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Dose-intensive cytostatic therapy and antibiotic treatment in allogeneic hematopoietic stem cell transplantation (allo-HSCT) cause severe abnormalities in a composition of gut microbiota as well as the emergence of antibiotic resistance. The data on the longitudinal recovery of major bacterial phyla and the expansion of genes associated with antibiotic resistance are limited. We collected regular stool samples during the first year after allo-HSCT from 12 adult patients with oncohematological disorders after allo-HSCT and performed 16SrRNA sequencing, multiplex PCR, conventional bacteriology and CHROMagar testing. We observed a decline in Shannon microbiota diversity index as early as day 0 of allo-HSCT (p = 0.034) before any administration of antibiotics, which persisted up to 1 year after transplantation, when the Shannon index returned to pre-transplant levels (p = 0.91). The study confirmed the previously shown decline in Bacillota (Firmicutes) genera and the expansion of E. coli/Shigella, Klebsiella and Enterococci. The recovery of Firmicutes was slower than that of other phyla and occurred only a year post-transplant. A positive correlation was observed between the expansion of E. coli/Shigella genera and blaKPC, blaCTX-M-1 and blaTEM (p < 0.001), Klebsiella spp. and blaOXA-48-like, blaNDM, blaCTX-M-1, blaTEM, and blaSHV (p < 0.001), Pseudomonas spp. and blaNDM (p = 0.002), Enterococcus spp. and blaOXA-48-like, blaNDM, blaCTX-M-1, blaSHV (p < 0.01). The correlation was observed between the expansion of Enterobacterales and and carbapenemase-positive CHROMagar samples (p < 0.001). Samples positive for carbapenem-resitant bacteria were at their maximum levels on day +30, and were gradually diminishing one year after allo-HSCT. From day +30 to +60, all isolated K. pneumoniae strains in fecal samples proved to be resistant to the main antibiotic groups (carbapenems, aminoglycosides, fluoroquinolones, third-generation cephalosporins). One year after HSCT, we documented the spontaneous decolonization of K. pneumoniae. The sensitivity of molecular biology techniques in the search for total and antibiotic-resistant Klebsiella seems to be superior to common bacteriological cultures. Future studies should be focused on searching for novel approaches to the efficient reconstitution and/or maintenance of strictly anaerobic microbiota in oncological patients.

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This study was aimed at understanding the distributions of the MICs (minimum inhibitory concentrations) of aztreonam-avibactam, ceftazidime-avibactam and meropenem with respect to Klebsiella pneumoniae isolates producing different types of carbapenemases and their combinations. K. pneumoniae isolates were collected between 2019 and 2022 from 37 hospitals. PCR was used to screen for blaKPC-, blaNDM- and blaOXA-48-like genes. MICs were determined by the broth microdilution method for meropenem, aztreonam-avibactam and ceftazidime-avibactam at a constant avibactam concentration of 4 mg l-1. MIC distributions were analyzed for groups of isolates based on the identified carbapenemases including their combinations. The AZT/AVI MIC50 and MIC90 for all NDM-positive isolates were 0.25 and 0.5, respectively, and for serine-carbapenemase-only producers, they were 0.25 and 1 mg l-1, respectively. The CZD/AVI MIC50 and MIC90 values for serine-carbapenemase-only producers were 1 and 4 mg l-1, respectively. The AZT/AVI MIC50 and MIC90 values for co-producers and single carbapenemase producers were the same (i.e., 0.25 and 1 mg l-1, respectively). The total proportion of meropenem-susceptible isolates (≤8 mg l-1) among all the carbapenemase producers was 25.1% (31.1% among single-carbapenemase producers and 9.2% among co-producers). The results support the use of aztreonam-avibactam for the empirical treatment of infections caused by any carbapenemase producers.

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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.

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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.

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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.

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The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) is a new threat to healthcare. In this study, we analyzed nine CR-hvKp isolates of different sequence-types (ST) recovered from patients with nosocomial infections in two hospitals in Saint Petersburg. Whole-genome sequencing showed that eight of them harbored large mosaic plasmids carrying resistance to carbapenems and hypervirulence simultaneously, and four different types of hybrid plasmids were identified. BLAST analysis showed a high identity with two hybrid plasmids originating in the UK and Czech Republic. We demonstrated that hybrid plasmids emerged due to the acquisition of resistance genes by virulent plasmids. Moreover, one of the hybrid plasmids carried a novel New Delhi metallo-beta-lactamase (NDM) variant, differing from NDM-1 by one amino acid substitution (D130N), which did not provide significant evolutionary advantages compared to NDM-1. The discovery of structurally similar plasmids in geographically distant regions suggests that the actual distribution of hybrid plasmids carrying virulence and resistance genes is much wider than expected.

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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.

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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.

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Fifteen hypermucoviscous isolates (13 blaNDM-1-positive) obtained from 11 oncology patients were analyzed by whole genome sequencing, and selected isolates were assessed in a murine model of sepsis. ST395/K2 isolates harboring rmpA, rmpA2, peg-344, aerobactin, enterobactin, yersiniabactin, type I fimbriae, etc. displayed maximal virulence in the mouse lethality assay (LD50 = 102 CFU). ST147/K20 isolates lacking yersiniabactins were relatively less virulent (LD50 = 104 CFU), ST395/K2 isolates lacking rmpA, rmpA2, peg-344, and aerobactin, but harboring yersiniabactin demonstrated minimal virulence (LD50 = 105 CFU). Isolates represent various paths and stages of evolution directed towards convergence of multidrugresistant classical Klebsiella pneumoniae and hypervirulent K. pneumoniae.

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An Escherichia coli sequence type 31 isolate co-harbouring mcr-1 and blaNDM-1 genes on the plasmids of Incl2 and IncC groups, respectively, was recovered from a newborn with ventilator-associated pneumonia in Moscow, Russia. The convergence of polymyxin and carbapenem resistance and its expansion beyond Southeast Asia is a serious threat to human health.

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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.

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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.

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BACKGROUND: Fecal microbiota transplantation (FMT) has been recently approved by FDA for the treatment of refractory recurrent clostridial colitis (rCDI). Success of FTM in treatment of rCDI led to a number of studies investigating the effectiveness of its application in the other gastrointestinal diseases. However, in the majority of studies the effects of FMT were evaluated on the patients with initially altered microbiota. The aim of our study was to estimate effects of FMT on the gut microbiota composition in healthy volunteers and to monitor its long-term outcomes. RESULTS: We have performed a combined analysis of three healthy volunteers before and after capsule FMT by evaluating their general condition, adverse clinical effects, changes of basic laboratory parameters, and several immune markers. Intestinal microbiota samples were evaluated by 16S rRNA gene and shotgun sequencing. The data analysis demonstrated profound shift towards the donor microbiota taxonomic composition in all volunteers. Following FMT, all the volunteers exhibited gut colonization with donor gut bacteria and persistence of this effect for almost ∼1 year of observation. Transient changes of immune parameters were consistent with suppression of T-cell cytotoxicity. FMT was well tolerated with mild gastrointestinal adverse events, however, one volunteer developed a systemic inflammatory response syndrome. CONCLUSIONS: The FMT leads to significant long-term changes of the gut microbiota in healthy volunteers with the shift towards donor microbiota composition and represents a relatively safe procedure to the recipients without long-term adverse events.

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ST239-MRSA-III is probably the oldest truly pandemic MRSA strain, circulating in many countries since the 1970s. It is still frequently isolated in some parts of the world although it has been replaced by other MRSA strains in, e.g., most of Europe. Previous genotyping work (Harris et al., 2010; Castillo-Ramírez et al., 2012) suggested a split in geographically defined clades. In the present study, a collection of 184 ST239-MRSA-III isolates, mainly from countries not covered by the previous studies were characterized using two DNA microarrays (i) targeting an extensive range of typing markers, virulence and resistance genes and (ii) a SCCmec subtyping array. Thirty additional isolates underwent whole-genome sequencing (WGS) and, together with published WGS data for 215 ST239-MRSA-III isolates, were analyzed using in-silico analysis for comparison with the microarray data and with special regard to variation within SCCmec elements. This permitted the assignment of isolates and sequences to 39 different SCCmec III subtypes, and to three major and several minor clades. One clade, characterized by the integration of a transposon into nsaB and by the loss of fnbB and splE was detected among isolates from Turkey, Romania and other Eastern European countries, Russia, Pakistan, and (mainly Northern) China. Another clade, harboring sasX/sesI is widespread in South-East Asia including China/Hong Kong, and surprisingly also in Trinidad & Tobago. A third, related, but sasX/sesI-negative clade occurs not only in Latin America but also in Russia and in the Middle East from where it apparently originated and from where it also was transferred to Ireland. Minor clades exist or existed in Western Europe and Greece, in Portugal, in Australia and New Zealand as well as in the Middle East. Isolates from countries where this strain is not epidemic (such as Germany) frequently are associated with foreign travel and/or hospitalization abroad. The wide dissemination of this strain and the fact that it was able to cause a hospital-borne pandemic that lasted nearly 50 years emphasizes the need for stringent infection prevention and control and admission screening.

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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 (ST239Kras) 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).

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ST8/SCCmecIV community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been a common threat, with large USA300 epidemics in the United States. The global geographical structure of ST8/SCCmecIV has not yet been fully elucidated. We herein determined the complete circular genome sequence of ST8/SCCmecIVc strain OC8 from Siberian Russia. We found that 36.0% of the genome was inverted relative to USA300. Two IS256, oppositely oriented, at IS256-enriched hot spots were implicated with the one-megabase genomic inversion (MbIN) and vSaß split. The behavior of IS256 was flexible: its insertion site (att) sequences on the genome and junction sequences of extrachromosomal circular DNA were all divergent, albeit with fixed sizes. A similar multi-IS256 system was detected, even in prevalent ST239 healthcare-associated MRSA in Russia, suggesting IS256's strong transmission potential and advantage in evolution. Regarding epidemiology, all ST8/SCCmecIVc strains from European, Siberian, and Far Eastern Russia, examined had MbIN, and geographical expansion accompanied divergent spa types and resistance to fluoroquinolones, chloramphenicol, and often rifampicin. Russia ST8/SCCmecIVc has been associated with life-threatening infections such as pneumonia and sepsis in both community and hospital settings. Regarding virulence, the OC8 genome carried a series of toxin and immune evasion genes, a truncated giant surface protein gene, and IS256 insertion adjacent to a pan-regulatory gene. These results suggest that unique single ST8/spa1(t008)/SCCmecIVc CA-MRSA (clade, Russia ST8-IVc) emerged in Russia, and this was followed by large geographical expansion, with MbIN as an epidemiological marker, and fluoroquinolone resistance, multiple virulence factors, and possibly a multi-IS256 system as selective advantages.

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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

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