Molecular genetic characteristics of the carbapenem resistant Klebsiella pneumoniae KP254 strain as a representative of the highly virulent strain evolutionary branch

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Here we provide molecular and genetic characteristics of the Klebsiella pneumoniae KP254 clinical strain belonging to clonal group 23 based on the genome-wide sequencing data. It is known that representatives of such clonal group exert highly virulent properties and cause community-acquired infections. Phenotypically, K. pneumoniae KP254 strain is characterized by multidrug resistance, including carbapenems. The determinants of antibiotic resistance (blaSHV-1, oqxAB, fosA) and pathogenicity encoding fimbriae 1, 3 types and the siderophore yersineobactin synthesis were found in the chromosome structure. However, there was uncovered the lack of conjugative element ICEKp1, the pathogenicity island KPHPI208, and the allantoin regulon genes which are often found in highly virulent strains. Analyzing nucleotide sequences in silico allowed to reveal the replicons of incompatibility group plasmids for FII, FIAHI1/FIIK, Col440I, ColpVC, FIBK, FIIpCRY. Combining contigs relative to reference sequences by using the BLASTN service allowed to identify two putative antibiotic resistance plasmids IncFII and IncFIIpCRY as well as one virulence plasmid IncFIBK. The determinants of the aerobactin siderophore, the RmpA2 mucoid phenotype regulator as well as heavy metal resistance genes constitute the virulence plasmid structure. The virulence plasmid nucleotide sequence coverage comprised 93% relative to the virulence plasmid pK2044 with 99.38% identity level; the genomic regions responsible for the salmochelin and RmpA protein synthesis were deleted. The set of antibiotic resistance determinants identified in the mobilome structure includes the genes for beta-lactamase LAP-2 (IncFIIpCRY plasmid) — a TEM-1 analogue, as well as extended-spectrum beta-lactamase CTX-M-55 (IncFII plasmid), both of which are rarely recorded in the Russian Federation. Additionally, widespread genes blaOXA-1, aac(3’)-IIa, ΔcatB4, aac (6’)-Ib-cr, tet(A), qnrSI, sul2, catA2 were also found in the plasmid DNA. The carbapenemase genes are absent in the resistome structure, whereas the examined strain exerts carbapenem resistance. The analysis of the ompK35 and ompK36 porin gene translated sequences revealed mutational changes which resulted in emerged stop codon within the ompK35 gene, whereas OmpK36 amino acid sequence contains a large number of substitutions, insertions, and deletions. The changes identified serve as one of the factors determining the carbapenem resistance. A synergistic effect may be accounted for by activity of the efflux pumps found in the structure of the K. pneumoniae KP254 genome, particularly AcrAB-TolC and KpnEF. Thus, the strain examined by us preserves the most significant signs specific to the highly virulent evolutionary branch Klebsiella strains, and at the same time, acquires the multidrug resistance genetic determinants.

About the authors

A. E. Alekseeva

I.N. Blokhina Scientific Research Institute of Epidemiology and Microbiology of Nizhny Novgorod

Author for correspondence.
ORCID iD: 0000-0001-6482-0268

Anna E. Alekseeva - PhD (Biology), Senior Researcher, Laboratory of Metagenomics and Molecular Indication of Pathogens, Blokhina I.N. Scientific Research Institute of Epidemiology and Microbiology of Nizhny Novgorod.

603950, Nizhniy Novgorod, Malaya Yamskaya str., 71.

Phone: +7 (831) 432-87-91; Fax: +7 (831) 469-79-20

Russian Federation

N. F. Brusnigina

I.N. Blokhina Scientific Research Institute of Epidemiology and Microbiology of Nizhny Novgorod

ORCID iD: 0000-0003-4582-5623

PhD (Medicine), Associate Professor, Head of the Laboratory of Metagenomics and Molecular Indication of Pathogens, Blokhina I.N. Scientific Research Institute of Epidemiology and Microbiology of Nizhny Novgorod.

Nizhny Novgorod.

Russian Federation

N. A. Gordinskaya

I.N. Blokhina Scientific Research Institute of Epidemiology and Microbiology of Nizhny Novgorod

ORCID iD: 0000-0002-4146-0332

PhD, MD (Medicine), Senior Researcher, Laboratory of Metagenomics and Molecular Indication of Pathogens, Blokhina I.N. Scientific Research Institute of Epidemiology and Microbiology of Nizhny Novgorod.

Nizhny Novgorod.

Russian Federation


  1. Алексеева А.Е., Бруснигина Н.Ф., Солнцев Л.А., Гординская Н.А. Молекулярное типирование клинических изолятов Klebsiella pneumoniae, продуцирующих бета-лактамазы расширенного спектра действия // Клиническая лабораторная диагностика. 2017. Т. 62, № 11. С. 699—704. doi: 10.18821/0869-20842017-62-11-699-704
  2. Комисарова Е.В., Воложанцев Н.В. Гипервирулентная Klebsiella pneumoniae — новая инфекционная угроза // Инфекционные болезни. 2019. Т. 17, № 3. С. 81—89. doi: 10.20953/17299225-2019-3-81-89
  3. Bertels F., Silander O.K., Pachkov M., Rainey P.B., van Nimwegen E. Automated reconstruction of whole-genome phylogenies from short-sequence reads. Mol. Biol. Evol, 2014, vol. 31, no. 5, pp. 1077—1088. doi: 10.1093/molbev/msu088
  4. Bhagirath A.Y., Li Y., Patidar R., Yerex K., Ma X., Kumar A., Duan K. Two component regulatory systems and antibiotic resistance in gram-negative pathogens. Int. J. Mol. Sci., 2019, vol. 20, no. 7: 1781. doi: 10.3390/ijms20071781
  5. Bialek-Davenet S., Criscuolo A., Ailloud F., Passet V., Jones L., Delannoy-Vieillard A., Garin B., Le Hello S., Arlet G., Nicolas-Chanoine M.-H., Decre D., Brisse S. Genomic definition of hypervirulent and multidrug-resistant Klebsiella pneumoniae clonal groups. Emerg. Infect. Dis, 2014, vol. 20, no. 11, pp. 1812—1820. doi: 10.3201/eid2011.140206
  6. Bulger J., MacDonald U., Olson R., Beanan J., Russo T.A. Metabolite transporter PEG344 is required for full virulence of hy-pervirulent Klebsiella pneumoniae strain hvKP1 after pulmonary but not subcutaneous challenge. Infect. Immun., 2017, vol. 85, no. 10: e00093-17. doi: 10.1128/IAI.00093-17
  7. Cabral L., Junior G.V.L., Pereira de Sousa S.T., Dias A.C.F., Lira Cadete L., Andreote F.D., Hess M., de Oliveira V.M. Anthropogenic impact on mangrove sediments triggers differential responses in the heavy metals and antibiotic resistomes of microbial communities. Environ. Pollut., 2016, vol. 216, pp. 460— 469. doi: 10.1016/j.envpol.2016.05.078
  8. Carattoli A., Hasman H. PlasmidFinder and in silico pMLST: identification and typing of plasmid replicons in whole-genome sequencing (WGS). Methods Mol. Biol, 2020, vol. 2075, pp. 285-294. doi: 10.1007/978-1-4939-9877-7_20
  9. Chen X., He L., Li Y., Zeng Z., Deng Y., Liu Y., Liu J.-H. Complete sequence of a F2:A-:B- plasmid pHN3A11 carrying rmtB and qepA, and its dissemination in China. Vet. Microbiol., 2014, vol. 174, no. 1-2, pp. 267-271. doi: 10.1016/j.vetmic.2014.08.023
  10. Chen Y.T., Chang H.Y., Lai Y.C., Pan C.C., Tsai S.F., Peng H.L. Sequencing and analysis of the large virulence plasmid pLVPK of Klebsiella pneumoniae. Gene, 2004, vol. 337, pp. 189-198. doi: 10.1016/j.gene.2004.05.008
  11. Cheong H.S., Chung D.R., Park M., Kim S.H., Ko K.S., Ha Y.E., Kang C.I., Peck K.R., Song J.H. Emergence of an extended-spectrum в-lactamase-producing serotype K1 Klebsiella pneumoniae ST23 strain from Asian countries. Epidemiol. Infect., 2017, vol. 145, no. 5, pp. 990-994. doi: 10.1017/S0950268816003113
  12. Chou H.C., Lee C.Z., Ma L.C., Fang C.T., Chang S.C., Wang J.T. Isolation of a chromosomal region of Klebsiella pneumoniae associated with allantoin metabolism and liver infection. Infect. Immun., 2004, vol. 72, no. 7, pp. 3783-3792. doi: 10.1128/IAI.72.7.3783-3792.2004
  13. Deng Y., He L., Chen S., Zheng H., Zeng Z., Liu Y., Sun Y., Ma J., Chen Z., Liu J.H. F33:A-:B- and F2:A-:B- plasmids mediate dissemination of rmtB-blaCTX-M-9 group genes and rmtB-qepA in Enterobacteriaceae isolates from pets in China. Antimicrob. Agents Chemother., 2011, vol. 55, no. 10, pp. 4926-4929. doi: 10.1128/AAC.00133-11
  14. Edelstein M., Pimkin M., Palagin I., Edelstein I., Stratchounski L. Prevalence and molecular epidemiology of CTX-M extended-spectrum в-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Russian hospitals. Antimicrob. Agents Chemother., 2003, vol. 47, no. 12, pp. 3724-3732. doi: 10.1128/aac.47.12.3724-3732.2003
  15. Fang C.T., Chuang Y.P., Shun C.T., Chang S.C., Wang J.T. A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J. Exp. Med., 2004, vol. 199, no. 5, pp. 697-705. doi: 10.1084/jem.20030857
  16. Gu D., Dong N., Zheng Z., Lin D., Huang M., Wang L., Chan E.W., Shu L., Yu J., Zhang R., Chen S. A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study. Lancet Infect. Dis., 2017, vol. 18, no. 1, pp. 37-46. doi: 10.1016/S1473-3099(17)30489-9
  17. Hobbs E.C., Yin X., Paul B.J., Astarita J.L., Storz G. Conserved small protein associates with the multidrug efflux pump AcrB and differentially affects antibiotic resistance. Proc. Natl. Acad. Sci. USA, 2012, vol. 109, no. 41, pp. 16696-16701. doi: 10.1073/pnas.12100931
  18. Holdsworth S.R., Law C.J. The major facilitator superfamily transporter MdtM contributes to the intrinsic resistance of Escherichia coli to quaternary ammonium compounds. J. Antimicrob. Chemother., 2013, vol. 68, no. 4, pp. 831—839. doi: 10.1093/jac/dks491
  19. Hu X., Gou J., Guo X., Cao Z., Li Y., Jiao H., He X., Ren Y., Tian F. Genetic contexts related to the diffusion of plasmid-mediated CTX-M-55 extended-spectrum beta-lactamase isolated from Enterobacteriaceae in China. Ann. Clin. Microbiol. Antimicrob., 2018, vol. 17, no. 1. doi: 10.1186/s12941-018-0265-x
  20. Izquierdo L., Coderch N., Pique N., Bedini E., Corsaro M.M., Merino S., Fresno S., Tomas J.M., Regue M. The Klebsiella pneumoniae wabG gene: role in biosynthesis of the core lipopolysaccharide and virulence. J. Bacteriol., 2003, vol. 185, no. 24, pp. 7213-7221. doi: 10.1128/jb.185.24.7213-7221.2003
  21. Kiratisin P., Apisarnthanarak A., Saifon P., Laesripa C., Kitphati R., Mundy L.M. The emergence of a novel ceftazidime-resistant CTX-M extended-spectrum beta-lactamase, CTX-M-55, in both community-onset and hospital-acquired infections in Thailand. Diagn. Microbiol. Infect. Dis., 2007, vol. 58, no. 3, pp. 349-355. doi: 10.1016/j.diagmicrobio.2007.02.005
  22. Kucken D., Feucht H., Kaulfers P. Association of qacE and qacEDelta1 with multiple resistance to antibiotics and antiseptics in clinical isolates of Gram-negative bacteria. FEMS Microbiol. Lett., 2000, vol. 183, iss. 1, pp. 95-98. doi: 10.1111/j.1574-6968.2000.tb08939.x
  23. Lai Y.C., Lin A.C., Chiang M.K., Dai Y.H., Hsu C.C., Lu M.C., Liau C.Y., Chen Y.T. Genotoxic Klebsiella pneumoniae in Taiwan. PLoS One, 2014, vol. 9, no. 5: e96292. doi: 10.1371/journal.pone.0096292
  24. Lev A.I., Astashkin E.I., Kislichkina A.A., Solovieva E.V., Kombarova T.I., Korobova O.V., Ershova O.N., Alexandrova I.A., Malikov V.E., Bogun A.G., Borzilov A.I., Volozhantsev N.V., Svetoch E.A., Fursova N.K. Comparative analysis of Klebsiella pneumoniae strains isolated in 2012—2016 that differ by antibiotic resistance genes and virulence genes profiles. Pathog. Glob. Health, 2018, vol. 112, no. 3, pp. 142-151. doi: 10.1080/20477724.2018.1460949
  25. Li J., Zhang H., Ning J., Sajid A., Cheng G., Yuan Z., Hao H. The nature and epidemiology of OqxAB, a multidrug efflux pump. Antimicrob. Resist. Infect. Control., 2019, vol. 8, no. 44. doi: 10.1186/s13756-019-0489-3
  26. Li X.-Z., Plesiat P., Nikaido H. The challenge of efflux mediated antibiotic resistance in gram-negative bacteria. Clin. Microbiol. Rev., 2015, vol. 28, no. 2, pp. 337-418. doi: 10.1128/CMR.00117-14
  27. Lin T.L., Lee C.Z., Hsieh P.F., Tsai S.F., Wang J.T. Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella pneumoniae strain isolated from a primary liver abscess. J. Bacteriol., 2008, vol. 190, no. 2, pp. 515-526. doi: 10.1128/JB.01219-07
  28. Ma L.-C., Fang C.-T., Lee C.-Z., Shun C.-T., Wang J.-T., Genomic heterogeneity in Klebsiella pneumoniae strains is associated with primary pyogenic liver abscess and metastatic infection. J. Infect. Dis., 2005, vol. 192, no. 1, pp. 117-128. doi: 10.1086/430619
  29. Moura A., Soares M., Pereira C., Leitao N., Henriques I., Correia A. INTEGRALL: a database and search engine for integrons, integrases and gene cassettes. Bioinformatics, 2009, vol. 25, no. 8, pp. 1096-1098. doi: 10.1093/bioinformatics/btp105
  30. Nzakizwanayo J., Scavone P., Jamshidi S., Hawthorne J.A., Pelling H., Dedi C., Salvage J.P., Hind C.K., Guppy F.M., Barnes L.M., Patel B.A., Rahman K.M., Sutton M.J., Jones B.V. Fluoxetine and thioridazine inhibit efflux and attenuate crystalline biofilm formation by Proteus mirabilis. Sci. Rep., 2017, vol. 7: 12222. doi: 10.1038/s41598-017-12445-w
  31. Pan Y.S., Liu J.H., Hu H., Zhao J.F., Yuan L., Wu H., Wang L.F., Hu G.Z. Novel arrangement of the blaCTX-M-55 gene in an Escherichia coli isolate coproducing 16S rRNA methylase. J. Basic. Microbiol., 2013, vol. 53, no. 11, pp. 928-933. doi: 10.1002/jobm.201200318
  32. Poirel L., Cattoir V., Soares A., Soussy C.J., Nordmann P. Novel Ambler class A beta-lactamase LAP-1 and its association with the plasmid-mediated quinolone resistance determinant QnrS1. Antimicrob. Agents Chemother., 2007, vol. 51, no. 2, pp. 631- 637. doi: 10.1128/AAC.01082-06
  33. Russo T.A., Olson R., Fang C.-T., Stoesser N., Miller M., MacDonald U., Hutson A., Barker J.H., La Hoz R.M., Johnson J.R. for the Hypervirulent Klebsiella pneumoniae Investigator Group (HVKPIG). Identification of biomarkers for differentiation of hypervirulent Klebsiella pneumoniae from classical K. pneumoniae. J. Clin. Microbiol., 2018, vol. 56 (9): e00776-18. doi: 10.1128/JCM.00776-18
  34. Russo T.A., Olson R., Macdonald U., Metzger D., Maltese L.M., Drake E.J., Gulick A.M. Aerobactin mediates virulence and accounts for increased siderophore production under iron-limiting conditions by hypervirulent (hypermucoviscous) Klebsiella pneumoniae. Infect. Immun., 2014, vol. 82, no. 6, pp. 2356-2367. doi: 10.1128/IAI.01667-13
  35. Russo T.A., Marr C.M. Hypervirulent Klebsiella pneumoniae. Clin. Microbiol. Rev., 2019, vol. 32 (3): e00001-19. doi: 10.1128/CMR.00001-19
  36. Siguier P., Perochon J., Lestrade L., Mahillon J., Chandler M. ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res., 2005, vol. 34, pp. D32-D36. doi: 10.1093/nar/gkj014
  37. Srinivasan V.B., Rajamohan G. KpnEF, a new member of the Klebsiella pneumoniae cell envelope stress response regulon, is an SMR-type efflux pump involved in broad-spectrum antimicrobial resistance. Antimicrob. Agents Chemother., 2013, vol. 57, no. 9, pp. 4449-4462. doi: 10.1128/AAC.02284-12
  38. Struve C., Roe C.C., Stegger M., Stahlhut S.G., Hansen D.S., Engelthaler D.M., Andersen P.S., Driebe E.M., Keim P., Krogfelt K.A. Mapping the evolution of hypervirulent Klebsiella pneumoniae. mBio, 2015, vol. 6 (4): e00630-15. doi: 10.1128/mBio.00630-15
  39. Sugawara E., Kojima S., Nikaido H. Klebsiella pneumoniae major porins OmpK35 and OmpK36 allow more efficient diffusion of в-lactams than their Escherichia coli homologs OmpF and OmpC. J. Bacteriol., 2016, vol. 198, no. 23, pp. 3200-3208. doi: 10.1128/JB.00590-16
  40. Surgers L., Boyd A., Girard P.M., Arlet G., Decre D. ESBL-producing strain of hypervirulent Klebsiella pneumoniae K2, France. Emerg. Infect. Dis., 2016, vol. 22, no. 9, pp. 1687-1688. doi: 10.3201/eid2209.160681
  41. Tsai Y.-K., Fung C.-P., Lin J.-C., Chen J.-H., Chang F.-Y., Chen T.-L., Siu L.K. Klebsiella pneumoniae outer membrane porins OmpK35 and OmpK36 play roles in both antimicrobial resistance and virulence. Antimicrob. Agents Chemother., 2011, vol. 55, no. 4, pp. 1485-1493. doi: 10.1128/AAC.01275-10
  42. Villa L., Garci'a-Fernandez A., Fortini D., Carattoli A. Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. J. Antimicrob. Chemother., 2010, vol. 65, no. 12, pp. 2518—2529. doi: 10.1093/jac/dkq347
  43. Wang L., Fang H., Feng J., Yin Z., Xie X., Zhu X., Wang J., Chen W., Yang R., Du H., Zhou D. Complete sequences of KPC-2-encoding plasmid p628-KPC and CTX-M-55-encoding p628-CTXM coexisted in Klebsiella pneumoniae. Front. Microbiol., 2015, 6: 838. doi: 10.3389/fmicb.2015.00838
  44. Weston N., Sharma P., Ricci V., Piddock L.J.V. Regulation of the AcrAB-TolC efflux pump in Enterobacteriaceae. Res. Microbiol., 2018, vol. 169, no. 7-8, pp. 425-431. doi: 10.1016/j.resmic.2017.10.005
  45. Wu K.M., Li L.H., Yan J.J., Tsao N., Liao T.L., Tsai H.C., Fung C.P., Chen H.J., Liu Y.M., Wang J.T., Fang C.T., Chang S.C., Shu H.Y., Liu T.T., Chen Y.T., Shiau Y.R., Lauderdale T.L., Su I.J., Kirby R., Tsai S.F. Genome sequencing and comparative analysis of Klebsiella pneumoniae NTUH-K2044, a strain causing liver abscess and meningitis. J. Bacteriol., 2009, vol. 191, no. 14, pp. 4492-4501. doi: 10.1128/JB.00315-09
  46. Yuan Y., Li Y., Wang G., Li C., Chang Y.F., Chen W., Nian S., Mao Y., Zhang J., Zhong F., Zhang L. BlaNDM-5 carried by a hypervirulent Klebsiella pneumoniae with sequence type 29. Antimicrob. Resist. Infect. Control, 2019, vol. 8: 140. doi: 10.1186/s13756-019-0596-1
  47. Zhao W.H., Hu Z.Q. Epidemiology and genetics of CTX-M extended-spectrum в-lactamases in Gram-negative bacteria. Crit. Rev. Microbiol., 2013, vol. 39, no. 1, pp. 79-101. doi: 10.3109/1040841X.2012.691460

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