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Background. At the height of the novel coronavirus infection, the risk of fungal coinfection increases due to a decrease in the immune status of patients and massive antibacterial therapy, which leads to a complication in the course of the underlying disease and increases the risk of an unfavorable outcome. Therefore, it is necessary not only to identify all associates that cause the infectious process, but also to determine their sensitivity to antibacterial and antifungal drugs in order to select an adequate treatment regimen for a patient.The aim of the study was to investigate the species composition of fungal associates in corona positive and corona negative patients with community-acquired pneumonia (CAP), as well as to determine the spectrum of sensitivity/resistance of yeasts and yeast-like fungi to antimycotic drugs.Material and methods. The species composition of the fungal microflora in sputum samples from 723 patients with CAP, sent from a health care facility in Rostov-on-Don, was investigated. The isolated cultures were identified using bacteriological and mass spectrometric methods. The sensitivity of yeasts to antimycotic drugs was determined by disk-diffusion method.Results. It has been established that yeast and yeast-like fungi were present in sputum samples in 31–32% of patients with CAP in etiologically significant quantities. 10 different types of yeast and yeast-like fungi were identified. Candida albicans was the predominant species in terms of the number of isolated cultures (83%). A re-examination of corona positive patients after their hospital stay revealed a tendency towards an increase in the number and intensity of manifestation of candidal lesions. Moreover, a change in Candida species has been registered, which entails a change in their sensitivity to antifungal drugs. A high percentage of strains resistant to fluconazole was identified after determining the sensitivity of fungi to antimycotics.Conclusion. The data obtained demonstrate that yeast and yeast-like fungi are the dominant associates in complicated forms of CAP. In this regard, it seems appropriate to provide individual microbiological support to patients during their treatment with the identification of all pathogens.

Relevance. In the context of the ongoing pandemic of coronavirus infection, the course of viral pneumonia is often complicated by the addition of bacterial microflora due to a decrease in the body's immune status. The causative agents of such a co-infection can exhibit multiple drug resistance, which significantly reduces the effectiveness of etiotropic therapy. In this regard, it seems expedient to provide microbiological support to patients in order to select the most optimal treatment regimens. Aim. To study the composition of bacterial pathogens’ species, that cause community-acquired pneumonia (CAP) in corona-positive (COVID-19+) and corona-negative (COVID-19–) patients and to determine the spectrum of their sensitivity/resistance to antibacterial drugs. Material and methods. The species composition of microorganisms in sputum samples from 723 patients with CAP, who were admitted from the healthcare facility in Rostov-on-Don in August and December 2020 were studied. The isolated cultures were identified using bacteriological and mass spectrometric methods. The sensitivity of bacteria to antibiotics was determined by the disk diffusion method. Results. It was shown that in August pneumococci and staphylococci prevailed in the spectrum of CAP pathogens, while in December the percentage of excretions of Acinetobacter spp. and S. haemolyticus increased. Various types of p. Candida yeast were found with a high degree of isolation, COVID-19 + patients showed a tendency towards greater contamination (I104 mcl/ml). Some pathogens (A.baumannii, S.haemolyticus, P.aeruginosa, S.maltophilia) are characterized by polydrug resistance, and some strains of these species are pan-resistant to all groups of antibiotics. Conclusion. The data obtained demonstrate that severe forms of community-acquired pneumonia can be caused by viral-bacterial and viral-bacterial-yeast combinations of pathogens, including bacteria with a narrow spectrum of sensitivity to antibacterial drugs.

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.

We studied 14 samples of gram-negative and gram-positive bacteria, unicellular yeasts and green algae using optical, atomic force and analytical scanning electron microscopy, thermal analysis, gas chromatography, infrared spectroscopy, inductively coupled plasma mass spectrometry and isotope mass spectrometry. In shape, the cells of microorganisms vary from rod-shaped to lenticular and coccoid, in size they range from mesonanometer to micrometer. A strong positive correlation was found between the extreme cell sizes. An admixture of inorganic chemical elements — Mg, Ca, Ba, Na, K, Cu, Zn, P, S, Cl and submicron-sized precipitates of mineral phases — carbonates, phosphate sulfates, hydrogen sulfate phosphates, hydrogen phosphates, halite, kaolinite, was found in the chemical composition of the studied microorganisms, metal alloys of brass composition, baddeleyite. Among the 45 microelements identified in microorganisms, there were 7 essential elements (E), 17 physiogenically active (FA) and 19 abiotic (AB). The total concentration of trace elements ranged from 0.003—0.26 wt. %. The value of the essential coefficient — E / AB — averaged 196 ± 153. Microorganisms were characterized by a mixed fat-protein elemental composition, they contained 14 amino acids belonging to the aliphatic, aromatic, basic, acidic, hydroxyl, imino and sulfur-containing groups. The total amino acid content ranged from 409 to 942 (682 ± 221) mg/g. Up to half of the amino acids were represented by the left (L) and right (D) enantiomers. The degree of racemization (D / L) ranged from 0.01 to 0.37. Yeast and chlorella were characterized by the most isotopically heavy composition of carbon in combination with relatively isotopically light nitrogen. In bacteria, a statistically lighter carbon was found in combination with a much heavier nitrogen. According to a number of properties — the chemical composition of organic matter, microelements, the degree of enrichment with antibiotic elements, the content of amino acids and the degree of their racemization — gram-negative and gram-positive bacteria differed. In general, the studied biological microorganisms were fundamentally different from the abiogenic organic substances found in meteorites and products of modern volcanism regarding their elemental and amino acid composition, carbon and nitrogen isotopes.

Comparative analysis of parameters of humoral and cell immunity in individuals, vaccinated against tularemia.Sera and blood samples of 258 immunized individuals were studied by indirect hemagglutination and leukocytolis with tularin reaction.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.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.

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.Beta-lactamase activity was determined in 30 strains of Francisella genus bacteria by quantitative iodometric method.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.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.

The two subspecies of Francisella tularensis, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. palaearctica (type B), differ from each other in biochemistry and virulence. Strains of F. tularensis subsp. tularensis are believed to be confined to North America, whereas strains of F. tularensis subsp. palaearctica occur in Europe, in Asia, and in North America. Moreover, the existence of two other subspecies, designated F. tularensis subsp. mediaasiatica and F. tularensis subsp. palaearcitica japonica, has been suggested for strains of F. tularensis isolated in the central Asian focus of the Soviet Union and in Japan, respectively. In the present study, strains biochemically classified as F. tularensis subsp. mediaasiatica or F. tularensis subsp. palaearctica japonica have been investigated by hybridization with probes specific to 16S rRNAs of the two main subspecies. Furthermore, the virulence and biochemical characteristics of the strains were compared with those of strains belonging to F. tularensis subsp. palaearctica and F. tularensis subsp. tularensis. It was found that 16S rRNAs of F. tularensis subsp. mediaasiatica and F. tularensis subsp. palaearctica japonica hybridize with the probe specific to a genotype proposed herein, genotype A (F. tularensis subsp. tularensis), which shows that strains genetically related to this subspecies are found outside North America. However, the central Asian strains differed from F. tularensis subsp. palaearctica and F. tularensis subsp. tularensis strains when investigated by fermentation of glucose. The results of the biochemical tests could not be unambiguously used for differentiation of strains into F. tularensis subsp. palaearctica or F. tularensis subsp. tularensis. These drawbacks suggest that classification of strains of Francisella on the basis of 16S rRNA analysis may be preferable to classification on the basis of biochemical analysis.

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.

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.

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.

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.

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.

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.

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 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.

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 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.

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.

Therapeutic efficacies of various drugs were studied comparatively in the treatment of experimental plague in albino mice at the stage of the infection generalization. It was shown that out of the tested drugs such as ciprofloxacin, amikacin, gentamicin, rifampicin and polymyxin B only ciprofloxacin provided a rather high therapeutic effect in the treatment of the plaque septic form. The in vitro and in vivo experiments demonstrated that ciprofloxacin had an antitoxic action on lipopolysaccharide (LPS) and the plague microbe toxin. In comparison to ciprofloxacin, polymyxin B had a higher neutralizing activity. It was found that the efficacy of the experimental plague treatment at the stage of the infection generalization increased with the use of combinations of the drugs with antitoxic and antibacterial activities (ciprofloxacin and polymyxin B).

Comparative study of the properties of initial capsular (cap+) and non-capsular (cap-) F. tularensis strains has revealed that the non-capsular variants have their specific biological features. The characteristic features of these variants are avirulence and inability to produce an immunogenic effect in sensitive laboratory animals. Cap- mutants, depending on the method of their obtaining, may exhibit different sensitivity to some antibacterial preparations. In contrast to the initial virulent strains, cap- variants are intensively phagocytosed by peritoneal macrophages of white mice and capable of multiplication and survival in these macrophages. F. tularensis non-capsular forms are characterized by higher superoxide-dismutase and neuraminidase activity than the initial strains, which may be the explanation of the phenomenon of their intracellular survival. At the same time the main biochemical signs of cap- strains (citrullinureidase, phosphatase and penicillinidase) remain unchanged, which determines the possibility of their classification with this subspecies.

New methods (selection in medium T with 20% Tween-80, inducing mutations leading to the appearance of resistance to triphenyl tetrazolium chloride or indoxyl phosphate) helped create a collection of F. tularensis stable non-capsular variants from strains of different subspecies. The characteristic features of all F. tularensis cap- variants include inability to serologic tests with erythrocytic diagnosticum, the absence of capsular antigens, sensitivity to normal human serum.

It was shown that the presence of subinhibitory concentrations of ampicillin, cefotaxime or gentamicin in the cultivation medium had a marked inhibitory effect on the catalase activity of plague microbe. The effect depended on the characteristic features of plague microbe strains and the incubation temperature. When the cells of a virulent strain of the plague microbe Y. pestis 1300 were cultivated at a temperature of 37 degrees C on a medium containing the subinhibitory concentrations of ampicillin or cefotaxime, the pathogen virulence for albino mice significantly decreased.

Natural strains of F. tularensis were characterized by sensitivity to 2,3,5-triphenyl tetrazolium chloride (TTC). Development of TTC resistance in the cells of F. tularensis was accompanied by changes in the biological properties of the culture, i.e. the colony morphology, antigenic structure, virulence and immunogenicity for laboratory animals. Moreover, there was a direct correlation between the levels of TTC resistance and resistance to chloramphenicol, erythromycin, tetracycline, furazolidone and rifampicin. The antibiotic resistant mutants of F. tularensis were in turn more resistant to TTC than the initial strains. This could be useful in isolation of polymarked strains of F. tularensis for genetic studies and investigation of the nature of the phenomenon of virulence in F. tularensis.

In two Francisella species (F. tularensis and F. novicida) neuraminidase activity, heretofore unknown, was detected. The enzyme exhibited specificity with respect to the substrates used in the investigation, neutralizing natural mucins, but not other compounds (glycoproteins and glycoproteins). All F. tularensis strains were found to have enzymatic activity irrespective of their subspecies, but neuraminidase activity was higher in the strains belonging to the American subspecies. Experimentally obtained F. tularensis noncapsular variants possessed higher neuraminidase activity than the capsular parent strains. The conclusion on the possible role of this enzyme in F. tularensis colonization of the host body was made.

In the causative agent of tularemia new markers correlating with different subspecies of this microbe have been detected. Thus, F. tularensis strains belonging to the American and Central Asian subspecies are characterized by phosphatase activity, which makes it possible to use the phosphatase test for their differentiation from the strains of the holarctic variety. F. tularensis subsp. mediasiatica are incapable of producing beta-lactamase which differentiates them from the representatives of the varieties holarctica and tularensis. These newly discovered signs are stable and do not depend on the virulence of the cultures under study and on the conditions of the cultivation of F. tularensis.

Superoxide dismutase (SOD) activity has been registered in representatives of the genus Francisella. The electrophoretic mobility of the enzyme and a number of its isoforms in F. tularensis are linked with the subdivision of this species into several subspecies. Avirulent and noncapsular variants of F. tularensis are characterized by a higher SOD activity than the initial virulent strains. Attempts to detect catalase activity in F. tularensis have failed.

With a large collection of the strains of F. tularensis isolated it has been recently shown that cultures belonging to holarctica and mediaasiatica circulate in the endemic foci of the USSR. By their biological and genetic properties the natural strains of F. tularensis were homogeneous and represented type cultures of F. tularensis. Various ecological conditions in the natural environment did not change within the last 20 years the sensitivity of the tularemia microbe to the antibacterial drugs.

Fifteen stable variants of Yersinia pestis strains exhibiting different degrees of virulence for white mice and guinea pigs were obtained. Multiple passages of the organisms at 37 degrees C in a fluid nutrient medium containing antiplague agglutinating serum were found to be the most efficient method for obtaining noncapsular forms of the plague agent. Acquisition of the Fra(-) phenotype both by wild and laboratory strains was not associated with a loss of the high-molecular plasmid by cells but was probably a result of mutational alterations in the plasmid genes.

In vitro study on antibacterial activity of amikacin in comparison to that of streptomycin revealed a high sensitivity of tularemia microbes of three geographical races to it. Amikacin showed a high therapeutic activity in treatment of albino mice infected with tularemia. The prospects of amikacin use in prophylaxis and treatment of tularemia are defined by its antibiotic activity against streptomycin-resistant forms of the tularemia causative agent.

Altered viable forms of F. tularensis with spheroplast specific damages of the surface structures were isolated after the culture exposure to lithium chloride (0.5 and 1%). Study of natural penicillin resistance in the spheroplasts and bacterial forms of F. tularensis revealed their difference: the spheroplasts of the strains tested had a lower resistance to beta-lactam antibiotics than the bacterial forms while the activity of spheroplast beta-lactamase did not differ from that of the enzyme of the bacterial form and equalled 224 to 252 U/ml of the cell suspension. Therefore, on the model of the lithium-induced spheroplasts it appeared possible to show that the damages of the surface structures of the cell walls of F. tularensis changed the penicillin resistance level which was indicative of involvement of the F. tularensis cell walls in the phenomenon of the natural resistance to beta-lactams.

A transparent nutrient medium T (tularemic) for cultivation of F. tularensis was developed on the basis of heart-brain infusion, yeast extract, bactotryptone, technical casamino acids, bactoagar, salts, L-cysteine and glucose. Introduction of an antibiotic (polymyxin B, 100 units/ml) to the medium composition provided selective conditions for growth of F. tularensis. Comparative study of the medium growth properties revealed that medium T by this parameter was not inferior to the Emel'ianova and Antsiferov media and by the yield of the bacterial mass was superior to them. A liquid variant of the medium (broth T) may be successfully used in various experiments requiring broth cultures of F. tularensis (interspecies conjugation, transformation, mutagenesis, etc.). The medium provides the use of wider temperature ranges for cultivation of F. tularensis. Because of transparency the medium may be used in identification of strains and their description by certain biochemical features. Medium T may be also used in studies with field materials (dead rodents, water).

beta-Lactamase was isolated from the cells of a penicillinase-producing strain of the tularemia causative agent. It was purified 100 times. By its properties (the substrate profile, Km, temperature optimum and thermostability) beta-lactamase differed from penicillinases isolated from other sources.

beta-Lactamase and penicillinacylase were detected in the tularemia causative agent. These enzymes participated in the mechanism of natural resistance of the microorganisms to penicillins. It was shown that the growth temperature had a significant effect on the beta-lactamase activity. The penicillin resistance marker was not eliminated with the use of acridine orange, ethidium bromide or sodium dodecylsulfate.