Metagenomic Analysis of Gingival Sulcus Microbiota and Pathogenesis of Periodontitis Associated with Type 2 Diabetes Mellitus.

Biofilm of the gingival sulcus from 22 patients with type 2 diabetes mellitus and periodontitis, 30 patients with periodontitis not complicated by diabetes mellitus (reference group), and 22 healthy volunteers without signs of gingival disease (control group) was studied by quantitative PCR. Quantitative analysis for the content of P. gingivalis, T. forsythia, A. ctinomycetemcomitans, T. denticola, P. intermedia, F. nucleatum/periodonticum, and P. endodontalis in the dental plaque was performed with a Dentoscreen kit. The presence of other bacterial groups was verified by metagenomic sequencing of the 16S rRNA gene to evaluate some specific features of the etiological factor for periodontitis in type 2 diabetes mellitus. Specimens of the Porphiromonadaceae and Fusobacteriaceae families were characterized by an extremely high incidence in combined pathology. The amount of Sphingobacteriaceae bacteria in the biofilm was shown to decrease significantly during periodontitis. Metagenomic analysis confirmed the pathogenic role of microbiota in combined pathology, as well as the hypothesis on a possible influence of periodontitis on the course and development of type 2 diabetes mellitus.

[MOLECULAR MECHANISMS OF DRUG RESISTANCE NEISSERIA GONORRHOEAE HISTORY AND PROSPECTS].

Neisseria gonorrhoeae (gonococcus) is a strict human pathogen, which causes gonorrhea--an infectious disease, whose origin dates back to more than two thousand years. Due to the unique plasticity of the genetic material, these bacteria have acquired the capacity to adapt to the host immune system, cause repeated infections, as well as withstand antimicrobials. Since the introduction of antibiotics in 1930s, gonococcus has displayed its propensity to develop resistance to all clinically useful antibiotics. It is important to note that the known resistance determinants of N. gonorrhoeae were acquired through horizontal gene transfer, recombination and spontaneous mutagenesis, and may be located both in the chromosome and on the plasmid. After introduction of a new antimicrobial drug, gonococcus becomes resistant within two decades and replaces sensitive bacterial population. Currently Ceftriaxone is the last remaining antibiotic for first-line treatment of gonorrhea. However, the first gonococcus displaying high-level resistance to Ceftriaxone was isolated in Japan a few years ago. Therefore, in the near future, gonorrhea may become untreatable. In the present review, we discuss the chronology of the anti-gonorrhea drugs (antibiotics) replacement, the evolution of resistance mechanisms emergence and future perspectives of N. gonorrhoeae treatment.

[Analysis of genetic determinants of multidrug and extensively drug-resistant Mycobacterium tuberculosis using oligonucleotide microchip].

Steadily growing resistance of the tuberculosis causative agent towards a broad spectrum of anti-tuberculosis drugs calls for rapid and reliable methods for identifying the genetic determinants responsible for this resistance. In this study, we present a biochip-based method for simultaneous identification of mutations within rpoB gene associated with rifampin resistance, mutations in katG, inhA, ahpC genes responsible for isoniazid resistance, mutations within the regions of gyrA and gyrB genes leading to fluoroquinolones resistance, and mutations in the rrs gene and the eis promoter region associated with the resistance to kanamycin, capreomycin and amikacin. The oligonucleotide microchip, as the core element of this assay, provides simultaneous identification of 99 mutations in the format "one sample--one PCR--one microchip", and it makes it possible to complete analysis of multi-drug-resistant and extensively drug-resistant tuberculosis within a single day. The tests on 63 Mycobacterium tuberculosis clinical isolates with different resistance profiles using the developed approach allows us to reveal the spectrum of drug-resistance associated mutations, and to estimate the significance of the inclusion of extra genetic loci in the determination of M. tuberculosis drug resistance.

[Microbiological and molecular genetic characteristics of coagulase-negative staphylococcal isolates from neonates in intensive care unit].

The problem of hospital-acquired infections due to coagulase-negative staphylococci (CoNS) in neonatal intensive care units is crucial over the last 20 years in the world. Neonates with very low or extremely low body weight belong to a special group of risks by the CoNS infection. However, in Russia CoNS up to now are frequently considered as contaminants and not as the main etiologic factors of pneumonia and sepsis in extremely premature infants. It was shown that hospital strains of CoNS causing fatal infections in extremely premature infants are always present in intensive care units.

[Susceptibility of gramnegative carbapenemase-producing bacteria to various group antibiotics].

The study involved 25 isolates of gramnegative carbapenemase-producing bacteria. 17 isolates of Klebsiella pneumonia produced carbapenemase NDM-1 and were highly resistant to cephalosporins (MIC>128 mcg/ml), carbapenems (MIC>16 mcg/ml), aminoglycosides and fluoroquinoiones, while among them 4 isolates preserved susceptibility to azthreonam and all of them were susceptible to tigecycline and polymyxin. 2 isolates of Acinetobacter genomospecies 13 produced NDM-1 and were resistant to all the beta-lactams and amikacin, while preserved susceptibility to gentamicin, co-trimoxazole, tigecycline and polymyxin, the susceptibility to ciprofloxacin being lowered. Carbapenemase VIM-4 was produced by 2 isolates of Enterobacter cloacae, which were highly resistant to cephalosporins and azthreonam, significant synergism being observed between cefepim and clavulanate. The resistance of the isolates to carbapenems was low (MIC 0.5-4.0 mcg/ml), they also being resistant to aminoglycosides and ciprofioxacin and susceptible to tigecycline and polymyxin. Carbapenemases KPC-2 were detected in 2 isolates of K.pneumoniae and in 1 isolate of E.cloacae. The above isolates were resistant to all the beta-lactams, ciprofloxacin, aminoglycosides and co-trimoxazole. I isolate of E.cloacae showed resistance to tigecychine and I isolate of K.pneumoniae was resistant to polymyxin. Carbapenemase OXA-48 was detected in 1 isolate of K.pneumoniae. It was resistant to all the beta-lactams, ciprofloxacin and co-trimoxazole and susceptible to aminoglycosides, tigecycline and polymyxin.

Molecular typing of Mycobacterium tuberculosis circulated in Moscow, Russian Federation.

The present study investigates epidemiological diversity and multidrug resistance spreading among Mycobacterium tuberculosis strains circulating in Moscow, Russian Federation. Among 115 M. tuberculosis strains selected randomly from the sputum of epidemiologically unrelated tuberculosis (TB) patients, multidrug-resistant (MDR) strains predominated. Mutations in the RRDR of the rpoB gene were detected in 64 (83.1%) of 77 rifampicin (RIF)-resistant strains. The Ser531→Leu substitution was prevalent among them (76.5%). Aberrations in the Ser315 codon of katG and/or in the inhA promoter region were found in 79 (84.0%) of 94 isoniazid (INH)-resistant strains. Strains belonging to the Beijing family prevailed. Seventy-one different patterns were identified using the 24-VNTR loci typing scheme. Three main 24-loci VNTR clusters included 34 strains which belonged to the Beijing family. The spoligotyping and 24-loci VNTR typing combination demonstrated maximal discriminatory power. Among the Beijing strains, the MDR phenotype was revealed more frequently than among the others. High genetic heterogeneity of the studied population was shown by the assessment of VNTR loci variability in the analyzed group and in the strains from other parts of Russia. Comparison of the 24-VNTR locus typing and spoligotyping data with revealed resistance-associated mutation allows us to make a suggestion that the active transmission of MDR strains and the independent appearance of drug resistance during chemotherapy occurred in the studied population simultaneously.

[Differentiation of α-hemolytic Streptococci by direct mass spectrometry profiling].

The modern phenotypic and genetic methods except for Multi Locus Sequence Typing do not allow the reliable differentiation within Mitis group of α-hemolytic streptococci. During this study the MALDI mass spectra were acquired for 28 clinical isolates initially identified as S. pneumoniae by routine bacteriological tests. Due to Multi Locus Sequence Typing these isolates were found to belong to two closely related species - S. pneumoniae (n = 22) and S. mitis (n = 6). Distribution of those isolates in accordance with cluster analysis of collected mass spectra matched to Multi Locus Sequence Typing data. The diagnostic model based on Genetic Algorithm classifier demonstrated the differentiation of α-hemolytic streptococci with 100% sensitivity and 94.6% accuracy. Statistical analysis of MS peak areas revealed 2 peaks which are different for S. mitis and S. pneumoniae groups.

[Population pattern of pneumococci with lower susceptibility to penicillin and prospects of antipneumococcal vaccination to control antibiotic resistance distribution].

Large-scale antipneumococcal vaccination is followed by changes in the serotype composition and level of antibiotic resistance in pneumococci. The aim of the study was to evaluate the serotype composition and population pattern of pneumococci with lower susceptibility to penicillin before large-scale antipneumococcal vaccination. Among 260 Streptococcus pneumoniae strains isolated in the Russian Federation within 2003-2007, serotypes 23F (37.2%) and 19F (13.9%) were the most frequent ones. 19.3% of the isolates belonged to serogroup 6, 3.6% of the isolates each belonged to serotype 3 and serogroup 18, 4.9% of the isolates belonged to serotype 14 and 2.2% of the isolates belonged to serotype 19A. 66.8% of the isolates belonged to serotypes of the 7-valent conjugated pneumococcal vaccine, 67.3 and 82.1% of the isolates belonged to the 10- and 13-valent conjugated pneumococcal vaccines respectively. The isolates with lower susceptibility to penicillin were characterized by significant clonality and 56.9% of them belonged to 4 global clonal complexes (CC81, CC156, CC320 and CC315). Inclusion of the conjugated antipneumococcal vaccine to the National Vaccination Time-Table of the Russian Federation could promote lower levels of antibiotic resistance in pneumococci.

[Investigation of molecular mechanisms of aminoglycoside resistance in Salmonella].

The spread of aminoglycoside resistance phenotype and respective genetic resistance determinants was evaluated in 243 Salmonella strains isolated within 1948-2010 and stored in the Culture Collection of the Russian State Research Institute for Control, Standardization and Certification of Veterinary Preparations (Moscow). The Salmonella strains showed resistance to streptomycin and gentamicin in 3.7% (n = 9) and 0.8% (n = 2) of the isolates respectively. Intermediate resistance to streptomycin was recorded in 9.9% (n = 24) of the isolates. To detect the genes responsible for the aminoglycoside resistance, primers for aadA1, aadA2, aadB, aphA1, aphA3, sat, strA, strB, aphA, aacC, rmtB, armA and rpsL genes amplification and sequencing were designed. The strains with lower susceptibility to streptomycin harbored aadA1, aadA2, strA, strB resistance genes encoding enzymes for aminoglicoside modification and rpsL mutant allele (K42N, G91D). Genetic mechanisms able to explain the gentamicin resistance development were not detected. Some strains carried genetic markers of streptomycine resistance but had no clinically sufficient resistance to it. In this regard, genetic testing is essential for prevention of drug resistance spreading due to horizontal transfer of genes in microbial population.

[A mass-spectrometric analysis of genetic markers of S. pneumoniae resistance to beta-lactam antibiotics].

Beta-lactam antibiotics remain the drugs of choice for treatment of S. pneumoniae infections in spite of growing level of resistance. The formation of S. pneumoniae resistance to these drugs is mediated by modifications of the penicillin-binding proteins (PBPs), the targets of the antibiotic action. A new approach to detection of mutations in PBP1A, 2B and 2X genes based on minisequencing reaction followed by MALDI-ToF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) mass spectrometry was developed in this study. The evaluation of these mutations prevalence in clinical S. pneumoniae isolates (n = 194) with different susceptibility level to beta-lactam antibiotics was performed. Twenty-four different combinations of mutations in PBPs (genotypes) were detected. All isolates susceptible to penicillin (n = 49, MIC > or = 0.06 > or = gamma/ml) carried no mutations in all analyzed loci. For 145 S. pneumoniae isolates with reduced susceptibility to penicillin (MIC > 0.06 > or = gamma/ml) the mutations in PBPs were detected in 133 (91.7 %) cases that testify to high diagnostic sensitivity of such approach. The isolates with MIC > or = 4 > or = gamma/ml (n = 20) carried multiple mutations in all analyzed genes that confirms cumulative effects of penicillin resistance formation. However, it was not possible to associate observed mutations in PBPs genes with decrease of susceptibility to cefotaxime that allows suggesting the entire difference in molecular mechanisms of formation of resistance to penicillins and cephalosporins. The offered method of S. pneumoniae genotyping is suitable for susceptibility testing to penicillin of individual isolates and for molecular monitoring of the resistance determinants in population.

[Molecular characteristic of methicillin-resistant Staphylococcus aureus strains isolated in Moscow clinics].

UNLABELLED: OBJECTIVES; Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen of nosocomial infection. The goal of this work was to evaluate the clonality of hospital-acquired MRSA (HA-MRSA) circulating in Russian Federation and to compare different multiplex PCR techniques with SNP-based approach for MRSA typing. METHODS: Epidemiologically unrelated MRSA isolates (n = 62) from Moscow hospitals were selected for typing. Genomic DNA from clinical isolates was purified using the DNA express kit (Lytech Ltd, Russia). Staphylococcus chromosomal cassette mec (SCCmec) typing was performed by PCR using the previously described methods. Seven loci from five housekeeping genes (arcC162, arcC210, aroE132, gmk123, tpi241, tpi243 and yqiL333) were used for SNP-typing. Detection of particular nucleotides in selected loci was carried out in the thermocyclic primer extension reaction, followed by mass spectrometry of the products. Standard MLST procedure was performed as reference method. RESULTS: The majority of the MRSA isolates (93.6%) belong to world-wide disseminated clonal complex (CC) 8. Three isolates (4.8%) belong to CC 1. All ST 239 isolates were found to carry SCCmec type III; ST 8 isolates, SCCmec type IV. CONCLUSION: Among Russian MRSA CC 8 isolates carrying SCCmec IV type are predominant. SNP-typing is powerful toll for studies of molecular epidemiology of MRSA.

[Mass spectrometry of nucleic acids in molecular medicine].

A stable streamlining trend in the field of medical diagnostics by practical adoption of high-tech and knowledge-intensive analytical systems providing for molecular level studies has appeared during the last few decades. An illustrative example of such technologies is mass spectrometry methods for analyzing biomolecules. This review is intended to brief the potential of the state-of-the-art inventory of spectrometry equipment and illustrate the application of mass spectrometry of nucleic acids (DNA and RNA) for solving practical problems related to the analysis of human genomic DNA and clinically significant microorganisms of bacterial and viral natures.

[HCV genome variability in acute and chronic viral hepatitis C].

AIM: To evaluate HCV genome variability in acute and chronic phases of viral hepatitis C. MATERIAL AND METHODS: The study of heterogeneity of HCV in acute hepatitis C has detected genetic heterogeneity and variability of individual HCV population circulating in the blood. Significant genetic heterogeneity of HCV was observed in 1b, 2a and 3a genotypes. Variability of HCV did not depend on virus load. Genetic HCV structure changed significantly both in patients with manifest ALT deviations and in normal ALT, mean number of HCV genetic variants in these groups being the same. No significant correlations were found between virus concentration in the patient's blood, its variability and ALT values. Genetic heterogeneity of interferon-sensitive region of gene NS5A subtype 1b HCV was studied in blood of 16 patients with chronic hepatitis C resistant to interferon therapy. RESULTS: It is shown that genetic heterogeneity and variability of an individual HCV population circulating in blood serum can not be a prognostic criterion in assessment of variants of acute hepatitis C course. No mutations in ISDR region were found in 25% of 16 patients studied. 75% cases had 1-3 replacements of amino acid sequences, most frequent mutation was replacements in position 2218 (histidin/arginin). The above results are close to those obtained in Japanese and European populations. Results of ISDR sequence-analysis conducted before treatment may predict efficacy of interferon-alpha2 treatment in an individual patient in future. Large-scale trials are necessary for detection of mutations responsible for resistance to interferon-alpha2 in patients living in Russia.

[Detection of mutations in codon 306 of the embB gene for molecular genetic characterization of clinical Mycobacterium tuberculosis strains].

A total of 254 Mycobacterium tuberculosis strains were used in the study. Among them, there were 183 ethambutol (EMB)-resistant strains, 13 multidrug resistant ones, but EMB-sensitive, and 39 strains sensitive to rifampicin (RIF), isoniazid (INZ), and EMB. All the strains were analyzed for genetic changes in three loci: embB306, rpoB, and katG/inhA promoter, which were associated with the formation of resistance to EMB, RIF, and INZ, respectively. The Mycobacterium tuberculosis strains were obtained from pulmonary tuberculosis patients living in the Central Region of the Russian Federation. Resistance to RIF, INZ, and EMB was revealed by the absolute concentration test. The inhibitory concentration (IC) of EMB was determined for all the strains. Genetic changes in the above loci were estimated by mini-sequencing, followed by mass-spectrometry recording MALDI-TOF products. The relative low frequency of embB306 mutations was observed among the EMB-resistant strains (about 41.5%). Mutations in codon 306 were detected only in strains with EMB IC > or = 2 mg/L. A statistical significant association was found between the frequency of embB306 mutations and the multidrug resistant phenotype. A combination of these mutations with the traditional genetic markers of multidrug resistance may be used for the more effective detection of multidrug-resistant strains.

A MALDI TOF MS-based minisequencing method for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains of Enterobacteriaceae.

A minisequencing method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) was developed for rapid identification of single nucleotide polymorphisms at bla(TEM) gene codons 104, 164 and 238 associated with extended-spectrum activity on TEM-type beta-lactamases. The method was validated by testing the Escherichia coli and Klebsiella pneumoniae strains possessing the known bla(TEM) gene sequences.

[Study on prevalence and molecular mechanisms of antibacterial resistance in Gram-negative pathogens of nosocomial infections in hospitals of Moscow and Perm'].

A significant increase of resistance in out-of-hospital and hospital pathogens to antimicrobials is being lately observed all over the world, including the Russian Federation. Within 2005-2007 clinical strains of potential producers of extended spectrum beta-lactamases were isolated in diverse medical centres. The isolates suspected to produce extended spectrum beta-lactamases by the criteria of CLSI (Clinical and Laboratory Standards Institute, USA) were included into the test with the polymerase chain reaction and sequence for detecting the genes of beta-lactamases TEM, SHV, CTX, OXA and MBL by the original primers. The total of 99 isolates of gramnegative bacteria suspected to produce extended spectrum beta-lactamases by the findings of the local laboratories were tested at the National Research Centre of Antibiotics: 44 strains of the family Enterobacteriaceae, 38 strains of P. aeruginosa and 17 strains of A. baumanii. The Enterobacteriaceae isolates were mainly multiple resistant (29.5%) carrying simultaneously the genes of 4 groups of beta-lactamases (TEM, SHV, CTX and OXA). The investigation revealed 1 strain of E. coli having the gene of metal-beta-lactamase VIM. The strains of A. baumanii mainly contained the genes of beta-lactamases OXA (23%). Group CTX-M1 prevailed (95%) among the detected genes CTX, 2 strains of group CTX-M9 were also isolated. The mechanisms of antibiotic resistance were not elucidated in 35 strains of P. aeruginosa (92%), gene TEM being detected only in 1 strain and gene MBL (metal-beta-lactamase) being detected in 2 strains. The prevalence of multiple resistant strains carrying the beta-lactamase genes OXA and TEM in the medical centres was shown. The most negative fact was isolation of the E. coli strain producing metal-beta-lactamase.

[MALDI-ToF mass-spectrometry in analysis of genetically determined resistance of Streptococcus pneumoniae to fluoroquinolones].

New fluoroquinolones with higher antipneumococcal activity are considered promising in the treatment of respiratory tract infections. Still, their wide use in clinical practice is connected with possible selection and rapid distribution of the resistance, requiring constant monitoring. Development of resistance to fluoroquinolones results from step-wise accumulation of mutations in the genes of DNA-gyrase and topoisomerase IV, the mutations of the first step being not always accompanied by a significant increase of the MIC of the new fluoroquinolones. Therefore, to detect the first signs of the resistance development, it is necessary not only to detect the susceptibility of the circulating Streptococcus pneumoniae strains phenotypically, but also to detect the genetic changes. In the present study the minisequent reaction followed by detection of the reaction products by MALD-ToF mass-spectrometry was used to reveal the mutations in the genes of the fluoroquinolone targets of 38 S. pneumoniae strains with different levels of the resistance to ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin. In the strains with high resistance to all the three fluoroquinolones (MIC 4-16 mcg/ml) there were detected mutations in GyrA (Ser81Tyr or Glu85Zys) and as well in ParC (Ser79Phe or Ser79Tyr). In the strains resistant to ofloxacin and ciprofloxacin (MIC 4-8 mcg/ml) with preserved susceptibility to levofloxacin and moxifloxacin, the mutations were detected only in GyrA (Ser114Gly). In the moderately resistant strains (MICs 4 and 2-4 mcg/ml respectively for ofloxacin and ciprofloxacin) there were detected the known mutations in ParC (Ser79Tyr or Ser79Phe or Asp83Tyr) and in GyrB (Glu475Lys) as well as the earlier not described mutations in ParE (ins Asn381a) and in Gyr B (Thr329Ala or Va1355Ile). The described method can be used in mass screening of S. pneumoniae strains for the presence of mutations in the genes of the fluoroquinolone targets.

[Detection of fluoroquinolone resistance SNPs in gyrA and parC genes of Neisseria gonorrhoeae using MALDI-TOF mass-spectrometry].

For many known mechanisms of the drug resistance in microorganisms are described genetic markers (specific changes in the genome of microorganism, in the majority of the cases representing single nucleotide polymorphism). The search for the new methods, which make possible to identify single nucleotide changes with the greater effectiveness and at smaller prime is actual for the solution of the problem of the identification of the resistant strains. In this work a new approach of the determination of single nucleotide polymorphisms is proposed. It is based on the reactions of mini-sequencing and/or sequencing with the subsequent Matrix-Assisted Laser Desorption/Ionisation Time Of Flight Mass-Spectrometry (MALDI-TOF MS) of the reaction products. The approach was tested on a clinical group of Neisseria gonorrhoeae strains to investigate specific single nucleotide polymorphisms in genes gyrA and parC (the genetic markers of the bacterium fluoroquinolone resistance). The results of the nucleotide polymorphism deter- mination was completely agreed with the data, obtained earlier with the use of a "gold standard" (sequencing with the classical gel-electrophoresis separation of the reaction products). There is specific interest in the method of sequencing of the short DNA sequences using MALDI-TOF MS. The new high-throughput approach of the single nucleotide polymorphisms determination in bacterial genes considerably increases the effectiveness of the methods of microorganism's identification, genotyping and determining the genetic markers of the drug resistance.

[A2144G is the main mutation in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance].

To detect point mutations A2115C, A2143G/C, and A2143G in the 23S rRNA gene of Helicobacter pylori associated with resistance of the microorganism to clarithromycin, a new powerful way of analysis was used. This method involved the reaction of minisequencing followed by MALDI-TOF mass spectrometry of reaction products. In ten analyzed clarithromycin-resistant clinical isolates of H. pylori obtained in Russia, the resistance was found to be mediated only by mutation A2144G in the 23S rRNA gene.

[Detection of direct markers of cytomegalovirus. Research of the spectrum and avidity of antiviral antibodies in infants and preschool children].

Thirty-three children aged 1 month to 3 years were examined within the case study. spELISA, immunoblot (IB), shell vial method (SVM) and PCR, were used for the detection of anti-CMV IgM and IgG, in the diagnosis of cytomegalovirus (CMV). Clinical signs of CMV infection (CMVI) were registered in 20 children (group 1); no CMVI specific signs were detected in the remaining 13 children (group 2). Class M antibodies were identified in 50% of group-1 sera. Around 80% of children in the group had anti-CMV-IgG. AI < 0.6 was in 3 (20%) of 15 examinees. Direct CMV markers (DNA and infection activity) were detected in 13 (65%) of 20 children. Sera of 13 children with non-specific symptomatology (group 2) had no anti-CNV-IgM, while IgG were found in 54% examinees in the group. The infectious active virus was not detected in a single baby. The used laboratory tools enhance the efficiency of CMVI diagnosis and denote a disease variation.