Strain specific motility patterns and surface adhesion of virulent and probiotic Escherichia coli.

Bacterial motility provides the ability for bacterial dissemination and surface exploration, apart from a choice between surface colonisation and further motion. In this study, we characterised the movement trajectories of pathogenic and probiotic Escherichia coli strains (ATCC43890 and M17, respectively) at the landing stage (i.e., leaving the bulk and approaching the surface) and its correlation with adhesion patterns and efficiency. A poorly motile strain JM109 was used as a control. Using specially designed and manufactured microfluidic chambers, we found that the motion behaviour near surfaces drastically varied between the strains, correlating with adhesion patterns. We consider two bacterial strategies for effective surface colonisation: horizontal and vertical, based on the obtained results. The horizontal strategy demonstrated by the M17 strain is characterised by collective directed movements within the horizontal layer during a relatively long period and non-uniform adhesion patterns, suggesting co-dependence of bacteria in the course of adhesion. The vertical strategy demonstrated by the pathogenic ATCC43890 strain implies the individual movement of bacteria mainly in the vertical direction, a faster transition from bulk to near-surface swimming, and independent bacterial behaviour during adhesion, providing a uniform distribution over the surface.

Effect of Non-Thermal Plasma on Proliferative Activity and Adhesion of Multipotent Stromal Cells to Scaffolds Developed for Tissue-Engineered Constructs.

We studied the effect of non-thermal argon plasma on proliferative activity of bone marrow multipotent stromal cells in vitro. Treatment of stromal cell suspension with pure argon did not affect their proliferation. The cells treated with non-thermal argon plasma and explanted in the treatment medium demonstrated growth inhibition by 30-40% in comparison with the control. Multipotent stromal cells treated with plasma and after centrifugation explanted in normal medium within 12 min demonstrated accelerated growth. The total cell growth from the pellet and supernatant significantly exceeded the control values. We also analyzed adhesion and proliferative activity of multipotent stromal cells treated with non-thermal plasma on bioresorbable carriers. The cells adhered and proliferated on all types of studied samples. Adhesion properties of scaffolds differed. Caprolactone was found to be the most suitable material for adhesion and proliferation of multipotent stromal cells.

[The role of technogenic and biological factors in spread of foodborne bacterial infections].

Epidemiologic aspects of ecology of foodborne infection causative agents that have taken root in contemporary technogenic loci created by humans in urbocenoses which include agrocomplexes of animal breeding, vegetable growing in open and closed ground are discussed. Soil and water sources with a wide transport network where microorganisms dwell in the environment under favorable for vital functions and growth conditions (humus deposits, temperature regimen, optimal pH, associations with hydrobionts, biofilm formation) are potent secondary reservoirs of infection causative agents. Some molecular-genetic mechanisms of polyhostality of Gram negative bacteria and listeria are examined.

Nonthermal plasma affects viability and morphology of Mycoplasma hominis and Acholeplasma laidlawii.

AIM: To study the effects exerted by argon microwave nonthermal plasma (NTP) on cell wall-lacking Mollicutes bacteria. METHODS AND RESULTS: 10(8) CFU ml(-1) agar plated Mycoplasma hominis and Acholeplasma laidlawii were treated with the nonthermal microwave argon plasma for 30-300 s. The maximal 10- and 100-fold drop was observed for A. laidlawii and Myc. hominis, respectively. Similarly treated Escherichia coli and Staphylococcus aureus demonstrated the 10(5) and 10(3) drop, respectively. Removal of cholesterol affected resistance of A. laidlawii. 10 mmol l(-1) antioxidant butylated hydroxytoluene decreased mortality by a factor of 25-200. UV radiation alone caused 25-85% mortality in comparison with the whole NTP. Exogenously added hydrogen peroxide H2O2 did not cause mortality. NTP treatment of Myc. hominis triggered growth of microcolonies, which were several tenfold smaller than a typical colony. CONCLUSIONS: Despite the lack of cell wall, A. laidlawii and Myc. hominis were more resistant to argon microwave NTP than other tested bacteria. Mycoplasma hominis formed microcolonies upon NTP treatment. A role of UV and active species was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The first study of NTP effects on Mollicutes revealed importance of a membrane composition for bacterial resistance to NTP. New specific Myc. hominis morphological forms were observed. The study confirmed importance of the concerted action of reactive oxygen species (ROS) with UV and other plasma bioactive agents for NTP bactericidal action.

[Study of possible involvement of MEK mitogen-activated protein kinase and TGF-ß receptor in planarian regeneration processes using pharmacological inhibition analysis].

Possible involvement of MEK mitogen-activated protein kinase and TGF-ß receptor in the processes of regeneration and morphogenesis in freshwater planarian flatworms Schmidtea mediterranea was studied using a pharmacological inhibitor analysis. It was found that pharmacological inhibitors of these kinases significantly inhibit the regeneration of the head end of the animals and that this effect is realized due to inhibition of proliferative activity of neoblasts, planarian stem cells. It is shown that that the inhibition of the studied protein kinases in regenerating planarians markedly disturbs stem cell differentiation and morphogenesis.

[Effect of low temperature plasma on various mycoplasma species].

AIM: Study the influence of low temperature (cold) electrolyte plasma (CEP) on survivability of some mycoplasma strains growing in agar as well as mycoplasma that most frequently contaminate transplantable human cell lines of normal and malignant origin with the aim of decontamination. MATERIALS AND METHODS: Mycoplasma hominis, Mycoplasma arginini and Aholeplasma laidlawii grown in agar and mycoplasma that contaminated transplantable human cell lines of normal (MT4) and malignant (HeLa) origin. Plasma source--Plasmatom device that generates CEP at normal atmosphere pressure and environment temperature. Exposure to plasma was carried out with adherence to the same modes for all the variants of biological substrate. The duration of exposure was selected randomly from 15 to 300 seconds. RESULTS: A pronounced bactericidal effect of high doses of CEP on all the tested mycoplasma variants exposed immediately after seeding into agar was shown. However after a passage a residual number of survived colonies was registered. Passage of colonies exposed in grown state even to high doses of CEP also showed survival of a residual number of bacteria in all the tested mycoplasma species. Exposure of M. hominis immediately after seeding to low doses of CEP resulted in formation of unusual mini-colonies identical to those isolated from humans infected by the same mycoplasma. During microbiological seeding into agar of cultural fluid from 2 spontaneously contaminated strains of transplantable human cells and exposed to CEP growth ofmycoplasma was not detected. CONCLUSION: CEP has pronounced bactericidal properties on various mycoplasma strains growing in both agar and contaminating eukaryotic cells. However even at high doses of exposure to CEP an insignificant part of bacterial cells growing in agar still survives. This may indicate a high degree of heterogeneity and adaptation of mycoplasma subjected to even such hard exposure as cold plasma with plasma-chemical mechanism of destruction of biological substrate.

[Bactericidal action of non-thermal plasma on biofilms formed in vitro and within a root channel].

AIM: Evaluation of efficiency of non-thermal plasma as bactericidal agent affecting biofilms formed in vitro and on walls of a root channel. MATERIALS AND METHODS: The multiple antibiotic resistant strain Staphylococcus epidermidis isolated from pulpitis was used. Biofilms formed in vitro on the plastic surface and ex vivo at the walls of the root canal were treated with plasma torch formed by argon:air (9:1) mixture eradiated with 100 kHz electrtomagnetic field. Bacterial viability was determined by plating and by differential Live/Dead labeling. RESULTS: The dose-dependent decrease in living bacteria was demonstrated. The three-step kinetics ofbacterial killing was observed. Total elimination ofup 10(9) CFU/sample was reached at exposition of 240 s or more. CONCLUSION: The non-thermal plasma effectively destroyed bacterial biofilms within root channels.

Non-thermal Plasma Causes p53-Dependent Apoptosis in Human Colon Carcinoma Cells.

Non-thermal plasma (NTP) consists of a huge amount of biologically active particles, whereas its temperature is close to ambient. This combination allows one to use NTP as a perspective tool for solving different biomedical tasks, including antitumor therapy. The treatment of tumor cells with NTP caused dose-dependent effects, such as growth arrest and apoptosis. However, while the outcome of NTP treatment has been established, the molecular mechanisms of the interaction between NTP and eukaryotic cells have not been thoroughly studied thus far. In this work, the mechanisms and the type of death of human colon carcinoma HCT 116 cells upon application of non-thermal argon plasma were studied. The effect of NTP on the major stress-activated protein p53 was investigated. The results demonstrate that the viability of HCT116 cells upon plasma treatment is dependent on the functional p53 protein. NTP treatment caused an increase in the intracellular concentration of p53 and the induction of the p53-controlled regulon. The p53-dependent accumulation of active proapoptotic caspase-3 was shown in NTP-treated cells. The study was the first to demonstrate that treatment of human colon carcinoma cells with NTP results in p53-dependent apoptosis. The results obtained contribute to our understanding of the applicability of NTP in antitumor therapy.

[Contribution of L,D-carboxypeptidases in virulence of facultative intracellular pathogenic bacteria Listeria monocytogenes].

AIM: Evaluate influence of mutation of Listeria monocytogenes genes coding murein-tetrapeptide L,D-carboxypeptidase Lmo0028 and Lmo1638 on dynamics of infectious process and interaction of purified muropeptides with NOD1 receptor. MATERIALS AND METHODS: Wild type EGDe strain and recombinant strains GIMins1638 H GIMins0028 obtained on its basis by site-specific mutagenesis were used. Infectious process dynamics was studied on the model of intravenous infection of BALB/c mice. Ligand-receptor interaction activity of muropeptides isolated from recombinant and parent strains were assayed on HEK293-hNOD1 cell line expressing NOD1 receptor and containing in their genome beta-galactosidase reporter gene under the control of NF-kappaB dependent promoter expression. RESULTS: Lack of Lmo0028 decelerates reproduction of listerias in animal liver starting from 24 hours and at later terms after the infection whereas lack of Lmo1638 leads to increase of microbial load 6 and 24 hours after the infection with no influence on further infection. Differences in activation of NOD1 receptor by muropeptides isolated from recombinant and parent strains were not detected. CONCLUSION: Despite high homology murein-tetrapeptide L,D-carboxypeptidase Lmo0028 and Lmo1638 make a different contribution to the development of infectious process caused by L. monocytogenes in BALB/c line mice. Lack of differences in NOD1 receptor activation may be associated with compensation of enzymatic functions in strains with mutation in each of the genes owing to the presence of homologous protein.

[New approaches to therapy of persistent infections: elimination of intracellular Chlamydai trachomatis by exposure to low temperature argon plasma].

AIM: Study microbicidal activity of low temperature argon plasma (LTP) that is a stream of partially ionized argon having macroscopic temperature of the environment against Chlamydia trachomatis obligate intracellular parasites. Study viability of host cells in parallel. MATERIALS AND METHODS: McCoy line cells infected with C. trachomatis (Bu-434/L2 strain) were exposed to LTP obtained by using atmospheric pressure plasma SHF generator. Intracellular localization of chlamydiae was visualized by luminescent microscopy. RESULTS: Exposure of infected McCoy line cells resulted in the destruction of chlamydia inclusions and practically complete elimination of intracellular bacteria. At the same time LTP exposure did not result in immediate death of host cells, an insignificant reduction of the number of cells was observed 24 hours after the exposure to LTP. CONCLUSION: The effect of LTP for elimination of intracellular chlamydia without significant changes in viability of eukaryotic host cells was demonstrated.

[Prospects for the use of low-temperature gas plasma as an antimicrobial agent].

Results of application of LTP at atmospheric pressure as an antibacterial agent during the last decade are considered with reference to physicochemical mechanisms of its bactericidal action. The principles of designing modern LTP sources are described in conjunction with the results of LTP application against pathogenic bacteria in vitro and in biofilms. The possibility to destroy biofilm matrix by LTP is estimated along with the results of its testing for the treatment of acute and chronic wound surfaces. Prospects for the development of "plasma medicine" in this country and abroad are discussed with special emphasis on its advantages, such as the absence of long-acting toxic compounds, small probability of spontaneous mutations accounting for resistance to LTP, relatively low cost of LTP sources, independence of LTP effect of the surface relief, painless application.

Effect of Sodium Chloride on Aggregation of Merocyanine 540 and Photosensitized Inactivation of Staphylococcus aureus and Pseudomonas aeruginosa.

Merocyanine 540 (MC540) is used as a photosensitizer for the inactivation of microorganisms. The following is already known about MC540: firstly, MC540 exists in distilled water in both monomeric and dimeric forms, and the addition of salts into a MC540 solution leads to the formation of large aggregates that can be detected by the resonance light scattering technique. Secondly, singlet oxygen can only be photogenerated by MC540 monomers. In the present work, we studied the effect of MC540 in the aggregated state on the rate of photosensitized inactivation ofStaphylococcus aureusandPseudomonas aeruginosa. To this end, bacteria either in MC540-containing distilled water or in a 0.25 M sodium chloride aqueous solution also containing MC540 are irradiated (546 nm). The results show that, in the presence of salt, the aggregation of MC540 greatly increases the efficiency of the MC540-photosensitized inactivation ofP. aeruginosaandS. aureus. In the presence of salt, the rates ofP. aeruginosaandS. aureusinactivation increase by factors of 10 and 30, respectively, in comparison with the rate of inactivation observed in the case of distilled water. Our results suggest that a salt-induced photosensitization mechanism can switch from the singlet oxygen to the free-radical pathway.

[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.

[Variability of the INV gene fragment encoding a functionally important domain of Yersinia pseudotuberculosis invasin].

A total of 84 Y. pseudotuberculosis isolates were studied. The isolates were obtained in Russian Federation in 1967-2008. The majority of Y. pseudotuberculosis isolates (n = 55) were of clinical origin and were isolated from feces of patients with the clinically and serologically proved diagnosis of pseudotuberculosis/Far East scarlet-like fever. These isolates included 18 isolates obtained from 3 outbreaks. Nine isolates were isolated from the internal organs of wild rodents. Other isolates were obtained from environmental sources. Ten Y. pseudotuberculosis isolates belonged to the serovar III and the other isolates belonged to the serovar I. The sequences of 600 b.p. fragment of the inv gene that encodes 667 through 866 invasin amino acids were determined for all isolates. Totally, 3 allelic variants were found. The most abundant allele 1 was found in 76 isolates. The allele 1 is represented in the database Genbank by the strain IP31758 isolated in the Far East of Russia (Eppinger et al., 2007). The allele 2 differed from allele 1 in 3 positions: G,2299N, O2300N, and O2302N. Substitutions in positions 2299 and 2302 were non-synonymous and resulted in amino acid substitutions Ser768 Thr and Val769 Ala. Six isolates carried allele 2. Allele 3 was found in two isolates different from allele 2 by a synonymous substitution G2324O. This allele is similar to the sequence found in Y. pestis strains, represented in the GenBank. The allelic distribution was not serovar specific: Y. pseudotuberculosis of serovar III and majority of serovar I isolates carried allele 1. The analysis of the allelic distribution among subpopulations formed on the base of a source of isolation revealed a statistically significant difference in spreading of alleles among clinical and wild rodent isolates (p < 0.05). Allele 1 prevailed over clinical isolates (95%), while allele 1 and allele 2 were disseminated equally among rodent isolates (55 % and 45 %, respectively).

[Influence of mutation L,D-carboxypeptidase-coding gene on dynamics of Listeria infection].

AIM: To study the progress of infection caused by mutant Listeria monocytogenes with deletion of Imo0028 gene coding L,D-carboxypeptidase (protein involved in metabolism of peptidoglycan). MATERIALS AND METHODS: Deletion of Imo0028 gene was performed with site-specific mutagenesis method using wild-type strain EGDe. Virulence was studied on mice of BALB/c line. RESULTS: Strain GIMd0028 with deletion of Imo0028 gene did not differ from parent strain on in vitro growth rate, cytotoxicity and production of listeriolysin O and phospholipase PlcA pathogenicity factors. Effective LD50 for wild-type strain EGDe was 1x10(4) CFU/ mouse and 2x10(5) CFU/mouse for intravenous and intraperitoneal inoculation respectively. Deaths of animals were observed after 3 - 7 days. LD50 for mutant strain GIMd0028 was 1x10(5) CFU/mouse for intravenous inoculation. It was not possible to determine LD50 for intraperitoneal inoculation, and infection progressed atypically slowly. For example, deaths of animals were observed during 22 days (time of experiment) starting from day 4 when strain GIMd0028 in dose 10(6) CFU/mouse was used for inoculation. In average, death of 1 - 2 mice out of 75 inoculated was observed. Presence of L. monocytogenes was confirmed by its isolation from liver and brain of dead mice. CONCLUSION: Disruption of function of Imo0028 gene coding L,D-carboxypeptidase protein, which takes part in metabolism of peptidoglycan, changes dynamics of listeriosis.

[Identification of proteins determining the virulence of Listeria monocytogenes using the bioinformation analysis].

The postgenomic stage of biotechnology allows the bioinformation approaches to be used for revealing previously unknown factors involved in different processes. In this work, bioinformation approaches were applied to analysis of factors involved in the L. monocytogenes virulence. Several open reading frames (ORFs) were identified in L. monocytogenes genome, which encode proteins with high level homology to bacterial peptidases and which meets the developed criteria. The influence of the ORFs on virulence was demonstrated by infection of mice with L. monocytogenes strains mutated in the studied genes. Growth rates, morphology, and production of surface proteins characterized the obtained strains.

[Differentiantion of Listeria monocytogenes strains isolated in the Far East and European part of Russia on the basis of polymorphism of genes encoding invasion factors].

Forty Listeria monocytogenes isolates obtained in European and Far East regions of Russia were differentiated on the basis of polymorphism of 5 markergenes. Total length of concatemers obtained after sequencing of internal fragments of genes inlA, inlB, inlC, inlE and prs was 3029 b.p. Comparative analysis of concatemers' sequences revealed 237 variable nucleotides. Totally, 25 sequence types were revealed, and isolates from European and Far East regions belonged to different types. On the dendrogram isolates split on 2 clusters, which correspond to early described phylogenetic lines of L. monocytogenes specie. Isolates obtained in European and Far East regions formed independent subclusters within main clusters. Fifteen clinical isolates of L. monocytogenes belonged to 7 different types. Analysis of epidemiologic data on time and place of isolates obtaining suggested that isolates of the same sequence type are epidemiologically related and might represent one strain; index of discrimination for proposed typing method was calculated as 0.982.

[Genes, encoding invasion factors in Listeria monocytogenes strains isolated in the European part and the far east of Russia].

The collection of 76 Listeria monocytogenes strains isolated from humans, animals and food products was screened with PCR to reveal genes, which encode invasion factors of the internalin family. Obtained results demonstrated the correlation between the strain specific polymorphism of the revealed internalin genes and the source of the strain.

[Effect of the constitutive activity of pathogenicity genes in Listeria monocytogenes].

The effect of the constitutive expression of pathogenicity factors in L. monocytogenes was studied on the model of the intraperitoneal infection of mice. The constitutive expression was due to a single amino acid substitution in the transcriptional factor PfrA, the master regulator of L. monocytogenes virulence genes. The effective lethal dose (LD50) for the strain with the constitutive expression of pathogenicity factors was 3 times lower than for the isogenic wildtype strain. When introduced in equal doses, this Listeria strain led to the death of the animals 1-2 days earlier than the wild-type stain. The study revealed that the constitutive expression of pathogenicity factors resulted in faster dissemination of bacteria in the body of the animal during the first 24 hours, but led to their earlier elimination from the internal organs at later stages of infection.

[A new complex approaches for the identification of Listeria isolated during production of fermented sausages].

Distribution L. monocytogenes and other Listeria spp. in raw meat and during manufacturing of fermented meat products is investigated. The high contamination of raw materials and semi finished foods--in 36.5% of samples, ready-to-eat sausages--31.8% by Listeria spp. is established. Detection L. monocytogenes in 9.7% cases from the surfaces of equipment indicates the intensive circulation of listeriosis agents on meat plants. For identification 49 isolated strains the approach providing application of most informative for L. monocytogenes phenotypical tests (mobility, ability to haemolysis, presence of specific lecithinase) and assay based on PCR of DNA sites, coding phospholipase, factors of invasion and citotoxicity is used. Efficiency of this circuit is confirmed with positive results PCR with species-specific for L. monocytogenes primers to genes PIcA and ActA only at strains, having lecithinase and haemolysis activity. Application of the given approach has allowed to reduce in three times number of the cultures initially identified as L. monocytogenes on a complex of biochemical tests.