[Changes in the phase variant spectra in the populations of lactic acid bacteria under antibiotic treatment].

Effect of the antibiotics kanamycin and ampicillin on growth and phase variation of the populations of four strains of lactic acid bacteria (Lactobacillus sp. M67AT, L. casei MB, Enterococcus faecium M, and E. faecium M3185) was studied. The presence of antibiotics in the medium resulted in a dose-dependent decrease in viable cell numbers and in partial or complete substitution of the dominant S variant with the minor Sm and Sb variants. The variants differed in colony morphology, as well as in some physiological, biochemical, biotechnological, and probiotic characteristics. The Sm type variants of all strains exhibited the highest resistance to antibiotics. High production of exopolysaccharides was found in Sb variants of lactobacilli and in S variants of enterococci. The highest antibacterial activity was found in Sm variants of lactobacilli, especially in Lactobacillus sp. M76AT The latter is biotechnologically the most promising strain, since all variants fermented milk yielding the products with uniformly pronounced functional and organoleptic properties. These patterns are of importance for the understanding of the mechanisms of antibiotic resistance and for selection of the variants with desired properties, as well as for quality control of the lactic acid bacteria starter cultures.

[Comparing performance of "TB-BIOCHIP", "Xpert MTB/RIF" and "genotype MTBDRplus" assays for fast identification of mutations in the Mycobacterium tuberculosis complex in sputum from TB patients].

The frequency of mutations causing drug resistance in MTB isolates were studied in the respiratory material obtained from TB-patients in the Moscow Region. In izoniazid-resistant isolates, the most prevalent mutation was found to be the Ser315Thr substitution in the katG gene (15.8%) whereas the most frequent mutations in multidrug-resistant isolates were Ser531Leu and Ser315Thr in the rpoB and katG genes (26.3%), or a combination of these two substitutions with a T15 mutation in the inhA gene (5.3%). We compared performance of three molecular assays--"TB-BIOCHIP" ("BIOCHIP-IMB", Ltd, Russia), Xpert MTB/RIF ("Cepheid", USA) and GenoType MTBDRplus ("Hain Life-science", Germany), with the efficiency of luminescent microscopy, and phenotypic drug-suscepibility testing in an automated system BACTEC MGIT 960 (Becton, Disckinson and Company, USA). Xpert MTB/RIF, TB-BIOCHIP and GenoType MTBDRplus detected MTB in sputum in 92, 78 and 49% of all culture-positive cases, respectively. The agreement between standard cultural data and molecular DST results for Xpert MTB/RIF (resistance towards rifampicin), for TB-BIOCHIP and GenoType MTBDRplus (resistance towards rifampicin and izoniazid) amounted to 100, 97 and 100% respectively. Summing up, Xpert MTB/RIF was concluded to be the most efficient assay for primary detection of MTB, whereas the TB-BIOCHIP was shown to be the only molecular assay sensitive enough for simultaneous detection of MTB DNA and for revealing multidrug resistance in sputum (i.e. resistance to both first-line anti-TB drugs, rifampicin and izoniazid).

[Treatment principles of brain arteriovenous malformations of supratentorial localization in children].

Treatment of arteriovenous malformations (AVM) in children is an important problem of children surgery. An aim of the study was to determine an optimal method of treatment. We analyzed the results of examination and treatment of 300 children, aged from 3 months to 18 years, with brain AVM who were admitted to the Burdenko Neurosurgery Institute in 1995-2010. AVM of large brain hemispheres were found in 223 children and 77 children had AVM of deep brain structures. The results of the following surgeries are reviewed: endovascular surgery (64 patients), dissections of AVM (87), radiosurgery (77), combined treatment (42). Surgeries were not performed in 30 cases. The total post surgery lethality was 0,7%. It has been concluded that the method of AVM treatment in children should be chosen individually for every child on the basis of the complex analysis of several criteria (age of the child, AVM localization and angioarchitectonics as well as a variant of clinical course of the disease).

[Isolation and identification of non-tuberculosis mycobacteria].

Seeding of 117,579 samples of clinical materials at the Central Microbiological Laboratory, Moscow Research and Practical Centre of Tuberculosis, resulted in isolation of 15,786 (13,4%) mycobacterial cultures, with 15,333 (97.1%) ones being identified as M. tuberculosis complex and 490 (3.1%) as non-tuberculosis mycobacteria (NTM). Samples collected in the Moscow region were dominated by MAC, M. kansasii, M. xenopi, and M. fotuitum. Cultivation on solid and liquid media in an automated regime was shown to be optimal for isolation of NTM. Comparison of mycobacteria identification by microbiological methods and by high performance liquid chromatography revealed excellent agreement between the results (96.1%), with the latter technique taking less time to obtain them. Agreement between identification of acid-resistant mycobacterial cultures using the MAIS-dif test system and microbiological methods was estimated at 93.5%. IMS biochips may also be used to detect species composition of myobacteria because only one case of disagreement with the results of microbiological methods was documented in the study of 108 cultures by the two techniques.

[Comparative data on the use of high performance liquid chromatography to identify mycobacteria isolated on liquid and solid media].

Two hundred and forty-two mycobacterial cultures isolated from clinical materials on the Lwenstein-Jensen solid medium and 208 cultures in the automatic Bactec MGIT 960 system (on the modified Middlebrook 7H9 liquid medium) were examined. Of them, there was M. tuberculosis (85 and 82, respectively), M. kansasii (26 and 24), MAC (46 and 38), M. xenopi (24 and 20), M. fortuitum (26 and 22), and M. chelonae/abscessus complex (20 and 18). Identification of mycobacterial cultures isolated on the solid medium by microbiological assay and high performance liquid chromatography (HPLC) showed the agreement of results of the cultures isolated on the liquid medium in 95.5 and 97.2%, respectively; that by these techniques revealed rapidly growing nontuberculosis mycobacteria in 95.8 and 95.2% of cases, respectively; slowing growing mycobacteria in 91.7 and 97.8%, and M. tuberculosis in 96.5 and 97.6%. Mycobacterial isolation on the Middlebrook 7H9 liquid medium in the automatic Bactec MGIT-960 system takes a shorter time than that on the solid (Lwenstein-Jensen) medium. The microbiological identification of mycobacteria lasts as long as 3 weeks while the use of HPLC reduces its time to 24 hours. The efficiency of HPCL does not depend on whether mycobacterial cultures are isolated on the solid or liquid media.

[The detection rate of different species of non-tuberculous mycobacteria in Moscow].

The centralized microbiological laboratory of the Moscow Scientific-and-Practical Center for Tuberculosis Control (MSPCTC) conducted studies to isolate and identify mycobacterial cultures from clinical materials (sputum, bronchial and bronchoalveolar lavages) that had come from Moscow tuberculosis facilities in August 2005 to November 2008. The isolated mycobacterial cultures were identified by microbiological studies, high-performance liquid chromatography, and restriction fragment length polymorphism analysis of the hsp65 gene by means of the MAIS Dif test system developed at the MSPCTC. During the investigation, 117579 inoculations were made; 15786 (13.4%) of mycobacterial cultures were isolated, which included 15333 (97.1%) M. tuberculosis complex (MAC) cultures and 490 non-tuberculous mycobacteria (NTM) ones that amounted to 3.1% of the total number of the mycobacterial cultures isolated in this period. Two thirds of mycobacteria were referred to as slowly growing NTM (according to the Runyon classification; among them there were predominant MAC (32.40%), M. kansasii (14.7%), and M. xenopi (13.9%). Among the rapidly growing mycobacteria, M. fortuitum (21%) and M. chelonae/abscessus complex (6.5%) were isolated. MAC was frequently detected in all the examined administrative districts of the city However, among the slowly growing NTMs, M. kansasii was identified in the South and West districts even at a greater rate; M. xenopi ranked first in the West district. Among the rapidly growing NTM, M. fortuitum was most common in all districts. M. chelonae/abscessus complex was next in the East district (these NTMs were undetectable in other districts).

[Identification of mycobacteria by high-performance liquid chromatography].

AIM: To compare results of mycobacteria identification by bacteriologic methods as well as by high-performance liquid chromatography (HPLC). MATERIALS AND METHODS: Two hundred and eighty strains of mycobacteria isolated from respiratory specimens and identified by bacteriologic methods and HPLC were studied. RESULTS: It was established that results of HLPC use were highly correlated with results of microbiologic methods of mycobacteria identification: for identification of M. tuberculosis complex the correlation was 97.0%, for nontuberculous (NTM) slowly growing mycobacteria--95.3%, for quickly growing NTM--96.2% (overall--96.1%). Results of identification of mycobacteria by HPLC were ready in significantly shorter time-frame (during 24 hours). CONCLUSION: HLPC method could be recommended for identification of mycobacteria in bacteriologic reference laboratories.

[Use of molecular genetic studies to determine the species of the mycobacteria mais and M. tuberculosis complex].

The genus Mycobacterium currently comprises more than 90 species of Mycobacterium, of which a third is able to induce human diseases. With a rise in the incidence of diseases induced by non-tuberculosis mycobacteria, tuberculosis caused by M. bovis that is characterized by a severe cause and a high frequency of poor outcomes cannot be remembered. The species of mycobacteria should be identified to establish a diagnosis and to prescribe adequate chemotherapy. For this purpose, cultural, biochemical, chromatographic, and molecular genetic studies are conducted. The present study using the hsp65 gene restriction fragment length polymorphism test on museum mycobacterial strains and strains isolated from the diagnostic material of patients with suspected tuberculosis by means of Hind61 restrictase has provided a clear differentiation of the restriction profiles of MAIS complex mycobacteria and some other species of non-tuberculosis mycobacteria. To determine the species of representatives of M. tuberculosis complex (M. tuberculosis, M. bovis, BCG M. bovis), the authors have successfully used the test system "TUB-dif" developed at the Institute of Molecular Genetics, Russian Academy of Sciences, by applying the chain polymerase reaction of the senX3-regX3 region.

Identification of mycobacteria of the MAIS Complex and M. tuberculosis by restriction fragment length polymorphism analysis of hsp65 gene.

Restriction fragment length polymorphism analysis of hsp65 gene was performed on museum strains of mycobacteria using Hin6I restrictase. Study of restriction profiles allowed us to distinguish mycobacterial species of the MAIS complex and several strains of nontuberculous mycobacteria.

[The characteristics of the sensitivity of Mycobacterium tuberculosis to rifampicin and isoniazid through determination of mutations in the genes rpoB, katG, inhA, oxyR, and kasA by different molecular biological assays].

Two hundred and two patients with different forms of pulmonary tuberculosis were examined to study the characteristics of sensitivity with the signs of multidrug resistance to rifampicin and isoniazid, by using a microbiological assay of the absolute concentrations and determining mutations in the genes rpoB, katG, inhA, oxyR, and kasA, by employing different molecular biological assays. Mycobacterium tuberculosis (MBT) DNA was isolated from both a diagnostic material (such as sputum, bronchial secretion), and clinical MBT isolates. By showing a higher sensitivity and a higher specificity, as cultural techniques, molecular biological assays of MBT drug sensitivity in patients with tuberculosis were ascertained to accelerate its diagnosis until the patient was admitted to a clinic.

[Probable nature of Rickettsia prowazekii virulence]. / Veroiatnaia priroda virulentnosti Rickettsia prowazekii.

A 29.5 kda outer membrane protein (OmpB) of R. prowazekii virulent Breinl strain is known to differ from its counterpart in attenuated Madrid E strain, while OmpB of this latter one and of its virulent variant EVir coincide in mobility. The infectivity of these strains for macrophages was previously shown to be different as well, and to correlate with their virulence. Previously cloned R. prowazekii Breinl strain, 1.644-bp insert from lambda gtll recombinant expressing as OmpB in inducer-independent fashion was sequenced and used as a query to search for similarity in non-redundant GenBank/EMBL/DDBJ Data Base aided by BLASTX mail server. Extensive homology of inferred 282-aa sequence to peptidyl-propyl cis/trans isomerase C (PPIase C) of E. coli belonging to parvulin family of rotamases (foldases), and related proteins such as Campylobacter jejuni cell binding factor 2 (Cbf2), B. subtilis PrsA, and Lactococcus lactis PrtM has been revealed.

New tumoricidal semisynthetic ether phospholipid, plasmanyl-(N-acyl)ethanolamine (PNAE(s)) and enhancement of its tumoricidal activity by calcium ions.

Recently we described a novel, nontoxic, natural ether phospholipid with selective antitumor activity, 1-0-alkyl-2-acyl-sn-glycero-3-phospho-(N-acyl)ethanolamine, i.e., plasmanyl-(N-acyl)ethanolamine (PNAE), isolated from ischemic tissue of chick embryo. The chemical structure of PNAE has been confirmed by partial synthesis. The semisynthetic preparation PNAE(s), 1-0-octadecyl-2-oleoyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolamine, exhibited tumoricidal activity against human tumor cells T24 and mouse sarcoma cells Mcll as well as in vivo against murine sarcomas S-180 and Mcll. PNAE(s) selectively destroyed tumor cell membranes as has been demonstrated by scanning electron microscopy. The antitumor activity of PNAE(s) in vitro and in vivo was significantly enhanced by Ca2+ ions. These results may be explained by selective damage of tumor cell membranes by PNAE(s), increasing also the tumor cell membrane permeability for exogenous Ca2+ ions and altering the intracellular calcium homeostasis in tumor cells. By increased concentration of Ca2+ in tumor cell cytosol selective damage and lysis of tumor cells was induced. The combined use of parenteral administration of PNAE(s) and peroral doses of calcium gluconate may provide a new approach to enhancement of antitumor activity of PNAE(s) by Ca2+ in a very selective and nontoxic antitumor therapy.