Method of Selection of Bacteria Antibiotic Resistance Genes Based on Clustering of Similar Nucleotide Sequences.

A new method for selection of bacterium antibiotic resistance genes is proposed and tested for solving the problems related to selection of primers for PCR assay. The method implies clustering of similar nucleotide sequences and selection of group primers for all genes of each cluster. Clustering of resistance genes for six groups of antibiotics (aminoglycosides, ß-lactams, fluoroquinolones, glycopeptides, macrolides and lincosamides, and fusidic acid) was performed. The method was tested for 81 strains of bacteria of different genera isolated from patients (K. pneumoniae, Staphylococcus spp., S. agalactiae, E. faecalis, E. coli, and G. vaginalis). The results obtained by us are comparable to those in the selection of individual genes; this allows reducing the number of primers necessary for maximum coverage of the known antibiotic resistance genes during PCR analysis.

[ABILITY OF STAPHYLOCOCCUS OF VARIOUS STRAINS TO CREATE BIOFILMS AND THEIR EFFECT ON HUMAN BODY CELLS].

The urgency of the staphylococcus research is due to its ability to cause severe infections: softtissue infections, endocarditis, sepsis, toxic shock syndrome, and food poisoning. Coagulase-positive Staphylococcus aureus is the main infection agent of intrahospital infections. This agent has many factors of pathogenicity, which are well known. Among the coagulase-negative staphylococcus (CNS) strains, S. haemolyticus and S. epidermidis are clinically important, because they cause infections in patients with weak immune system. The mechanisms of the CNS pathogenicity are insufficiently understood. The goal of this work was to evaluate the potential pathogenicity of clinical strains of CNS from their capacity to create biofilms and the character of their interaction with human body cells by the example of the HT-29 cell culture. The research was carried out in laboratory strain S. aureus ATCC 29213 and clinical strains S. haemolyticus SH39, S. epidermidis SE36-1 isolated from the neonatal autopsy materials. The visual tests of biofilm formation by each strain and testing of the impact of the strains on the cell culture HT-29 was carried out in this work. The two species of CNS form biofilms at a higher rate than S. aureus. Upon incubation for 2 h of HT-29 cells with staphylococcus strains tested in this work, adhesion of bacteria on cell surface was observed. The adhesion was most pronounced in case of S. aureus ATCC 29213 and S. haemolyticus SH39. Upon 3 h of incubation with S. aureus ATCC 29213 and S. haemolyticus SH39, destruction of cell HT-29 monolayer was observed. The incubation for 24 h with the 3 strains tested in this work caused complete destruction of cell HT-29 monolayer. The maximal toxic effect on HT-29 cells was inherent in the strain S. haemolyticus SH39. The aggregate of the results obtained in this work indicates the presence of the pathogenicity factors in the strains S. haemolyticus SH39, which require additional research.

[MASS-SPECTROMETRY IN MICROBIOLOGICAL PRACTICE OF SCIENTIFIC CENTRE OF OBSTETRICS, GYNECOLOGY AND PERINATOLOGY].

AIM: Comparative evaluation of species identification of microorganisms by MALDI-TOF mass-spectrometry and automatic biochemical analyzer VITEK2 Compact 30. MATERIALS AND METHODS: Species identification of 18,400 isolates of microorganisms (staphylococci, streptococci, enterococci, enterobacteria, nonfermenting gram-negative bacteria, lactobacilli, anaerobes, yeast fungi, neisseriae), isolated from vagina of pregnant and non-pregnant women and from newborns, was carried out. Identification of the isolated microorganisms was carried out by automatic bacteriologic analyzer VITEK2 Compact30 (BioMerieuX, France) and MALDI-TOF-MS analysis method on AutofleXIII (Bruker Daltonics, Germany) mass-spectrometer. RESULTS: Comparative identification of 2005 isolates of microorganisms was carried out. Sequencing of ribosomal RNA was used as a reference method. Authenticity of species identification my MALDI-TOF-MS analysis method was: for staphylococci (95.8%), enterococci (97.5%), enterobacteria (98.4%), nonfermenting gram-negative bacteria (93.6%), ß-hemolytiC staphylococci (93.8%), lactobacilli (92.8%), yeast fungi (99.9%). CONCLUSION: Introduction of MALDI-TOF-MS analysis technology into practical work of microbiological laboratories exceeds previously used methods of microbiological testing in terms of speed; cost and authenticity of identification of a wide spectrum of microorganisms.

[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.