Synthetic Oligopeptides Mimicking γ-Core Regions of Cysteine-Rich Peptides of Solanum lycopersicum Possess Antimicrobial Activity against Human and Plant Pathogens.

Plant cysteine-rich peptides (CRPs) represent a diverse group of molecules involved in different aspects of plant physiology. Antimicrobial peptides, which directly suppress the growth of pathogens, are regarded as promising templates for the development of next-generation pharmaceuticals and ecologically friendly plant disease control agents. Their oligopeptide fragments are even more promising because of their low production costs. The goal of this work was to explore the antimicrobial activity of nine short peptides derived from the γ-core-containing regions of tomato CRPs against important plant and human pathogens. We discovered antimicrobial activity in peptides derived from the defensin-like peptides, snakins, and MEG, which demonstrates the direct involvement of these CRPs in defense reactions in tomato. The CRP-derived short peptides appeared particularly active against the gram-positive bacterium Clavibacter michiganensis, which causes bacterial wilt-opening up new possibilities for their use in agriculture to control this dangerous disease. Furthermore, high inhibitory potency of short oligopeptides was demonstrated against the yeast Cryptococcus neoformans, which causes serious diseases in humans, making these peptide molecules promising candidates for the development of next-generation pharmaceuticals. Studies of the mode of action of the two most active peptides indicate fungal membrane permeabilization as a mechanism of antimicrobial action.

A Novel Bifidobacterium longum Subsp. longum T1 Strain from Cow's Milk: Homeostatic and Antibacterial Activity against ESBL-Producing Escherichia coli.

Background/Objectives: The global emergence of antibiotic-resistant zooanthroponotic Escherichia coli strains, producing extended-spectrum beta-lactamases (ESBL-E) and persisting in the intestines of farm animals, has now led to the development of a pandemic of extra-intestinal infectious diseases in humans. The search for innovative probiotic microorganisms that eliminate ESBL-E from the intestines of humans and animals is relevant. Previously, we received three isolates of bifidobacteria: from milk of a calved cow (BLLT1), feces of a newborn calf (BLLT2) and feces of a three-year-old child who received fresh milk from this calved cow (BLLT3). Our goal was to evaluate the genetic identity of BLLT1, BLLT2, BLLT3 isolates using genomic DNA fingerprinting (GDF), to study the tolerance, adhesion, homeostatic and antibacterial activity of BLLT1 against ESBL-E. Methods: We used a complex of microbiological, molecular biological, and immunological methods, including next generation sequencing (NGS). Results: GDF showed that DNA fragments of BLLT2 and BLLT3 isolates were identical in number and size to DNA fragments of BLLT1. These data show for the first time the possibility of natural horizontal transmission of BLLT1 through with the milk of a calved cow into the intestines of a calf and the intestines of a child. BLLT1 was resistant to gastric and intestinal stresses and exhibited high adhesive activity to calf, pig, chicken, and human enterocytes. This indicates the unique ability of BLLT1 to inhabit the intestines of animals and humans. We are the first to show that BLLT1 has antibacterial activity against ESBL-E strains that persist in humans and animals. BLLT1 produced 145 ± 8 mM of acetic acid, which reduced the pH of the nutrient medium from 6.8 to 5.2. This had an antibacterial effect on ESBL-E. The genome of BLLT1 contains ABC-type carbohydrate transporter gene clusters responsible for the synthesis of acetic acid with its antibacterial activity against ESBL-E. BLLT1 inhibited TLR4 mRNA expression induced by ESBL-E in HT-29 enterocytes, and protected the enterocyte monolayers used in this study as a bio-model of the intestinal barrier. BLLT1 increased intestinal alkaline phosphatase (IAP) as one of the main molecular factors providing intestinal homeostasis. Conclusions: BLLT1 shows promise for the creation of innovative functional nutritional products for humans and feed additives for farm animals that will reduce the spread of ESBL-E strains in the food chain.

The Extracellular Vesicles Containing Inorganic Polyphosphate of Candida Yeast upon Growth on Hexadecane.

The cell wall of Candida yeast grown on presence of hexadecane as a sole carbon source undergoes structural and functional changes including the formation of specific supramolecular complexes-canals. The canals contain specific polysaccharides and enzymes that provide primary oxidization of alkanes. In addition, inorganic polyphosphate (polyP) was identified in Candida maltosa canals. The aim of the work was a comparative study of the features of cell walls and extracellular structures in yeast C. maltosa, C. albicans and C. tropicalis with special attention to inorganic polyphosphates as possible part of these structures when grown on the widely used xenobiotic hexadecane (diesel fuel). Fluorescence microscopy with DAPI has shown an unusual localization of polyP on the cell surface and in the exovesicles in the three yeast species, when growing on hexadecane. Electron-scanning microscopy showed that the exovesicles were associated with the cell wall and also presented in the external environment probably as biofilm components. Treatment of hexadecane-grown cells with purified Ppx1 polyphosphatase led to the release of phosphate into the incubation medium and the disappearance of polyP in vesicles and cell wall observed using microscopic methods. The results indicate the important role of polyP in the formation of extracellular structures in the Candida yeast when consuming hexadecane and are important for the design of xenobiotic destructors based on yeast or mixed cultures.

Whole-Genome Sequencing and Biotechnological Potential Assessment of Two Bacterial Strains Isolated from Poultry Farms in Belgorod, Russia.

Bacteria, designated as A1.1 and A1.2, were isolated from poultry waste based on the ability to form ammonia on LB nutrient medium. Whole genome sequencing identified the studied strains as Peribacillus frigoritolerans VKM B-3700D (A1.1) and Bacillus subtilis VKM B-3701D (A1.2) with genome sizes of 5462638 and 4158287 bp, respectively. In the genome of B. subtilis VKM B-3701D, gene clusters of secondary metabolites of bacillin, subtilisin, bacilisin, surfactin, bacilliacin, fengycin, sactipeptide, and ratipeptide (spore killing factor) with potential antimicrobial activity were identified. Clusters of coronimine and peninodin production genes were found in P. frigoritolerans VKM B-3700D. Information on coronimine in bacteria is extremely limited. The study of the individual properties of the strains showed that the cultures are capable of biosynthesis of a number of enzymes, including amylases. The B. subtilis VKM V-3701D inhibited the growth of bacterial test cultures and reduced the growth rate of the mold fungus Aspergillus unguis VKM F-1754 by 70% relative to the control. The antimicrobial activity of P. frigoritolerans VKM V-3700D was insignificant. At the same time, a mixture of cultures P. frigoritolerans VKM B-3700D/B. subtilis VKM B-3701D reduced the growth rate of A. unguis VKM F-1754 by 24.5%. It has been shown that strain A1.1 is able to use nitrogen compounds for assimilation processes. It can be assumed that P. frigoritolerans VKM V-3700D belongs to the group of nitrifying or denitrifying microorganisms, which may be important in developing methods for reducing nitrogen load and eutrophication.

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.

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.

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.

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.

Capture of Essential Trace Elements and Phosphate Accumulation as a Basis for the Antimicrobial Activity of a New Ultramicrobacterium-Microbacterium lacticum Str. F2E.

Microbial interactions play an important role in natural habitat. The long-term coevolution of various species leads to the adaptation of certain types of microorganisms as well as to the formation of a wide variety of interactions such as competitive, antagonistic, pathogenic and parasitic relationships. The aim of this work is a comprehensive study of a new ultramicrobacterium Microbacterium lacticum str. F2E, isolated from perennial oil sludge, which is characterized by high antimicrobial activity and a unique ultrastructural organization of the cell envelope, which includes globular surface ultrastructures with a high negative charge. A previously undescribed mechanism for the antagonistic action of the F2E strain against the prey bacterium is proposed. This mechanism is based on the ability to preferentially capture essential microelements, in which charge interactions and the property of phosphate accumulation may play a significant role. The revealed type of intermicrobial interaction can probably be attributed to the non-contact type antagonistic action in the absence of any diffuse factor secreted by the antagonistic bacteria.

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.

Characterization of Soil Bacteria with Potential to Degrade Benzoate and Antagonistic to Fungal and Bacterial Phytopathogens.

The intensive development of agriculture leads to the depletion of land and a decrease in crop yields and in plant resistances to diseases. A large number of fertilizers and pesticides are currently used to solve these problems. Chemicals can enter the soil and penetrate into the groundwater and agricultural plants. Therefore, the primary task is to intensify agricultural production without causing additional damage to the environment. This problem can be partially solved using microorganisms with target properties. Microorganisms that combine several useful traits are especially valuable. The aim of this work was to search for new microbial strains, which are characterized by the ability to increase the bioavailability of nutrients, phytostimulation, the antifungal effect and the decomposition of some xenobiotics. A few isolated strains of the genera Bacillus and Pseudomonas were characterized by high activity against fungal phytopathogens. One of the bacterial strains identified as Priestiaaryabhattai on the basis of the 16S rRNA gene sequence was characterized by an unusual cellular morphology and development cycle, significantly different from all previously described bacteria of this genus. All isolated bacteria are capable of benzoate degradation as a sign of the ability to degrade aromatic compounds. Isolated strains were shown to be prospective agents in biotechnologies.

A Blue-Purple Pigment-Producing Bacterium Isolated from the Vezelka River in the City of Belgorod.

Violacein is a biotechnologically significant secondary metabolite due to its antibacterial, antifungal, and other properties. Isolation, research, and identification of violacein producing strains are of interest for the development of biotechnological processes, in order to enhance the biosynthesis of this compound. The purpose of the present work was to study the properties of a newly isolated bacterium capable of synthesizing blue-purple pigment. An aboriginal bacterium was isolated from the coastal zone of the Vezelka River in the city of Belgorod. Based on chemical and spectrophotometric studies of the crude ethanol extract, the pigment was identified as violacein, and the isolate was assigned to the group of violacein-forming bacteria, which includes bacteria of the genera Chromobacterium, Iodobacter, Janthinobacterium, Duganella, Collimonas, and Massilia. Based on cultural, morphological, tinctorial, physiological, and biochemical properties, as well as analysis of the 16S rRNA gene sequence, the new isolated strain was assigned to the genus Janthinobacterium. The isolated strain is capable of suppressing the growth of a number of fungal and bacterial phytopathogens. For representatives of the genus Janthinobacterium, their inhibitory influence on cyanobacteria was shown for the first time.

S-layer protein 2 of vaginal Lactobacillus crispatus 2029 enhances growth, differentiation, VEGF production and barrier functions in intestinal epithelial cell line Caco-2.

We have previously demonstrated the ability of the human vaginal strain Lactobacillus crispatus 2029 (LC2029) for strong adhesion to cervicovaginal epithelial cells, expression of the surface layer protein 2 (Slp2), and antagonistic activity against urogenital pathogens. Slp2 forms regular two-dimensional structure around the LC2029 cells,which is secreted into the medium and inhibits intestinal pathogen-induced activation of caspase-9 and caspase-3 in the human intestinal Caco-2 cells. Here, we elucidated the effects of soluble Slp2 on adhesion of proteobacteria pathogens inducing necrotizing enterocolitis (NEC), such as Escherichia coli ATCC E 2348/69, E. coli ATCC 31705, Salmonella Enteritidis ATCC 13076, Campylobacter jejuni ATCC 29428, and Pseudomonas aeruginosa ATCC 27853 to Caco-2 cells, as well as on growth promotion, differentiation, vascular endothelial growth factor (VEGF) production, and intestinal barrier function of Caco-2 cell monolayers. Slp2 acts as anti-adhesion agent for NEC-inducing proteobacteria, promotes growth of immature Caco-2 cells and their differentiation, and enhances expression and functional activity of sucrase, lactase, and alkaline phosphatase. Slp2 stimulates VEGF production, decreases paracellular permeability, and increases transepithelial electrical resistance, strengthening barrier function of Caco-2 cell monolayers. These data support the important role of Slp2 in the early postnatal development of the human small intestine enterocytes.

A cytological characterization of the parasitic action of ultramicrobacteria NF1 and NF3 of the genus Kaistia on chemoorganotrophic and phototrophic bacteria.

Two strains (NF1 and NF3) of free-living chemoorganotrophic bacteria have been isolated from multiyear oil slime and Pedilanthus tithymaloides rhizosphere and ascribed to the genus Kaistia of the class Alphaproteobacteria on the basis of the nucleotide sequences of 16S rRNA gene and phenotypic characteristics. These strains can be assigned to ultramicrobacteria as their populations are represented by two subpopulations: (1) ultrasmall cells, on average 200-300 nm in diameter and <0.1 microm(3) in volume, of up to 60% of the total number of cells in a population, and (2) cells 400-800 nm in diameter and 0.15-0.5 microm(3) in volume, of up to 40% of the total number of cells in a population. The interaction of the isolated ultramicrobacteria strains (IUMB) with different bacterial species has been studied in cocultures grown under starvation and in complete nutrient media. It has been found that IUMB can be facultative parasites on certain species of chemoorganotrophic and phototrophic bacteria. The interaction of IUMB with prey bacteria exhibits the extracellular type of parasitism and involves establishing stable cell-cell contacts between the parasites and their prey to cause destruction of host cells.