[Development of pulmonary chlamydia infection in inbred mice lines differentiated by genetically determinated sensitivity to tuberculosis infection].

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. The susceptibility does not depend on the Nramp1 gene, as I/St mice carry its resistant allele, but is controlled by little interacting QTL mapped to chromosomes 3, 9, 17. To find out whether the tuberculosis-susceptible I/St mice are susceptible to other intracellular bacteria taxonomically distant pathogen of Chlamydia pneumoniae was studied. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1r) demonstrated that the former were more susceptible to chlamydia, displaying a significantly shortened survival time following challenge (I/St, 9.2 +/- 1.2 days; A/Sn, 22.0 +/- 0 days (p < 0.001)). To estimate the degree of chlamydial multiplication in the lungs, we suggested a quantitative real-time polymerase chain reaction (PCR)-based method which allows enumeration of the parasite's genome equivalents in infected tissue from 1 to 16 days after challenge. The interstrain difference of chlamydia burden in lungs was observed only after 24 hours after infection. Multiplication of chlamydia in the lungs was controlled efficiently after day 4 of infection. The numbers of genome equivalents dropped slightly by day 8 both in I/St and A/Sn mice. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia despite their similar ability to control bacterial multiplication. Lung tissue of susceptible I/St mice was markedly infiltrated with macrophages (p < 0.01), which differed significantly from the lungs of resistant A/Sn mice. In agreement with higher macrophage content in the lungs, significantly more macrophage-derived proinflammatory cytokines TNF-? and IL-6 were detected in lung tissue homogenates obtained from I/St mice (p < 0.05). Because the prominent difference in survival time did not correlate with permanent difference in bacterial multiplication, we suggested that both infections trigger fatal pathological processes whose dynamics depends strongly upon the host genetics.

[Rate and level of contamination of potentially dangerous water supply facilities in Moscow region by Legionella pneumophila].

AIM: To assess rate and level of contamination by Legionella pneumophila of cooling water systems in industrial facilities as well as hot water supply systems of administrative buildings in Moscow region. MATERIALS AND METHODS: Cooling water systems of 8 industrial facilities and hot water supply systems of 12 administrative buildings or complexes located in Moscow or Moscow region were examined. Samples of water, washes and biofilms were studied by bacteriologic methods and RT-PCR. Results. Significant level of contamination of water systems by L. pneumophila was revealed in examined objects. Rate of contamination of cooling water systems in industrial facilities was 70%. The agent was detected in stagnant, end-capped, and rarely used segments of all hot water supply systems during decrease of water temperature to 36-52 degrees C. Visual detection of natural biofilms on the object correlated with high concentration of L. pneumophila in water samples. In some cases, associations of L. pneumophila with Pseudomonas aeruginosa were detected, including water samples from supply systems of 2 healthcare facilities. CONCLUSION: Obtained results confirm the importance of implementation of modem concept of legionellosis prevention in our country, based on regular quantitative monitoring for Legionella in potentially dangerous water objects and conduction of preventative measures if contamination exceeds acceptable level.

[Using real-time polymerase chain reaction for detection of Legionella in objects of the environment].

AIM: To assess efficacy of using the method of quantitative detection of Legionella in objects of the environment by real-time polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: For the development of the assay, genus-specific primers from gene coding 16S rRNAas well as species-specific primers for detection of Legionella pneumophila on the basis of mip gene sequence. For quantitative detection of L. pneumophila calibration samples of pGEM plasmid containing fragment of the mip gene in known concentration were used. Samples of water and biofilms obtained from cooling stacks of production plants, systems of autonomic water supply, humidification blocks of centralized systems of air conditioning were studied. RESULTS: Correlation of results obtained with RT-PCR and bacteriologic methods was shown during monitoring of potentially dangerous water objects as well as during epidemic outbreak of Legionella infection. Importance of samples preparation stage, during which considerable losses of DNA and inhibition of reaction could occur, is underlined. Disinfection measures on the studied objects significantly influenced on the results of the RT-PCR and can lead to false positive results. CONCLUSION: Obtained results confirm usefulness of testing of potentially dangerous water objects on the presence of Legionella based on the preliminary screening with RT-PCR for the 24 hours followed by bacteriologic testing of samples for 8 - 12 days.

[Polymerase chain reaction to determine and control drug-resistant Mycobacterium tuberculosis strains].

Real-time polymerase chain reaction was used to develop a one-stage procedure for molecular genetic analysis of Mycobacterium tuberculosis (MBT) DNA in order to determine mutations associated with drug resistance to the antituberculous agents: isoniazid and rifampicin. To analyze the spread of drug-resistance of the causative agent of tuberculosis in Russia, two thousand MBT strains were studied in 24 regions of all the federal districts. Testing 1406 MBT strains isolated by first detected and untreated patients revealed multidrug resistance (MDR) in 21.9% of cases. MRD was detected in 58.5% of the previously treated patients with MDR. The agreement of molecular genetic analysis of drug resistance with the results of cultural tests of 1096 strains was 94%.

[The gene encoding the outer membrane lipoprotein lipL32 as a genetic target for developing leptospira genotyping schemes].

The primers flanking the fragments sized 677 bp (external) and 204 (internal) were constructed on the basis of nucleotide sequences of the gene encoding the outer membrane lipoprotein LipL32. PCR-analysis was used to study the prevalence of the gene lipL32 among 79 Leptospiraceae family strains representing different genera and genomic species (77--genus Leptospira, 1--genus Leptonema, 1--genus Turneria). The two amplicons were detected in the pathogenic leptospires--L. interrogans (except L. inadai), but not in saprophytic--L. biflexa. In L. inadai only 204 bp-amplicon was detected. These test-systems can be successfully used to differentiate between two distinct ecological groups of leptospires. The gene encoding the lipL32 seems to be appropriate as an adequate genetic target for developing the leptospira genotyping systems (high prevalence, presence of both conservative and variable sites in its nucleotide schemes).

[Prevalence of the gene encoding the outer membrane lipoprotein LipL32 in leptospires of different taxons].

Primers flanking the fragment sized 677 bp have been constructed on the basis of nucleotide sequences of the gene encoding the outer membrane lipoprotein LipL32. PCR-analysis was used to reveal the prevalence of gene lipL32 among 73 Leptospiraceae family strains representing different genera and genomic species. The gene lipL32 appeared to be conservative across the pathogenic species. In contrast, it was not detected in the genome of nonpathogenic free-living leptospires. Thus the developed PCR test-system with primers LEP21/LEP22 may be efficiently used to differentiate these two distinct ecological groups of leptospires.

[Study on the polymorphism of the coagulase gene by the method of sequencing in methicillin-resistant Staphylococcus aureus strains, isolated in hospitals in different regions of Russia and Belarus].

This method was used for typing of 31 Staphylococcus aureus methicillin-resistant (MRSA) strains; of these, 27 were clinical isolates obtained in hospitals of different cities of Russia and Belarus and 4 were international epidemic strains EMRSA-1, -2, -3, -12. The sequencing of the variable area, located in the middle part of the coagulase gene between nucleotides 979-1355 and detected with the use of information technologies, was carried out. The results of this sequencing were compared with those of the earlier study on the polymorphism of the area of the same gene between nucleotides 1513-2188, carried out by the method of PCR-restrictive fragment length polymorphism. The sequencing of the part of the coagulase gene made it possible to confirm the presence of essential differences in the nucleotide sequences of the coagulase gene in international strains EMRSA-1, -3, -12, grounds for classifying clinical isolates of MRSA strains with two groups (4 and 5), as well as the genetic relationship of different phage types, isolated in different clinics. The study revealed considerable similarity in the nucleotide composition of strains EMRSA-2 and EMRSA-12 despite the fact that, according to the results of Cfol restriction of the 3'-end, they were classified with different groups; the study also revealed the identity of the nucleotide sequences of the coagulase gene in the cultures of group 5, isolated in hospitals of Moscow, St. Petersburg, Orenburg, and strain EMRSA-2, as well as methicillin-sensitive S. aureus strain 8325-4; in addition, in clinical isolates of group 4 and strain EMRSA-1 a considerable degree of homology was revealed. The study of two different loci made it possible to find out the strain with the recombinant form of the coagulase gene. The approach used in this study permitted the differentiation of the international epidemic strains EMRSA-1, -2, -3 and -12 into individual groups, which coincided with the results of Enright et al. (2002) who used multilocus sequencing.

[Development of a quantitative PCR variant, and use of it for assessing gene expression]. / Razrabotka kolichestvennogo varianta PTsR i primenenie ego dlia otsenki ékspressii genov.

The data on the development of a quantitative PCR technique are presented, including the synthesis of modified primers, the choice of conditions for the removal of reaction-mixture components from the amplified DNA, and the colorimetric detection of the PCR product. The application of this technique to the assessment of the level of gene expression in nonculturable bacterial forms characterized by a drastically reduced metabolism is described.

[The mechanism of action and the nature of the factors inducing the formation of uncultivatable forms of Salmonella typhimurium]. / Mekhanizm deistviia i priroda faktorov, indutsiruiushchikh obrazovanie nekul'tiviruemykh form u Salmonella typhimurium.

Approaches to the study of gene expression in the process of transition and prolonged stay of bacterial cells in the uncultivated state have been worked out with the use of the methods of reverse transcription and polymerase chain reaction. The differential expression of genes pqi and stn has been shown to occur in the uncultivated forms of S. typhimurium. The factor inducing the process of the transition of Salmonella cells into the uncultivated state has been found out.

[Development of polymerase chain reaction-based test systems for detecting leptospira in polytypical leptospirosis foci]. / Razrabotka test-sistemy na osnove polimeraznoi tsepnoi reaktsii dlia indikatsii leptospir v politipichnykh ochagakh leptospirozov.

Two highly sensitive test systems G and B, based on the polymerase chain reaction, were developed for indication of pathogenic Leptospira interrogans, including the serovariants appearing during outbreaks in polytypical foci of leptospirosis in the tropical zone of China. These test systems can be used for rapid diagnosis of leptospirosis in humans in foci with different etiological structure.

[Nonculturable forms of Yersinia pseudotuberculosis in the soils of a natural focus of pseudotuberculosis]. / Nekul'tiviruemye formy Yersinia pseudotuberculosis v pochvakh prirodnogo ochaga psevdotuberkuleza.

Soil samples taken from the natural focus of pseudotuberculosis, characterized by the persistent presence of Y. pseudotuberculosis, were studied. Bacteria were detected by means of polymerase chain reaction with the use of 2 test systems; one of them was specific to the chromosomal invasiveness gene (inv) and the other, to gene yopA, localized on plasmid pCad. In 10 out of 24 sample of genetic material of the total soil microflora, Y. pseudotuberculosis gene inv was detected, two of these samples being also containing gene yopA. The calculated concentration of Y. pseudotuberculosis varied 10(2)-10(4) microbial cells per 1 g of soil. Bacteriological studies yielded negative results, which is indicative of the existence of Y. pseudotuberculosis in the soil of the natural focus in the nonculturable form.

[Quantitative PCR-analysis: development of a system for determining the level of an amplified DNA fragment]. / Kolichestvennyi PTsR-analiz: razrabotka sistemy opredeleniia soderzhaniia amplifitsirovannogo fragmenta DNK.

A new technique is elaborated for quantitative evaluation of the polymerase chain reaction (PCR) results based on a system for yopA gene identification in Yersinia pseudotuberculosis fragment located on p45 plasmid. The analysis schedule includes amplification of the studied DNA sequence under the conditions when the reverse primer carries chemically bound biotin label on the 5'-end, hybridization of the labelled amplified fragment with the probe immobilized on the microplate surface, the probe being complementary to the inner portion of specific DNA, visualization of the label, and calculation of bound labelled DNA quantity. The lower threshold of analysis sensitivity is some picograms. It makes possible the analysis and detection of PCR product in the period of exponential increase of its content, i.e. in the initial period of reaction when the quantity of amplified DNA in the reaction mixture is not enough for reliable identification by ethidium bromide staining.