[Frequency of diseases caused by group A streptococci among invasive infections of soft tissues and characteristics of the causative agent].

AIM: Determine frequency of diseases caused by group A streptococci (GAS) among invasive infections of soft tissues; identify emm-types of the isolated streptococci, determine the presence of bacteriophage integrases and toxin genes in their genomes. MATERIALS AND METHODS: 4750 case histories of patients with soft tissue infections of the purulent-surgical department of the 23rd City Clinical Hospital.of Moscow "Medsantrud" in 2008 - 2011 were analyzed. 46 strains of GAS isolated from patients with invasive streptococcus infection (ISI) were studied. GAS identification was carried out by latex-agglutination method. GAS emm-type was determined by molecular-genetic methods, as well as the presence of bacteriophage integrases int2, int3, int4, int5, int6, int7, int49, bacteriophage toxins speA, speI, sla, speC/J, speL, speH, speC, ssa and speB gene present on the chromosomal DNA. RESULTS: 132 cases (2.8%) were attributed to invasive infections. In 46 cases of invasive infections (35%) GAS were isolated. 22 different emm-types of invasive GAS strains were detected. Only speB gene among all the toxin genes (as well as the expression of the gene--SpeB toxin) was detected in all the strains, whereas sla and speI genes were not detected in any of the strains. Genes of the other toxins (ssa, speL, speC, speA, speH, speC/J) occurred in a number of strains. Genes of phage integrases were detected among all the strains however in varying combinations (from 1 to 4 genes). CONCLUSION: Invasive infections caused by GAS are more frequently spread than had been previously assumed and a high degree of genetic heterogeneity of invasive GAS strains was detected.

[Invasive streptococcal (group A) infection of soft tissues in a Moscow surgical hospital].

AIM: To study features and risk factors of soft tissue's invasive streptococcal infection in surgical unit's patients: a) emm-types of the most often recovered streptococcus; b) human immune response for streptococcal antigens (streptolysin-O and the group A carbohydrate antigen); c) main patient's conditions that complicate the course of soft tissue's streptococcal infection. PATIENTS AND METHODS: 101 surgical unit's patients with soft tissue's infection, which group A streptococcal (GAS) nature, were examined. Emm-types were determined by comparison ofemm gene sequenced part with data of CDC Streptococcus Laboratory. ASO and specific anti-group-carbohydrate IgM were measured with photometry and ELISA4 accordingly. Patient's personal data were analyzed by standard statistical programs. RESULTS: Among 34 emm-types of all streptococcal isolates emm28 and emm64 were registered only in invasive infection. Emm-types 1, 49, 60, 66, 77, 84, 88 were most often. Among 23 sera from invasive GAS-infection's patients, 15(65%) were ASO-negative, 6 (26%) were ASO-low-positive, and 2 (8%) were positive, but negative for the anti-group-A-carbohydrate IgM. CONCLUSION: Lower levels of the humoral immune response as well as risk factors such as trauma, alcoholism, diabetes, chronic venous insufficiency contributed to development of severe forms of GAS-infection. The main streptococcal emm-types for soft tissue's invasive GAS infection in one of Moscow surgical unit's patients in 2008-2011 were registered.

[Humoral immune response in patients with angina and soft tissue infections of group a streptococci etiology].

AIM: Evaluate informativity of simultaneous determination of antibodies (AB) against extracellular (AB against streptolysin-O-ASL-O) and cellular (IgM against A-polysaccharide - A-PSC) antigens in patients with angina and soft tissue infections caused by serogroup A streptococci (SGA) and identify features of humoral immune response to SGA infection according to infectious process localization. MATERIALS AND METHODS. 2 groups of patients with bacteriologically confirmed SGA infection (50 cases of angina - group 1 and 51 case of soft tissue infection - group 2) were examined for the presence of ASL-O by using Architect ci8200 analyzer (Abbott, USA) and IgM against SGA A-PSC by EIA. RESULTS. In group 1, 23 (46%) individuals were recognized as positive by ASL-O level, and in group 2 - 20 (39%; p>0.05); conditionally significant exceeding of normal values (more than 1.5 times) was detected in 25% of patients of each group. Increased level of antibodies against SGA A-PSC was detected in 43 (86%) patients of group 1, and in 30 (59%) of patients of group 2 (p<0.05). In group 1 exceeding of normal values of anti-A-PSC IgM was noted mostly by 1.5 +/- 0.5 times (74%). In group 2 in 43% of patients the level of anti-A-PSC IgM was above normal more than 2 times and in most cases in uncomplicated variants of disease course. In 45% of patients with severe form of soft tissue infection this parameter did not exceed normal values (p<0.05). CONCLUSION. In acute period of disease with simultaneous determination of ASL-O and IgM against A-PSC sensitivity of serologic diagnostics of SGA etiology angina and SGA infection of soft tissues was established to reach 92% and 72%, respectively, and humoral immune response to cellular AG in each form of SGA has its features.

[Development of Streptococcus pyogenes cells inactivation for turbidimetric determination of bacteriolytic activity of phage-associated enzyme].

AIM: Development a method of treatment of Streptococcus pyogenes bacteria that does not disrupt the integrity of surface structure of cell and provides optimal reproducibility of enzyme preparation activity test results. MATERIALS AND METHODS: Museum cultures of S. pyogenes M29 and S. pyogenes T1 were used, as well as standard strain S.pyogenes T5 (ATCC) and 3 phage-associated lysine PlyC preparations (enzybiotics): 2 isolated from phage C1, third--recombinant enzyme obtained by cloning phage C1 DNA. 3 methods of S. pyogenes cells treatment were used: inactivation by chloroform, antibiotics and heating. RESULTS: Treatment of S. pyogenes cells by rifampicin and gentamicin allows simultaneous turbidimetric determination of enzyme preparations activity and streptococci lysis effectiveness with a good reproducibility of test results. Comparison of kinetic curves of streptococci lysis killed by heating with curves of live culture lysis showed that heat treatment of cells results in a decrease oflysis depth and a reduction of enzyme activity. Pattern and effectiveness of lysis of cells incubated with chloroform approached curve of live streptococci lysis, however this method did not exclude lysis of part of cells and required presence of equipment for work with chemical substances. CONCLUSION. S. pyogenes test culture inactivation method by 2-step treatment of culture with antibiotics that does not disrupt the integrity of surface structure of cells and provides optimal reproducibility of enzyme preparation activity test results was developed.

[Mechanism of development of epidemic process of group A streptococcal infection in children collectives].

AIM: To study the characteristics of group A streptococcal infection epidemic process in children aged 12 - 14 years arrived to summer camp "Orlenok" (Tuapse) from different regions of Russia. MATERIALS AND METHODS: Epidemiological (retrospective analysis of incidence of acute respiratory infections, tonsillitis, and scarlet fever), microbiological (isolation and identification of group A streptococci [GAS]), and molecular biological (pulse-electrophoresis, analysis of spe and emm genes) methods were used for the study. Objects of the study were GAS strains isolated from patients and carriers. RESULTS: Performed genotyping showed that cases of GAS infection in newly formed children collectives were caused by 2 - 3 epidemically important clones, which were genotypically heterogenous. CONCLUSION: Performed molecular biologic studies demonstrated polyclonal structure of GAS that determines the features of development of epidemic process.

[Persistence of Streptococcus pyogenes].

State-of-the-art data on persistence of Streptococcus pyogenes are presented. Survival and persistence of S. pyogenes in organism of the host are considered as a result of action of widely presented virulent functions of infectious agent aimed on evasion from defense mechanisms of the host. Information on adhesins, bacteriocins, IgA-proteases, molecules with invasive function, as well as with function of protection from pahocytosis is presented. Antigenic mimicry and L-forms of S. pyogenes are briefly characterized.

[Genetic characteristic of toxigenicity of group A streptococci cultures isolated from patients with tonsillitis].

Twenty-eight strains of group A streptococci (GAS) isolated from patients with lacunar and fibrinous-necrotic forms of tonsillitis during its 1st or recurrent episode were tested for presence of genes of erythrogenictoxins A, B, C, and Fusing PCR assay. Obtained results allow to consider that clinical features of the disease (severity, repeatedness, clinical form) can be determined by toxigenicity of GAS. Express identification of S. pyogenes on the basis of detection of erythrogenic toxins genes spe B and spe F (mf) can be used for etiologic confirmation of diagnosis, whereas detection of erythrogenic toxins genes spe A and spe C can be recommended for prediction of the disease's course.

[Bacteriophage enzymes for the prevention and treatment of bacterial infections: stability and stabilization of the Streptococcus pyogenes cell lysing enzyme].

The effect of various compounds on the activity and stability of a phage-associated enzyme lysing cells of streptococci of groups A and C (PlyC) was investigated. Substantial inhibition of the enzyme activity was revealed at an increased ionic strength (in the presence of NaCl) and upon the addition of carbohydrates (mono-, di-, and polysaccharides), i.e., agents stabilizing many enzymes. It was established that the enzyme activity was substantially reduced in the presence of positively charged polyelectrolytes and surfactants, whereas incubation with micelle-forming substances and negatively charged polyelectrolytes led to PlyC activation and stabilization. It was shown that, in the mycelial polyelectrolyte composition M16, the enzyme retained its activity for 2 months; while in a buffer solution under the same conditions (pH 6.3, room temperature), it practically completely lost its activity in 2 days. Characteristics of the enzyme thermal inactivation were found, in particular, its semiinactivation time at various temperatures; these allowed us to estimate its behavior at any temperature and to recommend conditions for its storage and use.

[Typing of group A streptococci isolated from urban population of different social status and age].

Pulse-electrophoresis, sequencing of emm genes coding protein M and PCR analysis of speA, speB, and speC genes were used for characterization of group A streptococci (GAS) isolated in different years in Moscow and Tuapse mostly from children and military staff. It has been shown that epidemic process of streptococcal infection caused by GAS in Moscow is based on circulation of many independent clones of Streptococcus pyogenes. Obtained data on complex typing of S. pyogenes would be useful for study of molecular epidemiology of diseases caused by GAS and improvement of epidemiologic surveillance.

[Lipoteichoic and teichoic acids of pathogenic streptococci: structure, functions, and role in interaction of the infectious agent with organism].

Features of structure, localization, and function of teichoic (TA) and lipoteichoic acids (LPA) of gram-positive bacteria are shortly characterized. Structures of polymers obtained from Streptococcus pyogenes, S. pneumoniae, S. agalactiae are described. The role of TA and LPA of pathogenic streptococci in different phases of infectious process and requirements for biological activity of these molecules are discussed.

[In vitro bactericidal action of a complex of natural cytokins on Streptococcus pyogenes].

The data on the antimicrobial in vitro action of a complex of natural cytokins (CNC)--the substance and the preparation "Superlymph"--on virulent museum and clinical macrolid-resistant group A beta-hemolytic streptococci, are presented. CNC suppressed the growth of museum test culture of streptococci in the concentration dependent manner. Moreover, the preparation "Superlymph" proved to be an effective antistreptococcal remedy in its action on erythromycin-resistant group A streptococcal (GAS) strains. The substance of the preparation induced the lysis of GAS cells, grown on lawn plates and in the system of spectrophotometric analysis. The effect thus established could be caused by cationic antimicrobial peptides (protegrins), contained in "Superlymph".

[Pulse electrophoresis of serogroup A streptocci isolated in Moscow].

Pulse electrophoresis of group A streptococci (GAS) isolated in 1998 - 2004 in Moscow from patients during outbreaks of tonsillitis in child institutions, military unit and also from sporadic cases in children and adults was performed. During analysis of 48 strains 16 pulse electrotypes were recognized. These data allow to consider that in different child institutions (and other groups) accumulation and spreading of most adapted to such environment variants of GAS are take place and population structure of GAS in such institutions is unique on molecular and other characteristics. This study showed that complex typing of GAS (use of pulse electrophoresis and PCR for detection of erythrogenic toxins A and C) will help to improve molecular-epidemiologic surveillance for streptococcal group A infection, development of recommendations on reduction of morbidity from this infection and also to decrease risk of its severe forms, complications and mortality.

[Genotypic emm-typing of streptococcal cultures, group A, isolated from patients with different clinical manifestations of streptococcal infection in Moscow].

The results of the intraspecific typing of group A streptococci (SGA), isolated from patients with different manifestations of group A streptococcal infection, carried out in Russia for the first time, are presented. The genotypic method of emm-typing, based on sequencing the DNA area coding the variable part of the molecule of SGA M-protein was used. The data obtained in this study made it possible to follow changes in the pattern of SGA M-types in Moscow and the specific features of SGA in comparison with the analogous data on other territories. New emm-types of SGA circulating on the territory of Russia were detected and described.

[Frequency of genes speA, speB, and speC in Streptococcus pyogenes strains and the identification of the infective agent by polymerase chain reaction]. / Chastota vstrechaemosti genov speABC v shtammakh Streptococcus pyogenes i identifikatsia vosbuditelia s pomoshch'iu PTSR.

In cultures of S. pyogenes isolated from patients and carriers in different territories of the Russian Federation the genes of erythorogenic toxins A, B and C (speA, speB and specC) were detected. The possibility of the identification of S. pyogenes by means of PCR on the basis of primers to erythrogenic toxin B was determined. Gene speB was detected in all S. pyogenes cultures under study and proved to be species specific. Genes speA and speC were detected, respectively, in 29.4% and 9.35% of the S. pyogenes cultures under study. A test system for the identification of S. pyogenes on the basis of primers to gene speB was developed. The prospects for the detection of genes speA and speC for intraspecific typing of this infective agent were evaluated.

[Susceptibility of beta-hemolytic streptococci to antibiotics and alternative drugs]. / Chuvstvitel'nost' beta-gemoliticheskikh streptokokkov k antibiotikam i al'ternativnym preparatam.

Antibiotic susceptibility of the circulating beta-hemolytic streptococci of serogroups A, B, C and G isolated from healthy and sick children and adults within 1987-1996 (more than 900 cultures) was studied. It was shown that the MICs of betalactam antibiotics did not change (within the susceptibility levels) with respect to the serogroup A, C and G streptococci. The number of the strains resistant to erythromycin and lincomycin increased the same as the frequency of the strains simultaneously resistant to chloramphenicol, tetracycline and gentamicin. Tomicid and solkarmon, drugs alternative to antibiotics, were found to be active against the above streptococci groups and efficient in the treatment and prophylaxis of streptococcoses. A suggestion that betalactams are not safe in the treatment of the toxic shock syndrome due to the group A streptococci is discussed.

[The hydrophobicity of Streptococcus pyogenes as a possible marker of its virulence]. / Gidrofobnost' Streptococcus pyogenes kak vozmozhnyi marker ego virulentnosti.

The hydrophobicity of 366 S. pyogenes cultures isolated from the pharynx of tonsillitis and scarlet fever patients and healthy children, as well as museum strain of type M29, was studied. The study revealed that the hydrophobicity of cultures isolated from scarlet fever patients exceeded that of cultures isolated from healthy carriers and tonsillitis patients. Among S. pyogenes cultures isolated in cases of prolonged carriership a considerable proportion of highly hydrophobic cultures was detected, which was probably indicative of an important role played by the hydrophobicity of streptococci in retaining them on the barrier epithelium. This was confirmed by the presence of correlation between the hydrophobicity of culture of S. pyogenes, type M29, and its adhesion to the fibronectin receptor and HEp-2 cells. For S. pyogenes of type M6, epidemic in the group of children under observation during the period of 1987-1988, the values characterizing its hydrophobicity were registered which significantly exceeded average hydrophobicity values. At the period of a rise in morbidity S. pyogenes cultures were characterized by significantly higher hydrophobicity. The variability of the hydrophobicity index and its relationship with the dissociation of cultures were established. The hydrophobicity index may be used as one of the virulence markers of S. pyogenes strains.

[The diagnostic and prognostic significance of the indices of the immune response to streptococcal group A antigens in rheumatism]. / Diagnosticheskoe i prognosticheskoe znachenie pokazatelei immunnogo otveta na antigeny streptokokka gruppy A pri revmatizme.

Serum samples obtained from 33 patients with acute rheumatic fever (ARF), 60 patients with rheumatic heart disease (RHD) and 30 adult healthy persons have been studied. Antibodies to streptococcal A polysaccharide (SAP) and lypoteichoic acid (LTA) were examined by ELISA, to streptolysin O by standard technique. SAP antibodies in high titers persisted from 5 to 12 months in ARF and rheumatic carditis patients especially when RHD was developing. High titers of antibodies to LTA and SLO persisted for 3 months since ARF onset, then fell (p < 0.05). ELISA proved sensitive in determination of antibodies to SAP and LTA. Its combination with determination of the titers to SLO antigens confirmed Streptococcus A infection involvement in emergence of ARF in all the cases including insidious or late-onset carditis and chorea. High levels of antibodies to SAP and LTA were more frequently detected in patients with RHD than in healthy persons (p < 0.001). Correlation between frequency of high levels of antibodies to SAP and bicillin 5 or erythromycin administration, duration of RHD was not established.