Ingredients
Genus: Enterococcus
Domain: Bacteria
Phylum: Firmicutes
Class: Bacilli
Order: Lactobacillales
Family: Enterococcaceae
Enterococcus belongs to the group of lactic acid bacteria (LAB), and is considered a ubiquitous microorganism, showing an astonishing potential to inhabit diverse environments, hostile biotopes, and even the gastrointestinal tracts of humans and warm-blooded animals. The main representatives of this genus are Enterococcus faecalis and Enterococcus faecium.
In 1985, members of the diverse genus Streptococcus were reclassified into Lactococcus, Enterococcus, Vagococcus, and Streptococcus based on biochemical characteristics, as well as molecular features. Prior to this reclassification, the species now known as E. faecalis and E. faecium were designated Streptococcus faecalis and Streptococcus faecium, respectively — a legacy naming convention that still appears in older clinical and commercial literature (e.g., Bioflorin/SF68 was historically called Streptococcus faecium SF68).
LAB are gram-positive, non-sporing, catalase-negative organisms that are devoid of cytochromes and of non-aerobic habit but are aerotolerant, fastidious, acid-tolerant, and strictly fermentative; lactic acid is the major end product of sugar fermentation. Among enterococcal species, E. faecium and E. faecalis are the predominant species of the human gastrointestinal system.
Several other species in the genus have been studied in food and probiotic contexts. Many enterococci strains have been described as useful for health functions or technological applications in food systems, including Enterococcus durans M4-5 (production of short-chain fatty acids), Enterococcus faecium M74 and Enterococcus durans KLDS 6.0930 (reduction of cholesterol levels in serum), Enterococcus mundtii ST4SA (production of bacteriocins), Enterococcus faecium LCW 44, and Enterococcus durans 6HL (production of antimicrobial compounds against Gram-positive and Gram-negative bacteria).
Currently, two strains belonging to the genus Enterococcus are widely identified as probiotics available on the market: Enterococcus faecium SF68® (NCIMB 10415, Cerbios-Pharma SA, Barbengo, Switzerland) and Enterococcus faecalis Symbioflor 1 (SymbioPharm, Herborn, Germany).
Quite a few enterococcal strains have been authorized as probiotics for use in pharmaceutical preparations or animal feed additives for decades, such as E. faecium Medilac-Vita (treatment of infantile enteritis, China), E. faecium SF68® (dietary supplementation for human and animal, Switzerland), E. faecium Cylactin® (feed additive for animal, Switzerland), E. faecalis Symbioflor® 1 (treatment of respiratory illness, Germany), and E. faecalis TH10 Dr. Ohhira® (dietary supplementation for human, Republic of Estonia).
The genus Enterococcus consists of lactic acid bacteria found predominantly in the gut of humans and animals. Among enterococcal species, E. faecium and E. faecalis are the predominant species of the human gastrointestinal system. Enterococci are also involved in the fermentation process of various foods, including cheeses and sausages.
Enterococcus species are widely present in traditional fermented foods due to their amazing capacity to withstand extreme temperatures, high salinity, and pH levels. They can grow in the presence of 40% (w/v) bile salts. Because they are "tough bugs," Enterococci can unusually survive for long periods on environmental surfaces and are tolerant to heat, chlorine, some alcohol preparations, and even in antibiotic fields.
Enterococcus faecalis is a bacterium which accompanies humans from the first days of life. As a commensal it produces vitamins, metabolizes nutrients, and maintains intestinal pH — all in exchange for a niche to inhabit.
The provenance of probiotics can be traced back to 2000 BC, when ancient civilizations began developing methods to preserve milk. Enterococci, as intrinsic components of spontaneously fermented dairy products, meats, and vegetable preparations, have been consumed by humans for millennia as part of the natural microbiota of those foods — though without identification or deliberate selection.
E. faecalis and E. faecium are deemed the most important strains used for food fermentation and spoilage, but have also been reported as probiotics for more than two subsequent decades without any reports of adverse effects.
Possibly due to their historic use as starter cultures and in food preservation, enterococci have been increasingly marketed as probiotics for animals and humans. The deliberate pharmaceutical use of E. faecium SF68 dates to the mid-twentieth century: the first product manufactured has been in use for as long as 70 years.
LAB strains have been isolated from traditional Tunisian fermented dairy products — Testouri cheese and Rigouta — and identified as Enterococcus faecalis by MALDI-TOF-MS and molecular assays. Such traditional fermented dairy products from North Africa, Eastern Europe, and the Middle East are among the food systems in which enterococci have historically been consumed without deliberate supplementation. Traditional fermented meat products such as Tunisian "Dried Ossban" have also been documented as sources of E. faecium strains with probiotic properties.
In the dietary supplement and pharmaceutical markets, Enterococcus-based products are available in several forms:
Enterococci produce small peptides that belong to the bacteriocin group and have antimicrobial properties. These include enterocin A, B, P, and ON-157 produced by E. faecium, and L50 made by E. faecalis. Enterocins exhibit broad antimicrobial activity, inhibiting the multiplication of bacteria such as Staphylococcus spp., Bacillus cereus, Listeria monocytogenes, Clostridium spp., E. coli, Pseudomonas aeruginosa, and Vibrio cholerae.
Many enterococci produce at least one bacteriocin, which is a ribosomally synthesized antimicrobial peptide active against a wide range of foodborne pathogens including Listeria spp. According to the classification established by Cotter et al. (2005), most enterocins are class II bacteriocins, defined as non-modified and heat stable.
Depending on the chemical structures and mechanism of action, some bacteriocins exhibit narrow-spectrum antimicrobial activities that only target bacteria of the same species or close phylogenetic groups, while others exhibit broader spectra. Bacteriocins have been considered a promising alternative to broad-spectrum antibiotics.
The probiotic bacterial strain E. faecium SF68 has been shown to alleviate symptoms of intestinal inflammation in human clinical trials and animal feed supplementation studies. Cell-free, whole-cell lysates of E. faecium SF68 showed a reversible, inhibitory effect on both NF-κB and JNK(AP-1) signaling pathway activation in intestinal epithelial cells, and abrogated the response to bacterial and other Toll-like receptor (TLR) ligands. This inhibitory effect on two central pro-inflammatory pathways represents a key mechanism by which SF68 may modulate intestinal inflammation.
In the case of Enterococcus faecalis, the pattern recognition receptors recognized typically by TLR2 are lipoteichoic acid (LTA) and/or lipoproteins. These interactions with innate immune receptors are foundational to the immunomodulatory potential of E. faecalis.
Enterococcal strains evaluated for probiotic properties have been found to be tolerant to the harsh conditions of the gastrointestinal tract (acidity and bile salt), with low to moderate biofilm formation, the ability to adhere to Caco-2/TC7 intestinal cells, and the ability to strengthen the intestinal barrier through the increase of transepithelial electrical resistance (TER).
Enterococci (e.g., E. faecium M-74, E. durans KLDS) are characterized by their ability to lower cholesterol levels. These bacteria produce a hydrolase which catalyzes the bile acid deconjugation process and assists in cholesterol integration into the bacterial cell wall or assists in precipitation if the environment is acidic.
Enterococcus durans M4-5 has been found to generate butyrate, a short-chain fatty acid (SCFA) that induces significant anti-inflammatory effects and contributes to the integrity of the intestinal epithelium.
Enterococcus species synthesize diverse antimicrobial metabolites, particularly enterocins and organic acids, which significantly suppress the growth of both pathogenic and spoilage organisms in food systems. By colonizing the intestinal mucosa and producing these compounds, enterococci can competitively exclude pathogens from binding sites and reduce their populations.
E. faecium SF68 is the most thoroughly studied enterococcal probiotic in human clinical trials for diarrhea. SF68® is a licensed pharmaceutical for the treatment and prevention of diarrhea in Austria, Italy, and Switzerland. However, evidence for its efficacy is based on small to medium-sized studies. Four studies on the treatment of acute diarrhea and the prevention of antibiotic-associated diarrhea have been analyzed: one randomized, double-blind, placebo-controlled trial (RCT) for treatment (n = 1,143), one open-label study for treatment (n = 5,093), one RCT for prevention (n = 1,397), and one open-label study for prevention (n = 4,340).
This analysis of two RCTs evaluating efficacy and safety of SF68 in the treatment of acute diarrhea and prevention of antibiotic-associated diarrhea found that both RCTs were positive studies for the primary endpoint of a decrease in diarrhea duration and a reduction in occurrence of new diarrhea cases, respectively. SF68 was safe and well-tolerated. With a total of 6,236 patients in the treatment trials and 5,737 subjects in the prevention studies, this represents by far the largest dataset on SF68, and, to the knowledge of the authors, the largest dataset ever reported for any probiotic in the treatment or prevention of acute diarrhea.
In the treatment arm and open-label studies, patients received SF68 twice daily (for prevention) or three times daily (for treatment) for 7 days. Primary endpoints were time to resolution of diarrhea (treatment) and percentage of development of diarrhea (prevention).
An earlier double-blind controlled study (Mitra and Rabbani, 1990) examined SF68 (then marketed as Bioflorin® under the name Streptococcus faecium SF68) in adults with acute diarrhea due to Vibrio cholerae and enterotoxigenic E. coli. Positive effects of E. faecium SF68 on the reduction and duration of diarrhea have been demonstrated in both humans and animals.
Evidence strength: For humans, the evidence for benefits from enterococcal probiotics remains ill-defined despite advertisements claiming their ability to increase general health and treat diverse medical conditions. Although a small cohort study showed protective efficacy against gastroenteritis by a probiotic product containing E. faecium T-110, no large, randomized, placebo-controlled clinical trials have been performed on which to base such claims. The large open-label datasets for SF68 are encouraging but open-label designs are inherently susceptible to bias.
Analyses of RCTs evaluating SF68 were positive for the primary endpoint of a decrease in diarrhea duration and a reduction in occurrence of new diarrhea cases. SF68 was safe and well-tolerated in these studies, which examined treatment and prevention of antibiotic-associated diarrhea.
Evidence strength: Preliminary to moderate. The RCT data are promising but the open-label nature of the larger observational cohorts and the lack of independent replication in large, pre-registered, double-blind trials limits current confidence levels.
Irritable Bowel Syndrome (IBS) is a common gastrointestinal disorder with symptoms including abdominal pain, flatulence, and irregular bowel movements. In an open-label trial with 85 IBS patients, a probiotic treatment containing enterococci, lactobacilli, and bifidobacteria improved stool characterization, urgency, distension, pain, duration, and frequency without any adverse drug reactions. The improvement of symptoms persisted when outcomes were measured 2 weeks after stopping treatment.
Evidence strength: Weak. The trial used a multi-strain formulation in an open-label design, making it impossible to attribute benefit specifically to the enterococcal component. No adequately powered, placebo-controlled, single-strain RCTs in IBS for any enterococcal probiotic have been identified in the peer-reviewed literature.
Lyophilized Enterococcus faecium M-74 was administered to 12 adult subjects in a daily oral dose of 5 × 109 bacteria for six weeks. The bacterium temporarily colonized the host intestine and its excretion with stool persisted for five weeks. In a study, the administration of E. faecium M-74 probiotic strain was associated with reduction of serum cholesterol concentration by 12% after 56 weeks.
E. faecium M-74 and E. faecalis ATCC19433 strains have been reported to reduce blood cholesterol, adhesion molecules, and regulate immune responses. Specifically, E. faecium M-74 strain and bound selenium remarkably decreased soluble intercellular adhesion molecule 1 (ICAM-1), CD54 on monocytes, and related markers.
In animal (murine) models, E. faecium GEFA01 exhibited a cholesterol removal rate (CRR) of 46.13% by coprecipitation, assimilation, and degradation of cholesterol in vitro, and significantly decreased body weight and levels of serum total cholesterol, LDL-C, hepatic triglycerides, and LDL-C, while increasing serum HDL-C levels in mice fed a high-cholesterol diet.
Evidence strength: The cholesterol-related evidence in humans is limited to small, uncontrolled or minimally controlled pilot studies (e.g., n = 12 in the M-74 oral supplementation study). Animal and in vitro data are more extensive but cannot be directly extrapolated to humans. No large, adequately powered RCTs specifically in human hypercholesterolemia have been identified.
The use of E. faecalis Symbioflor 1 as an immune regulator in the treatment of recurrent chronic sinusitis or bronchitis has been described. E. faecalis Symbioflor 1 is used to prevent and/or treat diarrhea in pigs, poultry, livestock, and pets, and to treat recurring illness in the human upper respiratory tract.
One of the most popular probiotic strains of Enterococcus faecalis is DSM 16440, used in Symbioflor-1. Genomic analysis showed a lack of large parts of the enterococcal chromosome, mainly containing virulence-associated genes, including genes encoding cytolysin, enterococcal surface protein, and gelatinase.
Evidence strength: The evidence for respiratory applications of Symbioflor-1 is based largely on clinical use in Germany over several decades, but published high-quality RCT data specifically for this application are limited in the accessible peer-reviewed literature. The genomic deletion of key virulence factors in DSM 16440 provides a notable safety rationale for this specific strain.
A Phase II study aimed at the prevention of febrile neutropenia (FN) by probiotic strain E. faecium M-74 enriched with selenium in leukemic patients enrolled 14 patients (6 males/8 females) with myelogenous leukemia treated by induction or consolidation chemotherapy. Patients received prophylaxis with E. faecium M-74 during one cycle of chemotherapy; the daily dose was 36 × 109 CFU three times daily.
No febrile episode or infection provoked by the tested strain was observed. Tolerance of therapy was excellent without significant adverse effects. The results demonstrated the safety of probiotic strain E. faecium M-74 enriched with selenium in leukemic patients with severe neutropenia. However, its administration was not effective in the prevention of febrile neutropenia, but this does not preclude the protective effect of other probiotic strains.
A mouse model showed that Enterococcus hirae helps shape the anti-cancer immune response. Cyclophosphamide (one of the drugs that stimulates anti-cancer immune response) changes the composition of the microbiota in the small intestine and induces the translocation of certain Gram-positive bacteria, including E. hirae, to the secondary lymphoid organs.
Evidence strength: Very preliminary. The Phase II clinical trial in neutropenic cancer patients showed safety but no efficacy for the primary endpoint. The murine cancer immunology data are animal/mechanistic only.
Mego et al. demonstrated the use of the probiotic E. faecium M-74 in the treatment of gastrointestinal complications in patients with myeloid leukemia. This represents one of the few human oncology-specific applications studied with enterococcal probiotics, though the evidence base consists of small, early-phase trials.
In a murine model, treatment with heat-killed E. faecalis EF-2001 decreased the expression of several cytokines, including COX-2, iNOS, IFN-γ, IL-1β, and IL-6 in inflamed colon compared to the DNBS (colitis-inducing agent) alone group; EF-2001 also suppressed colonic tissue destruction, strongly suggesting that it could alleviate inflammation associated with mouse IBD.
Importantly, the same organism has also demonstrated pro-inflammatory potential: germ-free interleukin-10 knockout (IL-10 KO) mice developed inflammatory bowel disease after colonization with a pure culture of Enterococcus faecalis; E. faecalis not only induced IBD (primarily in colon and rectum) but rectal dysplasia and adenocarcinoma were also found in the IL-10 KO mice.
Evidence strength: Preclinical only. These findings are in genetically modified, immunocompromised animal models and cannot be extrapolated to healthy humans or normal immunological contexts.
In a controlled study in young dogs, puppies were allotted to two groups receiving either a control diet or a diet supplemented with 5 × 108 CFU/day of probiotic E. faecium SF68 from weaning to 1 year of age. Fecal IgA and canine distemper virus vaccine-specific circulating IgG and IgA were higher in the probiotic group than in controls. There were no differences in CD4+ and CD8+ T cell percentages, but the proportion of mature B cells was greater in those fed the probiotic.
Evidence strength: This is a controlled animal study. No equivalent large human RCT for vaccine adjuvancy has been identified. The finding is mechanistically interesting but not directly applicable to human supplementation recommendations.
Dosages used in clinical and human studies vary substantially by strain and indication. The following are drawn directly from the cited literature:
No universally agreed therapeutic dosage exists for human enterococcal probiotic supplementation. No large, randomized, placebo-controlled clinical trials have demonstrated the safety and efficacy of any enterococcal probiotic in establishing a definitive dosing framework.
The paradoxical role of Enterococcus in food, health, and disease has sparked significant scientific debate. The potential of beneficial enterococci as probiotics must be carefully considered alongside their associated safety concerns.
Enterococci express many virulence traits including cytolysin, proteases, aggregation substance, capsular polysaccharide, enterococcal surface protein, biofilm formation, extracellular superoxide, intestinal translocation, and resistance to innate immunity that can lead to serious hospital-acquired infections.
Enterococci have been identified as opportunistic pathogens that cause various infections, among which approximately 80% are associated with Enterococcus faecalis. More importantly, the emergence and spread of vancomycin-resistant Enterococcus (VRE) isolates presents serious therapeutic difficulty, owing to a lack of effective antimicrobial therapy.
Enterococcal strains can carry plasmid-mediated resistance genes, which can be transferred between bacterial species, causing decreased susceptibility to common antibiotics. These plasmid-mediated genes in enterococci have contributed to vancomycin-resistant enterococci (VRE), which are problematic in the clinical setting.
Vancomycin-resistant enterococci (VRE) are bacterial strains of the genus Enterococcus that are resistant to the antibiotic vancomycin. Six different types of vancomycin resistance are shown by enterococcus: Van-A, Van-B, Van-C, Van-D, Van-E, and Van-G. The significance is that Van-A VRE is resistant to both vancomycin and teicoplanin, Van-B VRE is resistant to vancomycin but susceptible to teicoplanin, and Van-C is only partly resistant to vancomycin.
Probiotic and vancomycin exposure were significant risk factors for VRE colonization. The acquisition and transfer of resistance genes of bacteria may be mediated by probiotics. This concern has been raised particularly in neonatal intensive care settings.
Due to the plasmid- or transposon-mediated gene transfer ability of enterococci, surveillance monitoring and further studies regarding enterococcal consumption are warranted. Future studies that identify enterococcal strains safe to use in probiotics without virulence factors and antibiotic resistance are imperative for evidence-based decisions by health organizations and government agencies.
The Enterococcus genus neither has Generally Recognized as Safe (GRAS) status nor has it been included in the Qualified Presumption of Safety (QPS) list, implying that drastic legislation governs these microorganisms. Recently, EFSA has been developing the QPS system to regulate the use of microbial strains in foods and to require probiotic producers to conduct accountability and safety assessment for each enterococcal strain.
Enterococcus faecium is specifically excluded from EFSA QPS assessments based on an ambiguous taxonomic position or the possession of potentially harmful traits by some strains of the taxon, and therefore requires a specific safety assessment for each strain for which an application is made.
The EU council regulations 700/524/EEC and EG 1831/2003 allow the use of all Enterococcus species and strains in poultry, cattle, and pigs. In the USA, probiotics used as feed supplements (direct-fed microorganisms) encompass six enterococcal species considered as safe, including E. faecium but excluding E. faecalis.
Leading organizations in food safety and security such as the European Food Safety Authority (EFSA), the Advisory Committee on Novel Foods and Processes (ACNFP), and the Food Standards Agency (FSA) have allowed the use of certain strains of enterococci as food additives and supplements based on careful case-by-case appraisal. Every single strain must be considered, and health risks must be excluded for that specific strain.
Many studies have been conducted to evaluate the probiotic characteristics of Enterococcus strains, mainly E. faecium. Due to safety concerns, lack of safety information, and legislation, only a limited number are commercialized. Enterococcus has not yet obtained GRAS status.
One of the most popular probiotic strains, E. faecalis DSM 16440 (Symbioflor-1), shows a genomic deletion of large parts of the enterococcal chromosome, mainly containing virulence-associated genes, including those encoding cytolysin, enterococcal surface protein, and gelatinase. This illustrates that safety must be assessed at the strain level, not the species level.
Whole Genome Sequencing (WGS) has revealed the presence of tetracycline resistance and cytolysin genes in some E. faecalis strains, which led to high mortality of Galleria mellonella larvae in virulence tests — underscoring that genomic characterization is essential before any strain is designated as a probiotic.
Most VRE infections occur in hospitals and can cause urinary tract infections, bloodstream infections, and infections caused by catheters or surgery. Risk factors for VRE include previous treatment with vancomycin or other antibiotics, hospitalization, weakened immune system, surgical procedures, and medical devices such as urinary and intravenous catheters.
Based on reviewed data, enterococcal probiotic consumption has been reported beneficial for conditions or symptoms of multiple diseases without any apparent adverse effects in the reviewed studies. Nevertheless, the use of live enterococcal probiotics in hospitalized, immunocompromised, or critically ill patients carries inherently elevated risk given the documented nosocomial pathogenicity of this genus.
Both RCTs of SF68 were positive for the primary endpoint, and SF68 was safe and well-tolerated. Tolerance of therapy was excellent without significant adverse effects in the leukemia patient Phase II study; the results demonstrated the safety of E. faecium M-74 enriched with selenium in leukemic patients with severe neutropenia.
Health conditions that Enterococcus may help support.
Body systems that Enterococcus may help support.
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