Mobile Colistin Resistance Genetic Determinants of Non-Typhoid Salmonella enterica Isolates from Russia.

Polymyxin resistance, determined by mcr genes located on plasmid DNA, currently poses a high epidemiological threat. Non-typhoid Salmonella (NTS) are one of the key pathogens causing diarrheal diseases. Here, we report the isolation and whole genome sequencing of multidrug colistin-resistant/susceptible isolates of non-typhoid Salmonella enterica serovars carrying mcr genes. Non-typhoid strains of Salmonella enterica subsp. enterica were isolated during microbiological monitoring of the environment, food, and diarrheal disease patients between 2018 and 2020 in Russia (n = 586). mcr-1 genes were detected using a previously developed qPCR assay, and whole genome sequencing of mcr positive isolates was performed by both short-read (Illumina) and long-read (Oxford Nanopore) approaches. Three colistin-resistant isolates, including two isolates of S. Enteritidis and one isolate of S. Bovismorbificans, carried the mcr-1.1 gene located on IncX4 and IncI2 conjugative plasmids, respectively. The phenotypically colistin-susceptible isolate of S. Typhimurium carried a mcr-9 gene on plasmid IncHI2. In conclusion, we present the first three cases of mcr gene-carrying NTS isolates detected in Russia with both outbreak and sporadic epidemiological backgrounds.

Molecular surveillance of norovirus, 2005-16: an epidemiological analysis of data collected from the NoroNet network.

BACKGROUND: The development of a vaccine for norovirus requires a detailed understanding of global genetic diversity of noroviruses. We analysed their epidemiology and diversity using surveillance data from the NoroNet network. METHODS: We included genetic sequences of norovirus specimens obtained from outbreak investigations and sporadic gastroenteritis cases between 2005 and 2016 in Europe, Asia, Oceania, and Africa. We genotyped norovirus sequences and analysed sequences that overlapped at open reading frame (ORF) 1 and ORF2. Additionally, we assessed the sampling date and country of origin of the first reported sequence to assess when and where novel drift variants originated. FINDINGS: We analysed 16 635 norovirus sequences submitted between Jan 1, 2005, to Nov 17, 2016, of which 1372 (8·2%) sequences belonged to genotype GI, 15 256 (91·7%) to GII, and seven (<0·1%) to GIV.1. During this period, 26 different norovirus capsid genotypes circulated and 22 different recombinant genomes were found. GII.4 drift variants emerged with 2-3-year periodicity up to 2012, but not afterwards. Instead, the GII.4 Sydney capsid seems to persist through recombination, with a novel recombinant of GII.P16-GII.4 Sydney 2012 variant detected in 2014 in Germany (n=1) and the Netherlands (n=1), and again in 2016 in Japan (n=2), China (n=8), and the Netherlands (n=3). The novel GII.P17-GII.17, first reported in Asia in 2014, has circulated widely in Europe in 2015-16 (GII.P17 made up a highly variable proportion of all sequences in each country [median 11·3%, range 4·2-53·9], as did GII.17 [median 6·3%, range 0-44·5]). GII.4 viruses were more common in outbreaks in health-care settings (2239 [37·2%] of 6022 entries) compared with other genotypes (101 [12·5%] of 809 entries for GI and 263 [13·5%] of 1941 entries for GII non-GII.Pe-GII.4 or GII.P4-GII.4). INTERPRETATION: Continuous changes in the global norovirus genetic diversity highlight the need for sustained global norovirus surveillance, including assessment of possible immune escape and evolution by recombination, to provide a full overview of norovirus epidemiology for future vaccine policy decisions. FUNDING: European Union's Horizon 2020 grant COMPARE, ZonMw TOP grant, the Virgo Consortium funded by the Dutch Government, and the Hungarian Scientific Research Fund.

Global Spread of Norovirus GII.17 Kawasaki 308, 2014-2016.

Analysis of complete capsid sequences of the emerging norovirus GII.17 Kawasaki 308 from 13 countries demonstrated that they originated from a single haplotype since the initial emergence in China in late 2014. Global spread of a sublineage SL2 was identified. A new sublineage SL3 emerged in China in 2016.

Burden of Childhood Rotavirus Disease in the Outpatient Setting of the Russian Federation.

BACKGROUND: This is a prospective, multicentered study conducted in 9 large urban areas in Russia, in order to determine the burden of rotavirus gastroenteritis in children <5 years of age and the genotypes circulating during 1 rotavirus season. METHODS: From November 2012 to May 2013, surveillance was conducted in Moscow, Saint-Petersburg, Vologda, Krasnodar, Krasnoyarsk, Novosibirsk, Yaroslavl, Khanty-Mansiysk and Vladivostok. Children <5 years of age presenting at outpatient clinics with acute gastroenteritis (AGE) of less than 72 hours duration were enrolled in the study. Stool samples were tested for rotavirus and positive samples were P- and G-typed. Clinical symptoms were captured by physicians and parents on Day 1. Symptom severity was analyzed by Vesikari scoring system. The direct expenses of parents caused by AGE were obtained from questionnaires provided to parents by phone. RESULTS: A total of 501 were children enrolled. Stool samples were analyzed for 487 (97%) children, and 151 (31%) of those were rotavirus positive. Rotavirus gastroenteritis was associated with more severe clinical course (Vesikari score 11.4 ± 2.2) versus non-rotavirus gastroenteritis (Vesikari score 9 ± 3). The identified serotypes were G4P[8] 38.9%, G1P[8] 34.2%, G3P[8] 6%, G9P[8] 6%, G2P[4] 2% and G4P[4] 0.7%. The mean overall expenses of parents caused by rotavirus and non-rotavirus gastroenteritis were 143.7 USD and 128.8 USD, respectively. CONCLUSIONS: Rotavirus accounted for 31% of all AGE-related outpatient visits. The major rotavirus genotypes were G1P[8] and G4P[8]. Rotavirus gastroenteritis was associated with significantly more severe clinical symptoms than non-rotavirus gastroenteritis. The average costs of rotavirus cases for parents of children were elevated against the same indications for non-rotavirus. These findings underscore the need for a safe and effective rotavirus vaccine in Russia.

1-Phenylethynylpyrene (PEPy) as a novel blue-emitting dye for qPCR assay.

An alkyl azide derivative of 1-phenylethynylpyrene (PEPy) dye was prepared and used in the functionalization of oligonucleotides via click chemistry. Spectral and photo-physical properties of the PEPy-modified oligonucleotides as a single strand, and in perfect or mismatched duplexes, have been studied. A series of PEPy-Dabcyl fluorogenic TaqMan probes were synthesized and tested in qPCR. PEPy proved to be a superior substitute for AMCA as a short wavelength fluorescent dye for qPCR probes. PEPy probes were shown to significantly reduce Cq (a fractional PCR cycle used for quantification) vs. AMCA labeled probes, thus improving on the reliability of detection. Moreover, a larger increase of fluorescence during amplification was observed in the case of PEPy probes that makes this dye very suitable for an end-point PCR technique. This study broadens the panel of fluorescent dyes suitable for the use in probes for quantitative real-time PCR.

Solid- and solution-phase synthesis and application of R6G dual-labeled oligonucleotide probes.

A novel N-TFA-protected carboxyrhodamine 6G (R6G) phosphoramidite was synthesized for use in an automated DNA synthesis to prepare 5'-labeled oligonucleotides. Deprotection and purification conditions were optimized for 5'-labeled and dual-labeled oligonucleotide probes. As an alternative we synthesized an azide derivative of R6G for CuAAC post-synthetic oligonucleotide labeling. Dual-labeled probes obtained by both methods showed the same efficacy in a quantitative PCR assay. R6G-labeled probes demonstrated superior properties in a qPCR assay in comparison with alternative HEX, JOE and SIMA dyes due to more efficient fluorescence quenching by BHQ-1. We successfully used R6G dual-labeled probes for rotavirus genotyping.