Table 1. Comparative antagonistic activity of various probiotics against gram-negative pathogens

The green color highlights high antagonistic activity (minimum inhibitory concentration ≤ 2.5 lg CFU/mL

Compared to many strains of lactobacilli, the Enterococcus faecium L3 strain has the strongest anti-Helicobacter activity [12-14]. It is important to note that the anti-Helicobacter activity of L3 is equally expressed both in relation to the least pathogenic strains of Helicobacter and in relation to the most pathogenic strains of Helicobacter that cause stomach cancer.

The research revealed antiviral activity of the Enterococcus faecium L3 strain [16,17], and in certain cultures the antiviral effect of the supernatant of the Enterococcus faecium L3 biomass is comparable to the antiviral effect of acyclovir (Figure 2).

Figure 1. Antiviral activity of supernatants of bacterial cultures (the ordinate indicates the percentage of suppression of virus culture growth)

In an experiment with laboratory animals infected with the influenza virus, 100% mortality was observed in the group that did not receive Enterococcus faecium L3 treatment, while all the animals that received Enterococcus faecium L3 survived [18].

Mechanism of the antagonistic activity of the Enterococcus faecium L3 strain

Electron microscopy of Streptococcus pyogenes cells under the influence of bacteriocins of L3 strain.

E. faecium L3 produces enterocins A, B, and a two-component bacteriocin X-α - X-β, as well as lactobin. There is no other probiotic strain that has been previously described as having such a diverse and numerous collection of bacteriocins. The strain's effect on bacteria leads to the rupture of the cell wall due to the penetration of enterocin molecules through the cell wall. An example of such an effect is shown in electron microscopy image (Figure 3).

Symbiotic properties

The antagonistic mechanism through bacteriocins explains the symbiosis with the normal microbiota. When the L3 strain is grown together with representatives of the bifidoflora and with various lactobacilli, there is no inhibition of their growth. Moreover, when bifidobacteria are grown with the L3 strain, there is an enhanced growth of bacteria on the Blaurock medium [5], as the L3 strain has bifidogenic and lactogenic properties including the synthesis of a wide range of vitamins. Enterococcus faecium L3 is a good producer of vitamins. It outperforms other known vitamin-forming probiotic cultures in the quantity of vitamins, especially the B group vitamins. At the same time bacteriocins produced by L3 strain provide significant anti- viral effect against herpes and influenza viruses.

Interaction of the probiotic Enterococcus faecium L3 with the intestinal microbiota in in vivo experiments

Researchers evaluated the ability of the strain to restore the intestinal microbiota in experiments on laboratory animals that received antibiotics. The most well-studied model of dysbiosis in rats is when two antimicrobial drugs, metronidazole and ampicillin, were orally administered for 3 days, after which the E. faecium L3 was given to the animals. The restoration of the normal microbiota of the intestine was observed within 3-5 days [20].

The effect of Enterococcus faecium L3 strain on physiological processes in in vivo experiments

In biological experiments on laboratory animals, Enterococcus faecium L3 demonstrated the ability not only to suppress infection and restore normal gut microbiota, but also to positively affect digestion and metabolic processes. Digestion improved both due to changes in the spectrum of bacteria participating in digestion (when dysbiosis was eliminated) and due to the stimulation of its own digestive enzyme activity [21,22]. It was also found that antibiotics not only cause dysbiotic disorders in the intestine, but also lead to morphological changes in the intestinal mucosa: after 3-5 days of antibiotic use, villi of the small intestine epithelium are destroyed (Figures 3-5).

Figure 3. Normal mucosa of the small intestine of a rat (before taking antibiotics)

Figure 4. Destruction of the microvilli of the epithelium of the small intestine of rats after 3 days of antibiotic use.

Figure 5. Destruction of microvilli of the epithelium of the small intestine of rats after 5 days of antibiotics use

Enterococcus faecium L3 restores the structure of the intestinal mucosa within 3-5 days (Figure 6, 7), which is accompanied by the digestion normalization.

Figure 6. Restoration of the epithelium of the rat small intestine after 3 days of taking the probiotic Enterococcus faecium L3.

Figure 7. Restoration of the epithelium of the rat small intestine after 5 days of taking the probiotic Enterococcus faecium L3.

Normalization of the microbiota improved digestion, optimized metabolic processes in the animal's body, including increased resistance of the myocardium to ischemia. Taking E. faecium L3 also increased endurance of the animals [25]

Anti-inflammatory effect of Enterococcus faecium L3 strain in in vivo experiments

The anti-inflammatory effect of the Enterococcus faecium L3 strain was studied in an experimental model of infectious vaginitis in rats and mice. In the biological experiment, pathogenic streptococci and pathogenic staphylococci (mixed infection) were introduced vaginally, after which an inflammatory process developed in the animals. In the control group, which did not receive treatment, no self-healing was observed, and the infectious process spread to the surrounding organs.

Figure 8. Spread of inflammation to adjacent organs in untreated mice (left) compared to mice receiving L3 treatment (right)

Intravaginal administration of the probiotic strain Enterococcus faecium L3 led to the elimination of infection within 5 days, as in the comparison group receiving intravaginal erythromycin (Table 2).

Table 2. Indicators of vaginitis infection in mice infected with golden staphylococci for 5 days of treatment

However, in the group of animals receiving erythromycin, there was not a complete recovery of the vaginal mucosa: microtears and thickening of the mucosa and increased desquamation of the epithelium were observed in the mucosa and submucosa. In contrast to the antibiotic, the intra-vaginal use of the Enterococcus faecium L3 not only eliminated the infection but also restored the normal morphological structure of the vaginal mucosa.

The immunomodulating effects of Enterococcus faecium L3 in in vivo experiments

An important characteristic of probiotics is their ability to stimulate various reactions of innate immunity. This property is crucial in determining the nature of the action of the microbial preparation (pro- or anti-inflammatory). Experiments on laboratory animals have shown that L3 strain stimulates the anti-inflammatory arm of immunity (T2 polarization of lymphocytes), which is manifested in reduced expression of pro-inflammatory cytokines and pronounced production of IL10

In addition to the model of dysbiosis caused by antibiotics, similar effects were observed in the model of experimental autoimmune encephalomyelitis in animals. The intake of E. Faecium L3 strain reduced the effects of experimental autoimmune encephalomyelitis, and the leading cytokine effect was an increase in the expression of the anti-inflammatory cytokine IL10 and expression of the T cells CD3+CD161a+. Changes in immunological indicators were correlated with a positive clinical outcome and restoration of microbiota (Figure 9) [31-36]

Change in the clinical index of the development of symptoms of multiple sclerosis in laboratory rats after 14 and 28 days of treatment with L3.

The immune-modulating effect of Enterococcus faecium L3 was not inferior to the "Copaxone", which is used to treat multiple sclerosis. [96,97]
It should be noted that, while laboratory animal experiments have shown positive results with the use of either Copaxone or E. Faecium L3 for treatment, the use of both in combination was not as effective as using them separately.

Clinical application of probiotic Enterococcus faecium L3

The normalization of the microbiota in clinical conditions has been proven in numerous studies of patients with impaired microbiota, both when dysbiosis was the etiological factor in the development of diseases and when dysbiosis was a result of various treatment measures, including antibiotic therapy [38]. Thus, in patients with acute intestinal infections, Enterococcus faecium L3 effectively and quickly restores the microbiota, improves patients’ clinical indicators, shortens the treatment period and prevents development of complications, such as sepsis [39-41][42].

The use of the probiotic Enterococcus faecium L3 has been shown to be effective in the comprehensive treatment of patients with acute and chronic infections, including infections of the gastrointestinal tract [43-47], genitourinary system [48-50], and even tuberculosis [51].

The most widely studied area of Enterococcus faecium L3 effect is gastroenterology.

The series of clinical, randomized, independent studies, has shown that E. faecium L3 is capable of eradicating Helicobacter pylori in various pathological conditions, such as gastric ulcer disease, chronic gastritis, and non-specific ulcerative colitis [58-60]. It was found that the success of eradication largely depends on the H.pylori genotype: with the pathogenic "Cag+” in the genome of the pathogen, the standard eradication scheme, including antibiotics and a proton pump inhibitor, will be successful in only 45% of patients, and the addition of probiotics based on E.faecium L3 to the standard scheme increases the percentage of complete eradication to 55%. With less pathogenic strains of H.pylori in the patient, the standard H.pylori eradication scheme is effective in 90% of patients, and the addition of probiotics based on E.faecium L3 to the standard scheme guarantees complete eradication of H.pylori in 100% of patients.
E. faecium L3 is also effective as monotherapy for patients who are intolerant to standard eradication regimens based on antibiotics. In these patients L3 treatment leads to eradication of H. pylori in every second patient regardless of their genotype.

Figure 10. The percentage of H. pylori eradication depending on the pathogen genotype

As a component of comprehensive therapy, probiotics based on E.faecium L3 are successfully used in the treatment of IBD, hepatitis, and pancreatitis. In all these pathologies, there is a significant improvement in the composition of the intestinal microbiota, an acceleration of positive clinical dynamics and dynamics of biochemical and immunological indicators compared with patients who did not receive a probiotic [74-84].
Patients with chronic hepatitis who received probiotics based on E. faecium L3 had improved clinical and laboratory indicators and experienced a faster positive clinical progression compared to those who did not receive it. Additionally, significant improvements in brain function were observed in patients with encephalopathy [85].

E. faecium L3 have been shown to significantly improve metabolism in patients with chronic liver diseases and IBD, as well as in patients with metabolic disorders such as atherosclerosis. The improvement in gut microbiome leads to improved liver function and normalization of cholesterol and low-density lipoprotein levels [86][87]

The use of Enterococcus faecium L3 in the comprehensive treatment of patients with diabetes mellitus improved the gut microbiome, blood biochemistry, well-being, and mood in patients [88, 89]

The effectiveness of Enterococcus faecium L3 in comprehensive treatment of gynecological conditions such as vulvovaginitis, cervicitis, and bacterial vaginosis has been well established. Studies have also shown that there is an antagonistic relationship between Enterococci and Candida in the vaginal environment of reproductive age women, with increased Enterococci titers resulting in a decrease in the number of Candida. Furthermore, metabolites of E. faecium L3 have been shown to have strain-dependent fungistatic and fungicidal effects, suppress the formation of Candida biofilms, and reduce the adhesion of Candida species to mucosal epithelial cells.
The oral administration of Enterococcus faecium L3 in pregnant women has been shown to be safe and effective in preventing and treating genital infections, and even reducing the incidence of premature birth. A clinical trial involving pregnant women receiving the probiotic, showed that tL3not only prevented intestinal disorders and dysbiotic disorders but also led to a 30% reduction in premature rupture of the fetal bladder, thus reducing the likelihood of premature birth and improving the biochemical parameters of umbilical cord blood.

Sports

Positive metabolic effects of E. faecium L3 in athletes
In studies conducted with athletes of the Ukrainian Olympic team, the dynamics of biochemical indicators on the background of taking the probiotic Enterococcus faecium L3 was studied in the most detail. It was noted that a course of probiotic Enterococcus faecium L3 in qualified representatives of different groups of sports is accompanied by a positive effect on the expression of the syndrome of endogenous intoxication, which is one of the significant metabolic factors in reducing physical fitness. Enterococcus faecium L3 positively affects the prooxidant-antioxidant balance in cellular membranes, which improves the course of all metabolic processes in the athlete's body. A course of probiotic Enterococcus faecium L3 promotes the improvement of the functional state of the cardiovascular system, which is realized indirectly through the improvement of metabolic parameters, primarily endogenous toxicity, oxidative homeostasis at the membrane level, lipid metabolism, and also normalizes the indicators of the immune status and the balance of microflora.

TABLE 3 - Impact of the "L3 on specialized training indicators in athletes.

*p <0.05 compared to data before the mesocycle; ** p <0.05 compared to data in the control group at the corresponding stage of the study.

It was concluded that using Enterococcus faecium L3 in high-performance sports is beneficial. It helps to maintain proper metabolic processes and physical fitness. It is especially helpful during the special-preparatory stage of the training period. This conclusion is based on the positive effects L3 strain on performance [90-92].

The study also found that using E.Faecium L3 reduced disruption of heart repolarization in the V5-V6 area and reduced the incidence of early repolarization syndrome.

Immunological effects of L3 in clinical studies

Clinical studies of patients with chronic hepatitis and encephalopathies accompanied by liver dysfunction have shown that in the blood of patients taking L3, the level of IL10 significantly increased, which fully correlated with studies on laboratory animals [93].

A good clinical effect with the correction of dysbiotic disorders was observed in patients with autoimmune, including neurodegenerative diseases. Thus, in patients with multiple sclerosis and Parkinson's disease, Enterococcus faecium L3 significantly improved the composition of the gut microbiota [94,95]. There was a positive clinical dynamics and improvement in biochemical and immunological parameters, prolongation of the remission period, and improvement in the psycho-emotional condition of the patients. All patients had a high compliance with the probiotic. It turned out that L3 could significantly slow down the development of degenerative processes and improve the general condition of patients. It is important to note that in most of studies immunological effects of L3 strain on patients correlated with data on animals. The decrease in the production of proinflammatory cytokines and increase in production of regulatory cytokine IL-10 was always evident and statistically proved.

The use of Enterococcus faecium L3 in pediatrics.

Enterococcus faecium L3 strain is successfully used in both children and adults for the prevention and treatment of infectious diseases [99], and in gastroenterology for the prevention and treatment of functional disorders of the digestive organs [100]. The use of the probiotic Enterococcus faecium L3 improves the clinical picture, which is reflected in the disappearance of the abdominal pain syndrome, normalization of the frequency, form and consistency of the stool, reduction of meteorism, and the absence of adverse reactions. Coprographic indicators are improved as well. In the study of the intestinal microbiota of the observed children, it was found that the prescription of the E. faecium L3 reliably increases the content of pathogenetically significant indigenous butyrate-producing F. prausnitzii in the composition of the intestinal microbiota and leads to a elimination of opportunistic bacteria.
A positive clinical dynamics and improvement in the microbiota are observed in children with cholestasis [101], and inflammatory bowel diseases [102] when using Enterococcus faecium L3. Comprehensive rehabilitation of children with protein-energy deficiency is more effective when the L3 is added to the diet: children experience a twofold increase in weight gain (compared to the control group that did not receive E. faecium L3). Their dyspeptic symptoms completely disappear (in the control group, dyspepsia persists in 20% of children) [103].
In the treatment of skin pathologies (atopic dermatitis), a study was conducted with the L3 strain mixed with cocoa oil applied externally. It was shown that skin manifestations in 100% of cases were associated with colonization of the skin by Staphylococcus aureus. Treatment of the skin with the Enterococcus faecium L3 for 10 days significantly improved the condition of patients with dermatitis (figures 17, 18) [104].

Figure 17. The condition of the skin on the elbow folds of the patient K. before treatment

Figure 11. The condition of the skin on the elbow folds of the patient K. after 10 days of treatment with the probiotic strain Enterococcus faecium L3

Before

After

The Enterococcus faecium L3 strain is successfully used in the composition of complex probiotic preparations for the prevention of acute intestinal and respiratory infections in children [105], and for the treatment of allergic diseases [106, 107].

Neonatology

Impressive results have been obtained with the use of Enterococcus faecium L3 in neonatology: A liquid form of the L3 strain on a non-milk basis with a titer of not less than 10⁸ CFU/ml was given to deeply premature infants with low birth weight (LBW) and very low birth weight (VLBW) at a dose of 1.5 ml twice a day during feeding. This helped to prevent contamination of the gut with pathogenic and opportunistic bacteria, and improved the composition of the gut microbiota during antibiotic therapy [108-110]. L3 intake also prevented severe infectious complications such as "feed breakdowns", after which the child is put on parenteral nutrition and intravenous antibiotic therapy [111,112]. The use of L3 accelerated weight gain and improved physiological parameters, shortened the period of parenteral nutrition and the transition to full enteral feeding, thus reducing the child's hospital stay [113-116]. Further monitoring and probiotic support using Enterococcus faecium L3 helped to prevent recurrent respiratory infections, reduce the use of antibiotics and other medical interventions, as well as improve the overall health and development of premature infants [117-119].

The use of Enterococcus faecium L3 in oncology.

The ability of the strain to suppress infection, exert anti-inflammatory and immunomodulating effects makes it essential for cancer patients.

Medical studies in oncology clinics have demonstrated the benefit of using Enterococcus faecium L3 in cancer patients who receive chemotherapy or combined chemoradiotherapy. Thus, it was found that in patients receiving chemoradiotherapy and taking Enterococcus faecium L3, the toxicity decreased significantly, the immune system indicators improved, and the development of post-radiation reactions and complications was prevented [120-122].
But it was only after detailed study of the genome of the Enterococcus faecium L3 strain that some of the mechanisms for reducing the toxic effects of chemotherapy could be explained. For example, the L3 has a genetically confirmed ability (a gene called pepC, which encodes cysteine aminopeptidase, was found) to produce the enzyme - cysteine aminopeptidase. In clinical practice, this property is used to reduce the toxicity of bleomycin, that is, to reduce toxic complications when using chemotherapy for cancer treatment [123].

Additionally, it was found in a study that the use of Enterococcus faecium L3, along with enterococcal autoprobiotics, in the perioperative period in cancer patients was demonstrated to decrease the risk of infectious complications and increase the quality of life after major surgical interventions in the early postoperative period. This study also identified the presence of opportunistic and pathogenic bacteria in patients' microbiomes using PCR-DGGE analysis. Positive changes in the microbiome after taking Enterococcus faecium L3 and autoprobiotics in the perioperative period showed an increase in the content of the main producers of short-chain fatty acids - fecalibacteria and bifidobacteria, as well as a decrease in the content of opportunistic and pathogenic bacteria (Fusobacterium nucleatum, and others).

Figure 12. Dynamics of gut microbiome composition in CRC patients after probiotic and auto-probiotic therapy in the perioperative period.

The positive shifts in the composition of the microbiome in cancer patients were sustained for a long time. For example, such microbiological oncological markers as Fusobacterium nucleatum were not detected in patients 1-1.5 years after the operations and a repeat probiotic support course eliminated other opportunistic microorganisms (Enterobacter spp.). However, it should be noted that autoprobiotic strains were only obtained from half of the cancer patients due to serious dysbiotic disorders. Therefore, although autoprobiotic enterococci normalize the microbiocenosis in cancer patients faster than Enterococcus faecium L3, probiotic support with the E. faecium L3 (especially in case of inability to obtain autoprobiotic strains) should be recommended starting from the perioperative period and continuing with courses of probiotics 1-2 times a year for normalization of microbiocenosis, elimination of microbiological oncological markers, and thus - for the prevention of recurrences of oncological process.

In summary, the study found that using the probiotic strain Enterococcus faecium L3, along with other enterococcal autoprobiotics, in the form of a liquid, non-milk starter culture during the perioperative period for patients with cancer helped reduce the risk of infectious complications and improve quality of life after surgery. The study also found that this strain of bacteria has the ability to produce enzymes such as arginine deiminase, which breaks down arginine, and mannitol-1-phosphate-5-dehydrogenase, which breaks down mannitol and mannose, which gives it a direct anticancer effect by competing with cancer cells for nutrients. Additionally, the L3 strain was able to produce a peptide called Entap which also has anticancer effect. It is recommended to use E.faecium L3 support in the perioperative period, and continue with periodic treatments to maintain a normal microbiota, remove microbiological oncological markers, and prevent recurrence of the cancer.