Probiotic Potential, Safety Properties, and Antifungal Activities of Corynebacterium amycolatum ICIS 9 and Corynebacterium amycolatum ICIS 53 Strains.

The purpose of this study was to evaluate the probiotic characteristics and safety and to study the antifungal activity of C. amycolatum ICIS 9 and C. amycolatum ICIS 53 against Candida spp. The probiotic potential and safety properties were assessed by standard parameters. Both strains showed good survival at pH 3 for 3 h and high tolerance to 0.3% bile salts after 4 h of incubation. The indicators of hydrophobicity, autoaggregation, and surface tension for ICIS 9 were 89.43% (n-hexane) and 73.96% (xylene) and ranged from 13.13 to 39.86% and 34.27 mN/m, respectively. For ICIS 53, they were 59.95% (n-hexane) and 45.68% (xylene), from 35.58 to 51.53% and 32.40 mN/m, respectively. The strains ICIS 9 and ICIS 53 exhibited varying levels of coaggregation with all eight examined bacterial pathogens. The ICIS 9 strain was resistant to amikacin, amoxicillin, clarithromycin, chloramphenicol, ciprofloxacin, and gentamycin. ICIS 53 was resistant only to ciprofloxacin. The cell-free supernatant of strains ICIS 9 and ICIS 53 showed good antimicrobial and antibiofilm activity against 10 pathogenic vaginal and intestinal isolates of Candida spp. The CFS of ICIS 9 was more active against intestinal isolates, and the CFS of ICIS 53 showed good antimicrobial activity against vaginal isolates while inhibiting the growth of 2 out of 5 Candida spp. isolated from the intestine. Both of the strains were capable of reducing the biofilm formation of Candida fungi. In the case of the vaginal isolates of C. krusei V1, the results showed that the inhibition levels of ICIS 9 and ICIS 53 were 36.75 and 11.4%, respectively. In the case of C. albicans (V2, V3, V7, and V8), the inhibition of biofilm formation was no more than 7.07%. ICIS 9 and ICIS 53 also significantly inhibited biofilm formation of C. krusei 2613 intestinal isolates by 42.75 and 41.87%, respectively, with ICIS 9 inhibiting biofilm formation of C. albicans (2607, 2311, 2615, and 2615) from 3.38 to 15.69% and ICIS 53 from 5.95 to 23.48%. None of the strains showed DNase, haemolytic, or gelatinase activities. The results obtained revealed that ICIS 9 and ICIS 53 have safe properties and have the potential to be developed as probiotics.

A Novel High-Molecular-Mass Bacteriocin Produced by Enterococcus faecium: Biochemical Features and Mode of Action.

Discovery of a novel bacteriocin is always an event in sciences, since cultivation of most bacterial species is a general problem in microbiology. This statement is reflected by the fact that number of bacteriocins is smaller for tenfold comparing to known antimicrobial peptides. We cultivated Enterococcus faecium on simplified medium to reduce amount of purification steps. This approach allows to purify the novel heavy weight bacteriocin produced by E. faecium ICIS 7. The novelty of this bacteriocin, named enterocin-7, was confirmed by N-terminal sequencing and by comparing the structural-functional properties with available data. Purified enterocin-7 is characterized by a sequence of amino acid residues having no homology in UniProt/SwissProt/TrEMBL databases: NH2 - Asp - Ala - His - Leu - Ser - Glu - Val - Ala - Glu - Arg - Phe - Glu - Asp - Leu - Gly. Isolated thermostable protein has a molecular mass of 65 kDa, which allows it to be classified into class III in bacteriocin classification schemes. Enterocin-7 displayed a broad spectrum of activity against some Gram-positive and Gram-negative microorganisms. Fluorescent microscopy and spectroscopy showed the permeabilizing mechanism of the action of enterocin-7, which is realized within a few minutes.

[BIOLOGICAL ACTIVITY OF ANTIMICROBIAL PEPTIDES OF ENTEROCOCCUS FAECIUM].

AIM: Isolate bacteriocins from Enterococcus faecium metabolites and characterize their effect on cells of Gram positive (Listeria monocytogenes) and Gram negative (Escherichia coli) bacteria. MATERIALS AND METHODS: Methods of solid-phase extraction, ion-exchange and reversed phase chromatography were applied for isolation of bacteriocins from cultural medium of bacteria MALDI time-of-flight mass-spectrometry was used for characterization of the obtained preparations. The mechanism of biological effect of peptides was evaluated using DNA-tropic dyes (SYTO 9 and PI) with subsequent registration of fluorescence spectra: Atomic-force microscopy (AFM) was used for characterization of morpho-functional reaction of target cells. RESULTS: Peptide fractions with mass of 1.0 - 3.0 kDa were isolated from enterococci metabolites, that inhibit the growth of indicator microorganisms. E. faecium strain exoproducts were shown to increase membrane permeability during interaction with L. monocytogenes, that results in subsequent detectable disturbance of normal cell morphology of listeria. Alterations of E. coli surface during the effect of purified peptide fraction was detected using AFM. CONCLUSION: The studies carried out have revealed the effect of bacteriocins of enterococci on microorganisms with various types of cell wall composition and have confirmed the importance of bacterial barrier structure permeability disturbance in the mechanism of antimicrobial effect of enterocins.

[Antimicrobial compounds of enterococci].

The main studies of a group of antimicrobial compounds produced by Enterococcus genus bacteria: bacteriocins, organic acids, activated oxygen metabolites, enzymes, exopolysaccharides--are described in the review. Data on synergic interaction of these substances in killing of pathogens, formation of active substances from different components as a result of metabolic activity of enterococci are presented. Production of these compounds is a reflection of competitive habitation of bacteria under the conditions of multi-species microbiomes and ensures colonization resistance of host biotopes. A complementation of the group of bacterial antimicrobial compounds by compounds that do not have a direct effect on the growth of microorganisms but suppress pathogenic potential of infectious disease causative agents is assumed.

[Morphologic changes in listeria cells under the effect of metabolites of human intestinal microflora enterococci].

AIM: Characterization of morphologic changes in listeria cells under the effect of metabolites of human intestine microflora enterococci. MATERIALS AND METHODS: Culture of Listeria monocytogenes 88-BK cells was cultivated in the presence of enterococci waste products (experiment) or without them (control). The samples obtained were studied by atomic-force microscopy (AFM) in contact mode using scanning probe microscope SMM-2000. RESULTS: The character of morpho-functional reaction of indicator culture bacterial cells allows to assume the presence of cationic anti-microbial peptides with membrane-lytic mechanism of action in 30% of the studied enterococci strains. CONCLUSION: The use of atomic-force microscopy allows to detail the mechanisms of anti-microbial activity of bacteriocins of intestine microflora microorganisms against target bacteria under close to native conditions.

[Corneal ulcer caused by Serratia marcescens: case report].

A case of corneal ulcer caused by Serratia marcescens is reported in a patient with history of corneal microtrauma. Biological features (pathogenicity factors, antibiotic resistance) of isolated culture were characterized. Keratitis cases caused by this agent were analyzed.

[Study of features of the effect of cationic antimicrobial peptide nisin on Listeria monocytogenes bacteria by using atomic-force microscopy].

AIM: Characteristic of morpho-functional reaction of Listeria monocytogenes bacterial cells to the effect of cationic peptide nisin. MATERIALS AND METHODS: Culture of L. monocytogenes 88-BK bacterial cells was grown in the presence of nisin (experiment) and without it (control). The samples obtained were studied by atomic-force microscopy method in contact mode by using scanning probe of SM M-2000 microscope. Study of elastic properties of bacterial cells was carried out by using AFM in force spectroscopy mode. RESULTS: Microorganisms grown in the presence of sub-inhibitory concentrations of antibiotic were characterized by changes in cell morphology and mechanical properties that were recorded by using AFM as changes in size parameters of bacteria, visualized local lesions of surface with the increase of its roughness as well as decrease of mechanical durability of the cell wall. CONCLUSION: Use of atomic-force microscopy allows to detail mechanisms of biological activity of cationic peptide nisin on gram-positive bacteria, evaluate specter of changes of cell morphological and mechanical parameters that took place and were analyzed in the conditions close to native.

[Bacteriocinogenia of enterococci of human intestine microflora].

AIM: Characteristics of bacteriocinogenicity and bacteriocin-sensistivity of enterococci isolated from human feces. MATERIALS AND METHODS: Principle of delayed antagonism on solid nutrient mediu (120 x 120 cultures) was used for detection of bacteriocinogenicity of microorganisms. Factors of pathogenicity (production of hemolysin, gelatinase, DNase) as well as antagonism against Listeria genus bacteria were determined in highly active strains. RESULTS: Intrageneric antagonism was detected in 65% of bacterial cultures. Almost a quarter (23.1%) of bacteriocinogenic strains suppressed growth of more than 50 cultures of enterococci. This feature however was noted in members of 4 species, vast majority (77.8%) of the isolates with a wide specter of activity was attributed to Enterococcus faecium species. Under in vitro conditions these cultures displayed a pronounced anti-listeriosis effect, they did not have expression of pathogenicity factors. CONCLUSION: Higher prevalence of intra- and intergeneric antagonism in enterococci explains the significant importance of bacteria of this group of intestine microbiome in colonization resistance of host organism biotope.

[Mechanisms of resistance of enterococci to antimicrobial proteins and peptides].

Mechanisms of resistance of bacteria genus Enterococcus to the most important factors of innate immunity of the host--antimicrobial proteins and peptides--are described in the review. Data on enterococci lysozyme resistance associated with modification of peptidoglycan and changes in the net charge of the bacterial cell surface are presented. The role of enterococci sigma-factor with extra cytoplasmic SigV function is described. Evidence on microbial activation/degradation of neutrophil alpha-defensin (HNP-1), antibacterial peptide LL-37, cecropin, beta-lysine (thrombocytic cationic peptide) is presented. The accumulated experimental material is discussed from the position of persistence of enterococci--both pathogens causing various infectious processes and commensals composing a part of normal host microflora.

[Pathogenicity factors of enterococci of human intestinal microflora].

AIM: Characteristic of pathogenicity factors of enterococci isolated from human feces. MATERIALS AND METHODS: Production of hemolysin, gelatinase and DNase was determined in 161 enterococci cultures. RESULTS: Hemolytic activity detected in 14.9 +/- 2.8% of the studied cultures was the most prevalent characteristic; 22 of 24 hemolytic strains belonged to E. faecalis species. Human erythrocyte lysis was also caused by E. faecium and E. durans cultures (1 strain each). Other pathogenicity factors were detected solely in E. faecalis species members. Enterococci proteolytic activity associated with gelatinase enzyme production manifested on various substrates--both gelatin and milk. This property was detected in 7.5 +/- 2.1% cultures. Deoxyribonuclease was detected in 1 (1.2 +/- 0.9%) E. faecalis strain. A number of E. faecalis cultures possessing hemolytic activity additionally hydrolyzed gelatin (22.7 +/- 8.9% strains) and DNA (4.5 +/- 4.4% isolates). CONCLUSION: Though pathogenicity factors occur in enterococci of intestine microflora relatively rarely, separate cultures with expression of 2 or more pathogenicity factors may be essential in the development of endogenous infections especially in immune compromised patients.

[Features of microbiocenosis of the large intestine in dysbiotic disorders].

AIM: To determine features of intestinal microbiocenosis in dysbiosis as well as biological characteristics of isolated microflora in residents of Orenburg city. MATERIALS AND METHODS: 70 children one year old and 60 adult 1 - 60 years old were examined for dysbiosis. Bacteriologic identification of the large intestine's content was performed using method of serial dilutions. Isolated microorganisms were identified by routine methods. Assessment of the degree of dysbiotic disorder was conducted according to the standard guideline "Patients management protocol. Intestinal dysbiosis". Antibiotic resistance of Klebsiella species was determined by disc-diffusion method, antagonistic activity of lactobacilli--by plate culture method, and lysozyme activity--by agar bullet method. RESULTS: Dysbiotic disorders were registered in more than 90% of examined subjects. For patients of both age groups, stage I of intestinal dysbiosis was observed most often. Dysbiotic disorders were characterized by increased amount of bacteria from Klebsiella genus and yeast-like fungi from Candida genus. It was established that antibiotic resistance was widely prevalent in isolated strains of Klebsiella. At the same time representatives of normal microflora, i.e. lactobacilli, had a marked antagonistic activity against Klebsiella species, Staphylococcus aureus, Candida fungi and low level of lysozyme activity. CONCLUSION: Among the population of Orenburg, intestinal dysbiosis was widely prevalent and characterized by predominance of Klebsiella spp. and Candida spp. among opportunistic microflora. One of the rational methods of correction of compensated forms of intestinal dysbiosis is stimulation of growth of normal flora including lactobacilli, which have antagonistic activity.

[Multilocus sequence-typing of Enterococcus faecium fecal isolates].

AIM: Genetic characteristics of Enterococcus faecium strains isolated from human intestine in Russia. MATERIALS AND METHODS: Seven strains of E. faecium with antimicrobial activity against Gram-positive bacteria and yeast fungi were isolated from persons aged 4 months - 44 years. Using multilocus sequence-typing, sequences of internal fragments of genes of general metabolism (adk, atpA, ddl, gyd, gdh, purK, pstS) were determined. RESULTS: Number of alleles for each gene varied from 3 for gdh and pstS to 7 for atpA. Sequence-types of 4 out of 7 cultures of enterococci were described earlier, 3 strains were attributed to new sequence-types. CONCLUSION: Members of identified in this study sequence-types 32, 135, 170, 361 were isolated earlier in other countries from clinical samples (blood, faeces) and hospital environment. Diversity of sequence-types, sources of isolation and significant remoteness of regions where strains belonging to one sequence-type were isolated point to necessity of thorough study of E. faecium evolution.

[Formation of biofilms by fecal strains of enterobacteria and yeast fungi from Candida genus].

AIM: To assess the ability of bacteria and Candida genus' yeast fungi from human fecal microflora to form biofilms. MATERIALS AND METHODS: Thirty-one strains of enterobacteria and 45 strains of yeast fungi from Candida genus isolated from feces of patients during test on intestinal dysbiosis were used. In order to study the ability of microorganisms to form biofilms, level of binding of crystal violet by the formers in sterile 96-well polystyrene plates was studied. RESULTS: Ability to form biofilms was noted in 87.1% of enterobacteria strains. This characteristic was observed in lactose-positive and lactose-negative Escherichia coli and bacteria belonging to Klebsiella, Citrobacter, Serratia, Morganella, Providencia genuses. The highest mean value of the characteristic was noted in strains belonging to Enterobacter cloacae species (6.52+/-2.01). Overwhelming majority of fungal strains belonging to Candida genus formed static biofilms in wells of polystyrene plates. Mean value of the characteristic in group of Candida spp. (not albicans) was significantly higher compared with strains from C. albicans species (2.75+/-0.53 vs. 1.38+/-0.11, p<0.05). The highest value of the mentioned characteristic (6.75+/-1.65) was observed in yeast fungi belonging to C. krusei species. CONCLUSION: Obtained data widen our knowledge about armamentarium of biological characteristics of opportunistic microorganisms (bacteria and fungi) promoting their prolonged persistence in intestinal biotope.

[Species characteristic and factors of persistence of gut bifidoflora during healthy state and dysbiosis].

AIM: To determine features of species composition and factors of persistence of bifidobacteria of human gut microflora in healthy state and during dysbiosis. MATERIALS AND METHODS: Strains of bifidobacteria isolated from feces of patients with gut dysbiosis grades I - III (24 participants) and conditionally healthy persons (14 participants) were used for the study. Isolation and identification of microorganisms were performed by conventional methods. Antilysozyme activity was determined by photometric method and antilactoferrin sign--by immunoenzyme assay. RESULTS: Bifidobacteria isolated from intestinal biotope have differences in species composition, which depended from age and state of microbiocenosis of studied persons. Bifidobacterium bifidum was dominated between strains of bifidobacteria isolated from infants with eubiosis, whereas B. longum dominated in persons > 1 y.o. B. bifidum and B. adolescentis were mainly isolated in infants with gut dysbiosis, where as in children > 1 y.o. B. adolescentis was predominantly isolated. New characteristic of bifidoflora--antilactoferrin activity--was discovered for the first time. Expression of this sign, as well as antilysozyme activity, varied and depended from species of bifidobacteria. CONCLUSION: Obtained data widen our views on species composition of bifidobacteria and discover possible mechanisms of persistence of these microorganisms in intestinal biotope associated with inactivation of host defense factors--lysozyme and lactoferrin.

[Interaction of a causative agent with associative bacteria during salmonellosis].

AIM: To determine a composition of gut microflora during salmonellosis and to study the modification of persistent characteristics (antilysozyme activity, ALA) of symbiotic microorganisms in associations. MATERIALS AND METHODS: Bacteriologic study of feces was performed in 90 patients aged 18-39 years, which were divided to three groups: patients with salmonellosis in acute phase, reconvalescent patients, and conditionally healthy persons. Condition of gut microflorawas determined; microorganisms associated with Salmonella infection were isolated, and their influence on ALA of Salmonella was studied. RESULTS: Gut microbiocenosis was more diverse in patients compared with healthy persons. Significant reduction of bifidobacteria quantity (to 10(7) CFU/g of feces and less), especially in reconvalescent period, was noted. Association between bifidoflora deficiency and excessive increase of quantity of yeast fungi was revealed. It was determined that exometabolites of indigenous anaerobic microflora (bifidobacteria) promoted decrease of ALA of Salmonella, whereas opportunistic facultatively anaerobic microorganisms (enterobacteria, staphylococci) rendered mainly stimulating effect on the ALA of Salmonella. CONCLUSION: Obtained data reveal characteristics of bacterial interactions in associative symbiosis and provide insights about mechanisms of formation of pathobiocenosis and state of bacterial carriage.

[Microbial inhibitors of lysozyme].

Lysozyme is one of the most important factors of innate immunity, possessing anti-microbial action against a wide range of microorganisms due to cationic nature of protein and, in a lesser degree, due to muramidase activity. In the process of evolution, different mechanisms of lysozyme inhibition, defined as anti-lysozyme activity (ALA), were formed in microorganisms. The usage of the delayed antagonism principle allowed to reveal anti-lysozyme sign in microorganisms of different phylogenetic groups. In the review, data on the occurrence and level of anti-lysozyme activity in pathogens and representatives of normal microflora were presented; findings on nature and genetic determinants of lysozyme inhibitors are given. questions on drug regulation of ALA of the causative agents in infectious diseases and dysbiotic conditions are discussed.

[The role of anti-lactoferrin activity of bacteria in their persistence].

The role of anti-lactoferrin activity (ALfA) of bacteria in the persistence phenomenon, i.e. long survival of a causative agent in a host body, was studied in experimental staphylococcal infection. In experimental animals infected with isogenic clones of Staphylococcus aureus with different level of ALfA, longer terms were noted in bacterial recovery of staphylococci with this ability. Analysis on the occurrence and level of ALfA in clinical isolates of bacteria, isolated from different forms of infectious process, confirmed the significance of this sign in the persistence of a causative agent. High values of penetrance and expression of ALfA in bacteria were revealed in strains of microorganisms, isolated from chronic inflammatory diseases and from bacterial carriers.

[Algorithm for prediction of reconvalescent salmonellosis carriage].

Of total of 181 patients with Salmonella infection were examined by clinical and laboratory methods. As many as 124 indices characterizing biological properties of Salmonellae, a large intestine microbiocenosis status, systemic and local immunity, were investigated as well. Among these indices, the most revealing signs were defined to prognosticate the reconvalescent carrier state. Conclusion was made on the prevalence of immune mechanisms in the development of reconvalescent salmonella carriage and on the important role of the ability of a causative agent to inactivate the factors of host innate immunity in this process. Diagnostic prognosis algorithm for Salmonella bacterial carriage was worked out. Clinical testing confirmed its effectiveness.

[Influence of plasmid pR50 controlling bacterial protease activity on Salmonella experimental infection].

The comparative analysis of the infectious process and immunological parameters in (CBA x C57BL/6)F1 mice infected with S. typhimurium isogenic strains differing by the presence of plasmid pR50 determining protease activity, was carried out. A growth in the expression level of antilactoferrin, anticomplementary and anti-immunoglobulin activity in bacteria isolated from the spleen in the course of the infectious process was detected. In mice infected with S. typhimurium having plasmid pR50, in contrast to nonplasmid recipients, a higher level of contamination of organs, the suppression of spontaneous, stimulated production of interferon-gamma and the bactericidal properties of peritoneal macrophages were noted. The data obtained in this investigation suggested that the acquisition of R-plasmid of 50 MD, controlling protease activity and multiple medicinal resistance, contributed to the persistence of intracellular bacteria.

[Persistence factors of Francisella tularensis].

The study of the persistence potential of 64 F. tularensis strains isolated from different sources was carried out. The wide spread of the antilysozyme, antilactoferrin and anticomplementory activities of F. tularensis were detected. F. tularensis, isolated from ticks and water, were characterized by the highest level of the expression of antilysozyme activity, while anticomplementory and antilactoferrin activities of the infective agents were characteristic of those microorganisms which were isolated from rodents and their excrements.