Corynebacterium spp.: relationship of pathogenic properties and antimicrobial resistance.

Corynebacterium spp. are part of the human microbiome, but can cause the development of inflammatory diseases of various localization. Purpose - to evaluate the relationship between pathogenic properties and resistance to antimicrobial drugs (AMD) of Corynebacterium spp. from patients with inflammatory diseases of the respiratory tract. Strains of Corynebacterium spp. isolated from patients with inflammatory diseases of the respiratory tract (99 pcs.) and practically healthy individuals (33 pcs.). Isolates were identified by mass spectrometric method (MALDI-ToFMS), their adhesive and invasive activity on Hep-2 cells, cytopathic effect (CPE) in CHO-K1 cell culture, and resistance to antimicrobial drugs (AMD) were determined. Indicators of adhesion (3.65±0.679(CFU±m)x102/ml), invasion (1.72±0.230 (CFU±m)x102/ml), cytotoxicity (69.1±3.8% of dead CHO-K1 cells ) Corynebasterium spp. strains isolated from patients are higher (p≤0.05) than similar indicators in practically healthy people. 90.9% of isolates from patients had resistance to AMD, in most cases (57.6±4.9%) resistance to only one AMP was noted, less often to two (25.2±4.3%), three or more (8.08±2.7%). According to the results of correlation-regression analysis, pathogenic properties (adhesiveness, invasiveness, cytotoxicity) of Corynebacterium spp. strains isolated from patients are in close direct relationship with resistance to AMD. This indicates the importance of identifying strains of non-diphtheria corynebacteria resistant to AMDs, which, under the influence of developing resistance to AMDs, can increase their pathogenic potential, moving from commensalism to parasitism.

[Cypopathic effect of diphtheria pathogen in the composition of biofilm.]

When the nasopharynx is colonized with toxigenic strains of the diphtheria pathogen, toxin is released, which contributes to the death of epithelial cells. But in bacterial carriers, the development of the clinical picture of the disease does not occur. This is due to the peculiarities of the state of their immune system, as well as the peculiarities of the production of diphtheria exotoxin by corynebacteria in the biofilm. Goal. Determining the nature of the cytopathic effect of C. diphtheriae as part of a biofilm in CHO-K1 cell culture. The planktonic and biofilm (120- and 720-hour) cultures of the strains were studied: C. diphtheriae gravis tox+ № 665, C. diphtheriae gravis tox+ № 6765, C. diphtheriae mitis tox+ № 269, C. diphtheriae gravis tox+ isolated from a patient with a diagnosis Localized oropharyngeal diphtheria C. diphtheriae gravis with a silent tox-gene. Biofilm (120- and 720-hour) cultures of diphtheria pathogen strains were obtained according to the Watnik method. The cytopathic effect of corynebacterial strains was studied on a CHO-K1 cell culture, taking into account in an inverted microscope. When studying the cytopathic effect of planktonic cultures of toxigenic strains of corynebacteria, it was found that the number of living CHO-K1 cells after 24 hours was insignificant (25.3±1.2%) and sharply decreased (2.5±0.5%) after 72 hours of cultivation. Under the influence of biofilm and, especially, 720-hour cultures, a different cytopathic effect dynamics was found: the number of living cells after 24 hours remained significant (82.5±2.2%), while at 72-hour it decreased to 25.0±3.0%. In the study of filtrates of planktonic and biofilm cultures of C. diphtheriae strain with a «silent¼ tox-gene, similar patterns were revealed. However, the number of live CHO-K1 cells when exposed to the filtrate of a 720-hour biofilm culture was significantly higher (p≤0.05) than when studying toxigenic strains of corynebacteria. Considering the nature of the cytopathic action, it was found that planktonic cultures of toxigenic strains of corynebacteria are characterized by a change in the cell monolayer, manifested by their thinning and elongation. The study of 720-hour biofilm cultures at 72-hour exposure revealed the appearance of a large number of rounded cells (63-69%). The cytopathic effect, formed under the influence of filtrates of planktonic and biofilm cultures of C. diphtheriae with a «silent¼ tox-gene, as well as strains of non-diphtheria corynebacteria, is characterized by rounding of cells and the formation of symplasts. In the biofilm, the intensity of the cytopathic effect of toxigenic C. diphtheriae strains and C. diphtheriae strain with a silent tox-gene decreased. CPD, manifested by thinning and lengthening of CHO-K1 cells, is associated with the action of diphtheria exotoxin, and rounding is associated with corynebacterial enzymes and, apparently, fragments of surface structures - adhesins. Decreased release of toxin and enzymes beyond the C. bihfilm matrix is a significant cause of the «asymptomatic¼ carriage of diphtheria.

[THE ROLE OF QUORUM-SENSING IN REGULATION OF FORMATION OF BIO- FILMS BY VIBRIO CHOLERAE].

Materials regarding quorum-sensing that is the main regulator of inter-cellular communications in V cholerae are presented. Information transmission between separate vibrios is executed via autoinductors. Their interaction with regulatory proteins facilitates gene activation that take part in formation of biofilms of Vcholerae which ensures their survival and spread.

[ROLE OF BIOFILMS IN SURVIVAL AND PRESERVATION OF VIRULENCE OF CHOLERA VIBRIOS IN THE ENVIRONMENT AND HUMAN ORGANISM].

Materials regarding.biofilms of cholera vibrios are presented. Formation of biofilms is shown to be a significant pathogenicity factor and one of the main strategies, increasing survival of cholera vibrios in human organism and the environment.

[The immune-enzyme techniques of analysis in diagnostic of cholera.]

The optimal conditions of arrangement of direct alternative of flatbed enzyme-linked immunosorbent assay and dot-immunoanalysis with application of monoclonal peroxidase conjugates for express identification of comma bacillus of serogroups O1 and O139 both in hospital and field conditions without device support. The direct technique enzyme-linked immunosorbent assay in flatbed alternative shortens time of analysis up to 70-80 minutes and in case of dot enzyme-linked immunosorbent assay on membrane - up to 70-90 minutes. It is established that in case of analysis in conditions of room temperature (20-25 oC) sensitivity of techniques remains at initial level.

[Effect of plant extracts on cytotoxic activity of Vibrio cholerae hemolysin].

AIM: Study of plant extracts that have the ability to neutralize cytotoxic activity of hemolysin. MATERIALS AND METHODS: Preparations of purified and recombinant V. cholerae eltor hemolysin as well as supernatants of V. cholerae strains were used. Determination ofcytotoxic activity of hemolysin and neutralizing activity of plant extracts were carried out by using cell cultures CHO-K1 and CaCo2. RESULTS: Out of 9 water extracts only 3 - extracts of Rhei rhizome, Limonium gmelinii and Quercus robur neutralized hemolysin in cell culture CHO-K1 and CaCo2, whereas the other extracts--Humulus lupulus, Ocimum basilicum, Chelidonium majus, Juglans regia, Achillea milefolium and Hypericum perforatum did not have anti-cytotoxic effect. Neutralizing properties of extracts are exhibited during their co-incubation with hemolysin preparations and supernatants of V. cholerae strains already within 10 minutes. CONCLUSION: Plant extracts that have anti-cytotoxic activity against hemolysin are perspective for development oftherapeutic-prophylaxis preparations.

[The application of monoclonal peroxidase conjugates to identify comma bacillus of serum groups 01 and 0139 in the reaction of dot-immune analysis].

The source of monoclonal antibodies was chosen the cultural fluid of hybridoma-producers deposited in the specialized collection of cell cultures of vertebrates (St. Petersburg) with numbers RKKK(P) 386D and RKKK(P) 674D. The specific immunoglobulin (Ig) from cultural fluid was concentrated by precipitation with saturated solution of ammonium sulfate. The scheme of obtaining monoclonal antibodies included activation of peroxidase, conjugation of activated peroxidase with Ig, removal of unbounded proteins, storage and control. The preservation of activity of conjugates was supported with BSA (10%) or glycerin (50%). The last on is preferable to be applied for this purpose. The test of monoclonal antibody-01 and monoclonal antibody-0139 of peroxidase conjugates with kit of strains of comma bacillus 01 and 0139 demonstrated their strict specificity because they interacted only with corresponding serum groups under absence of crossed reactions with representatives of geterologic microorganisms. The direct dot-immune analysis is carried out during 1.5 hour and its sensitivity is within the limits 105-106. The application of diagnostic monoclonal peroxidase conjugates 01, 0139 in laboratory practice can promote the increase of specificity of serologic analysis of cholera and saving time-frame of its application.

[Influence of plant extracts on the activity of cholera toxin of Vibrio cholerae].

AIM: Study the activity of plant extracts against cholera toxin (CT) of Vibrio cholerae O1. MATERIALS AND METHODS: Antitoxic activity of plant extracts was determined by using enzyme immunoassay and CHO-K1 cell culture. RESULTS: 8 water extracts of plants were studied. Extracts of nut, tutsan, milfoil, basil do not have effect on CT activity in EIA or CHO-K1 cell culture. Celandine and rhubarb extracts do not reduce CT immunochemical activity but prevent elongation of CHO-K1 cells. Oak and hop extracts suppress binding in EIA of cholera toxin and GM1 receptors and insignificantly reduce its activity in cell culture. CONCLUSION: Antitoxic activityofplant extracts against CT is perspective for the development of preparations possessing inhibition effect.

[The tuberculous meningoencephalitis in patients with later stages of HIV infection].

Based on the examination and treatment of 120 patients with tuberculous meningoencephalitis (TM) in the later stages of HIV infection, the differences between these patients and patients without HIV infection were found. HIV-infected patients with TM had a more acute disease onset, more clinical symptoms of encephalitis confirmed by magnetic resonance imaging of the brain, more frequent presence of mycobacterium tuberculosis in the cerebrospinal fluid and higher level of resistance to tuberculosis drugs. The morphologic study demonstrated the domination of necrotic and exudative reactions over productive inflammation with destructive abscess-type lesions in the brain matter. The mortality was more than 2 times higher than that in the control group.

[Characteristics of Vibrio cholerae Cef (CHO cell elongating factor): bioinformatics analysis and experimental data].

Bioinformatics analysis of the primary and secondary structure of the Vibrio cholerae Cef (CHO cell elongating factor) protein was conducted. Similarity with triacylglycerol lipases and cytotonic toxins of other bacterial species was observed. Cef was predicted to be a heat-tolerant serine lipase with the Kunitz domain and leucine zipper. These data were confirmed experimentally. The Cefs of the two biotypes of V. cholerae O1, as well as O139 and nonO1/nonO139 serogroups, were purified from the recombinant Escherichia coli strains carrying corresponding cloned genes, and their physicochemical properties, biochemical and biological activities in vitro and in vivo were characterized. Biological activity against the cultured cells was not associated with estherase activity. Evidently, Cef is a bifunctional protein contributing both to pathogenicity of the cholera agent and to its competitive ability in different ecological niches.

Ultrastructural changes in cultured cells under the effect of Vibrio Cholerae hemagglutinin/protease.

Electron microscopic study of changes in cultured cells caused by Vibrio cholerae recombinant hemagglutinin/protease (HA/P) showed significant structural changes, most pronounced in HeLa and L-929 cells not forming a compact monolayer with tight junctions between the cells: formation of numerous vesicles on the cell surface and clasmatosis, vacuolation of the cytoplasm, swelling of mitochondria, clarification of their matrix and crist distortions, and increase in the number of lysosomes. Cytoplasm vacuolation predominated in MDCK culture, while clasmatosis was less intense. Addition of HA/P to CaCo2 cells forming a differentiated polarized monolayer, led to extension of cell-cell spaces not impairing tight junctions, swelling of mitochondria, cytoplasm vacuolation, and clasmatosis on the apical surface. These changes virtually completely coincided with those caused by the so-called NMDCY factor (non-membrane-damaging cytotoxin), described as new Vibrio cholerae toxin. These findings confirm our previous hypothesis about the identity of these factors.

[Use of various methods to isolate exopolysaccharide of eltor cholera Vibrios and its immunochemical characteristics].

AIM: Isolation of Vibrio eltor exopolysaccharide and study of its immunochemical properties. MATERIALS AND METHODS: Rugose variants of strains V. eltor 18895 and V. eltor 18843 obtained by us by selection in M9 medium were used in the study. Exopolysaccharides (EPS) were isolated by K. Kierek (2003), S.P. Zadnova (2004), N.P. Elinova (1984) methods and analyzed for carbohydrate, protein, nucleic acid content and lipopolysaccharide impurity. EPS, LPS, R-LPS structure was compared by high-pressure chromatography. Neutral sugars and amino sugars were identified by thin layer chromatography. Polyclonal antibodies were produced against EPS preparation isolated by N.P. Elinova (1984) method. Specific activity of obtained mice sera was tested by DIA method. RESULTS: EPS isolated by N.P. Elinova method (1984) was shown not to contain extraneous impurities. V. eltor EPS structure differs from LPS and R-LPS. Monosaccharide composition of EPS from ctx+ V. eltor 18895 strain is presented by a wider specter of carbohydrates including glucose, mannose, rhamnose, galacturonic acid. Use in DIA of specific sera produced against EPS from toxigenic strain did not reveal general epitopes with capsule polysaccharides of V. cholerae O139, V. parahaemolyticus and V. vulnificus. CONCLUSION: Use of EPS as an immunogen promoted production of sera that are specific against EPS and rugose variants of Vibrio cholerae eltor that can be used for their detection or characterization.

[The optimization of conditions of diagnostic fluorescent monoclonal immunoglobulins production to identify comma bacillus of serogroups O1 and O139].

The article considers, the issue of producing the species-specific fluorescent monoclonal immunoglobulins to detect comma bacillus of serogrups O1 and O139 in the reaction of direct immunfluorescence. It is established that not only ascitic but culture fluid too can be the source of monoclonal antibodies for producing fluorescent conjugates. The optimal conditions are selected to produce the fluorescent monoclonal immunoglobulins-monoclonal antibodies. The corresponding producing techology can be reproduced at any time in view of availability of hybrid-producers of monoclonal antibodies O1 and monoclonal antibodies O139 in the institute cryodepositoty. The results of testing the fluorescent preparations on homologous and heterologous strains demonstrated their strict specificiy and high sensibility regarding comma bacillus of serogroups O1 and O139. The new preparations favor significant increase of effectiveness of diagnostics of V. cholerae O1 and O139.

[GM1-dot-EIA for the detection of toxin-producing Vibrio cholerae strains].

A new variant of enzyme immunoassay (EIA) has been developed on the basis of GM1 gangliosides to detect the toxin-producing Vibrio cholerae strains--GM1-dot-EIA. Experiments were run using a nitrocellulose membrane to bind GM1 gangliosides and polyclonal antitoxic serum to detect cholerogen. GM1-dot-EIA testing identified cholera toxin in 11 of 13 supernatants of V. cholerae eltor ctx(+) strains isolated from man and in 3 of 7 supernatants of V. cholerae eltor ctx(+) strains isolated from water. These data agree with those obtained in CM1-EIA. There was no reaction with the supernatants of other microorganisms. The sensitivity of the technique was 10 ng/ml. Thus, the simple and specific GM1-dot-EIA may be recommended to detect toxin-producing V cholerae strains isolated from man and water.

[Evaluation of toxin producing abilities of non-O1/non-O139 serogroup Vibrio cholerae isolated from humans].

AIM: Determination of non-O1/non-O139 Vibrio cholerae toxin (CT) gene expression by using EIA, and biological effect of non-O1/non-O139 V. cholerae supernatant on cell cultures evaluation. MATERIALS AND METHODS: 39 V. cholerae strains from various serological groups were studied. Hemolytic activity of strains was determined by using Greig test, and cholera toxin production--in GM1-EIA and in continuous cell lines by registering cytotonic, cytotoxic and proteolitic effect. RESULTS: GM1-EIA method does not detect CT production in 29 museum strains of non-O1/non-O139 V. cholerae in vitro. CT was detected only in 1 non-O1/non-O139 V. cholerae strain supernatant with OD = 0.577 that is substantially lower than in O1 V. cholerae strains (OD = 2.176). In cell cultures non-O1/non-O139 V. cholerae supernatants diluted to 1:100 caused elongation only in single cells. CONCLUSION: Cytological model is a more sensitive technique to evaluate toxin producing abilities of non-O1/non-O139 V. cholerae strains and is appropriate for use.

[Toxicity and cytokine-inducing activity of lipopolysaccharide of virulent Yersinia pestis 231 strain].

AIM: Determine correlation between toxicity and cytokine inducing activity of parent and conformation modified forms of lipopolysaccharides (LPS) of virulent Yersinia pestis strain. MATERIALS AND METHODS: LPS was isolated by phenol method from Y. pestis 231 cells grown at 37 degrees C (LPS37). LPS37 was modified by "mice" toxin (MT) Y. pestis. Toxicity was controlled in mice. TNFalpha and IFNgamma cytokine production was determined by enzyme immunoassay. The study was performed in human monocytes U-937 cell line. TLR4 re-stimulation was performed after activation of monocytes by S-LPS and R-LPS of Escherichia coli. RESULTS: LPS37 conformation change of virulent Y. pestis 231 strain during formation of complex with "mice" toxin increases its toxicity for animals by 2 times. LPS37 and LPS37-MT induce TNFalpha and IFNgamma synthesis by human monocytes. LPS37 simultaneously activates MyD88-dependent as well as MyD88-independent signal pathways. Modified LPS37-MT form is a strong activator only of MyD88-dependent pathway and thereafter induces synthesis of predominately one of the cytokines--TNFalpha. Monocyte response to primary and recurrent activation by LPS37 and LPS37-MT corresponds to R- and S-LPS E. coli cytokine response profile. CONCLUSION: A direct correlation between toxicity of LPS37 and LPS37-MT and their TNFalpha-inducing activity was demonstrated in the study. LPS37 and LPS37-MT of Y. pestis 231 differentially activates TLR4 signal pathways of human monocytes.

[Effect of Yersinia pestis EV 76 lypopolysaccharides with different levels of toxicity on dynamics of TNF-alpha and INF-gamma synthesis by human monocytes].

UNLABELLED: AIM. To study dynamics of synthesis of TNF-alpha and INF-gamma by cell line U-937 human monocytes under the effect of Yersinia pestis EV 76 lypopolysaccharides (LPS) with different levels of toxicity: original LPS28 and LPS37 as well as their conformationally--changed variants with enhanced toxicity--complex of LPS with murine toxin (MT) of Y. pestis, and LPS modified by biologicall active compound (BAC) obtained from human erythrocytes. MATERIALS AND METHODS: Using phenol method, LPS were obtained from Y. pestis EV 76 cells grown at 28 and 37 degrees C. Production of cytokines was measured by ELISA. RESULTS: It was shown that original and modified forms of LPS28 and LPS37 induce synthesis of both TNF-alpha and INF-gamma by human monocytes. Expression of genes for two ways of synthesis of these cytokines points to activation and transmission of signal induced by all studied forms of Y. pestis EV 76 LPS through TLR4. Levels of activity of MyD88-dependent and MyD88-independent signaling pathways are different and depend from chemical structure of LPS28 and LPS37, conformation of their modified forms and duration of their exposition with monocytes. Dynamics ofcytokine synthesis corresponds to response of synergized TLR on activation with profound agonistic/antagonistic effect. CONCLUSION: It was determined that conformational modifications of Y. pestis EV76 LPS occurring due to effect of MT and BAC accompanied by quantitative, qualitative and temporal changes of TNF-alpha and INF-gamma synthesis by human monocytes and correlate with increase of their toxic properties.

[Identification of Vibrio cholerae serogroups O1 and O139 by monoclonal antibodies in the slide agglutination test].

The agglutinating properties of MCA-O1 of the IgG class and MCA-O139 of the IgM class towards epitopes of O-antigen of Vibrio cholerae O1 and accordingly Vibrio cholerae O139 were studied. The ascitic and cultural fluids by hybridomas F8G12 and D11 deposited in the specialized Collection of Cell Cultures of Vertebrates (Saint Petersburg) under RKKK (II) 386 D and RKKK (II) 674 D were the sources of monoclonal immunoglobulins. The advantage of diagnostic monoclonal immunoglobulins is that they are distinguished for strict specificity and their use in practical health care contributes to the higher specificity of a laboratory test for cholera and to its shorter performance.

[Comparative assessment of dot-immunoassay and immunochromatography methods for detection of 01 serogroup of vibrio cholerae].

AIM: Comparative study of sensitivity and specificity of immunochromatographic (IC) assay kit and dot-immunoanalysis for assessment of feasibility of their use for laboratory diagnostics of cholera. MATERIALS AND METHODS: Experimental lots of IC assay kit and dot-immunoassay (DIA) for detection of Vibrio cholerae serogroup 01 serovars Ogava and Inaba were constructed on the basis of species-specific monoclonal antibodies (MCA) conjugated with colloid gold (IC) and peroxidase (DIA). Hybridoma-producer of MCAwas obtained and stored in liquid nitrogen in Rostov-on-Don Research Institute for Plague Control. It was deposited in specialized collection of cell cultures of vertebrates in Institute of Cytology (Saint Petersburg). RESULTS: Strong specificity of IC assay kit and DIA relative to cholera vibrios 01 and absence of crossreactivity with closely related and heterologous microorganisms were shown. Minimal quantity of vibrios, which could be detected using IC assay kit and DIA, was 107 and 105-106 microbial cells respectively. CONCLUSION: Performance of IC assay takes 5-15 min, DIA--1.5 hour, they allow to visually assess the reaction, do not require instrumentation and in perspective both methods could be used on defined stages of scheme for laboratory analysis of cholera.

[The clinical and morphological features of the course of tuberculosis in HIV infection].

The development of an acutely progressing process of varying extent to the point of total damage to both lungs is typical of a patient with tuberculosis concurrent with HIV infection due to progressive immunodeficiency. There is an apparent need for dividing patients with comorbidity into 2 groups: (1) HIV/TB, in patients HIV infection is a primary disease; (2) TB/HIV, in whom tuberculosis is accordingly primary. These groups differ in clinical manifestations, forms of tuberculosis, and pathomorphological changes. Group 1 patients are mostly typified by the primary forms of tuberculosis with involvement of lymph nodes of all groups and by miliary processes at the sites of multiple organs (the lung, abdomen, and central nervous system). Most patients from Group 1 are observed to have fever, progressive intoxication, and morphologically necrotic foci without signs of differentiation and in the absence of typical granulomas. Multiple drug resistance is noted in more than 20% of the patients; in these patients, the efficiency of an intensive therapy phase in arresting bacterial discharge is 26.9%. In Group 2 patients, comorbidity takes a less acute course, pulmonary symptoms are less marked; there is a preponderance of infiltrative, disseminated, firocavernous pulmonary tuberculosis, and caseous pneumonia. In this group, the signs of a prior tuberculous process with phenomena of a slight or moderate productive reaction and with resolution elements are morphologically detectable. In late-stage HIV infection--AIDS, the patients from both groups develop a generalized tuerculous process. Both patient groups are typified by the severe progressive course with identical clinical and pathomorphological manifestations, which results in death.