Pecularities of Candida yeast identification by mass spectrometric analysis (MALDI-ToF MS).

To carry the comparative analysis of sample preparation methods for the most effective identification of Candida yeast by mass spectrometric analysis. 265 strains of yeast and yeast-like fungi isolated from the sputum of patients with pneumonia were investigated. The selected strains were identified by conventional methods (cultural, morphological, tinctorial, enzymatic properties) and MALDI-ToF MS using the Autoflex speed III Bruker Daltonics mass spectrometer (Germany) and Flex Control software. To evaluate the effectiveness of fungi species determinination, the comparative analysis of sample preparation was performed using 4 methods: direct application to the target, an extended direct application method, protein extraction using ethanol/formic acid or trifluoroacetic acid. The accelerated scheme of identification of fungi by the culture method does not provide clear and unambiguous results. When using mass spectrometric analysis, the reliability of the results depended on the sample preparation. A comparative study of the effectiveness of fungi species determination by various methods of the sample preparation of 50 clinical isolates was carried out. It was revealed that the extraction of cells using TFC acid does not lead to the appearance of the recordable protein spectra. The use of direct and extended direct application methods made it possible to establish the species only in 32-44% of the strains. The most effective method of sample preparation was the method using formic acid and ethanol, which allowed us to determine the species affiliation in 100% of the studied fungi (Score 2.0). Depending on the yeast species, a high statistical indicator (Score≥2.3) was registered for 42-100% of samples. The results of present study show that the use of MALDI-ToF MS is the most reliable and informative method of Candida spp.identification.

[ROLE OF VARIOUS ANTIGENIC PREPARATIONS OF FRANCISELLA TULARENSIS IN FORMATION OF ALLERGY REACTION IN HUMANS AND ANIMALS].

AIM: Study the role of LPS in induction of anti-tularemia immunity in humans and animals. MATERIALS AND METHODS: Activity of various antigenic preparations of tularemia microbe, including highly purified from protein and S- and R-LPS, was studied using leukocytolysis reaction with blood of vaccinated humans and guinea pigs and skin allergy test (guinea pigs). RESULTS: Only the whole cells of Francisella tularensis, killed in protein non-denaturating conditions and conserving full S-LPS structure (tularin⁺) were shown to be inductors of delayed-type hypersensitivity reaction. Alterations in LPS structure (tularin⁻) results in a significant decrease, and denaturation of bacterial proteins (during boiling) results in a complete loss of immune stimulating properties of the preparations. Purified LPS preparations and O-polysaccharide fraction of S-LPS are not able to activate cell-mediated immunity. CONCLUSION: The presence of LPS with the full structure affects the ability of antigenic preparations of F. tularensis to cause allergic reactions, and thus, form cell-mediated antitularemia immunity. LPS of F. tularensis can not be excluded as an adjuvant and provides the most effective presentation of epitopes of protein molecules for interaction with receptors of T-lymphocytes.

[Comparative analysis of parameters of humoral and cell specific immunity in individuals immunized with a live tularemia vaccine].

AIM: Comparative analysis of parameters of humoral and cell immunity in individuals, vaccinated against tularemia. MATERIALS AND METHODS: Sera and blood samples of 258 immunized individuals were studied by indirect hemagglutination and leukocytolis with tularin reaction. RESULTS: 73% of the examined individuals had both specific antibodies and positive values of cell immunity. The presence of anti-tularemia immunity was registered in 26 of 30 individuals immunized 10 - 20 years ago. However in 76 individuals (26%) we have detected discrepancies of the results of the 2 methods that complicate the evaluation of specific immunity status. As such, the use of only 1 method characterizing either humoral or cell immunity does not give objective information. CONCLUSION: The use of 2 methods directed on detection of both specific antibodies and delayed-type hypersensitivity reaction is reasonable for the increase of validity of results of anti-tularemia immunity status evaluation. Only positive immunologic parameters of both tests confirm the presence of immunity against Francisella tularensjs and the possibility of revaccination period delay.

[Features of resistance formation to beta-lactam antibiotics in Francisella tularensis subsp. mediasiatica].

AIM: Determination of penicillin resistance features in tularemia causative agents of the mediasiatica subspecies, stability evaluation of differences in strains of various taxa and development of a rapid method of F. tularensis intraspecies differentiation. MATERIALS AND METHODS: Beta-lactamase activity was determined in 30 strains of Francisella genus bacteria by quantitative iodometric method. RESULTS: All the strains regardless of subspecies membership were characterized by high resistance to beta-lactam antibiotics, and bacteria of the mediasiatica subspecies in contrast to other francisella did not synthesize beta-lactamase. Our attempts to induce beta-lactamase activity in vitro and in vivo in strains of this subspecies did not succeed. A method of intraspecies differentiation of F. tularensis by nitorcefin disks is proposed based on the distinctive feature. CONCLUSION: A high level of F. tularensis subsp. mediasiatica resistance to beta-lactams in vitro and their inefficiency during therapy of experimental tularemia due to a beta-lactamase negative strain suggests that F. tularensis beta-lactamase is not the leading factor in formation of native penicillin resistance of tularemia causative agent.

[Interaction of S- and R-lipopolysaccharides of Francisella tularensis with lypopolysaccharide-binding protein of human serum].

Investigation of ability of Francisella tularensis S- and R-lypopolysaccharide (LPS) preparations as well as the live bacteria with different chemotypes to interact with human lypopolysaccharide-binding protein (LBP) was carried out. It was found that LPS preparations derived from virulent(S-LPS) or isogenic avirulent mutant (R-LPS) strains of F. tularensis had markedly lower affinity to LBP as compared with typical S-LPS of Salmonella abortus and R-LPS of Yersinia pestis. It was shown that R-LPS preparation from avirulent mutant binds LPB more effectively than S-LPS from F. tularensis virulent strain. Differences in S- and R-LPS affinity were also confirmed for LPS represented by the live cells. Thus, bacteria with S-chemotype of LPS (F. tularensis 15/10) bound only 20.3% of LBP, whereas cells with R-LPS (F. tularensis 543 cap(-)) bound 39.9%. Such pattern was observed in experiments with both normal non-immune human serum and sera from people immunized with live tularemia vaccine. The latter indicates that opsonization of LPS by specific antibodies does not change its affinity to LBP. The observed more efficient binding of avirulent strain R-LPS to LBP is likely determines the more intensive host response directed to destruction and rapid elimination of the causative agent. At the same time, weak affinity of the vaccine and virulent strains S-LPS to LBP probably allows the bacterium to avoid activation of host defense mechanisms thus contributing to its long-term persistence in microorganism and development of specific immunity against tularemia.

[Genotypic heterogeneity and geographic diversity of collection strains of Francisella tularensis as determined using the VNTR variability analysis and DNA sequencing].

The analysis of the identification genotypes allowed the strains to be grouped into 61 variants from A to I with the incidence rate 0.002-0.142. The cluster analysis of the identification genotypes allowed the strains to be grouped into 9 clusters with different number of components. Actual existence of genotypic heterogeneity and geographic diversity of the F. tularensis strains was demonstrated in addition to territorial attribution of certain strains. The geoinformation system Tularemia was developed to provide spatioterritorial analysis of distribution of the genotypes of the strains. A specific feature of the geoinformation system is dynamic mode of its operation, which provides the ability of continuous addition of information not only by the expense of available data but also by the expense of creation of new layers. Any new information is automatically added to the geoinformation system, thereby providing both retrospective and operative analysis. The geoinformation system Tularemia should find promising application to the structure of the epidemiological method. The use of the system will bring the epidemiological control of tularemia to a qualitatively higher level.

[Vntr-genotyping of Francisella tularensis strains isolated in the former USSR territory and some European countries during epizootics in 1988 - 1989].

Retrospective VNTR-analysis of 159 Francisella tularensis subsp. holarctica strains isolated in December 1988 - February 1989 in former USSR and some European countries was carried out. Analysis of heterogenic genotypes of strains allow to subdivide them into 30 groups of variants by individual genotypes, while cluster analysis--to subdivide them in 7 clusters with different number of compositions. The predominance of genotype C1 strains isolated on the Rostov and Archangelsk regions and the Crimea was established. F. tularensis strains isolated in winter time 1988 - 1989 in different geographic regions were supposed to be resident cultures typical for their biotope in natural focus of disease.

[Phase variations of Francisella tularensis lipopolysaccharide in human infection and immunization].

The comparative study of the specificity of antibodies in human sera after tularemia infection and immunization with live tularemia infection was carried out with the use of passive hemagglutination and immunoblotting techniques. The sera of tularemia patients contained two different types of immunoglobulins: strictly specific to the antigenic epitopes of F. tularensis Iipopolysaccharide (LPS) and strictly specific to F. tularensis subsp. novicida LPS. Such phenomenon may be due to phase variations of the antigenic structure of F. tularensis LPS in the body of a slightly susceptible host. The immune sera of vaccinated were found to contain antibodies, strictly specific only to F. tularensis LPS. At the same time in one vaccinee by the presence of pronounced postvaccinal reactions was found sharply defined interaction between serum imunoglobulins and F. tularensis subsp. novicida LPS. As the result, the data on the possibility of the antigenic modification of F. tularensis in tularemia infection in humans were obtained. At the same time antigenic epitopes, characteristic of faintly pathogenic and closely related F. tularensis novicida LPS, appeared in the structure of F. tularensis LPS.

[Possible mechanisms of implementation of toxic potential of lipopolysaccharides of pathogenic bacteria].

The significance of variability of biological properties of lipopolysaccharides (LPS) is discussed in the paper within the pathogenesis of infectious process. On the basis of an analysis of published data and of results of independent research of two microorganisms (Yersinia pestis and Francisella tularensis) a conclusion is made on that a biologically inert LPS form (with a weak cytokine-inducing ability, apirogenicity and non-toxicity etc.) is typical of highly pathogenic bacteria. It is suggested that the above phenomenon is biologically expedient. Presumably, the inert LPS transforms to the active form inside a sensitive host and, according to an infection stage, each of them being functionally significant. It is the inert status of LPS that enables the pathogens, at the initial stages, to surmount freely the humoral and cell barriers of host. As the infection progressively aggravates and the proliferation of bacteria modifies itself due to LPS micro- and macroorganisms, its chemical structure and biopolymer conformation change. Both modification mechanisms enhance the LPS toxic potential. In case of a sensitive host, such variations transform the biologically inert LPS into a toxically active form with its function of endotoxin being realized. There is no LPS modification in a host insensitive to such infection, which entails either recovery or prolonged persistence of the pathogen inside the microorganism.

[Multilocus VNTR-typing of Francisella tularensis strains]. / Mul'tilokusnoe VNTR-tipirovanie shtammov Francisella tularensis.

In the analysis of F. tularensis genome with the use of the specially developed program "DNA" a great number of loci containing tandem repeats were found. For analysis, 3 of them were selected and designated as FtA, FtB, FtC. The study of DNA of 40 F. tularensis strains in the polymerase chain reaction with specific primers to these loci a great variability in the number of repeats was established, the presence of 17 alleles being found in locus FtA, 5 alleles in locus FtB and 5 alleles in locus FtC. The strains under study formed 24 variants of genotypes, whose occurrence varied from 0.025 to 0.125. Taking into account the variability of the detected loci and a great number of potential loci VNTR in the genome, further development of this method will facilitate the creation of local and general data bases of the strains, thus ensuring more effective genetic typing of F. tularensis.

[Genotyping of the Francisella tularensis strains isolated from natural foci of tularemia in the Rostov region by multilocus VNTR analysis]. / Genotipirovanie metodom mul'tilokusnogo VNTR-analiza shtammov Francisella tularensis, vydelennykh iz prirodnykh ochagov tuliaremii Rostovskoi oblasti.

On the basis of an analysis of the VNTR alleles' distribution in 109 strains of F. tularensis it was established that 19 genotypes of the disease causative agent circulated in the Rostov Region from 1945 to 2002. The microbe-provoked infection episodes can be divided into polyclonal, monoclonal and cluster ones. A retrospective analysis of the genotypes' distribution is indicative of that strains of similar or of closely-related genotypes circulate simultaneously in the studied territory. All investigated F. tularensis strains could be differentiated into two groups; strains, whose genotypes are encountered almost evenly within the entire Region's territory, belong to group 1; and strains of group 2 displayed a trend towards being geographically bound. Isolations of cultures with similar (close) genotypic features made in prolonged time periods suggest that a part of F. tularensis clones can persist for a long time in environmental foci. A set of strains described by genotype can provide a foundation for a database of the tularemic microbe culture within the geo-information system of the South Federative Okrug of Russia.

[The role of lipopolysaccharide in toxicity of Francisella genus bacteria]. / Rol' lipopolisakharida v toksichnosti bakterii roda Francisella.

It was demonstrated that the lipopolysaccharides (LPS) preparations, which were isolated from all representatives of Francisella Genus bacteria, i.e. F. tularensis, F. novicida, F. novicida-like and F. philomiragia by using the method of R.P. Darveau, R.E. Hancock (1983), were not toxic for white rats and white mice. A comparative study of toxicity of live F. tularensis bacteria (both wild and LPS-defective strains) made it possible to establish a direct correlation between the toxicity of microbes and LPS chemotype. It was found that only typical strains, which synthesize the wild-type S-LPS, caused the death of white rats and white mice in 24 hours after intraperitoneal contamination (10(9), 10(10) CFU/animal). Live bacteria of F tularensis R-mutants were not able to induce a lethal infection of rats and retained only residual virulence for mice. Other representatives of Francissela genus possessed less pronounced pathogenic properties. Thus, the toxic effect was registered, in case of white rats, only for F. novicida but not for F. novicida-like or F. philomiragia. At the same time, the two last mentioned species displayed a certain degree of virulence at high challenge doses (10(9), 10(10) CFU/animal) in respect to white mice. F. philomiragia, which generated lipoolygosaccharide (LOS) with an unusual structure, was found to be least pathogenic (25-75% of dead mice). The toxicity of bacteria, killed experimentally by different means (heating, UV-light, chloroform, acetone and formalin), was studied to define the role of bacterial proteins in the realisation of F. tularensis toxic potential in vivo. No lethal effect was exerted on experimental animals by killed microbes or purified LPS preparations. Finally, the study results show a priority role of the LPS molecule in the toxic effect of F. tularensis, which is possible in vivo only if structurally valuable molecules of live bacterial cells are available.

[Comparative analysis of the immune response of a rabbit to antigens to live and killed Francisella species bacteria]. / Sravnitel'nyi analiz immunnogo otveta krolika na antigeny zhivykh i ubitykh bakterii roda Francisella.

Serum antibodies were analyzed in rabbits immunized with live and formalin-killed Francisella (F. tularensis, F. novicida, F. novicida-like, and F. philomiragia). Passive hemagglutination test with erythrocytes sensitized by these bacteria' LPS showed much higher titers of species-specific antibodies in all sera to live microorganisms than sera to killed bacteria. The results of immunoblotting with purified LPS and bacterial lysates indicate that sera to live bacteria contained mainly immunoglobulins to species-specific antigenic epitopes of LPS O-polysaccharide chain and few antibodies to the protein component of the cell. By contrast, killed bacterial cells induced weak production of antibodies to S-LPS and a pronounced antibody response to protein antigens. Besides the quantitative differences, live and killed bacteria differed by the qualitative spectrum of immunodominant proteins. Serum to live F. tularensis 15/10 contained antibodies to at least 3 immunodominant antigens of the cell, while serum to killed bacteria contained antibodies to only two of these. Immunoglobulins to protein antigens, absent in homologous sera to live bacteria, were detected in the sera to killed F. novicida and F. novicida-like bacteria. Both sera to F. philomiragia had antibodies reacting with LPS epitopes and immunodominant complex containing protein. In contrast to other Francisella, F. philomiragia was found to synthesize an uncommon LPS representing two major lipooligosaccharides with different molecular weights and antigenic specificity. Therefore, immune response of the host to live and killed Francisella is different: live cells more effectively induce the production of antibodies to S-LPS epitopes, while killed ones to protein antigens.

[Species- and genus-specific antigenic epitopes of Francisella tularensis lipopolysaccharides]. / Vido- i rodospetsificheskie antigennye épitopy lipopolisakharidov Francisella tularensis.

Lipopolysaccharide (LPS) antigenic epitopes of natural virulent and isogenic avirulent Francisella tularensis strains and other species of the Francisella genus (F. novicida, F. novicida-like, and F. philomiragia) were studied by dot and immunoblotting. Polyclonal rabbit and human sera to virulent F. tularensis strains and monoclonal antibodies to F. tularensis LPS O-side chain were used for detecting species- and genus-specific LPS epitopes. Typical virulent F. tularensis strains produce two types of S-LPS with different antigenic specificity simultaneously. Antigenic determinants of two LPS types were located in LPS O-polysaccharide but not in the core oligosaccharide. The epitopes of the first LPS type were characterized by species specificity for F. tularensis in contrast to determinants of the second LPS type, which had epitopes common with F. novicida. Cross exhaustion of human and rabbit antitularemic sera by F. tularensis and F. novicida LPS showed that F. novicida LPS molecules contained at least two epitopes--highly specific for F. novicida and common with the second type of F. tularensis LPS. The immune response of rabbits and humans to F. tularensis LPS epitopes was different in principle. Sera from rabbits immunized with vaccine and virulent F. tularensis strains contained antibodies "recognizing" antigenic epitopes of two S-LPS forms of the bacterium: type 1 species-specific (in high titers) and type 2 epitopes common with F. novicida LPS (in low titers). In addition to these, sera from patients with tularemia contain immunoglobulins to species-specific epitopes of F. novicida LPS in high titers. Experiments on avirulent mutants showed that in some cases attenuation of F. tularensis can involve loss of species-specific LPS form, while S-LPS with epitopes common with F. novicida LPS will be retained. The difference in specificity of human and rabbit antitularemic antibodies is due to individual features in the host immune system.

[The use if Francisella tularensis lipopolysaccharide in the dot solid phase enzyme immunoassay]. / Ispol'zovanie preparatov lipopolisakharida Francisella tularensis v tochechnom tverdofaznom immunofermentnom analize.

To determine antitularemia antibodies in the sera of humans and animale, the possibility of using dot immunoassay with the use of F. tularensis lipopolysaccharide (LPS) as antigen-containing preparation was ascertained. Experiments demonstrated that this method made it possible to determine specific antitularemia antibodies in the sera of sick and immunized humans and animals. Investigetions carried out with the use of heterologous antisera to F. novicida, F. novicida-like and F. philomiragia, as well as Brucellf abortus, Vibrio cholerae and Yersinia enterocolitica, revealed that F. tularensis S-LPS was highly specific. The results obtained in this investigation are indicative of good prospects of using F. tularensis LPS in dot blotting for the laboratory diagnostics of tularemia in humans.

[The immunomodulating and antitoxic properties of preparations of lipopolysaccharides from representatives of the genus Francisella]. / Immunomoduliruiushchie i antitoksicheskie svoistva preparatov lipopolisakharidov predstavitelei roda Francisella.

The study of the biological properties of lipopolysaccharides (LPS) of bacteria of the genus Francisella (F. tularensis, F. novicida, F. novicida-like, F. philomiragia) revealed that the preparation of Francisella LPS possessed immunomodulating and antitoxic properties in the absence of toxicity. At the same time the structure of LPS (S or R) was found to produce an essential effect of the immunobiological activity of this molecule. Thus, the S-forms of LPS proved to be more effective as immunomodulators and the R-forms of LPS, as antagonists of classical endotoxins.

[Characterization of structure and antigenic activity of lipopolysaccharides of genus Francisella]. / Kharakteristika struktury i antigennoi aktivnosti preparatov liposakharidov razlichnykh predstavitelei roda Francisella.

Wild-type representatives of Francisella genus (F. tularensis, F. novicida, F. novicida-like, and F. philomiragia) produce S-type lipopolysaccharides (LPS) possessing different antigenic activity and common antigenic determinants in the core oligosaccharide. Electrophoretic analysis showed that F. philomiragia produced S-LPS containing two major molecular components with minor fractions between them, whereas S-LPS of F. tularensis, F. novicida, and F. novicida-like are characterized by the typical frequency distribution of molecules. A characteristic feature of Francisella LPS was the ability to form the dominant molecular components with similar electrophoretic mobility of major fractions of the original F. philomiragia LPS upon long storage in water solution. Natural virulent F. tularensis strains produce at least two types of S-LPS. Polysaccharide chains of type I S-LPS possess O-species-specific antigens, whereas the polysaccharide part of type II S-LPS has nonspecific antigenic epitopes. A decrease of F. tularensis virulence can be associated with impaired production of both S-LPS types or loss of S-LPS with O-species-specific antigenic activity.

[The phosphatase activity in representatives of the genus Francisella]. / Fosfataznaia aktivnost' u predstavitelei roda Francisella.

The comparative study of phosphatase activity in the representatives of the genus Francisella--F. tularensis, F. novicida, F. novicida-like, F. philomiragia--revealed that all the bacteria under study synthesized acidic phosphatase. The study also revealed that strains of the Central Asian subspecies possessed the highest enzymatic activity, while holarctic strains either produced no phosphatase at all, or produced less active phosphatase. Bacteria belonging to nonarctic F. tularensis subspecies and other representatives of this genus with the same area of circulation possessed the medium level of activity. Using isogenic F. tularensis variants as a model, we found out that the loss of virulence by bacteria was accompanied by an increase in phosphatase activity. Detergent (0.5% sodium dodecyl sulfate) was found to be capable of selectively inactivate this enzyme in the holarctic strains F. tularensis and F. novicida, F. novicida-like, F. philomiragia. The enzyme immunoassay of Francisella phosphatases made it possible to detect this enzyme in the nonarctic and Central Asian subspecies of F. tularensis and F. novicida, as well as in F. novicida-like, characterized by different electrophoretic mobility. No phosphatase could be visualized in F. tularensis and F. philomiragia holarctic strains.

[A rapid method for assessing the virulence of Francisella tularensis in vitro]. / Bystryi metod otsenki virulentnosti Francisella tularensis in vitro.

A rapid and simple method for the in vitro evaluation of the virulence of F. tularensis is proposed. The optimal parameters--the concentrations of bacteria and the substrate (phenolphthalein phosphate), the time incubation in the serum--have been developed and special conditions for strains of different subspecies have been selected. The proposed method makes it possible to greatly reduce the time of obtaining results and to exclude labor-consuming experiments on laboratory animals when working with large collections of F. tularensis laboratory and natural wild strains. Moreover, the method widens the possibilities of experimenters in the evaluation of the effect of some mutation on the virulence of the strain under study.

[In vitro influence of biologically active factors of microorganisms on antibiotic susceptibility of Yersinia pestis]. / Vliyanie in vitro biologicheski aktivnykh faktorov mikroorganizma na antibiotikochuvstvitel'noct' Yesinia pestis

To estimate the in vitro susceptibility of the plague microbe to chemotherapeutics, various experimental models with the maximum closeness to the host conditions were tested. The tests included the assay of the drug antibacterial activity against the plague microbe by the method of two-fold dilutions in biological fluids i.e. human normal (nonimmune) serum (HNS) and guinea pig heparinized blood. Hottinger broth was used as the control. It was shown that any system used for estimation of the drug MIC influenced the plague microbe susceptibility. Thus, the serum complement increased the antibacterial activity of cefotaxime, rifampicin, doxycycline, erythromycin and polymyxin B. In the blood of a susceptible host (guinea pigs) the activity of quinoxydine and dioxydine against the plague microbe markedly increased while the effect of benzylpenicillin, cefotaxime and furazolidone decreased. The data on the in vitro activity of the antibiotics in blood were comparable with those on their in vivo therapeutic efficacy.