[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.

Hepatitis B virus genetic typing using mass-spectrometry.

Mini-sequencing with subsequent result registration using MALDI-ToF mass-spectrometry was employed for hepatitis B virus genetic typing in Russian population. This approach was employed for hepatitis B virus genetic typing in HBsAg-positive patients with chronic hepatitis B, hepatitis of combined etiology and hepatic cirrhosis and allowed to show the prevalence of D genotype (83.3%) in all groups of patients. Other hepatitis B virus genotypes: genotype A (5.9%), genotype C (3.6%), and mixed infection with D and C (7.2%) were also found in patients with chronic hepatitis B and hepatic cirrhosis. All genotypes were found in patients with chronic hepatitis B and hepatic cirrhosis. Chronic hepatitis of combined etiology was noted only in patients with genotype D. Possibility of detection of mixed infection with hepatitis B viruses of various genotypes is a distinct advantage of mini-sequencing approach over direct nucleotide sequence evaluation for hepatitis B virus genetic typing.

[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.

Inhibitory effect of streptococci on the growth of M. catarrhalis strains and the diversity of putative bacteriocin-like gene loci in the genomes of S. pneumoniae and its relatives.

S. pneumoniae is a facultative human pathogen causing a wide range of infections including the life-threatening pneumoniae or meningitis. It colonizes nasopharynx as well as its closest phylogenetic relatives S. pseudopneumoniae and S. mitis. Both the latter, despite the considerable morphological and phenotypic similarity with the pneumococcus, are considerably less pathogenic for humans and cause infections mainly in the immunocompromized hosts. In this work, we compared the inhibitory effect of S. pneumoniae and its relatives on the growth of Moraxella catarrhalis strains using the culture-based antagonistic test. We observed that the inhibitory effect of S. mitis strains is kept when a hydrogen peroxide produced by cells is inactivated by catalase, and even when the live cells are killed in chloroform vapors, in contrast to the pneumococcus whose inhibiting ability disappeared when the cells die. It was suggested that this effect may be due to the production of bacterial antimicrobial peptides by S. mitis, so we examined the genomes of our strains for the presence of bacteriocin-like peptides encoding genes. We observed that a set of bacteriocin-like genes in the genome of S. mitis is greatly poorer in comparison with S. pneumoniae one; moreover, in one S. mitis strain we found no bacteriocin-like genes. It could mean that there are probably some additional opportunities of S. mitis to inhibit the growth of competing neighbors which are still have to be discovered.

[Resistance of Russian isolates of Neisseria gonorrhoeae to fluoroquinolones].

Fluoroquinolones still belong to the drugs of choice in the treatment of uncomplicated gonorrhea. At the same time, there have been more data on the spreading N. gonorrhoeae strains resistant to fluoroquinolones. A variety of mechanisms, like modification of the target of antibiotic's action (point mutations in genes gyrA and parC), a decreasing permeability of the bacterial cell membrane (amino-acid changes Por protein) and a growing efflux of antibiotic (mutations in the promoter or in the coding region of mtrR) mediate in the shaping resistance of the drugs. The MIC values for four fluoroquinolone-series antibiotics were determined and the gyrA, parC, por and mtrR genes were examined for resistance-responsible mutations in 32 studied clinical strains of N. gonorrhoeae. Strains with high resistance to fluoroquinolones were detected; 3 of them had no common changes in GyrA or ParC, however, amino acid changes and mutations were detected in Por protein and promoter or gene mtrR encoding region, respectively. The paper contains priority data on the detection (in Russia) of N. gonorrhoeae strains with high resistance to fluoroquinolones. Involvement of different mechanisms in the process of resistance shaping is discussed. The results are of practical importance for planning the antibacterial therapy of gonorrhoeae; they point out the need in regional testing of resistance in the N. gonorrhoeae population encountered in Russia.

[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.

[Detection of the markers of herpes simplex virus and cytomegalovirus in newborns and infants].

A total of 111 children suspected for herpesvirus infection were examined. In blood and urine samples the infectious activity of herpes simplex virus (HSV) and cytomegalovirus (CMV) was detected by the rapid culture method (RCM) and the presence of virus DNA--by the polymerase chain reaction (PCR). HSV and/or CMV were detected by two laboratory methods in 57 examined children (51%). Of these, in 18 children (16.2%) both HSV and CMV were detected. The coincidence of the results of the detection of HSV and CMV by these two methods was observed in 72.4% and 75.2% of cases respectively. The comparative analysis of the detection of anti-CMV IgG and IgM was made with the use of commercial test systems produced bythe following manufacturers: "Vector-Best" and "Bioservice" (Russia), "HUMAN" and "Boehringer" (Germany). The effective detection of both anti-CMV (IgG and IgM) was ensured by the test systems "Boehringer". The test system "Vector-Best" for anti-CMV IgG proved to be not inferior as regards sensitivity and specificity. The German test systems demonstrated the highest specificity in the detection of low-avid antibodies. The data obtained in this study indicate that the detection rate of HSV and CMV markers in newborns and infants suspected for herpesvirus infection was, on the average, 20 - 40%. Reliable diagnostics in newborns and infants is possible only in the presence of the combination of at least 2 serological tests (the determination of antivirus IgM and IgG avidity) and 2 methods for the detection of direct herpesvirus markers (PCR and RCM).

[Comparative efficacy of laboratory methods for cytomegalovirus detection in autopsy material]. / Sravnenie éffektivnosti laboratornykh metodov vyiavleniia tsitomegalovirusa v autopsiinom materiale.

Eighty eight autopsy specimens obtained from 30 fetuses, still-borns and infants died during the first year of life, all suspected for congenital virus infection at postmortem examination, were studied. The specimens were analyzed by 3 techniques: rapid culture method (RCM) for detection of cytomegalovirus (CMV) infectious activity, the immunocytochemical method for detection of CMV antigen in prints of organs and polymerase chain reaction (PCR) for detection CMV DNA. CMV was detected in 16 out of 26 specimens (61.5%) by PCR, in 43 out of 88 specimens (49%) by RCM and in 15 out of 64 specimens (23%) in prints. The comparison of immune reagents revealed that monoclonal antibodies (McAb) were more specific than polyclonal serum antibodies, as the latter yielded the positive reaction in 10 out of 26 cases (38%), found to be negative in PCR. The data thus obtained indicate that complex techniques, including PCR and RCM in combination with McAb, should be used for evaluation of CMV infection role in child mortality.

Discrimination between Streptococcus pneumoniae and Streptococcus mitis based on sorting of their MALDI mass spectra.

Accurate species-level identification of alpha-hemolytic (viridans) streptococci (VGS) is very important for understanding their pathogenicity and virulence. However, an extremely high level of similarity between VGS within the mitis group (S. pneumoniae, S. mitis, S. oralis and S. pseudopneumoniae) often results in misidentification of these organisms. Earlier, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been suggested as a tool for the rapid identification of S. pneumoniae. However, by using Biotyper 3.0 (Bruker) or Vitek MS (bioMérieux) databases, Streptococcus mitis/oralis species can be erroneously identified as S. pneumoniae. ClinProTools 2.1 software was used for the discrimination of MALDI-TOF mass spectra of 25 S. pneumoniae isolates, 34 S. mitis and three S. oralis. Phenotypical tests and multilocus gene typing schemes for the S. pneumoniae (http://spneumoniae.mlst.net/) and viridans streptococci (http://viridans.emlsa.net/) were used for the identification of isolates included in the study. The classifying model was generated based on different algorithms (Genetic Algorithm, Supervised Neural Network and QuickClassifier). In all cases, values of sensitivity and specificity were found to be equal or close to 100%, allowing discrimination of mass spectra of different species. Three peaks (6949, 9876 and 9975 m/z) were determined conferring the maximal statistical weight onto each model built. We find this approach to be promising for viridans streptococci discrimination.

Misidentification of alpha-hemolytic streptococci by routine tests in clinical practice.

Accurate species-level identification of viridans group streptococci (VGS) is very important for understanding of their pathogenicity and virulence. However, an extremely high level of the similarity between VGS, especially Streptococcus pneumoniae, Streptococcus mitis, Streptococcus oralis and Streptococcus pseudopneumoniae, often results in misidentification of these organisms, so there is an urgent need of novel approaches to species identification. A set of 50 randomly selected clinical isolates of alpha-hemolytic streptococci from upper respiratory tract were characterized by the routine phenotypic methods (alpha-hemolysis, colony morphology, Gram stain and optochin susceptibility). Modern proteomic and genetic approaches - the direct bacterial profiling (DBP) by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique and multilocus sequence analysis (MLSA) scheme (http://viridans.emlsa.net/) - were applied for the accurate species identification. After that all isolates were stored at -70°C. Later they were re-inoculated, and a number of additional tests (bile solubility, latex agglutination by commercial "Slidex® pneumo-kit" and repeated optochin test) were performed. A considerable discrepancy was discovered in the results of the different approaches. Looking in the future, one could say that MLSA-like schemes based on the analysis of the nucleotide sequences of seven or more loci of the bacterial genome, appeared to be the most useful instrument in the VGS discrimination, in contrast to the numerous one-target identification schemes, which have been introduced into practice by now.

Direct evaluation of drug resistance parameters in gonococcus.

We carried out complex genetic analysis of clinical samples containing N. gonorrhoeae DNA, the genotype and profile of drug resistance of this agent were evaluated. Changes in genes responsible for the formation of N. gonorrhoeae resistance to penicillins, fluoroquinolones, and spectinomycin were detected during minisequencing with subsequent MALDI-TOF mass spectrometry. The sensitivity of gonococcus was evaluated directly in the clinical sample without culturing.

Analysis of the contribution of molecular mechanisms into formation of gonoccocal resistance to tetracycline.

We applied complex genetic analysis for evaluation of tetracycline-resistance markers in 129 clinical strains of Neisseria gonorrhoeae from Central, Privolzhskii, and Siberian regions. For detection of mutations in rpsJ gene and MtrRCDE locus we first used minisequence reaction followed by identification of products by MALDI-TOF mass spectrometry. The incidence of detection of resistance markers among the analyzed strains were: tetM--3.1%, mutations in genes rpsJ--82.2%, penB--62.8%, and mtrR--54.3%. The analyzed genetic markers were not detected in 17.5% strains. tetM gene was detected in only 12.5% strains from the Central Region. No differences were revealed in regional distribution of other genotypes. Genotypes tetM(pres), rpsJ(mut), mtrR(mut), and rpsJ(mut), penB(mut), mtrR(mut) reliably predict tetracycline resistance. Microbiological and genetic testing of tetracycline resistance yielded similar results.

Analysis of genetic markers of N. gonorrhoeae resistance to beta-lactam antibiotics.

A complex method for detection of genetic markers of N. gonorrhoeae resistance to penicillin was developed. Mutations in penA and ponA genes were detected by minisequencing reaction with subsequent detection of reaction products by MALDI-TOF mass spectrometry. This approach was tested on 31 clinical strains of N. gonorrhoeae with minimum inhibitory concentration of penicillin from 0.03 to 8 microg/ml and higher. Mutations in penA and ponA genes in moderately resistant strains were shown (minimum inhibitory concentration up to 0.5 microg/ml) and mutations in penA, ponA, and penB genes in resistant strains (minimum inhibitory concentration more than 1.0 microg/ml). beta-Lactamase genes were detected in 4 strains with high resistance (minimum inhibitory concentration 4-8 and more microg/ml). Correlation between microbiological resistance and presence of respective mutations in the studied locuses was detected.

[Using the mass spectrometry analysis for hepatitis C virus typing].

Determination of the hepatitis C virus (HCV) genotype has become the standard procedure in laboratory diagnostics of HCV infection. Genotype elucidation has prognostic value assignment helps in assessing disease prognosis and promotes establishing appropriate duration of treatment. Now 11 major genotypes and more than 70 subtypes of HCV have been identified using the sequence variability within 5' non-coding region (5' NCR). In Russia the most common subtypes are 1a, 1b, 2a, 3a and more rare - 4 and 5 types. While the "gold standard" for testing is nucleic acid sequencing, a variety of other assays, including the line probe assay or type-specific amplification, has been developed to provide more rapid and cheaper forms of testing. The aim of this study was to determine the type-specific single nucleotide polymorphism (SNP) in 5' NCR HCV by the classical three-step minisequencing method with followed MALDI-TOF mass spectrometry detection The fragments of 5'NCR of HCV genomes were amplified by the nested RT PCR. The removal of excess nucleotides and primers was performed. Three oligonucleotide primers were design to detect two sets of type-specific SNP in 5' NCR HCV. The primer extension reaction was performed using modified thermostable DNA polymerase and in the presence of ddNTP. The molecular weights of primers extension reaction products were analyzed using MALDI-TOF mass spectrometry. The HCV genotype was determined according the presence in analyses sample the molecules with expected molecular weights. The suggested method was used to type HCV from 69 HCV-positive sera. The 1a genotype was determine in 4.5% samples, 1b - 48%, 2a - 4.5% 3a - 29%, 4 - 1.5%. The mixes of two genotypes were found in 13% samples. All data confirmed by direct nucleic acid sequence. Thus, the new method for HCV typing has been developed using the minisequencing reaction and mass spectrometry for the determination of nucleic acid molecular weight.

Fluoroquinolone-resistant Neisseria gonorrhoeae isolates from Russia: molecular mechanisms implicated.

OBJECTIVES: During a longitudinal study of the prevalence of antimicrobial resistance in Neisseria gonorrhoeae, a number of high-level fluoroquinolone-resistant isolates were obtained from the sexually transmitted diseases clinic in the Moscow region in 2002. The aim of the present study was to determine the molecular mechanisms of resistance and to assess the clonal relationship of these strains METHODS: For the 32 clinical strains of N. gonorrhoeae studied, the MIC values were determined for four fluoroquinolones. The gyrA, parC, por and mtrR genes were studied for the presence of mutations associated with fluoroquinolone resistance. RESULTS: We detected strains of N. gonorrhoeae showing high-level resistance to fluoroquinolones (21 strains, with MICs 1-32 mg/L). Mutations in gyrA and parC known to cause fluoroquinolone resistance were detected in a majority of strains. There were four strains (among 21) without known changes in gyrA and parC. However, amino acid changes in the Por protein and mutations in the promoter or encoding region of the mtrR gene were detected in three of them. One strain had no alteration in gyrA, parC, por or mtrR. CONCLUSIONS: The present study documents the first case of fluoroquinolone-resistant N. gonorrhoeae in Russia.

Molecular typing of N. gonorrhoeae strains prevalent in the Russian Federation.

Genetic polymorphism of Russian population of N. gonorrhoeae was detected and a system for genotyping of its clinical strains was introduced into practice. Comparative analysis of the prevalence of N. gonorrhoeae genotypes in Russia and abroad was carried out. For adaptation of the methods of molecular typing of N. gonorrhoeae strains and its approbation on clinical strains isolated in Russia 41 clinical strains of N. gonorrhoeae were typed. The predominance of PIB serovar (83%) was demonstrated.