[Refinement of taxonomic position of Lactobacillus genus probiotic strains by 16S rDNA and rpoA gene sequencing].

AIM: Revision of the species identification of collection lactobacilli strains based on 16S rDNA and rpoA gene sequencing. MATERIALS AND METHODS: 52 lactobacilli cultures that present mostly Gamaleya Research Institute of Epidemiology and Microbiology (GIMC) collection were studied. 16S rDNA gene fragments were amplified by using Lb16a, Lb16b, 16S-midford, 16S-midrev primers. 2 different reverse primers were used for the analysis of rpoA gene depending on lactobacilli species. DNA fragments sequencing was performed with 3130 Genetic Analyzer (Applied Biosystems/Hitachi) with primers used for amplification. RESULTS: The effectiveness of sequencing of 2 targets for differentiation of species within lactobacilli phylogenetic groups was shown. Species diversity was demonstrated for GIMC lactobacilli strain collection that includes members of 9 species. All the strains marked previously as L. acidophilus were determined to belong to L. helveticus. Strains belonging to recently discovered L. farraginis species that has promising application in agriculture were detected. CONCLUSION: Genetic passports of original strains of 9 species of lactobacilli that are promising for further research.

[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.

[Species composition and antibiotic resistance of lactobacillus in infants].

AIM: To study species composition and antibiotic resistance of indigenous bacteria from Lactobacillus genus in infants. MATERIALS AND METHODS: Twenty-six strains of lactobacilli isolated from feces of infants living in Kazan. Species membership of lactobacilli was determined using PCR with primers for identification of 7 species of lactobacilli: L. acidophilus, L. plantarum, L. rhamnosus, L. delbrueckii, L. casei, L. paracasei, and L. zeae. Susceptibility to antibiotics was determined by indicator paper discs method. RESULTS. In breastfed infants L. fermentum and L. rhamnosus dominated, whereas in artificially fed infants colonization with L. casei and L. paracasei was characteristic. L.acidophilus, L. plantarum, and L. zeae were not detected in infants. In 6 cases isolates could be identified only to genus characteristic. Lactobacilli were polyresistant and had from 8 to 15 markers of resistance. All strains were resistant to ciprofloxacin, co-trimoxazole, ceftriaxone, nitroxoline, metronidazole, furazolidone, and 95% of strains were resistant to vancomycin. In 60-70% of cases lactobacilli were susceptible to penicillin, linezolid, erythromycin and lyncomycin. CONCLUSION: Regional features of species composition and antibiotic resistance of lactobacilli in infants depending on type of feeding were revealed.

[Influence of Lactobacillus fermentum metabolites on ultrastructure of pathogenic Escherichia coli].

AIM: To study ultrastructural changes of hemolytic and enterohemorrhagic Escherichia coli during interaction with metabolites of Lactobacillus fermentum. MATERIALS AND METHODS: Strains of pathogenic hemolytic (Hly) and enterohemorrhagic Escherichia coli (O157:H7) as well as symbiotic bacteriocinogenic strain Lactobacillus fermentum 97 were used. Inhibition of growth of viable cells was performed by delayed antagonism method. Using electron microscopy, assessment of ultrastructural changes of hemolytic and enterohemorrhagic E. coli under the influence of diffusing in MPC-agar metabolites of lactobacilli. RESULTS: Changes pointing to profound destructive processes in bacterial cells were detected on ultrathin sections. Under the influence of diffusing metabolites of lactobacilli, the following changes were observed: destabilization of cell wall, expansion of periplasmatic space, and emergence of low electron density areas of cytoplasm in polar sections of cells with visualization of floccular material. Emergence of elongated paracrystallic packings and filamentous structures of different length, which deserve special study, was observed in cells of hemolytic E. coli. CONCLUSION: Bacteriocin-like products of lactobacilli during interaction with pathogenic E. coli cause profound destructive changes in the latter which lead to destruction of target cells.

[Differences in pattern of pathogenicity genes in Escherichia coli strains producing shiga-like toxin].

AIM: The detection by PCR of virulence markers in clinical strains of Escherichia coli strains producing the shiga-like toxin. MATERIALS AND METHODS: Thirty-four strains of STEC isolated from patients with acute intestinal infection were studied. PCR with primers to following 10 genes of pathogenicity: rfbE, eaeA, iha, saa, stx1, stx2, cdt, cubA, ehx and espP was utilized. Susceptibility of isolated to antibiotics was determined. Presence of class I integrons in bacterial cells was assessed. RESULTS: rfbE gene coding antigen of serogroup O157 was detected in 31 out of 34 isolates. Twenty-three (74.2%) strains of E. coli O157 had typical pattern of pathogenicity genes (rfbE, eaeA, stx1, stx2, ehx, and espP). In 12 out of 23 cultures (52.2%) sequence of iha gene was detected. Presence of cdt genes was revealed only in 2 clonical isolates belonging to serogroup O157 isolated in Moscow. Two out of 11 strains isolated in Tula region did not have rfbE and eaeA sequences but had sequences saa and subA, which were absent in other studied strains of STEC. Genes of class I integrons were not found in all studied strains although some of them were resistant to sulfonamides, tetracyclines, and aminoglycosides. CONCLUSION: Heterogeneity in pattern of pathogenicity genes was demonstrated in E. coli producing shiga-like toxin that points to circulation of different cultures among patients with acute intestinal infection, which is necessary to consider during analysis of clinical isolates.

[Bacteriocinogenic highly antagonistic strains of lactobacilli].

Ability to secrete bacteriocines and microcines was studied in 25 cultures of lactobacilli isolated from intestine of healthy children. Sixteen (64%) of them produced microcines with wide spectrum of antagonistic activity. Susceptibility of microcines-secreting cultures to antibacterial preparations, different dilutions of hydrochloric acid and bile was studied along with their acid-producing ability. Five strains without DNA-se, RNA-se, gelatinase, lecitinase and caseinolytic activity were selected from Sixteen microcines-producing cultures. Three of the selected strains carried plasmid DNA and two didn't have plasmids. Bacteria were characterized by tolerance to hydrochloric acid and bile - minimal inhibitory concentrations for them were 1,25 and 10% respectively. Strains without plasmids were susceptible to majority of wide-spectrum antibiotics and resistant to fluorochinolones. Microcines-producing lactobacilli with wide spectrum of antagonistic activity against pathogenic and conditionally pathogenic microorganisms and tolerance to physiological concentrations of hydrochloric acid and bile have a potential to be used in manufacturing of probiotics.

[PCR test systems for genetic identification of enterohemorrhagic Escherichia coli]. / PTsR test-sistemy dlia genoindikatsii énterogemorragicheskikh ésherikhii.

The analysis of modern data on the development of amplification test systems for the gene indication of enterohemorrhagic E. coli (EHEC) is presented. In this work the emphasis is laid on the importance of using specific primers whose nucleotide sequence is closely linked with genes controlling the key factors of EHEC pathogenicity; these factors include the determinants of the synthesis of adhesins and invasins (bfp, eae, tir), shiga-like toxins (stx1, stx2), enterohemolysin (ehx), serine protease (epsA) and specific LPS of O-antigen (rfb). The problem of using primers whose sequence is not linked with virulence genes, but which may also be used for the gene indication of E. coli O157:H7 (uid, fliC) is discussed.