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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.

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.

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.

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.

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.

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.

The resistance of methicillin-resistant staphylococci to phage 85 is due to the presence of a certain system restriction modification in microbial cells. The loss of the capacity for restricting phage DNA by the cell as the consequence of the loss of the mec determinant is not accompanied by the loss of its capacity for modifying phage DNA.

After the subcutaneous injection of type I (alpha) interferon into mice their survival rate in staphylococcal infection greatly increased. At the same time duration of staphylococcal persistence in these animals and the number of persisting staphylococci were found to decrease. After the injection of interferon the splenocytes of the treated animals showed a higher capacity for interferon production. During the whole experiment the characteristics of delayed hypersensitivity in these animals showed a tendency towards normalization in comparison with those in infected mice receiving no interferon.

The possibility of using phages, isolated from the lysogenic cultures of methicillin-resistant staphylococci and modified in methicillin-resistant cultures of phage 85, for the differentiation of nontypable staphylococcal strains has been studied. The variants of phage 85 cannot be used for the determination of differences between the strains of methicillin-resistant cultures; they are not suited for typing. For this purpose the collection of phages isolated from lysogenic methicillin-resistant cultures should be used.