zdorПроблемы здоровья и экологииHealth and Ecology Issues2220-09672708-6011Gomel State Medical University10.51523/2708-6011.2023-20-1-01zdor-2488Research ArticleКЛИНИЧЕСКАЯ МЕДИЦИНАCLINICAL MEDICINEМолекулярно-генетические маркеры резистентности и вирулентности инвазивных штаммов Klebsiella pneumoniae по данным полногеномного секвенированияMolecular genetic markers of resistance and virulence of invasive Klebsiella pneumoniae strains according to whole genome sequencing datahttps://orcid.org/0000-0003-2104-585XБондаН. А.BondaN. A.

Бонда Надежда Александровна, заведующий микробиологической лабораторией

Гомель

Nadezhda A. Bonda, Head of Microbiological Laboratory 

Gomel 

bondana8448@gmail.comhttps://orcid.org/0000-0003-0483-7329СтомаИ. О.StomaI. О.

Стома Игорь Олегович, доктор медицинских наук, доцент, ректор

Гомель

Igor O. Stoma, Doctor of Medical Sciences, Associate Professor, Rector 

Gomel 

rektor@gsmu.byhttps://orcid.org/0000-0002-1931-4224ОсипкинаО. В.OsipkinaO. V.

Осипкина Ольга Викторовна, заведующий научно-исследовательской лабораторией

Гомель

Olga V. Osipkina, Head of Research Laboratory 

Gomel 

olga.osipkina@mail.ruhttps://orcid.org/0000-0001-9542-3791ЗятьковА. А.ZiatskovA. A.

Зятьков Алексей Александрович, научный сотрудник научно-исследовательской лаборатории

Гомель

Aliaksei A. Ziatskov, Researcher at the Research Laboratory 

Gomel 

ziatskovaa@gmail.comhttps://orcid.org/0000-0002-6725-5353ШафоростА. С.ShaforostA. S.

Шафорост Александр Сергеевич, старший научный сотрудник научно-исследовательской лаборатории

Гомель

 

Alexander S. Shaforost, Senior Researcher at the Research Laboratory 

Gomel 

asofocl@mail.ruhttps://orcid.org/0000-0002-3952-6187КарповаЕ. В.KarpovaE. V.

Карпова Елена Васильевна, ассистент кафедры микробиологии, вирусологии и иммунологии

Гомель

Elena V. Karpova, Assistant Lecturer at the Department of Microbiology, Virology and Immunology

Gomel 

lena_2007_23@mail.ruhttps://orcid.org/0000-0002-9484-7848ТапальскийД. В.TapalskiD. V.

Тапальский Дмитрий Викторович, доктор медицинских наук, доцент, заведующий кафедрой микробиологии, вирусологии и иммунологии

Гомель

Dmitry V. Tapalski, Doctor of Medical Sciences, Associate Professor, Head of the Department of Microbiology, Virology and Immunology

Gomel 

tapalskiy@gsmu.byГомельский областной центр гигиены, эпидемиологии и общественного здоровьяGomel Regional Center for Hygiene, Epidemiology and Public HealthГомельский государственный медицинский университетGomel State Medical University202328032023201715Copyright © Бонда Н.А., Стома И.О., Осипкина О.В., Зятьков А.А., Шафорост А.С., Карпова Е.В., Тапальский Д.В., 20232023Бонда Н.А., Стома И.О., Осипкина О.В., Зятьков А.А., Шафорост А.С., Карпова Е.В., Тапальский Д.В.Bonda N.A., Stoma I.О., Osipkina O.V., Ziatskov A.A., Shaforost A.S., Karpova E.V., Tapalski D.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://journal.gsmu.by/jour/article/view/2488Цель исследования

Цель исследования. С использованием полногеномного секвенирования оценить генетические механизмы антибиотикорезистентности и вирулентности инвазивных штаммов Klebsiella pneumoniae, выделенных от госпитализированных пациентов.

Материалы и методы

Материалы и методы. Для двух устойчивых к карбапенемам множественно-антибиотикорезистентных инвазивных штаммов K. pneumoniae, а также двух чувствительных к карбапенемам инвазивных штаммов К. pneumoniae выполнено секвенирование в геномном секвенаторе MiSeq (Illumina). Проведена сборка геномных последовательностей и их аннотация. Выполнено определение сиквенс-типа, поиск плазмид и факторов вирулентности, генов антибиотикорезистентности и механизмов эффлюкса.

Результаты

Результаты. Штаммы K. pneumoniae относились к сиквенс-типам ST395, ST101, ST111, ST512 и имели гипермукоидный фенотип. Гены аэробактина iutA были обнаружены как у чувствительных, так и у резистентных к карбапенемам штаммов. У одного выделенного из крови штамма выявлены гены вирулентности fimH, fyuA, irp2. У двух штаммов выявлены гены карбапенемаз (blaKPC, blaNDM). Гены устойчивости к аминогликозидам и фторхинолонам выявлены у трех из четырех штаммов. У всех штаммов отмечено присутствие различных систем активного выведения антибиотиков из микробной клетки.

Заключение

Заключение. Показана возможность идентификации гипервирулентных штаммов K. pneumoniae при комплексном использовании фенотипического теста наряду с генотипированием hvKp. Результаты полногеномного секвенирования отражают значительную устойчивость выделенных из крови гипермукоидных штаммов K. pneumoniae к большинству антибиотиков, включая β-лактамы, аминогликозиды, фторхинолоны, фосфомицин, хлорамфеникол, полимиксины.

Objective

Objective. To evaluate genetic mechanisms of antibiotic resistance and virulence of invasive strains of Klebsiella pneumoniae isolated from inpatients using whole genome sequencing.

Materials and methods

Materials and methods. For two carbapenem-resistant multiple-antibiotic-resistant invasive strains of K.pneumoniae, as well as two carbapenem-sensitive invasive strains of K.pneumoniae, sequencing was performed using the MiSeq genomic sequencer (Illumina). Genomic sequences were assembled and annotated. Sequence type determination, search for plasmids and virulence factors, antibiotic resistance genes, and efflux mechanisms were performed.

Results

Results. K.pneumoniae strains belonged to sequence types ST395, ST101, ST111, and ST512 s and had a hypermucoid phenotype. The iutA aerobactin genes were detected in both sensitive and carbapenem-resistant strains. Virulence genes fimH, fyuA, and irp2 were detected in one strain isolated from blood. Carbapenemase genes (blaKPC, blaNDM) were detected in two strains. Aminoglycosides and fluoroquinolones resistance genes were detected in 3 of 4 strains. All strains showed the presence of different systems of active antibiotic elimination from the microbial cell.

Conclusion

Conclusion. The possibility of identifying hypervirulent strains of K.pneumoniae using a complex phenotypic test along with hvKp genotyping is shown. The results of full-genome sequencing reflect significant resistance of hypervirulent K.pneumoniae strains isolated from blood to most antibiotics, including β-lactams, aminoglycosides, fluoroquinolones, phosphomycin, chloramphenicol and polymyxins.

Klebsiella pneumoniaeгипервирулентностьантибиотикорезистентностьполногеномное секвенированиеKlebsiella pneumoniaehypervirulenceantibiotic resistancewhole genome sequencingReferencesTogawa A, Toh H, Onozawa K, Yoshimura M, Tokushige C, et al. Influence of the bacterial phenotypes on the clinical manifestations in Klebsiella pneumoniae bacteremia patients: a retrospective cohort study. J Infect Chemother. 2015 Jul;21(7):531-537. DOI: https://doi.org/10.1016/j.jiac.2015.04.004Togawa A, Toh H, Onozawa K, Yoshimura M, Tokushige C, et al. Influence of the bacterial phenotypes on the clinical manifestations in Klebsiella pneumoniae bacteremia patients: a retrospective cohort study. J Infect Chemother. 2015 Jul;21(7):531-537. DOI: https://doi.org/10.1016/j.jiac.2015.04.004Liu C, Guo J. Hypervirulent Klebsiella pneumonia (hypermucoviscous and aerobactin positive) infection over 6 years in the elderly in China: antimicrobial resistance patterns, molecular epidemiology and risk factor. Ann clin microbiol. 2019 Jan 21;18(1):4. DOI: https://doi.org/10.1186/s12941-018-0302-9Liu C, Guo J. Hypervirulent Klebsiella pneumonia (hypermucoviscous and aerobactin positive) infection over 6 years in the elderly in China: antimicrobial resistance patterns, molecular epidemiology and risk factor. Ann clin microbiol. 2019 Jan 21;18(1):4. DOI: https://doi.org/10.1186/s12941-018-0302-9Rossi B, Gasperini ML, Leflon-Guibout V, Gioanni A, de Lastours V, Rossi G, et al. Hypervirulent Klebsiella pneumonia in cryptogenic liver abscesses, Paris, France. Emerg Infect Dis. 2018 Feb;24(2):221-229. DOI: https://doi.org/10.3201/eid2402.170957Rossi B, Gasperini ML, Leflon-Guibout V, Gioanni A, de Lastours V, Rossi G, et al. Hypervirulent Klebsiella pneumonia in cryptogenic liver abscesses, Paris, France. Emerg Infect Dis. 2018 Feb;24(2):221-229. DOI: https://doi.org/10.3201/eid2402.170957Parrott AM, Shi J, Aaron J, Green DA, et al. Detection of multiple hypervirulent Klebsiella pneumoniae strains in a New York City hospital through screening of virulence genes. Clin. Microbiol.Infect. 2021 Apr;27(4):583-589. DOI: https://doi.org/10.1016/j.cmi.2020.05.012Parrott AM, Shi J, Aaron J, Green DA, et al. Detection of multiple hypervirulent Klebsiella pneumoniae strains in a New York City hospital through screening of virulence genes. Clin. Microbiol. Infect. 2021 Apr;27(4):583-589. DOI: https://doi.org/10.1016/j.cmi.2020.05.012Dong N, Yang X, Zhang R, Chan EW-C, Chen S. Tracking microevolution events among ST11 carbapenemase-producing hypervirulent Klebsiella pneumoniae outbreak strains. Emerg Microbes Infect. 2018 Aug 12;7(1):146. DOI: https://doi.org/10.1038/s41426-018-0146-6Dong N, Yang X, Zhang R, Chan EW-C, Chen S. Tracking microevolution events among ST11 carbapenemase-producing hypervirulent Klebsiella pneumoniae outbreak strains. Emerg Microbes Infect. 2018 Aug 12;7(1):146. DOI: https://doi.org/10.1038/s41426-018-0146-6Sanikhani R, Moeinirad M, Shahcheraghi F, Lari A, Fereshteh S, Sepehr A, et al. Molecular epidemiology of hypervirulent Klebsiella pneumoniae: a systematic review and meta-analysis. Iran J Microbiol. 2021 Jun;13(3):257-265. DOI: https://doi.org/10.18502/ijm.v13i3.6384Sanikhani R, Moeinirad M, Shahcheraghi F, Lari A, Fereshteh S, Sepehr A, et al. Molecular epidemiology of hypervirulent Klebsiella pneumoniae: a systematic review and meta-analysis. Iran J Microbiol. 2021 Jun;13(3):257-265. DOI: https://doi.org/10.18502/ijm.v13i3.6384Liao W, De Wang L, Li D, Du F-L, Long D, Liu Y, et al. High Prevalence of 16s rRNA methylase genes among carbapenem-resistant hypervirulent Klebsiella pneumoniae isolates in a Chinese Tertiary Hospital. Microb Drug Resist. 2021 Jan;27(1):44-52. DOI: https://doi.org/10.1089/mdr.2019.0482Liao W, De Wang L, Li D, Du F-L, Long D, Liu Y, et al. High Prevalence of 16s rRNA methylase genes among carbapenem-resistant hypervirulent Klebsiella pneumoniae isolates in a Chinese Tertiary Hospital. Microb Drug Resist. 2021 Jan;27(1):44-52. DOI: https://doi.org/10.1089/mdr.2019.0482RR, Judd LM, Froumine R, Tokolyi A, Gorrie CL et al. Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae. PLoS Genet. 2019 Apr 15;15(4):e1008114. DOI: https://doi.org/10.1371/journal.pgen.1008114RR, Judd LM, Froumine R, Tokolyi A, Gorrie CL, et al. Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae. PLoS Genet. 2019 Apr 15;15(4):e1008114. DOI: https://doi.org/10.1371/journal.pgen.1008114Feng Y, Lu Y, Yao Z, Zong Z. Carbapenem-resistant hypervirulent Klebsiella pneumonia of sequence type 36. Antimicrob Agents Chemother. 2018 Jun 26;62(7):e02644-17. DOI: https://doi.org/10.1128/AAC.02644-17Feng Y, Lu Y, Yao Z, Zong Z. Carbapenem-resistant hypervirulent Klebsiella pneumonia of sequence type 36. Antimicrob Agents Chemother. 2018 Jun 26;62(7):e02644-17. DOI: https://doi.org/10.1128/AAC.02644-17Yan J, Wang M, Zheng P, Tsai L, Wu J. Associations of the major international high-risk resistant clones and virulent clones with specific ompK36 allele groups in Klebsiella pneumonia in Taiwan. N Microbes New Infect. 2015 Feb;5:1-4. DOI: https://doi.org/10.1016/j.nmni.2015.01.002Yan J, Wang M, Zheng P, Tsai L, Wu J. Associations of the major international high-risk resistant clones and virulent clones with specific ompK36 allele groups in Klebsiella pneumonia in Taiwan. N Microbes New Infect. 2015 Feb;5:1-4. DOI: https://doi.org/10.1016/j.nmni.2015.01.002Hamzaoui Z, Ocampo-Sosa A, Martinez MF, Landolsi S, Ferjani S, Maamar E, et al. Role of association of OmpK35 and OmpK36 alteration and blaESBL and/or blaampC genes in conferring carbapenem resistance among non-carbapenemase-producing Klebsiella pneumoniae. Int J Antimicrob Agents. 2018 Dec;52(6):898-905. DOI: https://doi.org/10.1016/j.ijantimicag.2018.03.020Hamzaoui Z, Ocampo-Sosa A, Martinez MF, Landolsi S, Ferjani S, Maamar E, et al. Role of association of OmpK35 and OmpK36 alteration and blaESBL and/or blaampC genes in conferring carbapenem resistance among non-carbapenemase-producing Klebsiella pneumoniae. Int J Antimicrob Agents. 2018 Dec;52(6):898-905. DOI: https://doi.org/10.1016/j.ijantimicag.2018.03.020Li G, Shi J, Zhao Y, Xie Y, Tang Y, Jiang X, et al. Identification of hypervirulent Klebsiella pneumonia isolates using the string test in combination with Galleria mellonella infectivity. Eur J Clin Microbiol Infect Dis. 2020 Sep;39(9):1673-1679. DOI: https://doi.org/10.1007/s10096-020-03890-zLi G, Shi J, Zhao Y, Xie Y, Tang Y, Jiang X, et al. Identification of hypervirulent Klebsiella pneumonia isolates using the string test in combination with Galleria mellonella infectivity. Eur J Clin Microbiol Infect Dis. 2020 Sep;39(9):1673-1679. DOI: https://doi.org/10.1007/s10096-020-03890-zRusso TA, Olson R, Fang CT, Stoesser N, Miller M, MacDonald U, et al. Identification of biomarkers for differentiation of hypervirulent Klebsiella pneumonia from classical K. pneumoniae. J Clin Microbiol. 2018 Aug 27;56(9):e00776-18. DOI: https://doi.org/10.1128/JCM.00776-18Russo TA, Olson R, Fang CT, Stoesser N, Miller M, MacDonald U, et al. Identification of biomarkers for differentiation of hypervirulent Klebsiella pneumonia from classical K. pneumoniae. J Clin Microbiol. 2018 Aug 27;56(9):e00776-18. DOI: https://doi.org/10.1128/JCM.00776-18Hao M, Shi X, Lv J, Niu S, Cheng S, Du H, et al. In vitro activity of apramycin against carbapenem-resistant and hypervirulent Klebsiella pneumoniae isolates. Front. Microbiol. 2020 Mar 13;11:425. DOI: https://doi.org/10.3389/fmicb.2020.00425Hao M, Shi X, Lv J, Niu S, Cheng S, Du H, et al. In vitro activity of apramycin against carbapenem-resistant and hypervirulent Klebsiella pneumoniae isolates. Front. Microbiol. 2020 Mar 13;11:425. DOI: https://doi.org/10.3389/fmicb.2020.00425Shankar C, Jacob JJ, Vasudevan K, Biswas R, Manesh A, et al. Emergence of multidrug resistant hypervirulent ST23 Klebsiella pneumoniae: multidrug resistant plasmid acquisition drives evolution. Front. cell Infect. Microbiol. 2020 Nov 20;10:575289. DOI: https://doi.org/10.3389/fcimb.2020.575289Shankar C, Jacob JJ, Vasudevan K, Biswas R, Manesh A, et al. Emergence of multidrug resistant hypervirulent ST23 Klebsiella pneumoniae: multidrug resistant plasmid acquisition drives evolution. Front cell Infect Microbiol. 2020 Nov 20;10:575289. DOI: https://doi.org/10.3389/fcimb.2020.575289Gu D, Dong N, Zheng Z, Lin D, et al. A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study. Lancet Infect. Dis. 2018,18:37-46. DOI: https://doi.org/10.1016/S1473-3099(17)30489-9Gu D, Dong N, Zheng Z, Lin D, et al. A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study. Lancet Infect. Dis. 2018,18:37-46. DOI: https://doi.org/10.1016/S1473-3099(17)30489-9Gorrie CL, Mirceta, M, Wick RR, Edwards DJ, Thomson NR, Strugnell RA, et al. Gastrointestinal carriage is a major reservoir of Klebsiella pneumoniae infection in intensive care patients. Clin. Infect. Dis. 2017 Jul 15;65(2):208-215. DOI: https://doi.org/10.1093/cid/cix270Gorrie CL, Mirceta M, Wick RR, Edwards DJ, Thomson NR, Strugnell, RA, et al. Gastrointestinal carriage is a major reservoir of Klebsiella pneumoniae infection in intensive care patients. Clin Infect Dis. 2017 Jul 15;65(2):208-215. DOI: https://doi.org/10.1093/cid/cix270Sun QL, Gu D, Wang Q, Hu Y, Shu L, Hu J, et al. Dynamic colonization of isolates in gastrointestinal tract of intensive care patients. Front. Microbiol. 2019 Feb 11;10:230. DOI: https://doi.org/10.3389/fmicb.2019.00230Sun QL, Gu D, Wang Q, Hu Y, Shu L, Hu J, et al. Dynamic colonization of isolates in gastrointestinal tract of intensive care patients. Front Microbiol. 2019 Feb 11;10:230. DOI: https://doi.org/10.3389/fmicb.2019.00230Баранцевич ЕП. Продукция карбапенемаз нозокомиальными штаммами K. pneumoniae в Санкт-Петербурге. Клиническая микробиология и антимикробная химиотерапия. 2016;18(3):196-199.Barantsevich EP. Production of Carbapenemases in Klebsiella pneumoniae Isolated in Saint-Petersburg. Clin Microb Antichemother. 2016;18(3):196-199. (In Russ.).Lam MM, Wyres KL, Judd LM, Wick RR, Jenney A, Brisse S, et al. Tracking key virulence loci encoding aerobactin and salmochelin siderophore synthesis in Klebsiella pneumoniae. Genome Med. 2018 Oct 29;10(1):77. DOI: https://doi.org/10.1186/s13073-018-0587-5Lam MM, Wyres KL, Judd LM, Wick RR, Jenney A, Brisse S, et al. Tracking key virulence loci encoding aerobactin and salmochelin siderophore synthesis in Klebsiella pneumoniae. Genome Med. 2018 Oct 29;10(1):77. DOI: https://doi.org/10.1186/s13073-018-0587-5Тапальский ДВ, Петренев ДР. Распространенность Klebsiella pneumoniae - продуцентов карбапенемаз в Беларуси и их конкурентоспособность. Клиническая микробиология и антимикробная химиотерапия. 2017;19(2):139-144.Tapalski DV, Petrenev DR. Prevalence of carbapenemase-producing Klebsiella pneumoniae isolates in Belarus and their competitive ability Clin Microb Antichemother. 2017;19(2):139-144. (In Russ.).Тапальский ДВ, Осипов ВА, Евсеенко ЕО, и др. Металло-бета-лактамазы и карбапенемазы экстремально-антибиотикорезистентных энтеробактерий: распространение в Беларуси. Здравоохранение. 2017;(3):40-47.Tapalski DV, Osipov VA, Yevseyenko EO, et al. (2017) Metallo-beta-lactamases and carbapenemases among extreme antibiotic-resistant Klebsiella pneumoniae: occurrence in Belarus. Zdravoohranenie. 2017;(3):40-47. (In Russ.).

The authors declare that there are no conflicts of interest present.