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Papers by Tatiana Savinova

Клиническая микробиология и антимикробная химиотерапия, 2017

Molecular Genetics Microbiology and Virology, Sep 1, 2010

Despite the growing level of resistance to Streptococcus pneumoniae infections, β lactam antibiot... more Despite the growing level of resistance to Streptococcus pneumoniae infections, β lactam antibiot ics remain the drugs of choice treating these infections. The resistance of S. pneumoniae to these preparations is mediated by modifications of penicillin binding proteins (PBPs), which are the targets of antibiotics action. The new approach to detecting mutations in the PBP 1A, 2B, and 2X genes based on the minise quencing reaction followed by matrix assisted laser desorption/ionization time of flight (MALDI ToF) mass spectrometry has been developed in the present study. The evaluation of the prevalence of these mutations in clinical S. pneumoniae isolates (n = 194) with different levels of susceptibility to beta lactam antibiotics has been performed. In summary, 24 different combinations of mutations (genotypes) have been detected in PBPs. All penicillin susceptible isolates (n = 49, MIC ≤ 0.06 μg/ml) were characterized by the absence of mutations in all analyzed loci. In PBPs, mutations were detected in 133 (91.7%) out of 145 S. pneumoniae isolates with reduced susceptibility to penicillin (MIC > 0.06 μg/ml), which indicates the high diagnostic sen sitivity of this approach. Isolates with MIC 4 μg/ml (n = 20) possessed multiple mutations in all analyzed genes, which confirms the cumulative effects of the formation of penicillin resistance. At the same time, no association between the presence of mutations in PBP genes and decreased susceptibility to cefotaxime was shown, which makes it possible to suggest significant differences in molecular mechanisms of penicillins and cephalosporins resistance. The suggested method of S. pneumoniae genotyping is appropriate for the individ ual screening of the susceptibility of isolates to penicillin and the molecular monitoring of the resistance determinants in population.

Вестник Российского государственного медицинского университета, 2022

Nechuvstvitel'nye k antibiotikam shtammy Pseudomonas aeruginosa predstavlyayut soboj global&#... more Nechuvstvitel'nye k antibiotikam shtammy Pseudomonas aeruginosa predstavlyayut soboj global'nuyu problemu v zdravoohranenii. Issledovanie mekhanizmov vozniknoveniya rezistentnosti lezhit v osnove razrabotki sposobov bor'by s P. aeruginosa. Cel'yu raboty bylo issledovat' vozniknovenie kross-rezistentnosti u P. aeruginosa v processe adaptacii k populyarnomu antibiotiku meropenemu. Ob"ektami issledovaniya byli obrazcy P. aeruginosa, poluchennye pri roste referentnogo shtamma P. aeruginosa ATCC 27853 na srede s vozrastayushchej koncentraciej meropenema. CHuvstvitel'nost' izolyatov k karbapenemam i kolistinu opredelyali pri pomoshchi razvedeniya v agare, chuvstvitel'nost' k kolistinu ocenivali metodom serijnyh razvedenij. Bylo polucheno 93 izolyata P. aeruginosa, dva iz kotoryh imeli snizhennuyu chuvstvitel'nost' odnovremenno k karbapenemam (meropenem, imipenem) i kolistinu. Genomy izolyatov sekvenirovali na polnogenomnom sekvenatore MGISE...

Molecular Genetics, Microbiology and Virology, 2010

International Journal of Antimicrobial Agents, 2007

S622 17th ECCMID / 25th ICC, Abstracts accepted for publication only isolates. In centre I strain... more S622 17th ECCMID / 25th ICC, Abstracts accepted for publication only isolates. In centre I strains of VREF were prevalent in two departmentsintensive care and haematology. Isolates from intensive care department were more heterogeneous than from haematology department. vanB genes were detected in 9 (7%) of VREF isolates. Eight of them belonged to two PFGE types and were detected only in centre I, one remaining isolate was clonally unrelated and was isolated in centre II. Two isolates of the same PFGE type were carrying two different resistance genes (one-vanA, and one-vanB). Conclusion: The majority of VREF isolates belonged to the two predominant PFGE types. Clonal inter-and intrahospital dissemination was responsible for most of the spread of VREF.

Clinical Microbiology and Infection, 2013

Accurate species-level identification of alpha-hemolytic (viridans) streptococci (VGS) is very im... more 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 (bioM erieux) 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.

Bulletin of Russian State Medical University, 2022

Antibiotic-resistant strains of Pseudomonas aeruginosa are a global threat to public health. The ... more Antibiotic-resistant strains of Pseudomonas aeruginosa are a global threat to public health. The knowledge of mechanisms underlying antibiotic resistance is essential to counter P. aeruginosa infections. This study describes the phenomenon of meropenem-induced cross-resistance to colistin in the ATCC 27853 strain of P. aeruginosa. The study was conducted in the specimens of P. aeruginosa grown from the reference ATCC 27853 strain in the medium containing meropenem gradients. Susceptibility of the isolates to carbapenems and colistin was assessed using the agar dilution method; susceptibly to colistin was assessed using the broth microdilution method. A total of 93 P. Aeruginosa isolates were analyzed; of them two demonstrated reduced susceptibility to carbapenems (meropenem, imipenem) and colistin. Whole-genome sequencing of the isolates was performed on a MGISEQ-2000 platform. Missense mutations in the oprD and mexD genes and a nonsense mutation in the phoQ gene were detected. We c...

Sovremennye tehnologii v medicine, 2021

The aim of this work was to develop a new software tool for identifying gene mutations that deter... more The aim of this work was to develop a new software tool for identifying gene mutations that determine the porin-mediated resistance to antibiotics in gram-negative bacteria and to demonstrate the functionality of this program by detecting porin-mediated resistance to carbapenems in clinical isolates of Pseudomonas aeruginosa. Materials and Methods. The proposed algorithm is based on searching for a correspondence between the reference and the studied genes. When the sought nucleotide sequence is found in the analyzed genome, it is compared with the reference one and analyzed. The genomic analysis is then verified by comparing between the amino acid sequences encoded by the reference and studied genes. The genes of the susceptible P. aeruginosa ATCC 27853 strain were used as the reference nucleotide sequences encoding for porins (OprD, OpdD, and OpdP) involved in the transport of carbapenems into the bacterial cell. The complete genomes of clinical P. aeruginosa isolates from the PATRIC database 3.6.9 and our own collection were used to test the functionality of the proposed program. The analyzed isolates were phenotypically characterized according to the CLSI standard. The search for carbapenemase genes in the studied genomes of P. aeruginosa was carried out using the ResFinder 4.1. Results. The developed program for detecting the genetic determinants of non-plasmid antibiotic resistance made it possible to identify mutations of various types and significance in the porin genes of P. aeruginosa clinical isolates. These mutations led to modifications of the peptide structure of porin proteins. Single amino acid substitutions prevailed in the OpdD and OpdP porins of carbapenem-susceptible and carbapenem-resistant isolates. In the carbapenem-resistant strains, the gene encoding for OprD porin was found heavily modified, including insertions and/or deletions, which led to premature termination of porin synthesis. In several isolates resistant to meropenem, no mutations were detected in the gene encoding for OprD, which might be associated with alternative mechanisms of resistance to carbapenems. Conclusion. The proposed software product can become an effective tool for deciphering the molecular genetic mechanisms of bacterial chromosomal resistance to antibiotics. Testing the program revealed differences between the occurrences of mutations significant for carbapenem resistance in the oprD, opdD, and opdP genes.

Russian Journal of Bioorganic Chemistry, 2011

The modern phenotypic and genetic methods except for Multi Locus Sequence Typing do not allow the... more 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.

Microbiology Spectrum

I n some chronic infections, Pseudomonas aeruginosa is a key pathogen determining the course of t... more I n some chronic infections, Pseudomonas aeruginosa is a key pathogen determining the course of the disease. The type III secretion system (T3SS) is an important virulence factor of P. aeruginosa which can be inactivated under chronic conditions, such as cystic fibrosis (CF) (1). In a recent study by Karash et al. (2), the authors described the impact of several regulatory proteins on T3SS gene expression in two P. aeruginosa strains isolated from a chronic cutaneous ankle wound in a patient with keratitis-ichthyosis-deafness (KID) syndrome. They hypothesized that T3SS inactivation might promote the persistence of P. aeruginosa in the chronic infection locus. In these two KID isolates, the exsA, vfr, and cyaB gene sequences were identical to those in a reference strain, whereas fimV and bifA contained frameshift mutations (2). This article attracted our attention while we were examining the T3SS genes in 88 P. aeruginosa genomes recovered from CF patients from 29 regions in Russia using wholegenome sequencing (GenBank, BioProject accession numbers PRJNA786945, PRJNA770198). In these isolates, we analyzed genes involved in the T3SS contact toxin and protein synthesis and regulation in the context of Karash's hypothesis of their regulatory role in T3SS inactivation. The target gene sequences were compared by BLASTn using megablast default parameters (expect threshold = 0.05, word size = 28, match/mismatch scores = 1, 22, gap cost = linear, filter = low complexity regions). The T3SS gene sequences from the ATCC 27853 and PAO1 strains were used as the reference. Analysis of the T3SS genes is presented in Table 1. All examined P. aeruginosa isolates contained frame-disrupting (in-frame and out-of-frame indels) mutations in one or more T3SS structural genes. None of the 88 isolates possessed a set of T3SS structural genes which was identical to the reference or carried only synonymous or missense mutations. Next, we analyzed the gene sequences that Karash et al. (2) considered important for T3SS regulation, including exsA (primary activation of T3SS transcription), vfr (critical activation of exsA expression), cyaB (activation of exsA expression), fimV (regulation of adenylate cyclase CyaB), and bifA (regulation of cyclic-di-GMP phosphodiesterase). In exsA and cyaB, only synonymic substitutions and one or two amino acid substitution-inducing mutations were found. In 1 of the 88 isolates, the vfr gene harbored a frameshift mutation leading to a premature stop codon; another isolate contained a 5-bp deletion which resulted in a frameshift in vfr. The remaining 83/88 vfr sequences were either wild-type or contained synonymous substitutions or one amino acid substitution-inducing mutation. The bifA gene was more altered, harboring 11 significant mutations, including three nonsense mutations, seven out-of-frame deletions and insertions, and one in-frame deletion. In 1/88 isolates, a nonsense mutation was discovered in fimV. In addition, in 35/88 genomes, fimV possessed 9-or 15-nucleotide insertions with putative roles in regulator inactivation. Among our collection of 88 CF isolates, 56 different sequence types (STs), including 7 novel STs, were detected. The five most prevalent STs included ST235 (n = 8), ST274

Russian Clinical Laboratory Diagnostics, 2019

The growing prevalence of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa in nosocomia... more The growing prevalence of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa in nosocomial pathogen populations has been attributed to their clonal spread, and/or horizontal transfer of MBL determinants in mobile genetic elements, including integrons. To characterize the genetic background of the beta-lactamase VIM-2 encoding gene in the population of carbapenem-resistant (Carba-R) P. aeruginosa clinical isolates.The detection of class 1 integrons was performed by PCR. Typing of the class 1 integrons containing the blaVIM gene cassette was performed by the PCR-restriction fragment length polymorphism (RFLP) approach followed by sequencing of variable regions of class 1 integrons. Five types of the blaVIM-2-carrying integrons were identified: ST654-isolates accounting for more than 50% of the Carba-R population harbored In56; ST235-isolates contained In559 (26% Carba-R isolates); ST111-isolates (19% Carba-R isolates) were characterized by carrying In59-like integron; two ST23...

Epidemiology and Infection, 2017

SUMMARYClonal changes of serotype 19A pneumococci have been appreciated in conjunction with growi... more SUMMARYClonal changes of serotype 19A pneumococci have been appreciated in conjunction with growing prevalence of this serotype after implementation of the seven-valent pneumococcal conjugate vaccine (PCV7). In the present study, we characterized serotype 19A pneumococci collected in Russia within a decade preceding the implementation of PCV vaccination and described their clonal evolution. We retrospectively analyzed non-invasive serotype 19A isolates collected in 2002–2013. All isolates were subjected to multilocus sequence typing, antimicrobial susceptibility testing, determination of macrolide resistance genotype, molecular detection of pilus islet (PI) carriage, sequencing of penicillin-binding protein (PBP) genes. A total of 49 serotype 19A isolates represented 25 sequence types, of which 14 were newly described. The majority of isolates were distributed among clonal complex (CC) 663 (28%), CC230 (25%), CC156, and CC320 (14% each). CC663 and CC156 dominated in 2003, but were r...

Diagnostic Microbiology and Infectious Disease, 2021

The dissemination of multiple-drug resistant high virulent strains of P. aeruginosa in patients w... more The dissemination of multiple-drug resistant high virulent strains of P. aeruginosa in patients with cystic fibrosis is of concern worldwide. Herein, we describe genomic characteristics of ST235 isolates recovered from cystic fibrosis patients in Russia. Successful core-genome background and acquired resistance determinants provide spreading of high-risk clones in cystic fibrosis populations.

Russian Clinical Laboratory Diagnostics, 2021

Cystic fibrosis (CF) is a common genetic disease, manifested by airway obstruction and chronic re... more Cystic fibrosis (CF) is a common genetic disease, manifested by airway obstruction and chronic respiratory infection. The most prevalent infectious agent in airways of CF patients is Pseudomonas aeruginosa. This study aimed to determine sequence-types, antimicrobial resistance phenotypes and genes defining adaptive antibiotic resistance in P. aeruginosa isolates recovered from CF patients in Russia. In total, 84 P. aeruginosa strains from 64 CF patients were analyzed. Susceptibility to antibiotics was determined by disk diffusion test. Whole-genome sequencing (WGS) was performed on MGISEQ-2000 platform. SPAdes software, Galaxy, ResFinder, PubMLST were used for analysis of WGS data. Examined P. aeruginosa isolates belonged to 53 different sequence-types (STs), including 6 new STs. High-risk epidemic clone ST235 (10%) and clonal CF P. aeruginosa strains ST17, ST242, ST274 (7%) were detected. Non-susceptibility to ticarcillin-clavulanate, cefepime, imipenem was observed in 63%, 12% and...

International Journal of Antimicrobial Agents, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Clinical Microbiology and Antimicrobial Chemotherapy, 2018

Цель. Охарактеризовать популяционную структуру и определить молекулярногенетические меха низмы ре... more Цель. Охарактеризовать популяционную структуру и определить молекулярногенетические меха низмы резистентности к карбапенемам госпитальных штаммов P. aeruginosa, выделенных в Москве в 20122016 гг. Материалы и методы. Карбапенеморезистентные изоляты были выделены в двух педиатрических стационарах г. Москвы. Чувствительность изолятов P. aeruginosa к антибактериальным препаратам определяли с помощью Етестов и дискодиффузионным методом. Для генотипирования изолятов использовали метод мультилокусного сиквенстипирования (МЛСТ). Выявление генов металло бета лактамаз (МБЛ) проводили методом ПЦР в режиме реального времени. Результаты. Все исследованные изоляты обладали множественной лекарственной устойчивостью. Методом МЛСТ был выявлен 21 уникальный сиквенстип (ST). В структуре популяции доминировали представители пяти сиквенстипов (ST111, ST235, ST446, ST654 и ST2592), суммарно составляв шие 78% выборки карбапенеморезистентных P. aeruginosa. У 50 (57%) исследованных изолятов был детектирован ген МБЛ blaVIM2; генов других карбапенемаз, включая blaNDM и blaIMP, выявлено не было. Выводы. Генетическая структура исследованной популяции карбапенеморезистентных P. aeruginosa отличается разнообразием неродственных сиквенстипов с доминированием небольшого числа международных клонов высокого эпидемического риска, включая ST654, ST111 и ST235. Ведущим механизмом резистентности к карбапенемам у исследованных изолятов стала продукция карбапе немазы типа VIM2.

Diagnostic Microbiology and Infectious Disease, 2019

Serotype distribution and antimicrobial resistance were analyzed in 632 nasopharyngeal pneumococc... more Serotype distribution and antimicrobial resistance were analyzed in 632 nasopharyngeal pneumococcal isolates collected at a single pediatric center in 2010-2017 before and following the introduction of the 13-valent pneumococcal conjugated vaccine (PCV13) in Russia in 2014. The mean prevalence of PCV13 serotypes was 77.7% in 2010-2015 with a significant decline to 58.5% in 2017, which was accompanied by an elevation in serotype 15B/C prevalence (15.1% in 2017), 66% and 26% of 15B/C-pneumococci related to ST1025 and ST1262, respectively. The rate of oxacillin, erythromycin, and clindamycin resistance has increased by 15-20 percentage points from 2010 to 2016, approaching a 40-45% prevalence in 2016. The resistance rates significantly increased over time only in a group of PCV13 serotypes. The growing resistance among serotype 14 pneumococci was associated with expansion of a multidrug-resistant clone of ST143. These results emphasize the need for close monitoring of the constantly changing pneumococcal population.

Journal of Global Antimicrobial Resistance, 2019

Alteration of the porin-encoding gene oprD by insertion sequences (ISs) is one mechanism conferri... more Alteration of the porin-encoding gene oprD by insertion sequences (ISs) is one mechanism conferring carbapenem resistance in Pseudomonas aeruginosa. Here we describe a carbapenem-resistant clinical P. aeruginosa isolate 36-989 harbouring a novel IS (ISPa195) in oprD. Methods: Minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by the broth microdilution method. Carbapenemase activity was assessed using a MALDI-TOF/MS-based assay of meropenem hydrolysis. Efflux-dependent carbapenem resistance was evaluated using an assay with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The oprD gene and IS sequence were analysed by the Sanger method. Whole-genome sequencing was performed on an Illumina HiSeq 2500 platform. Results: Antimicrobial susceptibility testing demonstrated that P. aeruginosa 36-989 was resistant to imipenem (MIC = 32 mg/L) and meropenem (MIC = 16 mg/L). No carbapenemase activity was detected, however an efflux-mediated component of carbapenem resistance was revealed. A new IS element (ISPa195) was found in the oprD gene of P. aeruginosa 36-989. ISPa195 was 1190 bp in length, belonging to the IS3 family, and contained two open reading frames that overlapped through a ribosomal slippage to translate the full-size transposase enzyme. There was an IS-associated 284-bp deletion in the oprD gene; no direct repeats at flanking regions of the IS were detected. Conclusion: The absence of direct repeats at flanking regions in combination with the IS-associated deletion distinguished ISPa195 from other ISs previously detected in oprD. Carbapenem resistance in P. aeruginosa 36-989 was conferred by a combination of oprD alteration and carbapenem efflux.

Microbial Drug Resistance, 2017

Carbapenem-nonsusceptible (Carba-NS) Acinetobacter baumannii has emerged as an important cause of... more Carbapenem-nonsusceptible (Carba-NS) Acinetobacter baumannii has emerged as an important cause of nosocomial infections. In the present study, we characterized 91 Carba-NS A. baumannii isolates collected from patients of surgical departments and intensive care units at three hospitals in Moscow in 2012-2015. Multilocus sequence typing (MLST) using the Oxford (Oxf) scheme identified 16 sequence types (STs) of three clonal complexes (CCs), including CC92 Oxf (67%), CC109 Oxf (1%), CC944 Oxf (29%), and the singleton ST1100 Oxf (3%). CC944 Oxf was composed of ST944 Oxf (n = 16) and two of its newly described single locus variants ST1103 Oxf (n = 3) and ST1104 Oxf (n = 7); all the three STs were identical to the Pasteur (Pas) MLST scheme ST78. All CC944 Oxf /ST78 Pas isolates were bla OXA-40-like positive and all but one isolate harbored a bla CTX-M-like gene. ST944 Oxf was the only ST found in each of the three study hospitals. Biofilm growth capacity was similar among Carba-NS and nonclonal carbapenem-susceptible isolates. Our data demonstrate the predominance of two clonal lineages among Carba-NS A. baumannii. One of these, the uncommon bla OXA-40-like /bla CTX-M-like-positive clone of CC944 Oxf /ST78 Pas , seems to be endemic in Russia.

Microbial Drug Resistance, 2021

The pneumococcal population structure and drug resistance patterns are constantly changing worldw... more The pneumococcal population structure and drug resistance patterns are constantly changing worldwide. In this study, we described serotypes and antimicrobial susceptibility among 478 multiple-drug resistant (MDR) pediatric nasopharyngeal pneumococci recovered in 2010-2017. The majority of isolates (89.3%; n = 427) carried pneumococcal conjugate vaccine (PCV)13 serotypes, predominantly 6A/B, 14, 19A/F, and 23F. A non-PCV13 serotype capsule was detected in 44 (9.2%) MDR pneumococci, including serotypes 23A (n = 8), 13 (n = 7), 28F (n = 6), 11A (n = 5), and serogroup 35 (n = 10) isolates. The remaining seven (1.5%) MDR isolates were nontypeable. The majority of non-PCV13-serotype isolates were resistant to tetracycline, erythromycin, and clindamycin; most harbored both the ermB and mef genes. Among the 44 serotyped MDR non-PCV13 isolates, multilocus sequence typing analysis revealed 24 different sequence types (STs). ST2754 was the most abundant lineage demonstrating an unusual association with serotypes 13 (n = 7) and 9N (n = 1). The whole-genome sequencing-based analysis demonstrated that the serotype 13/ST2754 lineage was closely related to the serotype 13/ST2754 isolate recovered in Africa (Malawi) in 2013, possessed a Tn6002-like transposon carrying the erm(B) and tet(M) genes, and harbored additional virulence determinants, including arginine metabolism genes and a putative bacteriocin locus. Such a favorable genetic background may provide competitive advantages and potential for spreading and expansion of this clone among pneumococci. These data warrant further molecular monitoring of the genetic composition of the changing pneumococcal population.