Аннотация
Klebsiella pneumoniae относится к группе ESKAPE патогенов и представляет крайне серьёзную проблему для здравоохранения, поскольку является причиной возникновения вспышек нозокомиальных инфекций из-за способности формирования множественной лекарственной устойчивости (МЛУ). Для решения данной проблемы необходима разработка новых подходов, основанная на знаниях фундаментальных свойств устойчивости к антимикробным препаратам (АМП) и к бактериофагам. Цель исследования: апробировать подходы поиска CRISPR-кассет в геномах клинических изолятов Klebsiella pneumoniae с помощью молекулярно-генетических методов исследования для дальнейшей идентификации бактериофагов на основе биоинформатического анализа их спейсерного состава. На первом этапе проведён биоинформатический поиск и анализ разнообразия CRISPR/Cas-систем во всех полных нуклеотидных последовательностях геномов Klebsiella pneumoniae из базы NCBI, что составило 1311 штаммов. На основе полученных данных разработаны олигонуклеотидные праймеры для проведения ПЦР с целью выявления CRISPR-кассет в геномах 100 клинических изолятов Klebsiella pneumoniae, полученных от детей, госпитализированных в областную инфекционную больницу г. Иркутска. В результате в 17,0% исследуемых штаммов выявлены и расшифрованы локусы, содержащие CRISPR-кассеты. Из них в 73,3% случаев идентифицирована одна кассета, в 26,7% две. На основе выявленных спейсерных последовательностей CRISPR-кассет проведён скрининг бактериофагов. В 71,4% случаев отмечалось полное соответствие спейсеров протоспейсерам фагов из известных баз данных, характерных для бактерий рода Klebsiella. Получен полный спектр бактериофагов с указанием их номера доступа в GenBank, к которым, предположительно, исследуемые клинические изоляты обладают устойчивостью. Данные методы исследования могут лечь в основу разработки персонифицированных подходов в диагностике и лечении сложных инфекционных заболеваний.
Annotation
Klebsiella pneumoniae belongs to the ESKAPE group. It poses a serious problem for global health, as it is capable of developing multidrug resistance and causes outbreaks of nosocomial infections. To solve this problem, it is necessary to develop new approaches based on knowledge of the fundamental properties of resistance to antibacterial drugs and bacteriophages. Purpose of the study: to test approaches to searching for CRISPR cassettes in the genomes of clinical isolates of Klebsiella pneumoniae using developed locus-specific primers for identifying bacteriophages based on bioinformatics analysis of their spacer composition. At the first stage, a bioinformatic search and analysis of the diversity of CRISPR/Cas — systems was carried out in all full genome sequences of Klebsiella pneumoniae from the NCBI database, which amounted to 1311 strains. Further, based on the data obtained, oligonucleotide primers were developed for PCR to detect CRISPR — cassettes in the genomes of 100 clinical isolates of Klebsiella pneumoniae obtained from children who were treated at the regional infectious diseases hospital in Irkutsk. As a result, in 17.0% of the studied strains, loci containing CRISPR — cassettes were identified and deciphered. Of these, in 73.3% of cases one cassette was identified, in 26.7% two. Based on the deciphered spacer sequences of CRISPR — cassettes, bacteriophages were screened. As a result, in 71.4% of cases, there was complete correspondence of spacers to phage protospacers from known databases specific to bacteria of the genus Klebsiella. Thus, a complete spectrum of bacteriophages was obtained, indicating their GenBank accession number, to which the studied clinical isolates were presumably resistant. These research methods can form the basis for the development of personalized approaches to the diagnosis and treatment of complex infectious diseases.
Key words: Klebsiella pneumoniae; spacer; antibiotic resistance; CRISPR/Cas-system; bacteriophage; targeted.
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