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Objective. To characterize the genomes of oxacillin-susceptible mecA-positive Staphylococcus aureus ST59 isolated in St. Petersburg. Materials and Methods. Nine oxacillin-susceptible mecA-positive of S. aureus isolates (OS-MRSA) of ST59 were included in the study. The isolates were obtained from children who showed no clinical signs of staphylococcal infections during nasal screening of S. aureus in St. Petersburg in 2018–2019. One isolate was obtained from an adult patient with skin and soft tissue infection (SSTI). The susceptibility to antibiotics and whole genome sequencing were performed. The analysis included 242 genomes of S. aureus ST59 from open access databases. Results. By employing the broth serial dilution and VITEK, the isolates' phenotypic susceptibility to oxacillin was determined. The cefoxitin inhibition zones ranged from 17 to 22 mm. All isolates showed a penicillinclavulanate MIC ≤ 0.5 µg/mL. Isolates obtained from carriers belonged to the ST59-t1950-SCCmec Vb (seb+) genotype whereas the isolate obtained from SSTI belonged to the ST59-t437-SCCmec Vb (seb/ lukF/lukS+) genotype. Nucleotide position -33 (C/T) of mecA promoter and mutations in PBP2a (S225R + E246G) were present in all isolates. Based on phylogenetic analysis and Bayesian clustering the ST59 genomes were divided into four clusters and all Russian genomes belonged to the East Asian ST59 sublineage. The PVL toxin was present in the genomes of the first cluster of the East Asian ST59 sublineage. Pairwise comparisons of nucleotide substitutions among the genomes of Russian isolates showed a high similarity: median 13, interquartile range 8–18. All ST59 clusters were characterized by the presence of enterotoxin B, as well as mutations in PBP2a (S225R and E246G) and the promoter regions of the mecA gene (-7 G/A or -33 C/T). The genomes of the Russian isolates differed from the globally spread ST59 by specific mutations at the following loci (relative to the reference genome of S. aureus M013TW): lactose catabolism regulator RS03495 (N168D), ribosomal protein L28 (V47A), putative glyoxalase RS07825 (V42A), and the hypothetical protein RS13235 (K32E). Conclusions. Russian MRSA-ST59 isolates belong to the East Asian sublineage and are characterized by the presence of the enterotoxin B gene. Oxacillin susceptibility and borderline resistance to cefoxitin are specific characteristics of MRSA-ST59. OS-MRSA phenotypes have a risk of improper sensitivity testing leading to ineffective antibiotic treatment. Detection of mecA gene is the most accurate method for differentiating between MSSA and MRSA.

Background. Ceftaroline is a new broad-spectrum cephalosporin with in vitro activity against Gram-positive pathogens, including MRSA, and common Gram-negative pathogens. It is approved for the treatment of acute complicated bacterial skin and skin-structure infections (SSSIs) and community-acquired bacterial pneumonia. Objective. To determine in vitro activity of ceftaroline and other clinically available antimicrobials against major bacterial pathogens from different regions of Russian Federation. Materials and Methods. A total of 2778 consecutive, non-duplicate isolates were collected during multicentre microbiological in vitro study from 36 geographically distinct cities of Russia during 2008–2012. Only isolates considered to be clinically significant by microbiologist or clinical practice specialist were included in the study. Among tested strains 1000 were Staphylococcus aureus (including 612 MRSA), 954 — Streptococcus pneumoniae, 338 — beta-hemolytic streptococci, 85 — Haemophilus influenzae, 401 — Enterobacteriaceae, mainly ESBL-negative strains of E.coli (n=259) and K. pneumoniae (n=50); the remaining 88 strains were represented by Citrobacter spp., Enterobacter spp., Klebsiella spp., Morganella spp., Proteus spp., Serratia spp. All the isolates were subsequently confirmed using Microflex LT MALDI Biotyper System. The isolates were stored in glycerol-supplemented tryptic soy broth (TSB) at –70 °C until analysis. Minimum inhibitory concentrations (MICs) of antimicrobials were determined by the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. Results. Overall susceptibility rates to ceftaroline were 89,7% for S. aureus (100% for MSSA and 83,2% for MRSA — when using EUCAST or CLSI breakpoints; 0% for MSSA and 0,8% MRSA — when using PK/PD suggested breakpoint of ≤2 mg/l for susceptible strains), 100% for beta-hemolytic streptococci, 99,5% for S. pneumoniae, 75,8% for Enterobacteriaceae (resistant strains were mainly non-E.coli and non-K. pneumoniae AmpC-producing isolates), 98,8% — for H. influenzae. Conclusions. The results of the study demonstrate that ceftaroline has high in vitro activity against key bacterial pathogens and could be considered as an option for the therapy of community-acquired bacterial pneumonia and SSSIs in Russia.

The aim of this study is to describe the phenotypic and genetic properties of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) isolates and their beta-lactam resistant derivatives obtained after selection with oxacillin. A collection of hospital- (HA-) and community-acquired (CA-) MRSA was screened for oxacillin susceptibility. Antibiotic susceptibility testing, population analysis profile (PAP), mecA expression analysis, and whole genome sequencing (WGS) were performed for 60 mecA-positive OS-MRSA isolates. Twelve high-level beta-lactam resistant derivatives selected during PAP were also subjected to WGS. OS-MRSA were more prevalent among CA-MRSA (49/205, 24%) than among HA-MRSA (11/575, 2%). OS-MRSA isolates belonged to twelve sequence types (ST), with a predominance of ST22-t223-SCCmec IVc and ST59-t1950-SCCmec V lineages. OS-MRSA were characterized by mecA promoter mutations at - 33 (C→T) or - 7 (G→T/A) along with PBP2a substitutions (S225R or E246G). The basal and oxacillin-induced levels of mecA expression in OS-MRSA isolates were significantly lower than those in control ST8-HA-MRSA isolates. Most of the OS-MRSA isolates were heteroresistant to oxacillin. High-level beta-lactam resistant OS-MRSA derivatives selected with oxacillin carried mutations in mecA auxiliary factors: relA (metabolism of purines), tyrS, cysS (metabolism of tRNAs), aroK, cysE (metabolism of amino acids and glycolysis). Cefoxitin-based tests demonstrated high specificity for OS-MRSA detection. The highest positive predictive values (PPV > 0.95) were observed for broth microdilution, the VITEK® 2 automatic system, and chromogenic media. Susceptibility testing of CA-MRSA requires special attention due to the high prevalence of difficult-to-detect OS-MRSA among them. Mis-prescription of beta-lactams for the treatment of OS-MRSA may lead to selection of high-level resistance and treatment failures., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Vancomycin and daptomycin are first-line drugs for the treatment of complicated methicillin-resistant Staphylococcus aureus (MRSA) infections, including bacteremia. However, their effectiveness is limited not only by their resistance to each antibiotic but also by their associated resistance to both drugs. It is unknown whether novel lipoglycopeptides can overcome this associated resistance. Resistant derivatives from five S. aureus strains were obtained during adaptive laboratory evolution with vancomycin and daptomycin. Both parental and derivative strains were subjected to susceptibility testing, population analysis profiles, measurements of growth rate and autolytic activity, and whole-genome sequencing. Regardless of whether vancomycin or daptomycin was selected, most of the derivatives were characterized by a reduced susceptibility to daptomycin, vancomycin, telavancin, dalbavancin, and oritavancin. Resistance to induced autolysis was observed in all derivatives. Daptomycin resistance was associated with a significant reduction in growth rate. Resistance to vancomycin was mainly associated with mutations in the genes responsible for cell wall biosynthesis, and resistance to daptomycin was associated with mutations in the genes responsible for phospholipid biosynthesis and glycerol metabolism. However, mutations in walK and mprF were detected in derivatives selected for both antibiotics.

Coagulase-negative staphylococci (CoNS) for a long time were considered avirulent constituents of the human and warm-blooded animal microbiota. However, at present, S. epidermidis , S. haemolyticus , and S. hominis are recognized as opportunistic pathogens. Although linezolid is not registered for the treatment of CoNS infections, it is widely used off-label, promoting emergence of resistance. Bioinformatic analysis based on maximum-likelihood phylogeny and Bayesian clustering of the CoNS genomes obtained in the current study and downloaded from public databases revealed the existence of international linezolid-resistant lineages, each of which probably had a common predecessor. Linezolid-resistant S. epidermidis sequence-type (ST) 2 from Russia, France, and Germany formed a compact group of closely related genomes with a median pairwise single nucleotide polymorphism (SNP) difference of fewer than 53 SNPs, and a common ancestor of this lineage appeared in 1998 (1986-2006) before introduction of linezolid in practice. Another compact group of linezolid-resistant S. epidermidis was represented by ST22 isolates from France and Russia with a median pairwise SNP difference of 40; a common ancestor of this lineage appeared in 2011 (2008-2013). Linezolid-resistant S. hominis ST2 from Russia, Germany, and Brazil also formed a group with a high-level genome identity with median 25.5 core-SNP differences; the appearance of the common progenitor dates to 2003 (1996-2012). Linezolid-resistant S. hominis isolates from Russia demonstrated associated resistance to teicoplanin. Analysis of a midpoint-rooted phylogenetic tree of the group confirmed the genetic proximity of Russian and German isolates; Brazilian isolates were phylogenetically distant. repUS5 -like plasmids harboring cfr were detected in S. hominis and S. haemolyticus ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gostev, Leyn, Kruglov, Likholetova, Kalinogorskaya, Baykina, Dmitrieva, Grigorievskaya, Priputnevich, Lyubasovskaya, Gordeev and Sidorenko.)

Selective pressure of beta-lactams is thought to be responsible for mutation selection in methicillin-susceptible Staphylococcus aureus (MSSA). We used next-generation sequencing to compare the genomes of beta-lactamase-positive (SA0707) and -negative (SA0937) MSSA isolates with their derivatives obtained after selection with oxacillin, ceftaroline, or meropenem. Selection with oxacillin and ceftaroline caused a rapid and significant (6-8 times) increase in the minimum inhibitory concentration (MICs) of oxacillin, penicillin, amoxicillin/clavulanate, and ceftaroline against the derivatives of both isolates, associated with growth impairment. Selection with meropenem caused a limited increase in the MICs of all beta-lactams against both isolates. During the initial stages of selection (after 5-15 passages), mutations were detected only in some reads, which indicated the heterogeneity of the population; however, during the later stages, either the population reversed to the wild type or fixation of the mutation was observed in the entire population. Selection with different beta-lactams caused diverse mutational events, but common mutations were detected in gdpP, all penicillin-binding proteins, cell wall regulators ( vraST, graR ), and deletions in the promoter region of pbp4 . Therefore, the disk diffusion test with cefoxitin does not reveal resistance associated with these mechanisms in some cases, which can lead to the failure of beta-lactam therapy.

In this study, we identified the relationship between the genetic lineage of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) sequence type 22 (ST22) from Russia and other regions. Sixty ST22 isolates from Russia were characterised through whole-genome sequencing. To evaluate the phylogenetic relationship of Russian isolates with the global ST22 population, we analysed 1283 genomes obtained from NCBI's GenBank. The phylogenetic tree of the ST22 global population consisted of three main clusters (A, B and C). The first (cluster A) was represented by EMRSA-15 isolates, the second (cluster B) by heterogeneous isolates from different regions harbouring different sets of virulence genes, and the third (cluster C) by isolates from the Middle East previously recognised as 'Gaza clone' and similar isolates from Russia. Presence of the toxic shock syndrome toxin (tsst) and elastin-binding protein S (ebpS) genes as well as the hypothetical proteins NCTC13616_00051 and NCTC13616_00047 were the most useful factors in discriminating ST22 lineages. Although the CA-MRSA 'Gaza clone' was mainly recovered from carriers, its widespread occurrence is a cause for concern. Differentiation of the 'Gaza clone' from other MRSA lineages is necessary for planning infection control measures., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Russia introduced PCV13 in 2014. We studied the serotype composition of S. pneumoniae isolated from the nasopharynx of healthy children younger than 6 years in St. Petersburg, Smolensk, Perm, Krasnoyarsk, Khanty-Mansiysk and Khabarovsk, between 2016 and 2018. 2.4% of children had completed a 3-dose course of PCV13, while 25.6% had received 1 or 2 doses. Pneumococcal DNA detection by PCR demonstrated S. pneumoniae in 37.2% of samples with regional variation between sites (27.3 to 56.9%). There was little difference between vaccinated, partially vaccinated and un-vaccinated children. Children who had received at least 1 dose of PCV13 had lower carriage rates of vaccine serotypes than their unvaccinated peers (49.9 vs. 61.4%; p < 0.001). Children who had received at least 1 dose of PCV13 showed increased carriage rates of non-vaccine serotypes (50 vs 38.6%; P < 0.001). Especially serogroup 15AF was more prevalent among fully immunized children than among their peers (12.5 vs 2.7%; P < 0.05)., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The pathways in the development of ceftaroline resistance of methicillin-resistant Staphylococcus aureus (MRSA) isolates belonging to the ST8, ST239, and ST228 were evaluated. Ceftaroline-resistant derivatives were isolated through selection during 40 passages. Ceftaroline MIC measurements and whole-genome sequencing were performed after 5, 20, and 40 passages. In two ST8 derivative isolates, ceftaroline MIC increased up to 128 mg/L. Mutations were acquired in gdpP and graS in one isolate after 20 passages and in gdpP in another after 40 passages. MIC for two ST239 derivatives increased to 128 mg/L. Substitutions in Pbp4 and polymorphisms in the upstream region of pbp4 were identified in both derivatives after 40 passages. In one isolate, additional mutation in gdpP and deletion in graR were detected. In an ST228 derivative, MIC increased to 32 mg/L with one mutation in penicillin-binding protein 2a (Y446N) detected after five passages and a second (E447K) after 20 passages. Three pathways in the development of ceftaroline resistance were identified. For ST8 and ST239 derivatives mutations were detected in gdpP and pbp4 , respectively, whereas in ST228 - in mecA . Most derivatives harbored additional mutations whose potential role in the development of resistance has not been determined.

Objective: To determine the carriage and the serogroup distribution of Neisseria meningitidis in military academy applicants in the Russian Federation., Design: This was a prospective, observational study of adults aged >18years from a military academy; applicants who had samples taken on arrival (Day 1), and applicants who had samples taken after passing exams (Day 30) and 60days after arrival. N. meningitidis serogrouping was determined by slide agglutination tests of isolates and real-time PCR., Results: Samples were provided by 671 applicants on Day 1 and 261 applicants on Day 30, with 232 of these also providing samples on Day 60. N. meningitidis was detected in 16.2% of samples from Day 1, 7.7% of samples from Day 30 and 15.9% of samples from Day 60. Serogroup composition was most diverse at Day 1, with serogroups B and W dominant (40% [17/43 isolates] and 9% [4/43], respectively; 30% [13/43] ungroupable); by Day 60, there was a low diversity, with 58% (14/24 isolates) serogroup W., Conclusions: While carriage of N. meningitidis in this study appeared stable, there was an increase in carriers of serogroup W in this population. Given recent increases in outbreaks attributed to serogroup W, further monitoring may be considered., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The aim of this study was to investigate the patterns of antimicrobial resistance and molecular features of methicillin-resistant Staphylococcus aureus (MRSA) isolates in Russia. Isolates recovered from hospital patients (n=480), healthy medical personnel (n=25), and healthy carriers (n=13) were included in the study. Hospital-acquired MRSA (HA-MRSA) demonstrated high resistance to ciprofloxacin, gentamicin, and chloramphenicol (76%-92%), moderate - to tetracycline, erythromycin, clindamycin, and rifampicin (38%-54%), and low - to fusidic acid, co-trimoxazole, mupirocin, and daptomycin (2%-7%). Elevated MIC (2.0μg/ml) of vancomycin was detected in 26% of isolates. All isolates were susceptible to linezolid and tigecycline. Multilocus sequence typing (MLST) revealed that CC8 isolates (ST8+ST239) constituted 83.1% of HA-MRSA and that this genetic lineage dominated in all regions from Krasnoyarsk to Saint Petersburg. A local ST239 variant harboring the tst gene (ST239 Kras ) was detected in Krasnoyarsk. The other HA-MRSA isolates belonged to clonal complex 5 (CC5) (21 isolates, 12.2%) and CC22 (2, 1.2%). The majority of CC5 isolates were affiliated with sequence type 228 (ST228) and were characterized with decreased susceptibility to ceftaroline (MIC=2μg/ml). We also detected, for the first time in Russia, livestock-associated MRSA (LA-MRSA) from clusters CC398 and CC97 in humans. Among the 2053 healthy persons screened for nasal carriage of S. aureus, the bacteria were isolated from 426 (21%); among them, 13 carried isolates identified as community-associated MRSA (CA-MRSA). Eleven of 13 CA-MRSA isolates belonged to ST22 (spa types t223, t3243, and t3689; SCCmec types IVa and IVc, agr type I, tst-positive) and were similar to the EMRSA-15/Middle Eastern variant (Gaza strain)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A total of 473 strains of Enterobacteriaceae, including Escherichia coli, Klebsiella spp., Proteus spp., Citrobacter spp., Enterobacter spp., Serratia spp. and Providencia spp., were isolated from patients admitted to intensive care units and surgical units in Russia. About 90% of the isolates carried factors resistant to beta-lactams. The isolation rates of the extended-spectrum beta-lactamase (ESBL) producer defined in this study among E. coli, Klebsiella spp. and Proteus spp. were 45%, 48% and 17%, respectively. In the settings with high prevalence of the ESBL producer, flomoxef, which belongs to the oxacephem subgroup, and carbapenems retain their activity. The MIC₅₀ of flomoxef, meropenem and imipenem against total isolates were 1 µg/mL, ≤ 0.063 µg/mL and 0.25 µg/mL, respectively. Fifty-five carbapenem-resistant strains were isolated in this study. The carbapenem resistant rates of E. coli, Klebsiella spp. and Proteus spp. were 3%, 16% and 29%, respectively

Bioinformatic analysis of the data on the genome sequencing of the isolates of the Streptococcuspneumoniae clonal complex SS320 from the Russian Federation, as well as the data on SS320 isolates from public sources in the penicillin resistant isolates resulted in detection of 139 missense mutations in 45 genes. In addition to the mutations in the genes of the main penicillin-binding proteins (PSB - PBP1A, PBP2B and PBP2X) there was detected high frequency of mutations in the genes of the (division and cell wall) dcw-cluster, as well as in RegR protein belonging to the transcription regulators of the LacI/GaIR family. Development of resistance to beta-lactams in S.pneumoniae is defined not only by modification of the PSB, but also by adaptive changes in the metabolic pathways involved in the bacterial cell growth and division.

Ceftaroline is a unique cephalosporin with activity against methicillin resistant Staphylococcus aureus (MRSA). It was approved for clinical use in the USA, Europe and Russian Federation since 2010 for the treatment of the skin and soft tissue infection and community-acquired pneumoniae. In the present study there was used molecular typing of 24 isolates of MRSA with reduced susceptibility to ceftaroline. For 8 isolates belonging to different genetic lines (ST8, ST239 and ST228) and requiring MICs there were determined antibiotic concentrations preventing formation of resistant mutants (mutant prevention concentration) and the ranges of the mutant selection window (MSW). The last majority of the isolates with reduced susceptibility to ceftaroline (MIC of 2 mcg/ml) belonged to the clonal line ST228. The whole genome sequencing of two isolates of ST228 showed that they belonged to the epidemic South Germany genetic line and were characterized by the presence of mutations in PBP2a (N146K) and PBP2 (C197Y) responsible for reduced susceptibility. The highest rates of MPC (32 mcg/ml) and MSW (2-16 mcg/ml) were observed in the clinical isolates belonging to the genetic line ST8. The isolates of ST239 and ST228 had the selection window within 2-4 mcg/ml. No dependence of the MIC and MPC/MSW levels was detected.

Antibiotic susceptibility of 119 coagulase-negative staphylococci isolated at hospitals of St. Petersburg and Moscow was investigated and estimated at the local laboratories as oxacillin resistant. The following species were identified: Staphylococcus epidermidis, S. haemolyticus, S. hominis, S.capitis, S. simulans, S. pettenkoferi, S. lentus, S. carnosus and S. warneri. The oxacillin resistance was confirmed in 79.8% of the isolates. The frequency of the associated resistance to non-beta-lactams was much higher in the oxacillin resistant isolates vs. the oxacillin susceptible ones. When the CLSI and EUCAST susceptibility criteria were used, 1-3% difference in the resistance levels was recorded. Among the oxacillin resistant isolates the frequency of resistance to gentamicin, ciprofloxacin, erythromycin, moxifloxacin, tetracycline and clindamycin equaled 90, 88, 88, 63, 43 and 26% respectively. Two linezolid resistant isolates of S. epidermidis with lower susceptibility to tedizolid were isolated. Eight isolates of S. epidermidis showed lower resistance to mupirocin. The MIC of ceftarolin for oxacillin resistant coagulase-negative staphylococci varied from 0.5 to 2.0 mcg/ml, while for the oxacillin susceptible ones it was lower than 0.25 mcg/ml. No resistance to tigecyclin and vancomycin was observed.

The results of the multicentre trial on estimation of MRSA antibiotic susceptibility to 17 antibiotics are presented. 474 nonrepeting isolates of MRSA (mecA+), collected in 2011-2014 in 10 cities of the Russian Federation were used in the trial. The antibiotic susceptibility was determined by the method of serial microdilutions in broth with estimation of the MICs in accordance with the international standards CLSI 2014 and EUCAST 2014. The highest levels of the MRSA resistance were stated against ciprofloxacin--92%(MIC50 32 mcg/ml), gentamicin--85% (MIC50 128 mcg/ml), erythromycin--54% (MIC50 32-mcg/ml) and clindainycin - 45% (MIC50 0.03 mcg/ml), as well as against rifampicin--38% (MIC50 0.06 mcg/ml). The frequency of MRSA isolated at the vancomycin dose of 2 mcg/ml equaled 26%. No correlation of the decrease in susceptibility to vancomycin and rifampicin was observed. In 5% of MRSA isolated from infected surgical wounds in patients with bone infection or sepsis, there was observed a decrease in the susceptibility to ceftarolin (MIC 2-4 mcg/ml). Co-trimoxasole, fusidic acid (MIC50 0.06 mcg/ml) and mupirocin (MIC50 0.5 mcg/ml) showed high antibacterial activity, 93-98% of the isolates being susceptible to the drugs. No resistance to linezolid and tigecycline was detected. By the associate resistance spectrum, most of the MRSA isolates were characterized by resistance to drugs of 3-7 groups (56%). The phenotypes with simultaneous resistance to drugs of 8-10 groups amounted to 6%. As a whole, 70 variants of associate resistance combinations were detected.

The surveillance of the serotype pattern and antibiotic resistance of S. pneumoniae in various geographical regions is required for the validity of rational etiotrophic therapy of pneumococcal infections and the choice of the optimal vaccines for their prophylaxis. 250 S. pneumoniae isolates from children with acute otitis or pneumonia and healthy carriers in St. Petersburg in 2010-2013 were investigated. The analysis of the serotype pattern of the pneumococci showed that 13-valent conjugate vaccine was the most active (86.1% of pneumococci causing pneumonia and 86.4% of pneumococci causing acute otitis). The isolates were higly resistant to beta-lactams and macrolides. By the EUCAST criteria, the decrease in the susceptibility to penicillin, cefotaxime, erythromycin and ceftarolin was observed in 32.4%, 14%, 33.2 and 6% of the isolates respectively. 22.4% of the isolates showed associate resistance to penicillin and erythromycin.. No resistance to moxifloxacin was detected. The frequency of resistance to tetracycline, co-trimoxasole and chloramphenicol in various patients ranged within 30-50%. The prevalence of the antibiotic resistance was mainly characteristic of the isolates serotypes 19A, 19F, 14 and serogroup 6.