[The modern characteristics of species identification of coagulase-positive bacteria of Staphylococcus genus.]

The development of molecular techniques of research in the end of XX century permitted to broaden nomenclature of species forming genus Staphylococcus that nowadays numbers 51 species and 27 sub-species. The pathogenic species of genus have a capacity to coagulate blood plasma of mammals forming group of coagulase-positive staphylococci including 7 species: S. aureus, S. delphini, S. intermedius, S. pseudintermedius, S. lutrae, S. schleiferi ssp. сoagulans, S hyicus. In clinical practice, S.aureus is considered as the most virulent among staphylococci. The cumulated data testifies increasing etiologic significance of other representatives of group of coagulase-positive staphylococci in human and animal infection pathology. The keen attention is needed to be paid to Staphylococcus intermedius of group (SIG), uniting three close kindred species: S. pseudintermedius, S. intermedius, S. delphini. Among them the most broadly prevailed are methicillin-resistant clones of S. pseudintermedius, capable to bring on in patient various pyoinflammatory diseases. The laboratory methods based on phenotype tests, provide no opportunity to differentiate coagulase-positive staphylococci because of significant similarity of phenotype characteristics in certain representatives of this group. Te comparative analysis was implemented concerning efficiency of various methods of species identification of coagulase-positive staphylococci: biochemical, molecular genetic (multi-primer polymerase chain reaction for identifying differences in gene structure of thermonuclease, analysis of polymorphism of lengths of restricting fragments of catalase gene and their sequencing), matrix-activated laser desorptional/ionizing time-of-flight mass-spectrometry (MALDI-ToF MS) with various modes of probe preparation. The analysis was applied to 117 isolates of representatives of SIG, separated from ill and healthy individuals of small domestic animals, clinical isolates form patients of hospitals. The multi-primer polymerase chain reaction permitted to identify 97% of isolates, analysis of polymorphism of lengths of restricting fragments of catalase gene - 100% of isolates that confirms efficiency of molecular genetic methods of analysis. The MALDI-ToF MS requires replenishment data base of mass-spectrometer and application of the mode of preliminary protein extraction of samples fo increasing efficiency of species identification of coagulase-positive staphylococci.

[SPECIFIC FEATURES OF ECOLOGY, PATHOGENIC PROPERTIES, AND ROLE OF THE STAPHYLOCOCCUS INTERMEDIUS GROUP MEMBERS IN ANIMAL AND HUMAN INFECTIOUS PATHOLOGY.]

The changes in the nomenclature of species in the genus Staphylococcus, including the most pathogenic cluster of the coagulase-positive staphylococci, are represented. Presently, besides S. aureus, this cluster consists of 6 species: S. intermedius, S. schleiferi ssp. coagulans, S. lutrae, S. hyicus, S. pseudintermedius, and S. delphini. A particular attention was paid to the Staphylococcus intermedius group (SIG), which includes three closely related coagulase-positive bacterial species: S. intermedius, S. pseudintermedius, and S. delphini. The hosts of SIG species are various mammals and birds, which live in a close contact with humans. The current knowledge about the virulence factors and pathogenicity for animals and humans are analyzed. The diffic6lties of the species identification, the features of ecology and epidemiology, antimicrobial resistance were reviewed. The biological features of S. pseudintermedius, which has the greatest similarity with S. aureus, are considered in the context of the properties of newly emerging pathogens.

Molecular Mechanisms of Ceftaroline Susceptibility Reduction in Methicillin-Resistant Staphylococcus aureus.

Ceftaroline is a unique cephalosporin with activity against methicillin resistant Staphylococcus aureus (MRSA). It was approved for clinical use in the USA, Europe and Russian Federation since 2010 for the treatment of the skin and soft tissue infection and community-acquired pneumoniae. In the present study there was used molecular typing of 24 isolates of MRSA with reduced susceptibility to ceftaroline. For 8 isolates belonging to different genetic lines (ST8, ST239 and ST228) and requiring MICs there were determined antibiotic concentrations preventing formation of resistant mutants (mutant prevention concentration) and the ranges of the mutant selection window (MSW). The last majority of the isolates with reduced susceptibility to ceftaroline (MIC of 2 mcg/ml) belonged to the clonal line ST228. The whole genome sequencing of two isolates of ST228 showed that they belonged to the epidemic South Germany genetic line and were characterized by the presence of mutations in PBP2a (N146K) and PBP2 (C197Y) responsible for reduced susceptibility. The highest rates of MPC (32 mcg/ml) and MSW (2-16 mcg/ml) were observed in the clinical isolates belonging to the genetic line ST8. The isolates of ST239 and ST228 had the selection window within 2-4 mcg/ml. No dependence of the MIC and MPC/MSW levels was detected.

[Structural Polymorphism of Genome Islands Encoding Resistance to Beta-Lactams in Coagulase-Negative Staphylococci Isolated at Hospitals of the Russian Federation].

Coagulase-negative staphylococci (CoNS) are considered as a reservoir of mobile genetic elements and first of all of the staphylococcal cassette chromosome mec (SCCmec), defining staphylococci resistance to beta-lactams. Types II, IV, IVa, V, VII and VIII SCCmec were detected among 95 staphylococcal strains isolated in different regions of the Russian Federation. Subtypes C1a, C1b, C1c and C1 SCCmec were also identified (class B mec complex and two complexes of ccr1 and ccr2 genes recombinases). Some other cassette types carrying A, C1 and C2 classes of the mec complexes in combination with various recombinase genes were detected. The S.epidermidis isolates mainly formed cassettes carrying mec complex B, while the S. haemolyticus isolates had cassettes carrying classes C1 and C2 mec complex. Out of 9 isolates of S. hominis 5 isolates carried a new type cassette: class A mec complex in combination with the complex of the recombinase ccr1 genes. SCCmec was not identified in S. capitis and S. pasteuri. Their representatives carried either mec complex (1 isolate of S. pasteuri) or the recombinase complexes (2 isolates of S. capitis). The detected SCCmec variants in CoNS could be a source of emergence of new genetic lines of MRSA.

[Detection of Staphylococcus aureus toxins using immuno-PCR].

A highly sensitive test system, based on the method of immuno-PCR, was developed for the detection of two staphylococcal toxins: enterotoxin A (SEA) and toxic shock syndrome toxin (TSST). A key element of the developed systems was obtaining supramolecular complexes of bisbiotinylated oligodeoxynucleotides and streptavidin, which were used as DNA tags. Specificity studies showed no cross-reactivity when determining SEA and TSST. The sensitivity of detection of these toxins in the culture supernatants S. aureus was not less than 10 pg/mL.

[The architectonics of microbe ecology in the purulent surgery department of municipal clinical hospital].

S. aureus, S. pyogenes, P. aerugenosa, E. coli, P mirabilis, A. baumannii, S. epidermidis, K. pneumoniae, E. faecalis, E. cloacae are the priority pathogens of various forms of pyoinflammatory diseases. They form the architectonics of microbe ecology of the purulent surgery department of multi-type hospital of regional level with S.aureus subsp.aureus playing dominating role. In case of unchanged specter of priority pathogens of pyoinflammatory and pyoseptic diseases the number of gram-positive coccuses decreases at the expense of decrease of number of streptococcuses and staphylococcuses. At the same time, the number of enterobacteria and gram-negative non-fermentative bacteria increases. The resistance ofgram-positive coccuses increases regarding erythromycin, clyndamicin and cyprofloxacin. The resistance of gram-negative bacilli increases regarding ciprofloxacin, cephalosporins of III-IV generations, amikacin. The resistance is the highest among clinical isolates MRSA, K. pneumoniae, A. baumannii. The vancomycin is active regarding all gram-positive pathogens. The carbapenems are active regarding all enterobacteriae. The carbapenems, cefoperazone/tazobactam, cefepime are most active regarding non-fermentative glucose oxidizing bacteria. The netimicin is active regarding A.baumannii. The polymyxine is active regarding P. aerugenosa. The circulation of S. aureus hospital strain of particular genotype is established confirming the propagation of epidemic S. aureus strains in Moscow multi-type medical institutions. The strains are genetically affined to epidemic strains in European and other countries according the international data base (http://SpaServer.ridom.de). The genetic typing of S. aureus ssp. aureus out-hospital hemocultures detected their considerable genetic variety. The epidemic relationship between isolates from different patients is not established. The algorithms of rationale antibacterial chemotherapy of pyoinflammatory and pyoseptic diseases are developed to be implemented in the purulent surgery department of municipal clinical hospital of Moscow.

[Development of Staphylococcus Haemolyticus multilocus sequencing scheme and its use for molecular-epidemiologic analysis of strains isolated in hospitals in Russian federation in 2009-2010].

AIM: Development of Staphylococcus haemolyticus strain typing method based on multilocus sequencing for resolving problems of molecular epidemiology. MATERIALS AND METHODS: 102 strains of coagulase negative staphylococci (CNS) isolated in hospitals of various specialization in N. Novgorod and Moscow were studied. Species identification of strain was performed by using tuf gene fragment sequencing, S. haemolyticus strain differentiation--by MLST results. eBURST approach was used for cluster analysis of MLST data; structural changes in tagatose-6-phosphate kinase were studied by using InterProScan platform and SWISS-MODEL site programs; MLST scheme gene allele variability analysis was performed by using MEGA4.0 program package. RESULTS: In the 102 strains sampled CNS was detected in 28 strains of the S. haemolyticus species. The MLST scheme developed for the first time for S. haemolyticus including mvaK, rphE, tphK, gtr, arcC, triA, aroE genes allowed the differentiation of the sampled strains by 11 genotypes. Strains with ST 3, 8, 6, 1, 4, 5 and 11 differed by highest epidemiologic significance. Cluster and phylogenetic analysis of the data obtained showed a high adaptive ability of the nosocomial S. haemolyticus strains. Multiresistance to antibacterial preparations was detected in the analyzed strains. CONCLUSION: The MLST method developed was effective in the differentiation of S. haemolyticus strains that circulate in hospitals and threaten both neonates and hospitalized adult patients.

Prevalence of genes encoding exfoliative toxins among Staphylococcus hyicus isolated in Russia and Germany.

In the present study, previously characterized Staphylococcus hyicus isolated in Russia (n=23) and Germany (n=17) were investigated for the prevalence of the exfoliative toxin encoding genes exhA, exhB, exhC and exhD by multiplex PCR resulting in the detection of exhD positive strains among the S. hyicus isolated from pigs with exudative epidermitis in Russia and the detection of exhC and exhD for one and two strains isolated from exudative epidermitis in Germany respectively. The toxin gene negative strains were generally isolated from apparently healthy pigs, from other animals and from specimens where the relation between the isolation of S. hyicus and the clinical symptoms remained unclear. Partial sequencing of the toxin genes of selected exhC and exhD positive strains and comparing the sequencing results with sequences of exhC and exhD reference strains revealed an almost complete identity. The results of the present study were in agreement with the findings of Andresen and Ahrens (J. Appl. Microbiol., 96, 2004, 1265) and Andresen (J. Vet. Rec., 157, 2005, 376) that the presented multiplex PCR could be used to investigate S. hyicus for toxinogenic potential and that there is an association between the presence of toxin genes in S. hyicus strains from exudative epidermitis. However, comparable with the S. hyicus strains isolated in Germany which were investigated previously by Andresen (J. Vet. Rec., 157, 2005, 376), exhD seems to predominate in S. hyicus strains from Russia.

[Detection of the genes of pyrogenic toxins of superantigens in clinical isolates of methicillin resistant Staphylococcus aureus].

The content of methicillin resistant S. aureus (MRSA) genes, coding the synthesis of staphylococcal enterotoxins A, B, C (sea, seb, sec) and the toxin of the toxic shock syndrome (tst-H) which was classified with pyrogenic toxins of superantigens (PTSAgs), was studied with the use of PCR amplification. The study revealed the specific features of the content of genes sea and sec, detected in epidemic strains, identified earlier and found to circulate in Russian hospitals. Among the isolates, genetically related to international epidemic strain EMRSA-1, isolates containing no gene sea were detected, while among the isolates genetically related to strain EMRSA-2, isolates containing not only gene sea, but also gene sec were detected, which was indicative of the tendence of this epidemic strain in the direction of further acquisition of pathogenicity genes. As revealed in further studies, among the cultures obtained in bacteriemia, 88% contained gene sea. Two out of three isolates obtained from patients with the symptoms of toxic shock also contained this gene. The differences in the content of genes PTSAgs (sea, seb, sec and tst-H) could serve as a genetic criterium for the differention of isolates circulating in a hospital, as well as for a more complete characterization of the epidemic strains MRSA. The determination of the given genetic markers in genetic strains in circulating strains will make it possible to prognosticate the structure, severity and outcomes of hospital infections. The conditions of PCR amplification for the determination of genes sea, seb, sec and tst-H, as well as multiplex PCR for the determination of genes sea and seb, were developed.

Identification of Staphylococcus hyicus by polymerase chain reaction mediated amplification of species specific sequences of superoxide dismutase A encoding gene sodA.

A species specific PCR test, based on manganese-dependent superoxide dismutase A encoding gene sodA, was developed for the identification of Staphylococcus hyicus, an important bacterial pathogen in pigs. The designed primers allowed a rapid and reliable identification of phenotypically characterized S. hyicus, isolated in Russia, Germany and Denmark. No cross reactivities could be observed investigating staphylococcal reference strains representing 18 different species and subspecies. The use of the described primers might improve a future diagnosis of this bacterial pathogen.

[A study of the polymorphism of mec dna in methycillin-resistant strains of Staphylococcus aureus isolated at permanent stations in different regions of Russia].

Polymorphism of the chromosome staphylococcus cassette mec (SCCmec), a mobile and heterological genetic element providing resistance to beta-lactam antibiotics was studied in methycillin-resistant strains of Staphylococcus aureus (MRSA) isolated at permanent stations situated in different regions of Russia. Type SCCmec was identified using the PCR method by determining allotypes of 3 different structural genetic complexes incorporated in the cassettes mec. It was found that the isolates studied in this work contained 3 different types of SCCmec: I, III, and IVb. Both isolates containing 2 different copies of SCCmec and isolates containing defective copies of SCCmec were identified. It was demonstrated that determination of the SCC-mec type provided an opportunity to differentiate the isolates studied in this work from one another. The isolates attributed to the same genotype variant (identified by polymorphism of coagulase gene) but isolated at different hospitals located in different regions of Russia were found to contain the same type of the chromosome staphylococcus cassette mec, whereas the isolates of different coagulase groups (i.e., different genotype variants) contained different types of SCCmec. It was found that at least 2 epidemic strains circulated in the permanent hospitals of Russia. The strains differ from one another by the polymorphism of the coagulase gene and the mec DNA polymorphism. According to results of studies of several molecular markers (including mec DNA), these strains proved to be identical to the international strains EMRSA-1 and EMRSA-2. Possible mechanisms of MRSA formation and circulation in Russia and CIS countries are discussed.

[Molecular genetic typing of methicillin-resistant Staphylococcus aureus strains, isolated in hospitals of different regions of the Russia and Belarus].

The study of the length of the amplification products of the coagulase gene with the subsequent restriction analysis (the method of PCR--restrictive fragment length polymorphism, or RFLP) was used for typing 90 S. aureus strains. Among the strains under study, 78 were methicillin-resistant S. aureus (MRSA) strains, including 74 obtained in 1986 - 2002 in hospitals of different cities of the Russia and Belarus, as well as epidemic strains EMRSA-1, -2, -3, -12, obtained from the National Laboratory of Health, London (UK). The use of this method made it possible to type all the strains under study, which were differentiated into 9 groups by means of endonuclease Sfo1 and 7 groups by means of Alu1. Majority of clinical MRSA strains, belonged, according to the type of restriction, to groups 4 and 5. The study of the coagulase gene by the method of PCR - RFLP made it possible: to analyze the epidemic situation in hospitals for a period of several years; to compare the properties of strains isolated in different hospitals; to establish the genetic relationship of strains, isolated in 1998 - 2002, with strains, isolated in 1986 - 1990. The results of the study suggest that at least two epidemic MRSA strains, genetically similar to international strains, circulate in hospitals of Russia.

[A system of step-by-step differentiation of methicillin-resistant Staphylococcus aureus]. / Sistema poétapnogo differentsirovaniia metitsillinorezistentnykh shtammov Staphylococcus aureus]

One hundred and eighty four strains of methicillin resistant Staphylococcus aureus (MRSA) isolated in pyosurgical and burn departments of Moscow, Minsk, Omsk, Tbilisi, Vologda, Smolensk and Dushanbe were differentiated with using phages of the International Set (BIS), two collections of experimental phages and two-probe fingerprinting. More than 50 per cent of the isolates could not be typed by the BIS phages. The Experimental Collection of the N.F. Gamaleya Research Institute of Epidemiology and Microbiology (Moscow) was more useful in the differentiation in comparison to the Experimental Collection of the London Health Centre. A new approach applied by us to the establishment of our collection i.e. screening of cultures with a definite specificity of the system of the restriction-modification (by a modified phage 85) followed by their differentiation with respect to the specificity of the prophages (by the induced phages with the respective modification specificity) provided reliable and reproducible results. Our collection made it possible to classify the phage type 85 strains which as was confirmed by the fingerprinting data belonged to 3 different genotype variants. The fingerprinting had no advantages over the typing by the phages of the more extended phage set and was recommended for differentiation of the strains (14 per cent) not sensitive to the phages used. A step-by-step scheme for typing MRSA easy for use in clinical and epidemiological laboratories is described.

[The epidemiological surveillance of hospital infections linked to methicillin-resistant Staphylococcus aureus]. / Epidemiologicheskii nadzor za vnutribol'nichnymi infektsiiami, sviazannymi s metitsillinrezistentnymi Staphylococcus aureus.

As revealed in the realization of the epidemiological surveillance of hospital infections caused by methicillin-resistant S. aureus (MRSA) in different types of hospital, MRSA strains causing purulent inflammatory diseases belong to different clones. The complex marking of MRSA made it possible to determine the presence of the same clone in different hospitals and to detect the outbreaks of hospital infections caused by different clones of MRSA in one hospital. It was found necessary to supplement the commercial international phage-typing set with pages permitting the detection of the specific system of restriction-modification in MRSA.

[Additional differentiation of methicillin resistant strains of Staphylococcus aureus typed by the international phage bank]. / Dopolnitel'naia differentsiatsiia metitsillinorezistentnykh shtammov Staphylococcus aureus, tipiruemykh fagama mezhdunarodnogo nabora.

In the methicillin resistant strains of Staphylococcus aureus (MRSA) typed by the International Set phages the host specificity of the restriction-modification of the phage 85 DNA was determined, the finger printing of the cell DNA was carried out with using two probes and the lytic spectrum of the phages induced in them was studied. Four clones with different specificity of the restriction-modification system (rm89, rm108, rm121 and rm947) differing from that of strain PS 85 which is the host of phage 85 were detected. The strains belonging to the modification types m89, m108 and m121 contained prophages (within the respective groups) with similar lytic spectra when tested with the use of the PS strains of the International Collection and had cross antiphage immunity. Six phage variants were detected among the phages induced in the strains with the modification type m947 which could be indicative of the clone heterogeneity.

[Experimental substantiation of population heterogeneity in methicillin resistant Staphylococcus aureus]. / Eksperimental'noe obosnovanie geterogennosti populiatsii metitsillinrezistentnykh Staphylococcus aureus.

A comprehensive intraspecies typing of the cultures of MRSA collected during inspection of drug resistance in causative agents of intrahospital infections was performed. The following parameters were investigated: antibiotic resistance, toxin production, sensitivity to the phages of the International Set and the phages of an experimental collection providing the isolation of strains with definite specificity of the restriction-modification system. Different clones of methicillin resistant S. aureus were found to be circulating on the territory of the CIS.

[A new collection of phages for typing methicillin resistant Staphylococcus aureus]. / Novaia kollektsiia fagov dlia tipirovaniia metitsillino-rezistentnykh Staphylococcus aureus.

A new collection of phages for typing methicillin-resistant S. aureus (MRSA) is proposed. The collection includes phage 85 (modified by the MRSA strain), capable of selecting strains with the similar specificity of the restriction-modification system, and 9 MRSA-induced phages. The latter differentiate MRSA strains according to the specificity of prophages present in bacterial cells. The use of this phage collection has permitted the typing of MRSA strains insensitive to the phages of the international collection. Among these cultures an epidemic strain has been detected and the source of its spread in the burn center has been established.

[Conjugative plasmid transfer in Staphylococcus aureus?]. / Kon''iugativnyi perenos plazmid u Staphylococcus aureus?

The microbiological and electron-microscopic study of the transfer of conjugative (pG873) and nonconjugative (rms7 and pE994) plasmids in two systems, on nitrocellulose filters and in a fluid culture medium, was carried out. In both systems the low frequency of the transfer of plasmid pG873 or the absence of the transfer of plasmids rms7 and pE994 were observed when nonlysogenic recipients were used for crossing. The presence of prophage in recipient cells increased the rate of the detection of gentamicin-sensitive transconjugants 100-fold and provided to reveal the transfer rms7 and pE994 plasmids. The electron-microscopic study of specimens with lysogenic recipients revealed a picture which can be interpreted as the fusion of two cells. Such picture was not observed in crossings with a nonlysogenic recipient and in preparations obtained from separate donor and recipient cultures.

[Phage-mediated conjugative transfer of plasmids in Staphylococcus aureus]. / Oposredovannyi fagom kon''iugativnyi perenos plazmid u Staphylococcus aureus.

It was shown possible to transfer nonconjugative plasmids during joint cultivation of the donor and recipient cells by transduction and phage-mediated conjugation. In the latter case it was necessary that the phage in the medium was free and the prophage was present in the recipient cells. Differences in the regularities of the transfer of the nonconjugative plasmids mobilized by the conjugative plasmid or phage were observed.

[Effect of prophages on transfer frequency of conjugative plasmid G 873]. / Vliianie profagov na chastotu peredachi kon''iugativnoi plazmidy G 873.

Transfer of the conjugative plasmid G873 on filters and mixed cultivation of the donor and recipient cells in liquid media is described. In the both systems the use of the lysogenic recipient cells (phages of serogroups B and F) in the crossings increased mor than 100-fold the frequency of plasmid transfer. The conjugative transfer of the plasmid in the mixed cultivation system was proved. The conjugative transfer required the presence (while not obligatory) of calcium chloride and was restricted by the serum factors.