Protective Properties of S-layer Protein 2 from Lactobacillus crispatus 2029 against Candida albicans Infections.

Previously, the protective role of the S-layer protein 2 (Slp2) of the vaginal Lactobacillus crispatus 2029 (LC2029) strain against foodborne pathogens Campylobacter jejuni, Salmonella enterica serovar Enteritidis, and Escherichia coli O157:H was demonstrated. We demonstrate the new roles of the Slp2-positive LC2029 strain and soluble Slp2 against C. albicans infections. We show that LC2029 bacteria can adhere to the surface of the cervical epithelial HeLa cells, prevent their contact with C. albicans, and block yeast transition to a pathogenic hyphal form. Surface-bound Slp2 provides the ability for LC2029 to co-aggregate with various C. albicans strains, including clinical isolates. C. albicans-induced necrotizing epithelial damage is reduced by colonization with the Slp2-positive LC2029 strain. Slp2 inhibits the adhesion of various strains of C. albicans to different human epithelial cells, blocks yeast transition to a pathogenic hyphal form, and prevents the colonization and pathogenic infiltration of mucosal barriers. Only Slp2 and LC2029 bacteria stimulate the production of protective human ß-defensin 3 in various epithelial cells. These findings support the anti-Candida albicans potential of the probiotic LC2029 strain and Slp2 and form the basis for further research on their ability to prevent and manage invasive Candida infections.

Ligilactobacillus salivarius 7247 Strain: Probiotic Properties and Anti-Salmonella Effect with Prebiotics.

The Ligilactobacillus salivarius 7247 (LS7247) strain, originally isolated from a healthy woman's intestines and reproductive system, has been studied for its probiotic potential, particularly against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) as well as its potential use in synbiotics. LS7247 showed high tolerance to gastric and intestinal stress and effectively adhered to human and animal enterocyte monolayers, essential for realizing its probiotic properties. LS7247 showed high anti-Salmonella activity. Additionally, the cell-free culture supernatant (CFS) of LS7247 exhibited anti-Salmonella activity, with a partial reduction upon neutralization with NaOH (p < 0.05), suggesting the presence of anti-Salmonella factors such as lactic acid (LA) and bacteriocins. LS7247 produced a high concentration of LA, reaching 124.0 ± 2.5 mM after 48 h of cultivation. Unique gene clusters in the genome of LS7247 contribute to the production of Enterolysin A and metalloendopeptidase. Notably, LS7247 carries a plasmid with a gene cluster identical to human intestinal strain L. salivarius UCC118, responsible for class IIb bacteriocin synthesis, and a gene cluster identical to porcine strain L. salivarius P1ACE3, responsible for nisin S synthesis. Co-cultivation of LS7247 with SE and ST pathogens reduced their viability by 1.0-1.5 log, attributed to cell wall damage and ATP leakage caused by the CFS. For the first time, the CFS of LS7247 has been shown to inhibit adhesion of SE and ST to human and animal enterocytes (p < 0.01). The combination of Actigen prebiotic and the CFS of LS7247 demonstrated a significant combined effect in inhibiting the adhesion of SE and ST to human and animal enterocytes (p < 0.001). These findings highlight the potential of using the LS7247 as a preventive strategy and employing probiotics and synbiotics to combat the prevalence of salmonellosis in animals and humans caused by multidrug resistant (MDR) strains of SE and ST pathogens.

Influence of Dietary Addition of Mineral Shungite and Fucus vesiculosus on Production Performance, Egg Quality, Nutrients Digestibility, and Immunity Status of Laying Hens.

The main purpose of this study was to assess the impact of using the thermally modified mineral adsorbent shungite (MAS) and the dried seaweed meal Fucus vesiculosus (DSM) with different doses in Brown Nick cross laying hens' diet on their productivity, nutrient digestibility, morphological and blood profile, immunity status, and egg quality. A total of 261,720 hens were used in this experiment at the age of 63 weeks, and they were randomly divided into 5 groups (feeding program) with six repetitions of 8724 chickens in each. The first served (control) as a control group where laying hens were fed the basal diet that was used on the farm only; the second and the third groups represented MAS+ and MAS++, where they received the basal diet supplemented by 0.1% and 0.25% (or 1.0 kg/t and 2.5 kg/t of feed) of the mineral adsorbent shungite (MAS) which was provided in the feed in powder form (5 microns) and was added to the feed at the feed mill; the fourth and fifth groups represented DSM+ and DSM++, which received the basal diet provided with 0.1% and 0.25% (or 1.0 kg/t and 2.5 kg/t of feed) of dried seaweed meal of F. vesiculosus algae (DSM). The average egg weight over the entire period of the experiment revealed significant differences between the experimental groups and represented in the control group 65.20 vs. 66.88, 66.87 and 68.10 and 68.13 g in the MAS+ and MAS++, and DSM+ and DSM++ groups, respectively. Once the dried seaweed meal F. vesiculosus (DSM) was used, the crude protein increased significantly (p < 0.05) in egg yolk by 2.64 and 2.67%, carotenoids by 1.13 and 1.20 mg/g DM. The inclusion of both MAS and DSM feed additives revealed a significant decrease in the level of crude fat (lipids) in their liver when compared with the control group. The level of erythrocytes (RBCs) increased (p < 0.05) in the MAS+ and MAS++ and DSM+ and DSM++ groups when compared to the control group. Similarly, a significant increase was noted in hemoglobin when DSM was supplemented when compared to the control one. Moreover, the number of heterophils increased (p < 0.05) in groups of MAS and DSM when compared to the control group. The percentage of phagocytic activity increased significantly by 5.39, 6.90, and 7.18% in MAS++, DSM+, and DSM++, respectively, relative to the control group. On the other hand, the phagocytic number decreased (p < 0.05) by 1.15 and 1.12 conditional units in MAS+ and MAS++ and by 1.03 and 0.83 conditional units in DSM+ and DSM++ when compared to the control group, respectively. Consequently, the inclusion of thermally modified mineral adsorbent shungite and the dried seaweed meal F. vesiculosus with different doses in Brown Nick cross laying hen diets improves the egg weight and egg quality, crude protein, carotenoids and vitamin A in the egg mass, the utilization of lysine and methionine nutrients, hemoglobin content, immunity status, while decreases the incidence of fatty liver occurrence.

Limosilactobacillus fermentum 3872 That Produces Class III Bacteriocin Forms Co-Aggregates with the Antibiotic-Resistant Staphylococcus aureus Strains and Induces Their Lethal Damage.

LF3872 was isolated from the milk of a healthy lactating and breastfeeding woman. Earlier, the genome of LF3872 was sequenced, and a gene encoding unique bacteriocin was discovered. We have shown here that the LF3872 strain produces a novel thermolabile class III bacteriolysin (BLF3872), exhibiting antimicrobial activity against antibiotic-resistant Staphylococcus aureus strains. Sequence analysis revealed the two-domain structural (lysozyme-like domain and peptidase M23 domain) organization of BLF3872. At least 25% residues of this protein are expected to be intrinsically disordered. Furthermore, BLF3872 is predicted to have a very high liquid-liquid phase separation. According to the electron microscopy data, the bacterial cells of LF3872 strain form co-aggregates with the S. aureus 8325-4 bacterial cells. LF3872 produced bacteriolysin BLF3872 that lyses the cells of the S. aureus 8325-4 mastitis-inducing strain. The sensitivity of the antibiotic-resistant S. aureus collection strains and freshly isolated antibiotic-resistant strains was tested using samples from women with lactation mastitis; the human nasopharynx and oral cavity; the oropharynx of pigs; and the cows with a diagnosis of clinical mastitis sensitive to the lytic action of the LF3872 strain producing BLF3872. The co-cultivation of LF3872 strain with various antibiotic-resistant S. aureus strains for 24 h reduced the level of living cells of these pathogens by six log. The LF3872 strain was found to be able to co-aggregate with all studied S. aureus strains. The cell-free culture supernatant of LF3872 (CSLF3872) induced S. aureus cell damage and ATP leakage. The effectiveness of the bacteriolytic action of LF3872 strain did not depend on the origin of the S. aureus strains. The results reported here are important for the creation of new effective drugs against antibiotic-resistant strains of S. aureus circulating in humans and animals.

Role of Dietary Inclusion of Phytobiotics and Mineral Adsorbent Combination on Dairy Cows' Milk Production, Nutrient Digestibility, Nitrogen Utilization, and Biochemical Parameters.

Our research purpose was to study the effect of the inclusion of a combination of phytobiotics in the form of dry Fucus vesiculosus grits (FG) and a mineral adsorbent from the heat-treated mineral shungite (TMS) on milk productivity, nutrient digestibility, and biochemical parameters of the Suksun dairy cows. A total of 80 dry-hardy cows of the Suksun breed were divided into four groups (20 heads each), balanced primarily by breed, age, body weight, body condition score, and indicators of milk yield for the previous lactation. The selected cows were with an average live body weight of 512.0 ± 1.28 kg, BCS 3.0-3.5, and parities of 6250 kg milk. The control group (CON) were fed the basic ration only; the second (TMS), third (FG), and fourth (TMS + FG) groups were fed the basic ration provided by 50 g of the mineral adsorbent from heat-treated shungite, 100 g of Fucus grits (Fucus vesiculosus), 50 g of the mineral adsorbent from heat-treated shungite, and 100 g of dry grits from Fucus vesiculosus, respectively. The total protein content in milk was significantly higher in the group receiving Fucus vesiculosus by 0.05% and the group receiving a combination of mineral adsorbent and Fucus vesiculosus by 0.03%. The percentage of milk fat content recorded the highest significant value in (TMS) group when compared to the control and represented (4.37 vs. 3.95). The group of cows that received (TMS + FG) revealed a significant difference in the digestibility of both ether extract and crude fiber when compared to the control group and represented (54.74 vs. 51.71 and 60.68 vs. 55.15%), respectively. The cows supplemented with a mineral adsorbent or a combination of mineral adsorbent and Fucus vesiculosus revealed a significant difference in the digestibility of ether extract and crude fiber in the group receiving TMS + FG by 3.0% (p < 0.05) and 5.5% (p < 0.05), respectively. The intake of nitrogen with the diet increased in (FG) and (TMS + FG) groups by 11.3 g (p < 0.05) and 13.4 g (p < 0.05) of nitrogen. There was an increase (p < 0.05) in the concentration of rumen ammonia in the control group compared to the other groups. The glucose content of those cows that received FG and TMS + FG combination increased (p < 0.05) by 0.76 mmol/L and 0.90 mmol/l in relation to the control group. The globulin, albumin/globulin ratio, and the level of triglycerides revealed a significant difference between the different experimental groups. In brief, the inclusion of a combination of phytobiotics in the form of dry Fucus vesiculosus grits and a mineral adsorbent from the heat-treated mineral shungite in Suksun dairy cows' diets improved milk composition, digestibility of nutrients, utilization of nitrogen, and did not cause deleterious effects on blood biochemical indicators.

Anti-Salmonella Defence and Intestinal Homeostatic Maintenance In Vitro of a Consortium Containing Limosilactobacillus fermentum 3872 and Ligilactobacillus salivarius 7247 Strains in Human, Porcine, and Chicken Enterocytes.

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. Ligilactobacillus salivarius strain 7247 (LS7247) was isolated at the same time from the intestines and reproductive system of a healthy woman. The genomes of these strains contain genes responsible for the production of peptidoglycan-degrading enzymes and factors that increase the permeability of the outer membrane of Gram-negative pathogens. In this work, the anti-Salmonella and intestinal homeostatic features of the LF3872 and LS7247 consortium were studied. A multi-drug resistant (MDR) strain of Salmonella enteritidis (SE) was used in the experiments. The consortium effectively inhibited the adhesion of SE to intact and activated human, porcine, and chicken enterocytes and reduced invasion. The consortium had a bactericidal effect on SE in 6 h of co-culturing. A gene expression analysis of SE showed that the cell-free supernatant (CFS) of the consortium inhibited the expression of virulence genes critical for the colonization of human and animal enterocytes. The CFS stimulated the production of an intestinal homeostatic factor-intestinal alkaline phosphatase (IAP)-in Caco-2 and HT-29 enterocytes. The consortium decreased the production of pro-inflammatory cytokines IL-8, TNF-α, and IL-1ß, and TLR4 mRNA expression in human and animal enterocytes. It stimulated the expression of TLR9 in human and porcine enterocytes and stimulated the expression of TLR21 in chicken enterocytes. The consortium also protected the intestinal barrier functions through the increase of transepithelial electrical resistance (TEER) and the inhibition of paracellular permeability in the monolayers of human and animal enterocytes. The results obtained suggest that a LF3872 and LS7247 consortium can be used as an innovative feed additive to reduce the spread of MDR SE among the population and farm animals.

Limosilactobacillus fermentum Strain 3872: Antibacterial and Immunoregulatory Properties and Synergy with Prebiotics against Socially Significant Antibiotic-Resistant Infections of Animals and Humans.

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. The high-quality genome sequencing of LF3872 was performed, and a gene encoding a unique bacteriocin was discovered. It was established that the bacteriocin produced by LF3872 (BLF3872) belongs to the family of cell-wall-degrading proteins that cause cell lysis. The antibacterial properties of LF3872 were studied using test cultures of antibiotic-resistant Gram-positive and Gram-negative pathogens. Gram-positive pathogens (Staphylococcus aureus strain 8325-4 and S. aureus strain IIE CI-SA 1246) were highly sensitive to the bacteriolytic action of LF3872. Gram-negative pathogens (Escherichia coli, Salmonella strains, and Campylobacter jejuni strains) were more resistant to the bacteriolytic action of LF3872 compared to Gram-positive pathogens. LF3872 is a strong co-aggregator of Gram-negative pathogens. The cell-free culture supernatant of LF3872 (CSLF3872) induced cell damage in the Gram-positive and Gram-negative test cultures and ATP leakage. In the in vitro experiments, it was found that LF3872 and Actigen prebiotic (Alltech Inc., Nicholasville, KY, USA) exhibited synergistic anti-adhesive activity against Gram-negative pathogens. LF3872 has immunoregulatory properties: it inhibited the lipopolysaccharide-induced production of proinflammatory cytokines IL-8, IL-1ß, and TNF-α in a monolayer of Caco-2 cells; inhibited the production of IL-12 and stimulated the production of IL-10 in immature human dendritic cells; and stimulated the production of TGF-ß, IFN-γ, and IgA in the immunocompetent cells of intestinal Peyer's patches (PPs) in mice. These results indicate the possibility of creating a synbiotic based on LF3872 and a prebiotic derived from Saccharomyces cerevisiae cell wall components. Such innovative drugs and biologically active additives are necessary for the implementation of a strategy to reduce the spread of antibiotic-resistant strains of socially significant animal and human infections.