SARS-CoV-2 Recombination and Coinfection Events Identified in Clinical Samples in Russia.

Recombination is one of the mechanisms of SARS-CoV-2 evolution along with the occurrence of point mutations, insertions, and deletions. Recently, recombinant variants of SARS-CoV-2 have been registered in different countries, and some of them have become circulating forms. In this work, we performed screening of SARS-CoV-2 genomic sequences to identify recombination events and co-infections with various strains of the SARS-CoV-2 virus detected in Russia from February 2020 to March 2022. The study included 9336 genomes of the COVID-19 pathogen obtained as a result of high-throughput sequencing on the Illumina platform. For data analysis, we used an algorithm developed by our group that can identify viral recombination variants and cases of co-infections by estimating the frequencies of characteristic substitutions in raw read alignment files and VCF files. The detected cases of recombination were confirmed by alternative sequencing methods, principal component analysis, and phylogenetic analysis. The suggested approach allowed for the identification of recombinant variants of strains BA.1 and BA.2, among which a new recombinant variant was identified, as well as a previously discovered one. The results obtained are the first evidence of the spread of recombinant variants of SARS-CoV-2 in Russia. In addition to cases of recombination we identified cases of coinfection: eight of them contained the genome of the Omicron line as one of the variants, six of them the genome of the Delta line, and two with the genome of the Alpha line.

Genomic Variability of SARS-CoV-2 Omicron Variant Circulating in the Russian Federation during Early December 2021 and Late January 2022.

Analysis of genomic variability of pathogens associated with heightened public health concerns is an opportunity to track transmission routes of the disease and helps to develop more effective vaccines and specific diagnostic tests. We present the findings of a detailed genomic analysis of the genomic variability of the SARS-CoV-2 Omicron variant that spread in Russia between 8 December 2021 and 30 January 2022. We performed phylogenetic analysis of Omicron viral isolates collected in Moscow (n = 589) and downloaded from GISAID (n = 397), and identified that the BA.1 lineage was predominant in Russia during this period. The BA.2 lineage was also identified early in December 2021. We identified three cases of BA.1/BA.2 coinfections and one case of Delta/Omicron coinfection. A comparative genomic analysis of SARS-CoV-2 viral variants that spread in other countries allowed us to identify possible cases of transmission. We also found that some mutations that are quite rare in the Global Omicron dataset have a higher incidence rate, and identified genetic markers that could be associated with ways of Omicron transmission in Russia. We give the genomic variability of single nucleotide variations across the genome and give a characteristic of haplotype variability of Omicron strains in both Russia and around the world, and we also identify them.

Molecular Epidemiology of mcr-1-Positive Escherichia coli and Klebsiella pneumoniae Isolates: Results from Russian Sentinel Surveillance (2013-2018).

BACKGROUND: The dissemination of mobile colistin resistance (mcr) genes is a serious healthcare threat because polymyxins represent "last-line" therapeutics for multi-drug-resistant Gram-negative pathogens. This study aimed to assess the prevalence of colistin resistance and mcr genes and characteristics of clinical Escherichia coli (Eco) and Klebsiella pneumoniae (Kpn) isolates and plasmids carrying these genes in Russia. METHODS: A total of 4324 Eco and 4530 Kpn collected in the frame of sentinel surveillance in 2013-2018 were tested for susceptibility to colistin and other antibiotics using the broth microdilution method. mcr genes were screened by real-time PCR. Phylogeny, genomic features and plasmids of mcr-positive isolates were assessed using whole-genome sequencing and subsequent bioinformatic analysis. RESULTS: Colistin resistance was detected in 2.24% Eco and 9.3% Kpn. Twenty-two (0.51%) Eco and two (0.04%) Kpn from distant sites carried mcr-1.1. Most mcr-positive isolates co-harbored ESBLs and other resistance determinants to various antibiotic classes. The mcr-positive Eco belonged to 16 MLST types, with ST359 being most common; Kpn belonged to ST307 and ST23. mcr-1.1 was carried mainly in IncI2 (n = 18) and IncX4 (n = 5) plasmids highly similar to those identified previously in human, animal and environmental isolates. CONCLUSION: This study demonstrated a dissemination of "typical" mcr-bearing plasmids among diverse Eco and Kpn genotypes and across a wide geographic area in Russia. Given the frequent association of mcr with other resistance determinants and potential clinical impact, the continual surveillance of this threat is warranted.

Antimicrobial resistance profiles and genes in uropathogenic Enterobacteriaceae in reproductive-age women in St. Petersburg, Russia: implication for treatment of pyelonephritis in pregnancy.

BACKGROUND: Urinary tract infections (UTIs) are common in women, and during pregnancy can cause significant morbidity. Growing and greatly varying antimicrobial resistance (AMR) of Enterobacteriaceae, responsible for most UTIs, necessitates regular local AMR surveillance. In obstetric population, where beta-lactams are the mainstay for treatment of severe UTIs, particular focus should be placed on beta-lactam resistance. This study aimed to evaluate AMR rates and frequency of extended spectrum beta-lactamase (ESBL) and carbapenemase genes in uropathogenic Enterobacteriaceae among reproductive-age women in St. Petersburg, Russia. MATERIALS/METHODS: Urine samples were collected from consecutive reproductive-age women, who attended the D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology from October 2017 to November 2019, and cultured according to routine procedures. Susceptibility to antibiotics and ESBL production was determined using the disc diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. All urine samples and Enterobacteriaceae isolates were tested for ESBL and carbapenemase genes using real-time PCR. RESULTS: Enterobacteriaceae were detected in 91 (56 pregnant and 35 non-pregnant) of 119 (76%) included women. The vast majority of Enterobacteriaceae strains were susceptible to nitrofurantoin, fosfomycin and meropenem (99-100%). The frequency of strains susceptible to penicillins and cephalosporins ranged from 59% to 82%; 78% of strains were susceptible to ciprofloxacin. ESBL production was phenotypically detected in 15 (16%) Enterobacteriaceae strains, with CTX-M genes revealed in all cases. In all corresponding urine samples, CTX-M genes were also detected. The remaining 104 urine samples were negative for CTX-M genes. In none of the isolates and urine samples, carbapenemase genes were present. CONCLUSIONS: The frequency of ESBL producing Enterobacteriaceae was relatively high (16%), with CTX-M genes detected in all cases in both urine and urine cultures. Rapid PCR detection of CTX-M genes directly in urine samples from women with pyelonephritis can be valuable for timely informing treatment choices.

Identification of Clinically Significant Prostate Cancer by Combined PCA3 and AMACR mRNA Detection in Urine Samples.

PURPOSE: Preclinical evaluation of PCA3 and AMACR transcript simultaneous detection in urine to diagnose clinical significant prostate cancer (prostate cancer with Gleason score ≥7) in a Russian cohort. PATIENTS AND METHODS: We analyzed urine samples of patients with a total serum PSA ≥2 ng/mL: 31 men with prostate cancer scheduled for radical prostatectomy, 128 men scheduled for first diagnostic biopsy (prebiopsy cohort). PCA3, AMACR, PSA and GPI transcripts were detected by multiplex reverse transcription quantitative polymerase chain reaction, and the results were used for scores for calculation and statistical analysis. RESULTS: There was no significant difference between clinically significant and nonsignificant prostate cancer PCA3 scores. However, there was a significant difference in the AMACR score (patients scheduled for radical prostatectomy p=0.0088, prebiopsy cohort p=0.029). We estimated AUCs, optimal cutoffs, sensitivities and specificities for PCa and csPCa detection in the prebiopsy cohort by tPSA, PCA3 score, PCPT Risk Calculator and classification models based on tPSA, PCA3 score and AMACR score. In the clinically significant prostate cancer ROC analysis, the PCA3 score AUC was 0.632 (95%CI: 0.511-0.752), the AMACR score AUC was 0.711 (95%CI: 0.617-0.806) and AUC of classification model based on the PCA3 score, the AMACR score and total PSA was 0.72 (95%CI: 0.58-0.83). In addition, the correlation of the AMACR score with the ratio of total RNA and RNA of prostate cells in urine was shown (tau=0.347, p=6.542e-09). Significant amounts of nonprostate RNA in urine may be a limitation for the AMACR score use. CONCLUSION: The AMACR score is a good predictor of clinically significant prostate cancer. Significant amounts of nonprostate RNA in urine may be a limitation for the AMACR score use. Evaluation of the AMACR score and classification models based on it for clinically significant prostate cancer detection with larger samples and a follow-up analysis is promising.

Purification, crystallization and preliminary X-ray analysis of uridine phosphorylase from Salmonella typhimurium.

The structural udp gene encoding uridine phosphorylase (UPh) was cloned from the Salmonella typhimurium chromosome and overexpressed in Escherichia coli cells. S. typhimurium UPh (StUPh) was purified to apparent homogeneity and crystallized. The primary structure of StUPh has high homology to the UPh from E. coli, but the enzymes differ substantially in substrate specificity and sensitivity to the polarity of the medium. Single crystals of StUPh were grown using hanging-drop vapor diffusion with PEG 8000 as the precipitant. X-ray diffraction data were collected to 2.9 A resolution. Preliminary analysis of the diffraction data indicated that the crystal belonged to space group P6(1(5)), with unit-cell parameters a = 92.3, c = 267.5 A. The solvent content is 37.7% assuming the presence of one StUPh hexamer per asymmetric unit.