DFT Study of Nitroxide Radicals. 1. Effects of solvent on structural and electronic characteristics of 4-amino-2,2,5,5-tetramethyl-3-imidazoline-N-oxyl

Imidazoline-based nitroxide radicals are often used as spin probes for medium acidity and polarity in different systems. In this work, using the density functional theory (DFT) approach, we have studied how physico-chemical characteristics (geometry, atomic charges and electron spin density distribution) of pH-sensitive spin label 4-amino-2,2,5,5-tetramethyl-3-imidazoline-N-oxyl (ATI) depend on protonation and aqueous surroundings. Our calculations demonstrate that ATI protonation should occur at the nitrogen atom of the imidazoline ring rather than at the amino group. Protonation of ATI leads to a decrease in a spin density on the nitrogen atom of the nitroxide fragment >N-O. For simulation of ATI hydration effects, we have constructed a water shell around a spin label molecule by means of gradual (step-by-step) surrounding of ATI with water molecules (n = 2-41). Calculated spin density on the nitrogen atom of the nitroxide fragment increased with an extension of a water shell around ATI. Both protonation and hydration of ATI caused certain changes in calculated geometric parameters (bond lengths and valence angles). Investigating how structural and energy parameters of a system ATI-(H2O)n depend on a number of surrounding water molecules, we came to the conclusion that a hydrogen-bonded cluster of n ≥ 41 water molecules could be considered as an appropriate model for simulation of ATI hydration effects.Comment: 30 pages, 11 figures, 6 table

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

AbstractAccurate 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

Typing of Lymphogranuloma Venereum Chlamydia trachomatis Strains

We analyzed by multilocus sequence typing 77 lymphogranuloma venereum Chlamydia trachomatis strains from men who have sex with men in Europe and the United States. Specimens from an outbreak in 2003 in Europe were monoclonal. In contrast, several strains were in the United States in the 1980s, including a variant from Europe

MLVA Subtyping of Genovar E Chlamydia trachomatis Individualizes the Swedish Variant and Anorectal Isolates from Men who Have Sex with Men

This study describes a new multilocus variable number tandem-repeat (VNTR) analysis (MLVA) typing system for the discrimination of Chlamydia trachomatis genovar D to K isolates or specimens. We focused our MLVA scheme on genovar E which predominates in most populations worldwide. This system does not require culture and therefore can be performed directly on DNA extracted from positive clinical specimens. Our method was based on GeneScan analysis of five VNTR loci labelled with fluorescent dyes by multiplex PCR and capillary electrophoresis. This MLVA, called MLVA-5, was applied to a collection of 220 genovar E and 94 non-E genovar C. trachomatis isolates and specimens obtained from 251 patients and resulted in 38 MLVA-5 types. The genetic stability of the MLVA-5 scheme was assessed for results obtained both in vitro by serial passage culturing and in vivo using concomitant and sequential isolates and specimens. All anorectal genovar E isolates from men who have sex with men exhibited the same MLVA-5 type, suggesting clonal spread. In the same way, we confirmed the clonal origin of the Swedish new variant of C. trachomatis. The MLVA-5 assay was compared to three other molecular typing methods, ompA gene sequencing, multilocus sequence typing (MLST) and a previous MLVA method called MLVA-3, on 43 genovar E isolates. The discriminatory index was 0.913 for MLVA-5, 0.860 for MLST and 0.622 for MLVA-3. Among all of these genotyping methods, MLVA-5 displayed the highest discriminatory power and does not require a time-consuming sequencing step. The results indicate that MLVA-5 enables high-resolution molecular epidemiological characterisation of C. trachomatis genovars D to K infections directly from specimens

Identification of Streptococcus pneumoniae by a real-time PCR assay targeting SP2020.

Real-time PCR targeting lytA (the major autolysin gene) and piaB (permease gene of the pia ABC transporter) are currently used as the gold-standard culture-independent assays for Streptococcus pneumoniae identification. We evaluated the performance of a new real-time PCR assay - targeting SP2020 (putative transcriptional regulator gene) - and compared its performance with the assays previously described. A collection of 150 pneumococci, 433 non-pneumococci and 240 polymicrobial samples (obtained from nasopharynx, oropharynx, and saliva; 80 from each site) was tested. SP2020 and lytA-CDC assays had the best performance (sensitivity of 100% for each compared to 95.3% for piaB). The specificity for lytA and piaB was 99.5% and for SP2020 was 99.8%. Misidentifications occurred for the three genes: lytA, piaB and SP2020 were found in non-pneumococcal strains; piaB was absent in some pneumococci including a serotype 6B strain. Combining lytA and SP2020 assays resulted in no misidentifications. Most polymicrobial samples (88.8%) yielded concordant results for the three molecular targets. The remaining samples seemed to contain non-typeable pneumococci (0.8%), and non-pneumococci positive for lytA (1.7%) or SP2020 (8.7%). We propose that combined detection of both lytA-CDC and SP2020 is a powerful strategy for the identification of pneumococcus either in pure cultures or in polymicrobial samples

Multicenter evaluation of the vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of gram-positive aerobic bacteria

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting

Видовая идентификация и анализ генетических маркеров лекарственной устойчивости стрептококков с помощью количественной мультиплексной полимеразной цепной реакции у пациентов с хронической обструктивной болезнью легких

The aim of this study was to develop a new diagnostic tool for Streptococcus identification and simultaneous macrolide resistance genetic marker detection in chronic obstructive pulmonary disease (COPD) patients. Methods. The "Streptopol +" experimental diagnostic panel (Lytech Ltd) was used to test laboratory collection of Streptococcus strains and DNA samples isolated from oropharyngeal swabs of 89 patients with stable COPD with length of disease ≥ 12 months, smoking history ≥ 10 pack / years, no exacerbations during the previous 4 weeks and no antibiotic therapy during the previous 12 weeks prior to study entry. Resluts. The "Streptopol +" experimental set allowed detection of drug resistance genetic markers and Streptococcus species identification based on a multiplex real-time PCR. Streptococcus was determined reliably in 83 (83 / 89; 93.3 %) samples obtained from COPD patients; Streptococcus was not found in one sample; 5 samples were in a "gray" zone with low DNA titers. Mainly, Streptococcus was identified as S. viridans, mitis group which was revealed to be as a reservoir for macrolide resistance genetic determinants. This increases a risk of macrolide resistance and therapeutic failure of these drugs. The mef genes were detected in all samples (89 / 89; 100 %), ermB gene was less frequent (81 / 89; 91.0 %). Conclusion. The "Streptopol +" experimental diagnostic panel has shown a high specificity and sensitivity to diagnose Streptococcus infection in COPD patients. На лабораторной коллекции клинических штаммов стрептококков апробирована и использована для тестирования клинических образцов ДНК, изолированных из орофарингеальных мазков от пациентов со стабильной хронической обструктивной болезнью легких, экспериментальная диагностическая панель "Стрептопол+" (ООО НПФ "Литех"). В исследование включены пациенты (n = 89) с анамнезом заболевания ≥ 12 мес., индексом курения ≥ 10 пачко-лет, отсутствием обострений на протяжении предшествующих 4 нед. и терапии антибактериальными препаратами в течение 12 нед. до взятия клинического материала. При использовании экспериментального набора "Стрептопол+" обнаружены генетические маркеры лекарственной устойчивости и проведена видовая идентификация стрептококков по принципу мультиплексной полимеразной цепной реакции в реальном времени. Достоверно стрептококки определялись в 83 (93,3 %) образцах, в 1 образце стрептококков не обнаружено, 5 образцов были отнесены в "серую" зону, характерную для низких титров ДНК (преимущественно зеленящие стрептококки группы mitis). Выявлено, что все они являются резервуаром генетических детерминант резистентности к макролидам, что повышает риск микробиологической резистентности и терапевтической неэффективности препаратов данной группы. Присутствие генов mef было зафиксировано в 89 (100 %) образцах; ген ermB обнаружен в 81 (91,0 %) образце.

Chlamydia trachomatis Strains Show Specific Clustering for Men Who Have Sex with Men Compared to Heterosexual Populations in Sweden, the Netherlands, and the United States

High-resolution genotyping of Chlamydia trachomatis improves the characterization of strains infecting different patient groups and sexual networks. In this study, multilocus sequence typing (MLST) and ompA sequence determination were used for an analysis of C. trachomatis strains from 203 men who have sex with men (MSM) from Sweden, the Netherlands, and the United States. The results obtained were compared with data from 153 heterosexual women from Sweden and the Netherlands. The overlap in MLST/ompA profiles between MSM from Sweden and the Netherlands was 68%, while the overlap between heterosexual populations from these countries was only 18%. The distribution of genotypes in MSM from the United States was less similar to that in MSM from the European countries, with 45% and 46% overlaps for MSM in Sweden and the Netherlands, respectively. Minimum-spanning-tree analysis of MLST/ompA sequence types identified two large clusters that contained almost exclusively samples from MSM and comprised 74% of all MSM samples. Three other clusters were predominated by samples from women but also contained MSM specimens. Of 19 detected variants of the MLST target CT144, three variants were highly associated with MSM. Our study supports the hypotheses of both tissue tropism as well as epidemiological network structures as explanations for the linkage between specific genetic variants and sexual orientation

Applications of MALDI-TOF Mass Spectrometry in Clinical Diagnostic Microbiology

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) represents one of the most accurate, reliable, and fast methods for the identification of bacterial strains from positive cultures, and therefore it has largely replaced all other previously used approaches for microbial identification. The main application of MALDI-TOF MS in clinical microbiology laboratories is the identification of bacteria from colonies recovered from solid culture media. This chapter discusses specific identification procedures that are needed for some bacteria, such as Actinomycetes and Mycobacteria. The performance of MALDI-TOF MS identification relies on the number of mass spectra that reach the quality allowing identification and the number of correct identifications. MALDI-TOF MS has also been proposed for Staphylococcus aureus strain typing or for the detection of biomarkers of the most virulent toxigenic isolates. MALDI-TOF MS could also be used for Mycobacterium