The Inhibitory Activity of Lactobacillus plantarum Supernatant against Enterobacteria, Campylobacter, and Tumor Cells.

Cell-free supernatant of Lactobacillus plantarum exhibit a strong antimicrobial effect against a number of pathogenic enterobacteria (E. coli, Shigella flexneri, Salmonella typhimurium, Proteus mirabilis, and Campylobacter jejuni). The degree of growth inhibition in broth culture reached a high level for all tested bacteria. The highest rates were noted for P. mirabilis (by 13 times) and the lowest for S. flexneri (by 5 times) and C. jejuni (by 4.5 times). Significant antiproliferative effect of the supernatant on cells of tumor-derived epithelial cell lines was shown. The highest degree of inhibition (by 22 times) was observed for HT-29 cells (colon carcinoma). Thus, inclusion of probiotics in traditional treatment schemes can increase the effectiveness of antibacterial and antitumor drug therapy.

Molecular Genetic Characterization of Streptococcus pyogenes Strains Isolated from Patients with Various Manifestations of Streptococcal Infection.

In 82 clinical strains of Streptococcus pyogenes (group A streptococci) isolated from patients with various manifestations of streptococcal infection, emm-typing revealed 27 emm-types (n=77) with a predominance of emm-89 (n=15; 18%), emm-75 (n=9; 11%), and emm-1 (n=6; 7%); types emm-3, emm-12, and emm-58 (n=4; 5% each) were found with almost equal frequency; other types were less common. The superantigen genes speC, speG, speH, speI, speJ, speK, speL, speM, smeZ, and SSA were identified in S. pyogenes strains using multiprimer PCR; the genes of the superantigen SpeA and cysteine proteinase SpeB were detected using real-time PCR. All the studied S. pyogenes strains contained superantigen genes, and 98% of the strains had several (from 2 to 7) genes. The number of variants of these sets reached 37; 2% of the strains contained only one superantigen gene. The distribution frequencies of superantigen genes in the studied strains were: speA - 43%; speC - 38%; speG - 93%; speH - 13%; speI - 6%; speJ - 24%; speK - 13%; speL and speM - 11% each; smeZ - 98%; SSA - 15%. All studied S. pyogenes strains contained the speB gene. Our studies have demonstrated that the sets of superantigen genes of group A streptococci are characterized by pronounced diversity to some extent associated with emm-type.

Antibacterial Activity of Lactobacillus plantarum Supernatant on Non-Fermenting Gram-Negative Bacteria.

We studied the effect of the L. plantarum strain supernatant on the growth of culture and biofilm of non-fermenting bacteria of the genera Pseudomonas, Achromobacter, and Burkholderia. To obtain a supernatant, the culture of L. plantarum was grown for 48 h at 37°C on a Lactic broth nutrient medium with casein peptone, then centrifuged and filtered through a 0.22-µm Millipore filter. Antimicrobial activity was determined by broth microdilution assay. The inhibitory effect of the supernatant on the growth of bacteria of all three genera was demonstrated. The maximum inhibition was observed for P. aeruginosa (by 13 times compared to the control). For bacteria of the Achromobacter and Burkholderia genera, the inhibition was less pronounced: by 7 and 6 times, respectively. The supernatant also inhibited biofilm formation by P. aeruginosa and A. ruhlandii, but did not affect formed biofilm. Thus, the L. plantarum supernatant obtained by us exhibited pronounced antimicrobial activity against non-fermenting bacteria, the causative agents of nosocomial infections, especially in immunocompromised individuals, very often in cystic fibrosis patients.

Inhibitory Effect of Copper and Zinc Ions on the Growth of Streptococcus pyogenes and Escherichia coli Biofilms.

Analysis of the effect of copper and zinc ions on Streptococcus pyogenes and Escherichia coli biofilms revealed significant differences in the effect of these metals in the form of sulfates or chlorides on biofilm formation. Zinc ions in low doses (salt concentration 0.005 M) inhibited the growth of S. pyogenes biofilms by 1.5 times. After increasing salt concentration to 0.05-0.5 M, the growth of biofilm was reduced by 2.5 times in comparison with the positive control. In case of E. coli biofilms, the inhibition was more pronounced: zinc sulfate in a concentration of 0.005 M reduced its growth by 4.6 times in comparison with the positive control. After increasing salt concentration, the growth of E. coli biofilm decreased by 6.8 times. In case of zinc chloride, zinc ions produced weaker effect and reduced biofilm growth by 2.2 and 5 times, respectively. Copper salts in a concentration of 0.005 M had practically no effect on the growth of S. pyogenes biofilm; with increasing salt concentration, the degree of inhibition was close to the effect of zinc. In case of E. coli biofilm, we observed a slight inhibition of the growth by low doses of copper ions (by 1.4-1.3 times); with increasing salt concentration the effect increased by 5.6 and 2.2 times for copper sulfate and chloride, respectively. Copper and zinc cations had no effect on mature biofilm.

Morphology and Ultrastructure of Group A Streptococcus Biofilms.

Light and electron microscopy enables researchers to study the ultrastructure of GAS biofilms formed on abiotic surfaces. Chains of streptococci surrounded by a bluish film are seen under a light microscope after alcian blue staining of preparations grown on coverslips. The extracellular matrix (indicator of biofilm maturity) becomes visible on ultrathin sections in transmission electron microscopy after additional staining with alcian blue; filamentous structures, characteristic of biofilm, are observed in intercellular spaces. The data obtained by scanning electron microscopy also demonstrate the presence of biofilm.

Antimicrobial Activity of Supernatant of Lactobacillus plantarum against Pathogenic Microorganisms.

We studied ntimicrobial activity of L. plantarum strain against different pathogens: Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes, Staphylococcus aureus. It was shown that supernatant of 48-h L. plantarum culture in liquid nutrient medium exhibits inhibitory activity against gram-positive and gram-negative pathogenic microorganisms. Supernatant of 24-h culture exhibited lower activity, while supernatant of 72-h culture produced no inhibitory effect. Boiling and proteinase K treatment did not affect activity of the preparation, i.e. antimicrobial activity of the supernatant was not associated with protein or peptide component. These data were confirmed by the results observed after ultrafiltration of the preparation: the growth of E. coli, P. aeruginosa, and S. aureus was inhibited by the low-molecular-weight fraction, but not high-molecular-weight fraction of the supernatant. On the other hand, the high-molecular-weight fraction suppressed the growth of streptococcus by 3 times. We hypothesized that L. plantarum supernatant obtained in our experiments contained at least two antimicrobial components with different molecular weights.

Optical and Electron Microscopic Study of the Morphology and Ultrastructure of Biofilms Formed by Streptococcus pyogenes.

Our study confirmed the capacity of S. pyogenes strains to form biofilms on abiotic surfaces. Chains of streptococci surrounded by bluish film were seen under a microscope after alcian blue staining of the preparations grown on slides. On ultrathin sections in transmission electron microscope, the extracellular matrix (indicator of biofilm maturity) became visible after staining with alcian blue. Microscopy of the sections shows structures characteristic of a biofilm in spaces between the cells. Scanning electron microscopy also demonstrates the presence of a biomembrane. Importantly that type 1M strain forming in fact no membranes when cultured on plastic plates (Costar) formed biofilms on the glass. It seems that the conditions for the biofilm formation on the plastic and on the glass differ, due to which the exopolymeric matrices formed on different surfaces vary by biochemical composition.

Effects of Miramistin and Phosprenil on Microbial Biofilms.

Effects of Miramistin and Phosprenil on biofilms of S. pyogenes, S. aureus, E. coli, L. acidophilus, and L. plantarum were studied. Significant differences in the effects of these substances on mature biofilms of microorganisms and the process of their formation were observed. Miramistin had significant inhibiting effects on the forming of biofilms and on the formed biofilms of all studied microorganisms. Treatment with Miramistin inhibited biofilm formation by 2-3 times compared to the control. This effect was found already after using of Miramistin in the low doses (3.12 µg/ml). Inhibition of the growth of a formed biofilm was observed only after treatment with Miramistin in the high doses (25-50 µg/ml). Phosprenil in the high doses (15-30 mg/ml) inhibited the forming of biofilms, especially the biofilms of S. pyogenes and L. plantarum (by 3-4.5 times). Treatment of formed biofilms with the agent in doses of 6.0 and 0.6 mg/ml was associated with pronounced stimulation of its growth in S. pyogenes, S. aureus, and L. acidophilus.

Interleukin-6 in Sera of Mice Inoculated with Group A Streptococcus and Cultural Supernatants.

Dynamics of IL-6 level was studied in blood serum of CBA mice receiving intraperitoneal injections of killed and live culture of group A Streptococcus and its supernatants. It was found that administration of killed culture was followed by a significant rise in IL-6 level (by 4.7 times in 1 h and by 9.3 times in 5 h in comparison with the control). By 24 h, cytokine content was below the control. The highest levels of IL-6 were found after treatment with supernatants of Streptococcus cultures (by 10.5 times in 1 h and by 14.9 times in 5 h, in comparison with the control). Administration of live culture was accompanied by an increase in IL-6 concentration by 3.2 times in 3 h. In this experimental series, the maximum level of IL-6 was found in 48 h (by 5.2 times), and then it gradually decreased below the control. Different dynamics of changes in IL-6 level after administration of killed and live cultures of group A Streptococcus may suggests that they activate different signal pathways.

Changed Serum Cytokine Profile in Mice in Response to Streptococcus A Culture.

Comparative analysis of serum cytokine profiles of CBA mice was carried out 1, 5, 24, and 48 h after intraperitoneal injection of killed culture of different streptococcus A types. The production of cytokines in response to different streptococcus types varied. The highest level was recorded in response to types 1M and 3T+M, more often detected in invasive streptococcal infection. The highest levels of IL-2 were recorded in response to 1M (47-fold increase in comparison with the control) and 3T+M streptococcus types (more than 10-fold increase). Injections of these types also led to an increase of IFN-γ level (15.6 and 11.3 times, respectively). The level of TNF-α increased less (3.6 times in response to 3T+M and 2.6 times in response to 1M type). The levels of IL-5, IL-10, and IL-12 increased 2-3-fold. Injections of 1T and 5M types led to just a 2-fold increase of cytokine levels. These data indicated induction of the immune response trend by mainly Th1 or mixed Th1/Th2 pattern in response to group A streptococcus antigens.

[Biofilm formation by group A Streptococci of various types and study of antibiotics effect on this process].

AIM: Evaluation of the ability to form biofilms by various M, T and MT-types of group A streptococci (GAS), as well as study of the effect of various antibiotics on biofilm formation. MATERIALS AND METHOD: 43 strains of various M and T type GAS were studied. The cultures were grown in Todd-Hewitt broth with the addition of 0.5% yeast extract. Comparative evaluation of the ability to form biofilm was carried out using photometry. Benzylpenicillin, oxacillin, cepha- losporin, cefuroxime and ceftriaxone antibiotics were used at various concentrations. RESULTS: GAS differ significantly by their ability to form biofilms. The highest ability was noted in 8 strains--2M, 9M, 12M, 13M, 19M, 30M, 36M-types and 6MT type. Simultaneous introduction of GAS cultures and antibiotics into the culture well, except for ceftriaxone, resulted in growth inhibition of both plankton cells and biofilms. CONCLUSION: The ability of GAS to form biofilm depends on streptococci serotype. During simultaneous introduction of GAS with antibiotics into the well, the biofilm does not form.

[Diagnostic effectiveness of nutrient media for the bacteriological detection of enterobacteria].

The effectiveness of new nutrient media intended for the differential isolation of pathogenic and opportunistic enterobacteria (SS agar), as well as for the preliminary identification of enterobacteria, was determined on a wide range the strains of the family Enterobacteriaceae. The comparative evaluation of SS agar and Ploskirev's bactoagar was carried out. The newly developed medium was shown to have advantage in its differentiating and inhibiting properties. The clinical trials of the media, carried out in this study, revealed high detection rate and exceedingly exact differentiation of lactose-negative lactose-positive enterobacteria, as well as 100% coincidence of the results obtained on the nutrient medium for the preliminary identification of enterobacteria and on laboratory-made control media.

[A dried nutrient medium for the isolation of Vibrio cholerae]. / Sukhaia pitatel'naia sreda dlia vydeleniia Vibrio cholerae.

A dried differential nutrient medium for the isolation of V. cholerae has been developed. The medium is sufficiently sensitive, has pronounced differentiating properties and greatly inhibits the appearance of microbial associations. During the cultivation of V. cholerae with the use of this medium the cultural, morphological and agglutination properties of the initial strains are retained.

[Effect of R plasmids on the growth characteristics of Shigella sonnei and Escherichia coli]. / Izuchenie vliianiia R-plazmid na rostovye kharakteristiki Shigella sonnei i Escherichia coli.

Revealing of growth characteristics in plasmid and plasmid-free strains was studied with the use of two different hosts: E. coli 15-3 and Sh. sonnei 11-941 containing conjugative R plasmids differing in the set of the resistance markers. It was shown that the R plasmids had no noticeable effect on the period of the lag phase and the time of the microbial cell generation. It was also shown that the number of the viable cells in separate cultures of the plasmid-free strain of Sh. sonnei 11-941 and its plasmid variants was of the same order. Counting of the viable cells in mixed cultures of the plasmid-free strain of E. coli 15-3 and its plasmid variant on the complete nutrient medium revealed an insignificant increase (by 10 per cent) in the proportion of the bacteria carrying the plasmid after 6-hour growth (during early stationary growth phase) and later, up to 24 hours. The results of the study suggested that development of nutrient media for microbial strains containing R plasmids does not require additional cultivation conditions.