The aim of the study was to characterize toxigenic strains of Corynebacterium diphtheriae by examining 12 toxigenic strains of C.diphtheriae isolated in Russia between January, 2017 to June, 2019. The morphological, toxigenic and biochemical properties of C.diphtheriae was studied. Genotyping of C.diphtheriae strains was performed by using MLST and dtxR gene sequencing with subsequent phylogenetic analysis. Results. Toxigenic strains of C.diphtheriae were isolated in the Novosibirsk, Samara and Chelyabinsk regions, the Khanty-Mansi autonomous okrug - Yugra as well as the republic Northern Ossetia - Alania. Among these strains, 5 of them were isolated from diphtheria patients (moderate disease found in one case, mild course – remaining patients) and 7 strains were isolated from bacterial carriers. In two cases C.diphtheriae from diphtheria patients were identified as ST25 sequence type, gravis variant; in one case – ST8 type, gravis variant; two cases – ST67 sequence type, mitis variant. In asymptomatic carriers of tox-positive C.diphtheriae strains they belonged to ST25 sequence type, gravis variant – in two cases, ST67 type, mitis variant – in four cases. A sequencing type was not identified in one case. All sequence types were widespread globally being presented by a large number of isolates in the PubMLST and characterized by a substantial amount of derivative sequence types. At the same time, they belonged to different clonal complexes and differed markedly from each other contributing to their reliable difference as assessed by MLST. Study of gene dtxR sequence diversity showed that all allelic variants were typical for the representatives of these a sequence types. New alleles of gene dtxR were not revealed in strains examined. It was shown that non-synonymous substitution C440T leading to A147V amino acid substitution was found solely in one allele distributed in ST8, ST185, ST195 and ST451 types suggesting at late mutation. In contrast, the polymorphism C640A resulting in the amino acid substitution L214I was found not only in the same allele, but also in the basal tree branches indicating that isoleucine was in the ancestral sequence of the protein.
ПОЛИМОРФИЗМ ГЕНА DTXR У СОВРЕМЕННЫХ ШТАММОВ СORYNEBACTERIUM DIPHTHERIAEСчитается, что патогенез Corynebacterium diphtheriae основан на воздействии дифтерийного токсина на синтез белка в клетках человека. Регуляция синтеза токсина находится под контролем белка DtxR. Данный белок осуществляет железоопосредованную репрессию гена дифтерийного токсина, а также координирует работу множества других ге-нов, необходимых для нормальной жизнедеятельности C. diphtheriae. Ген, кодирующий DtxR, можно использовать для анализа популяционной структуры вида, так как он присутствует в геноме как токсигенных, так и нетоксигенных штам-мов. В работе было изучено 45 штаммов C. diphtheriae, выделенных на территории Российской Федерации в 2010-2015 гг. Анализ этих штаммов показал, что ген dtxR обнаруживается у всех представителей вида и является высоко-консервативным участком генома С. diphtheriae. Большинство выявленных полиморфизмов были синонимичны (16 из 18 однонуклеотидных замен). Несмотря на низкий уровень филогенетического сигнала, аллельный вариант dtxR был ассоциирован с биологическими признаками штамма (биовар, токсигенность). Полученные данные свидетельствуют о высокой активности отрицательного отбора, направленного на поддержание в популяции существующей последова-тельности белка, и позволяют рекомендовать наработку фрагментов гена dtxR методом полимеразной цепной реакции в качестве дополнительного метода идентификации возбудителя, что особенно актуально в условиях растущего числа заболеваний, ассоциированных с нетоксигенными штаммами C. diphtheriae. Финансирование: исследование выполнено в рамках Отраслевой научно-исследовательской программы Роспотребнадзора на 2016-2020 гг. «Проб-лемно-ориентированные научные исследования в области эпидемиологического надзора за инфекционными и паразитарными болезнями» по про-ектам № А16-116021550311-2 «Изучение роли микробиоценозов ротоглотки и крови человека при дифтерии, коклюше и других инфекционно-вос-палительных заболеваниях» и № АААА-А16-116101810127-7 «Разработка молекулярно-генетических методов лабораторной диагностики дифтерии и коклюша». The pathogenic mechanism used by Corynebacterium diphtheriae is attributed to the ability of the diphtheria toxin to disrupt protein synthesis in human cells. Diphtheria toxin production is regulated by the DtxR protein. The latter is involved in the ironmediated repression of the toxin gene and coordinates activities of other genes essential for the survival of C. diphtheriae. The DtxR-encoding gene occurs in both toxigenic and non-toxigenic strains; therefore it can be used to analyze the population structure of the species. In our work we have studied 45 strains of C. diphtheriae isolated in the Russian Federation in 2010-2015. These strains were analyzed to reveal that gene dtxR is a highly conservative region of С. diphtheriae genome that can be found in all members of the studied species. The majority of the discovered polymorphisms were synonymous (16 of 18 single nucleotide polymorphisms identified). In spite of the low phylogenetic sign...
Objective: to assess anti-pertussis immunity in schoolchildren aged 7-17 who complained of a prolonged cough during the 11-year follow-up period.Materials and methods. The study included 1046 patients aged 7 to 17 years who applied to the Consultative and Diagnostic Center of the G.N. Gabrichevsky Research Institute of Epidemiology and Microbiology with complaints of prolonged cough in the period from 2010 to 2020. Blood serums were examined in ELISA with the determination of IgM, IgG, IgA antibodies using RIDASCREEN test system (Germany).Results. An active infection with the detection of IgM and/ or IgA, IgG antibodies above threshold levels was detected in 51,3% of children with prolonged cough, while annually in a fairly high percentage throughout the follow-up period. Active pertussis infection, established based on the detection of IgM, IgG, IgA antibodies above thresholds in blood serum samples, prevailed in children 12-15 years old, accounting for more than 60% in children with prolonged cough. Antipertussis immunity as a result of childhood vaccination or previous disease was detected in 16.
Aim. Characteristics of clonal composition of Corynebacterium diphtheriae strain populatior in Russia using MLST, as well as evaluation of a possibility of using of this method during execution of monitoring of diphtheria infection causative agent strains. Materials and methods. C. diphtheriae strains, isolated in Russia in 1957 - 2015 and sent to Gabrichevsky MRIEM reference centre for diphtheria and pertussis, were studied. Genotyping of C. diphtheriae using MLST wa: carried out based on sequencing of «housekeeping» gene fragments. ST identification was carriec out according to PubMLST. Results. C. diphtheriae strains of 36 sequence-types (ST) were identified on the territory of Russia - 27 previously known and 9 novel, detected for the first time. 2 sequence types ST25 and ST8 (22% and 18%) dominated. Inter-relation between phenotype properties (toxigenicity and biovar) and membership of C. diphtheriae strains in certain sequence-types was shown - toxigenic and non-toxigenic C. diphtheriae strains of various biovars were characterized by certain sequence-types. Changes of clonal composition of C. diphtheriae population in dynamics of epidemic process of diphtheria infection were shown. Conclusion. Use of MLST allowed to characterized clonal composition of C. diphtheriae strains’ population in Russia and has shown perspectives of use of this method to characterize population of diphtheria causative agent, detect epidemically significant strains and decipher foci of diphtheria infection.
Community-acquired bloodstream infections (CBSIs) occur in the out-of-hospital setting (44%) and increase the overall mortality from bloodstream infections (BSIs) by 7.2% per year. The development of CBSIs depends on both comorbid and polymorbid diseases and the patients’ age. The causes of CBSIs are: respiratory, hepatobiliary gastrointestinal and urogenital tracts and dental interventions. The etiology of CBSIs is characterized by the isolation of coagulase-negative staphylococci (CNS) (32%), E. coli (27%). To investigate community-acquired bloodstream infection in therapeutic patients. The study included out-of-hospital patients (n=382). 4.5 ml of blood were taken intravenously into a closed vacuum system in order to obtain a buffy coat of blood, which was put on glasses for microscopy and Petri dishes with blood agar for cultivating under aerobic and anaerobic conditions. Microorganisms were identified by mass spectrometry. Microscopy of blood smears was used for rapid diagnosis of infection in the bloodstream. BSI was diagnosed in 183 (48.0%) out of 382 out-of-hospital patients. The etiology of CBSIs was studied on 297 isolated strains of microorganisms. CBSIs rather often complicated the underlying disease in women and young people. The spectrum of CBSI pathogens included aerobic and anaerobic bacteria and fungi. Gram-positive cocci with the leadership of S.epidermidis (25.7%) were more often isolated among bacteria. 70% of all isolated pathogens grew under anaerobic conditions. CBSIs were characterized by polymicrobiality (33.5%) of two to four different microorganisms in one blood culture; the species of associates of polymicrobial blood cultures are shown. Microscopic examination of blood smears revealed microorganisms in 97.1% of cases, including associations of bacteria with fungi (66.9%). CBSIs occurred after contour plastic, in diseases of the respiratory system, genitourinary system, oral cavity, skin and subcutaneous tissue. Microbiological examination of the buffy coat is an alternative microbiological method of CBSIs diagnosis, which includes microscopy and blood cultivating and has a high diagnostic efficiency (97.1% and 48% respectively). It can become an option for replacing imported blood culture automated systems.
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