Mobilization of the nonconjugative virulence plasmid from hypervirulent Klebsiella pneumoniae

Springer Science and Business Media LLC - 2021
Yanping Xu1,2, Jianfeng Zhang3, Meng Wang3, Meng Liu3, Guitian Liu3, Hongping Qu4, Jialin Liu4, Zixin Deng3, Jingyong Sun5, Hong‐Yu Ou3, Jieming Qu2,1
1Department of Pulmonary and Critical Care Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
2Shanghai Key Laboratory of Emergency Prevention, Diagnosis and Treatment of Respiratory Infectious Diseases, Shanghai, 200025, China
3State Key Laboratory of Microbial Metabolism, Joint International Laboratory on Metabolic & Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, China
4Department of Critical Care Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
5Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Tóm tắt

Abstract Background

Klebsiella pneumoniae, as a global priority pathogen, is well known for its capability of acquiring mobile genetic elements that carry resistance and/or virulence genes. Its virulence plasmid, previously deemed nonconjugative and restricted within hypervirulent K. pneumoniae (hvKP), has disseminated into classic K. pneumoniae (cKP), particularly carbapenem-resistant K. pneumoniae (CRKP), which poses alarming challenges to public health. However, the mechanism underlying its transfer from hvKP to CRKP is unclear.

 Methods

A total of 28 sequence type (ST) 11 bloodstream infection-causing CRKP strains were collected from Ruijin Hospital in Shanghai, China, and used as recipients in conjugation assays. Transconjugants obtained from conjugation assays were confirmed by XbaI and S1 nuclease pulsed-field gel electrophoresis, PCR detection and/or whole-genome sequencing. The plasmid stability of the transconjugants was evaluated by serial culture. Genetically modified strains and constructed mimic virulence plasmids were employed to investigate the mechanisms underlying mobilization. The level of extracellular polysaccharides was measured by mucoviscosity assays and uronic acid quantification. An in silico analysis of 2608 plasmids derived from 814 completely sequenced K. pneumoniae strains available in GenBank was performed to investigate the distribution of putative helper plasmids and mobilizable virulence plasmids.

 Results

A nonconjugative virulence plasmid was mobilized by the conjugative plasmid belonging to incompatibility group F (IncF) from the hvKP strain into ST11 CRKP strains under low extracellular polysaccharide-producing conditions or by employing intermediate E. coli strains. The virulence plasmid was mobilized via four modes: transfer alone, cotransfer with the conjugative IncF plasmid, hybrid plasmid formation due to two rounds of single-strand exchanges at specific 28-bp fusion sites or homologous recombination. According to the in silico analysis, 31.8% (242) of the putative helper plasmids and 98.8% (84/85) of the virulence plasmids carry the 28-bp fusion site. All virulence plasmids carry the origin of the transfer site.

 Conclusions

The nonconjugative virulence plasmid in ST11 CRKP strains is putatively mobilized from hvKP or E. coli intermediates with the help of conjugative IncF plasmids. Our findings emphasize the importance of raising public awareness of the rapid dissemination of virulence plasmids and the consistent emergence of hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) strains.

Từ khóa


Tài liệu tham khảo

Navon-Venezia S, Kondratyeva K, Carattoli A. Klebsiella pneumoniae: a major worldwide source and shuttle for antibiotic resistance. FEMS Microbiol Rev. 2017;41(3):252–75. https://doi.org/10.1093/femsre/fux013.

Yang X, Dong N, Chan EW-C, Zhang R, Chen S. Carbapenem resistance-encoding and virulence-encoding conjugative plasmids in Klebsiella pneumoniae. Trends Microbiol. 2020.

Heiden SE, Hübner N-O, Bohnert JA, Heidecke C-D, Kramer A, Balau V, et al. A Klebsiella pneumoniae ST307 outbreak clone from Germany demonstrates features of extensive drug resistance, hypermucoviscosity, and enhanced iron acquisition. Genome Med. 2020;12(1):113. https://doi.org/10.1186/s13073-020-00814-6.

Russo TA, Marr CM. Hypervirulent Klebsiella pneumoniae. Clin Microbiol Rev. 2019;32(3). https://doi.org/10.1128/CMR.00001-19.

Chen Y, Chang H, Lai Y, Pan C, Tsai S, Peng H. Klebsiella pneumoniae CG43 plasmid pLVPK, complete sequence. GenBank AY378100: NCBI Nucleotide; 2020. https://www.ncbi.nlm.nih.gov/nuccore/AY378100

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 CG43. Gene. 2004;337:189–98.

Russo TA, Olson R, Fang C-T, Stoesser N, Miller M, MacDonald U, et al. Identification of biomarkers for differentiation of hypervirulent Klebsiella pneumoniae from classical K. pneumoniae. J Clin Microbiol. 2018;56(9). https://doi.org/10.1128/JCM.00776-18.

Lam MMC, Wyres KL, Wick RR, Judd LM, Fostervold A, Holt KE, et al. Convergence of virulence and MDR in a single plasmid vector in MDR Klebsiella pneumoniae ST15. J Antimicrob Chemother. 2019;74(5):1218–22. https://doi.org/10.1093/jac/dkz028.

Gu D, Dong N, Zheng Z, Lin D, Huang M, Wang L, 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(1):37–46. https://doi.org/10.1016/S1473-3099(17)30489-9.

Xie Y, Tian L, Li G, Qu H, Sun J, Liang W, et al. Emergence of the third-generation cephalosporin-resistant hypervirulent Klebsiella pneumoniae due to the acquisition of a self-transferable blaDHA-1-carrying plasmid by an ST23 strain. Virulence. 2018:838–44.

Zhang R, Lin D, Chan EW-C, Gu D, Chen G-X, Chen S. Emergence of Carbapenem-resistant serotype K1 hypervirulent Klebsiella pneumoniae strains in China. Antimicrob Agents Chemother. 2016;60(1):709–11. https://doi.org/10.1128/AAC.02173-15.

Turton J, Davies F, Turton J, Perry C, Payne Z, Pike R. Hybrid resistance and virulence plasmids in “high-risk” clones of Klebsiella pneumoniae, including those carrying blaNDM-5. Microorganisms. 2019;7(9). https://doi.org/10.3390/microorganisms7090326.

Dong N, Lin D, Zhang R, Chan EW-C, Chen S. Carriage of blaKPC-2 by a virulence plasmid in hypervirulent Klebsiella pneumoniae. J Antimicrob Chemother. 2018;73:3317–21.

Huang Y-H, Chou S-H, Liang S-W, Ni C-E, Lin Y-T, Huang Y-W, et al. Emergence of an XDR and carbapenemase-producing hypervirulent Klebsiella pneumoniae strain in Taiwan. J Antimicrob Chemother. 2018;73(8):2039–46. https://doi.org/10.1093/jac/dky164.

Yang X, Wai-Chi Chan E, Zhang R, Chen S. A conjugative plasmid that augments virulence in Klebsiella pneumoniae. Nat Microbiol. 2019;4(12):2039–43. https://doi.org/10.1038/s41564-019-0566-7.

Xie M, Chen K, Ye L, Yang X, Xu Q, Yang C, et al. Conjugation of virulence plasmid in clinical Klebsiella pneumoniae strains through formation of a fusion plasmid. Adv Biosyst. 2020;4:e1900239.

Li R, Cheng J, Dong H, Li L, Liu W, Zhang C, et al. Emergence of a novel conjugative hybrid virulence multidrug-resistant plasmid in extensively drug-resistant Klebsiella pneumoniae ST15. Int J Antimicrob Agents. 2020;55:105952.

Qu H, Ou H. Klebsiella pneumoniae subsp. pneumoniae strain RJF293 plasmid pRJF293, complete sequence. BioProject PRJNA307277. NCBI Nucleotide. 2016; https://www.ncbi.nlm.nih.gov/nuccore/?term=PRJNA307277.

Wang X, Xie Y, Li G, Liu J, Li X, Tian L, et al. Whole-genome-sequencing characterization of bloodstream infection-causing hypervirulent Klebsiella pneumoniae of capsular serotype K2 and ST374. Virulence. 2018;9(1):510–21. https://doi.org/10.1080/21505594.2017.1421894.

Bi D, Jiang X, Sheng Z-K, Ngmenterebo D, Tai C, Wang M, et al. Mapping the resistance-associated mobilome of a carbapenem-resistant Klebsiella pneumoniae strain reveals insights into factors shaping these regions and facilitates generation of a “resistance-disarmed” model organism. J Antimicrob Chemother. 2015;70(10):2770–4. https://doi.org/10.1093/jac/dkv204.

Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27(5):722–36. https://doi.org/10.1101/gr.215087.116.

Qu J, Ou H. Klebsiella pneumoniae subsp. pneumoniae strain:RJBSI76 genome sequencing. BioProject PRJNA681750. NCBI Nucleotide. 2021; https://www.ncbi.nlm.nih.gov/nuccore/?term=PRJNA681750.

Qu J, Ou H. Klebsiella pneumoniae subsp. pneumoniae strain:RJBSI76-pV genome sequencing. BioProject PRJNA682095. NCBI Nucleotide. 2021; https://www.ncbi.nlm.nih.gov/nuccore/?term=PRJNA682095.

Qu J, Ou H. Escherichia coli strain:J53-p1-pV-hybrid-1 genome sequencing. BioProject PRJNA692573. NCBI Nucleotide. 2021; https://www.ncbi.nlm.nih.gov/nuccore/?term=PRJNA692573.

Qu J, Ou H. Escherichia coli strain:XL10-pF-pV-hybrid-1 genome sequencing. BioProject PRJNA692574. NCBI Nucleotide. 2021; https://www.ncbi.nlm.nih.gov/nuccore/1968731931.

Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068–9. https://doi.org/10.1093/bioinformatics/btu153.

Carattoli A, Zankari E, García-Fernández A, Voldby Larsen M, Lund O, Villa L, et al. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother. 2014;58(7):3895–903. https://doi.org/10.1128/AAC.02412-14.

Lam MMC, Wick RR, Wyres KL, Holt KE. Genomic surveillance framework and global population structure for Klebsiella pneumoniae. bioRxiv. 2020; 2020.12.14.422303.

Li J, Tai C, Deng Z, Zhong W, He Y, Ou H-Y. VRprofile: gene-cluster-detection-based profiling of virulence and antibiotic resistance traits encoded within genome sequences of pathogenic bacteria. Brief Bioinform. 2018;19:566–74.

Li X, Xie Y, Liu M, Tai C, Sun J, Deng Z, et al. oriTfinder: a web-based tool for the identification of origin of transfers in DNA sequences of bacterial mobile genetic elements. Nucleic Acids Res. 2018;46:W229–34.

Alikhan N-F, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011;12(1):402. https://doi.org/10.1186/1471-2164-12-402.

Sullivan MJ, Petty NK, Beatson SA. Easyfig: a genome comparison visualizer. Bioinformatics. 2011;27(7):1009–10. https://doi.org/10.1093/bioinformatics/btr039.

Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37(Web Server):W202–8. https://doi.org/10.1093/nar/gkp335.

Palacios M, Broberg CA, Walker KA, Miller VL. A serendipitous mutation reveals the severe virulence defect of a Klebsiella pneumoniae fepB mutant. mSphere. 2017;2.

Li L, Stoeckert CJJ, Roos DS. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 2003;13(9):2178–89. https://doi.org/10.1101/gr.1224503.

Letunic I, Bork P. Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 2019;47(W1):W256–9. https://doi.org/10.1093/nar/gkz239.

Wyres KL, Wick RR, 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;15(4):e1008114. https://doi.org/10.1371/journal.pgen.1008114.

Walker KA, Miner TA, Palacios M, Trzilova D, Frederick DR, Broberg CA, et al. A Klebsiella pneumoniae regulatory mutant has reduced capsule expression but retains hypermucoviscosity. MBio. 2019;10(2). https://doi.org/10.1128/mBio.00089-19.

Walker KA, Treat LP, Sepúlveda VE, Miller VL. The small protein RmpD drives hypermucoviscosity in Klebsiella pneumoniae. MBio. 2020;11(5). https://doi.org/10.1128/mBio.01750-20.

Smillie C, Garcillán-Barcia MP, Francia MV, Rocha EPC, de la Cruz F. Mobility of plasmids. Microbiol Mol Biol Rev. 2010;74(3):434–52. https://doi.org/10.1128/MMBR.00020-10.

Lawley TD, Klimke WA, Gubbins MJ, Frost LS. F factor conjugation is a true type IV secretion system. FEMS Microbiol Lett. 2003;224(1):1–15. https://doi.org/10.1016/S0378-1097(03)00430-0.

Furuya N, Nisioka T, Komano T. Nucleotide sequence and functions of the oriT operon in IncI1 plasmid R64. J Bacteriol. 1991;173(7):2231–7. https://doi.org/10.1128/jb.173.7.2231-2237.1991.

O’Connor MB, Kilbane JJ, Malamy MH. Site-specific and illegitimate recombination in the oriV1 region of the F factor. DNA sequences involved in recombination and resolution. J Mol Biol. 1986;189:85–102.

Kopotsa K, Osei Sekyere J, Mbelle NM. Plasmid evolution in carbapenemase-producing Enterobacteriaceae: a review. Ann N Y Acad Sci. 2019;1457:61–91.

Bi D, Zheng J, Li J-J, Sheng Z-K, Zhu X, Ou H-Y, et al. In silico typing and comparative genomic analysis of IncFIIK plasmids and insights into the evolution of replicons, plasmid backbones, and resistance determinant profiles. Antimicrob Agents Chemother. 2018;62(10). https://doi.org/10.1128/AAC.00764-18.

Shu L, Dong N, Lu J, Zheng Z, Hu J, Zeng W, et al. Emergence of OXA-232 carbapenemase-producing Klebsiella pneumoniae that carries a pLVPK-Like virulence plasmid among elderly patients in China. Antimicrob Agents Chemother. 2019;63(3). https://doi.org/10.1128/AAC.02246-18.

Wong MH-Y, Chan EW-C, Chen S. IS26-mediated formation of a virulence and resistance plasmid in Salmonella enteritidis. J Antimicrob Chemother. 2017;72(10):2750–4. https://doi.org/10.1093/jac/dkx238.

Bouet J-Y, Bouvier M, Lane D. Concerted action of plasmid maintenance functions: partition complexes create a requirement for dimer resolution. Mol Microbiol. 2006;62(5):1447–59. https://doi.org/10.1111/j.1365-2958.2006.05454.x.

Liu G, Bogaj K, Bortolaia V, Olsen JE, Thomsen LE. Antibiotic-induced, increased conjugative transfer is common to diverse naturally occurring ESBL plasmids in Escherichia coli. Front Microbiol. 2019;10:2119. https://doi.org/10.3389/fmicb.2019.02119.

Kim S, Yun Z, Ha U-H, Lee S, Park H, Kwon EE, et al. Transfer of antibiotic resistance plasmids in pure and activated sludge cultures in the presence of environmentally representative micro-contaminant concentrations. Sci Total Environ. 2014;468–469:813–20.

Anthony KG, Sherburne C, Sherburne R, Frost LS. The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F-like plasmids. Mol Microbiol. 1994;13(6):939–53. https://doi.org/10.1111/j.1365-2958.1994.tb00486.x.

Pérez-Mendoza D, de la Cruz F. Escherichia coli genes affecting recipient ability in plasmid conjugation: are there any? BMC Genomics. 2009;10(1):71. https://doi.org/10.1186/1471-2164-10-71.

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;199(5):697–705. https://doi.org/10.1084/jem.20030857.

The NCBI. Nucleotide/Protein database. National Center for. Biotechnol Inform (NCBI). https://www.ncbi.nlm.nih.gov/nucleotide/. Accessed 3 Mar 2021.

Thông tin
Thông tin xuất bản

Springer Science and Business Media LLC

Thông tin tác giả