the antimicrobial activity and safety of E. italicus GGN10, which was isolated from Tunisian bovine raw milk. The results showed its antimicrobial activity, stability over a wide range of pH, the production of enterocins (e.g., EntA, EntB), the absence of pathogenic islands, and the production of enzymes such as acid phosphatase and aminopeptidase. These positive attributes of this strain make them an important agent to use in flavor and texture development [122, 127, 128]. Likewise, the genomic analysis of the E. italicus DSM 15952 T-type strain (Accession PRJNA61487, ID 61487) showed the presence of genes responsible for the bifunctional enzyme, which is involved in glutathione production [121]. The aforementioned beneficial properties and its tolerance potential make it a promising agent for NGP potential. In contrast, the antibiotic resistance genes such as tetS (which encodes for a ribosomal protection protein) and tetK tetracycline-resistance were also determined in E. italicus [119].

3.14 Enterococcus xiangfangensis

E. xiangfangensis (named from the China district where it was isolated) is a non-motile species of Enterococcus, having a 1–1.5 mm size and giving circular and smooth colonies on the growth medium, that is, MRS agar [117, 118]. Its physiological properties include growth at 37 to 45°C, growing best at a basic pH of 11.0, tolerating a maximum of 5% NaCl, no ammonia being produced from arginine, and having the highest production of L-lactate, catalase negative, and homofermentative potential. It gives positive tests for pyrrolidonyl arylamidase and leucine arylamidase, while giving negative tests for α-galactosidase, β-galactosidase, β-glucuronidase, alkaline phosphatase, and arginine dihydrolase [117].

E. xiangfangensis has a complex sugar profile and showed both positive and negative tests against different metabolites. Despite being positive against different sugar metabolites, its tolerance potential, particularly against low pH and high salinity, is not good. Hence, it is ambiguous whether its NGP potential is really targeted or not. There are no data available to explore its NGP potential, so its genomic analysis may help identify its correct position from the perspective of NGPs.

3.15 Enterococcus phoeniculicola

E. phoeniculicola was first isolated by Law-Brown and Meyers from the uropygial gland of wild red-billed wood hoopoes (Phoeniculus purpureus). Sequence analysis of the 16S rDNA showed 97.3 and 97.5% sequence similarities with E. faecium and E. avium, respectively [114]. It is separated from these species by its metabolic profile (i.e., it is unable to form acid from lactose, D-mannitol, and D-sorbitol) and its lower tolerance potential against 40% bile or 6.5% NaCl [114].

E. phoeniculicola positive attributes are not well known. Its next-generation probiotic potential is not elucidated yet. Highlighting its metabolic profile indicates positive potential, but the tolerance potential creates a difficult dilemma. The investigation of its safety and genome analysis will help to identify its positons in the NGP.

3.16 Enterococcus plantarum

E. plantarum was isolated from plants as non-motile, ovoid-shaped cocci during enterococcal identification and characterization from plant sources. Its 16S rDNA sequence shows similarities to the E. faecalis group while separated from its close species via phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) gene sequence analysis. This species is unpigmented despite being showing a yellowish color on an agar plate [114, 129]. The biochemical properties include growth at different temperatures and pH, tolerance to 6.5% NaCl, and positive for Group-D antigen, and produce acetoin, leucine arylamidase, pyrrolidonyl arylamidase, urease, β-mannosidase, and β-glucosidase [129].

Till date, enterococci from plant sources are not very well investigated, and limited studies have shown their insight [130]. Luckily, the draft genome sequence of strain E. plantarum TRW2 (NCBI GenBank number SAMN07453839) was recently performed, which revealed 132 contigs (≥500 bp), 3,383,441 bp with a G + C content of 35.8% and N50 length of 89,597 bp [130]. The strains harbored no antibiotic resistance genes and neither any virulence traits [130]. So far, the strain is not investigated for its next-generation probiotic potential, but it is anticipated that it has NGP potential, as shown by its apparent safety profile.

3.17 Enterococcus quebecensis

E. quebecensis was recently identified by Sistek and his colleagues from a well water sample in Canada [131]. It is separated from other enterococcal species via sequences of a set of genes (i.e., 16S rRNA, rpoA, pheS, tufA, and atpD), DNA fingerprinting, and DNA–DNA hybridization [132]. It belongs to the E. faecalis group and has a close relationship with E. moraviensis [133]. Its biochemical characteristics include a negative test for the Voges-Proskauer test (no acetoin), peculiar temperature ranges for growth, catalase-negative, and not being grown in the presence of 6.5% NaCl. It hydrolyzes aesculin, leucine β-naphthylamide, L-pyrrolidonyl β-naphthylamide, and deaminated arginine [114, 132, 133, 134].

The next-generation probiotic potential of this species has not been described so far. However, the biochemical properties of the species give a positive insight into their use as probiotics, but their low tolerance to salts and temperatures, etc., can limit the NGP potential. Hence, it is important to investigate the genomic properties of the strains and explore their NGP potential.

3.18 Enterococcus rivorum

E. rivorum was identified by Niemi and collaborators in 2012 from pristine waters in Finland. This species shows a close relationship to the E. faecalis group [135]. E. rivorum showed less growth at high temperature (45°C) in the presence of 6.5% NaCl and has variable potential to produce Group-D antigen [114]. Although it can also tolerate high temperatures of 60°C for 30 minutes and 40% bile [135], it also showed positive tests for acid production from N-acetylglucosamine and the Voges-Proskauer test, and can produce acid from glycerol, ribose, galactose, D-glucose, D-fructose, methyl α-D-glucoside, cellobiose, maltose, sucrose, trehalose, and starch [135].

The next-generation probiotic potential of E. rivorum is little explored. A study conducted by Bhagwat et al. showed that several other enterococcal species, besides E. rivorum, have the potential to be used as probiotics. Their results indicated that E. rivorum strains have adherence potential, tolerance to pancreatic enzymes, produce hydrogen peroxide, and possess antimicrobial potential [136].

3.19 Enterococcus rotai

E. rotai is a yellow-pigmented, urease-producing enterococcal species that is isolated from different environmental sources such as water and plants and placed in the E. faecalis group [114, 134, 137]. It shows a close relationship with E. lemimoi [138]. Its biochemical properties include growth at pH 9.6 and 42°C (not grown on 45°C), no gas production, and the production acid phosphatase, chymotrypsin esterase, β-glucosidase, and β-mannosidase [133, 137].

Enterococcus rotai strains show resistance to clindamycin, chloramphenicol, oxacillin, and ofloxacin but show susceptibility to imipenem and gentamicin [114, 137]. The complete genome sequence of E. rotai LMG 26678 T was performed by Lauer et al. and submitted to GenBank under the accession number CP013655 [139]. Resistance to antibiotics makes the species unfit for next-generation probiotic potential, but its tolerance and hydrolytic potential, and the complete known genome can help explore in the race of NGP potential enterococci.

3.20 Enterococcus silesiacus

E. silesiacus was identified by Švec and collaborators from water in the Czech Republic and placed in the group E. faecalis [140]. This enterococcal species gives non-pigmented, shiny, and round colonies on an agar plate that can elongate in all directions. It is a non-motile species with best growth observed in Todd-Hewitt agar and brain heart infusion (BHI) agar in comparison with MRS medium [114, 140]. It produces arginine dihydrolase, pyrrolidonyl arylamidase, acetoin, and β-galactosidase, while hydrolyzing glycerol, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-maltose, D-lactose, and D-trehalose [140].

Its next-generation probiotic potential has not been elucidated, so far. However, its hydrolytic potential and tolerance capacity may help to select it as a next-generation probiotic candidate. Lauer et al. performed the complete genome sequence of the type strain E. silesiacus LMG 23085 T and submitted it to the GenBank under the accession number CP013614 [139].

3.21 Enterococcus termitis

E. termitis was isolated by Švec from the gut of the termites and placed in the E. faecalis group, which shows a close association with E. dunnyi [129, 134, 138, 140]. Besides basic enterococcal characteristics, it gives shiny and circular colonies that are smooth, non-motile, and non-pigmented. It shows more similar biochemical properties to E. silesiacus and has a genome size of 4.7 Mb [138]. It does not produce pyrrolidonyl arylamidase, arginine α-galactosidase, β-galactosidase, and β-glucuronidase, while hydrolyzing glycerol, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, D-cellobiose, D-maltose, D-lactose, and D-trehalose [134, 140]. E. termitis LMG 8895 T is considered a type strain that is obtained from a termite’s gut [114].

The next-generation probiotic potential of E. termitis is not fully known yet, but Hamad et al. identified the incidence of this species in cheese whey, indicating its potential applications in the cheese industry. The strain E. termitis LMG 8895 showed greater proteolytic activity, produced exopolysaccharides, and tolerated high nisin concentrations. There was also no hemolytic activity or vancomycin resistance observed [141]. These potential probiotic properties indicate the NGP potential of this species.

3.22 Enterococcus thailandicus/E. Sanguinicola

E. thailandicus was isolated in Thailand from sausage in 2008. The 16S rRNA and rpoA gene sequence analysis show more similarities to the E. faecium group [114, 142]. E. thailandicus is also described as E. sanguinicola in the literature [143, 144]. Characteristically, it is a non-motile, ovoid, non-spore-forming enterococcal species that gives red colonies on Slanetz-Bartley agar, while being considered a non-pigmented microbe [142].

The next-generation probiotic potential of this species has been elucidated in some studies, which showed promising results. Li et al. for the first time identified the antimicrobial potential of E. thailandicus against C. difficile infections (CDI) [145, 146]. E. thailandicus d5B, which is isolated from Chinese children’s fees, also showed enhanced colonization ability and had the basics probiotic properties, like free from virulence traits and antibiotic resistance [145]. In the study by Beukers et al. no intact prophages were detected, while an array of CRISPR-Cas was identified [113]. Likewise, strain E. thailandicus TC1 was also declared a potential probiotic strain as described [143]. Hence, these data showed that E. thailandicus has a promising NGP potential that needs to be explored in more detail.

3.23 Enterococcus ureasiticus

E. ureasiticus was first isolated from a well water sample and shows positive urease activity hence, its name. Sequence analysis showed its relationship with the E. faecalis group but can harbor variable rpoA, pheS, tufA, and atpD gene sequences than 16S rDNA analysis alone [114, 132]. It grows in the presence of 5% CO₂ and a wide range of temperatures, while no known strain is grown in a high salinity of 6.5% NaCl. Likewise, no extracellular polysaccharide production was observed. It produces acids from ribose, lactose, trehalose, sucrose, maltose, methyl, and tagatose, while no acid is produced from L-arabinose, D-arabitol, raffinose, and melibiose. It shows the potential to produce different enzymes and can hydrolyze different substances [132, 133, 134].

The next-generation probiotic potential of E. ureasiticus has not been explored yet. Its biochemical properties indicate a positive indication for probiotic usage, but its NGP potential needs to be identified.

3.24 Enterococcus ureilyticus

E. ureilyticus which is isolated from plants and water, is a yellow-pigmented, urease-producing, non-motile, non-spore-forming, ovoid shaped, facultative anaerobic enterococcal specie that is placed in the E. faecalis group on the basis of its 16S rDNA sequence [137, 147]. In contrast, it shows distinct characteristics based on pheS and rpoA gene sequence analysis, whole-cell protein electrophoresis, and DNA-DNA hybridization techniques [137]. It shows tolerance to high pH and salts and is able to grow in wide temperature ranges, but growth is limited at 45°C. It produces pyrrolidonyl arylamidase, leucine arylamidase, acetoin, and acid phosphatase, but not produces catalase, NH3, and arginine dihydrolase. It also has the potential to hydrolyze different sugars such as ribose, galactose, D-glucose, D-fructose, D-mannose, sorbose, cellobiose, maltose, sucrose, and trehalose [114, 133, 134, 137].

The next-generation probiotic potential of this species has not been explored yet. Some strains of this specie show resistance to chloramphenicol, oxacillin, and tetracycline, and susceptibility to imipenem [137], indicating the presence of antibiotic resistance, which indirectly lowers the NGP potential. However, its NGP potential will be explored based on in vitro assays and probiogenomic analysis.

3.25 Miscellaneous enterococcal species

The following are some enterococcal species that are less known and have not yet been explored for their next-generation probiotic potential.

Enterococcus caccae was isolated from a human stool sample and considered a separate specie in the E. faecalis group based on 16S rDNA sequence analysis, DNA-DNA hybridization, and whole-cell protein analysis. It also shows high similarities with E. moraviensis [97, 148]. Its peculiar behavior comprises its non-motile and non-pigmented nature, catalase-negative, ability to hydrolyze arabinose, lactose and mannitol, low G + C contents (32.5%), and tolerance to bile, NaCl, and temperature [114, 133, 148]. There is no such study available showing the next-generation probiotic potential of E. caccae. Firrman et al. studied the effect of quercetin (a polyphenol present in fruits) on E. caccae, and it was identified that quercetin can inhibit the growth of E. caccae [149].

Enterococcus aquimarinus was identified by Švec et al. [150, 151] from sea water [131]. 16S rDNA sequence analysis shows the closest relationship with E. saccharolyticus, E. sulfureus, and E. italicus [133, 150]. Its distinguishing features include polymorphism in the pheS gene, being unable to hydrolyze ribose, good growth at 42°C, and able to digest L-arabinose [114, 133, 150]. Its next-generation probiotic potential is not described.

Enterococcus camelliae was isolated in Thailand from fermented tea leaves and considered a separate specie based on its 16S rDNA genome sequence, rpoA genes, and DNA–DNA hybridization analysis. It showed a close relationship with E. italicus [99, 131, 152]. It is a non-pigmented, non-motile, catalase-negative species that has the capacity to produce acid from D-galactose and lactose, grows at a wide range of pH and temperatures, and shows no hydrolysis of arginine and gelatin [152]. It causes no acidification, coagulation, reduction, or liquefaction of litmus milk, and has a mean G + C content of 37.8% [114, 133, 152]. Currently, there are no such data found that show its next-generation probiotic potential.

Enterococcus canis was first identified by De Graef and colleagues from rectal swabs and chronic otitis in dogs that showed 99% similarity with E. faecium [97, 112, 124]. The distinguishing features include arginine hydrolysis, non-motile and non-pigmented nature, growing at 6.5%, no hydrolysis of starch, being unaffected by the presence of CO₂, and hydrolyzing some sugars [112, 114]. To the best of our knowledge, there are no data focusing on the next-generation probiotic potential of E. canis.

Enterococcus devriesei, an animal origin novel enterococcal specie first identified by Švec et al., has a close relationship (99% sequence identity) with E. pseudoavium [116, 151]. It belongs to the E. avium group and besides the groups characteristics, its distinguishing properties include hydrolysis of lactose, non-motile and non-pigmented nature, no growth at 45°C, tolerate to 8% NaCl, and hydrolysis of common sugars. The G + C contents are 40% [114, 117, 151]. It was also isolated from Spanish goats and characterized by safety measurements. The results showed antimicrobial activity against common pathogens and were not involved in any human infections, hence indicating its positive potential in different applications [153]. The next-generation probiotic potential is not attempted, but we anticipate this species based on its safety profile and potential antimicrobial value.

Enterococcus hermanniensis was first reported by Koort et al. from broiler meat and tonsils that have close phylogeny with E. pallens (99%) and was placed in the E. avium group [116, 133, 154]. It was distinguished from related species by the analysis of its 16S rDNA sequence, whole-cell protein, and DNA-DNA hybridization techniques. Other silent features include non-production of acids from common substrates, non-motile, non-pigmented, and catalase-negative nature, tolerance to 4% NaCl solution, no growth at 45°C, no production of Group-D antigen, acid production from mannitol, and mean G + C contents of 37.1% [114, 154]. To the best of our knowledge, there are no data available that describe the next-generation probiotic potential of this species.