Analysis of virulence genes of 376 <i>Klebsiella pneumoniae</i> strains_West China Medical Journal

Authors：

YANG Peng ¹ , KUANG Linghan ² , LUO Mei ³ , XIE Jing ¹ ,  LI Xinli ⁴

1. Department of Laboratory Medicine, Sichuan Provincial Fourth People’s Hospital, Chengdu, Sichuan 610016, P. R. China;
2. Department of Laboratory Medicine, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
3. Department of Laboratory Medicine, People’s Hospital of Xide County, Xide, Sichuan 616750, P. R. China;
4. Department of Laboratory Medicine, the Sixth People’s Hospital of Chengdu, Chengdu, Sichuan 610051, P. R. China;

Corresponding author：

LI Xinli, Email: 499409348@qq.com

Keywords：

Klebsiella pneumoniae; Virulence; Gene; Drug-resistance

DOI：

10.7507/1002-0179.202004415

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Objective To study the distributions of virulence genes of Klebsiella pneumoniae (KP) and the distribution of hypervirulent KP (HvKP), and assess the performance of a single gene to predict HvKP.Methods Polymerase chain reaction (PCR) method was used to analyze 12 virulence-related genes (entB, irp2, iroN, iucA, mrkD, fimH, c-rmpA, p-rmpA2, p-rmpA, wzy-K1, allS and peg-344) and drug-resistance gene bla_KPC among 376 clinical KP strains collected from January 2016 to December 2018. Sequence types (ST) of KP were determined after sequencing and comparison, following the detection of 7 house-keeping genes (gapA, infB, mdh, pgi, phoE, rpoB and tonB) by PCR method. Statistical analyses were made for the distributions of virulence genes of KP and the distribution of HvKP with GraphPad Prism 8 software.Results Among the 376 KP strains, the positive rates of entB, irp2, iroN, iucA, mrkD, fimH, c-rmpA, p-rmpA2, p-rmpA, wzy-K1, allS and peg-344 were 100.0%, 76.9%, 22.1%, 28.2%, 97.6%, 97.1%, 1.6%, 24.5%, 21.0%, 7.4%, 4.8% and 31.6%, respectively. The positive rates of the aforementioned virulence genes in the bla_KPC-positive group (n=167) were 100.0%, 94.0%, 7.2%, 16.8%, 97.0%, 96.4%, 0.0%, 15.0%, 6.6%, 0.0%, 0.0% and 21.0%, respectively, and those in the bla_KPC-negative group (n=209) were 100.0%, 63.2%, 34.0%, 37.3%, 98.1%, 97.6%, 2.9%, 32.1%, 32.5%, 13.4%, 8.6% and 40.2%, respectively; there was no statistically significant difference in entB, mrkD or fimH between the two groups (P>0.05), the positive rate of irp2 was higher in the bla_KPC-positive group than that in the bla_KPC-negative group (P<0.05), and the positive rates of the rest virulence-related genes were lower in the bla_KPC-positive group than those in the bla_KPC-negative group (P<0.05). The rate of HvKP in the bla_KPC-negative group was higher than that in the bla_KPC-positive group (38.3% vs. 18.0%, P<0.05). As a marker of HvKP, iucA showed high sensitivity and specificity (90.9% and 97.7%), followed by p-rmpA2 (83.6% and 100.0%) and iroN (73.6% and 99.2%). ST11 accounted for 87.4% in the bla_KPC-positive group, while ST23, ST20, ST54 and ST29 were the four primary types in the bla_KPC-negative group, accounting for 23.4% totally.Conclusions Different virulence genes mean different distributions in KP. bla_KPC-negative KP is more virulent than bla_KPC-positive KP. iucA and p-rmpA2 could serve as good predicators of HvKP. Armed with extreme virulence and drug-resistance, bla_KPC-positive HvKP is of great clinical concern.

Citation： YANG Peng, KUANG Linghan, LUO Mei, XIE Jing, LI Xinli. Analysis of virulence genes of 376 Klebsiella pneumoniae strains. West China Medical Journal, 2021, 36(8): 1037-1043. doi: 10.7507/1002-0179.202004415 Copy

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2.	Lee CR, Lee JH, Park KS, et al. Global dissemination of carbapenemase-producing Klebsiella pneumoniae: epidemiology, genetic context, treatment options, and detection methods. Front Microbiol, 2016, 7: 895.
3.	Zhang Y, Zeng J, Liu W, et al. Emergence of a hypervirulent carbapenem-resistant Klebsiella pneumoniae isolate from clinical infections in China. J Infect, 2015, 71(5): 553-560.
4.	Fu P, Tang Y, Li G, et al. Pandemic spread of bla_KPC-2 among Klebsiella pneumoniae ST11 in China is associated with horizontal transfer mediated by IncFII-like plasmids. Int J Antimicrob Agents, 2019, 54(2): 117-124.
5.	Russo TA, Marr CM. Hypervirulent Klebsiella pneumoniae. Clin Microbiol Rev, 2019, 32(3): e00001-19.
6.	Zhang S, Zhang X, Wu Q, et al. Clinical, microbiological, and molecular epidemiological characteristics of Klebsiella pneumoniae-induced pyogenic liver abscess in southeastern China. Antimicrob Resist Infect Control, 2019, 8: 166.
7.	Wang JL, Chen KY, Fang CT, et al. Changing bacteriology of adult community-acquired lung abscess in Taiwan: Klebsiella pneumoniae versus anaerobes. Clin Infect Dis, 2005, 40(7): 915-922.
8.	Chang WN, Huang CR, Lu CH, et al. Adult Klebsiella pneumoniae meningitis in Taiwan: an overview. Acta Neurol Taiwan, 2012, 21(2): 87-96.
9.	Gu D, Dong N, Zheng Z, 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.
10.	Siu LK, Huang DB, Chiang T. Plasmid transferability of KPC into a virulent K2 serotype Klebsiella pneumoniae. BMC Infect Dis, 2014, 14: 176.
11.	Lee CR, Lee JH, Park KS, et al. Antimicrobial resistance of hypervirulent Klebsiella pneumoniae: epidemiology, hypervirulence-associated determinants, and resistance mechanisms. Front Cell Infect Microbiol, 2017, 7: 483.
12.	Russo T, Olson R, Fang CT, et al. Identification of biomarkers for differentiation of hypervirulent Klebsiella pneumoniae from classical K. pneumoniae. J Clin Microbiol, 2018, 56(9): e00718-e00776.
13.	Compain F, Babosan A, Brisse S, et al. Multiplex PCR for detection of seven virulence factors and K1/K2 capsular serotypes of Klebsiella pneumoniae. J Clin Microbiol, 2014, 52(12): 4377-4380.
14.	Lam MMC, Wick RR, Wyres KL, et al. Genetic diversity, mobilisation and spread of the yersiniabactin-encoding mobile element ICEKp in Klebsiella pneumoniae populations. Microb Genom, 2018, 4(9): e000196.
15.	Yu VL, Hansen DS, Ko WC, et al. Virulence characteristics of Klebsiella and clinical manifestations of K. pneumoniae bloodstream infections. Emerg Infect Dis, 2007, 13(7): 986-993.
16.	Stahlhut SG, Tchesnokova V, Struve C, et al. Comparative structure-function analysis of mannose-specific FimH adhesins from Klebsiella pneumoniae and Escherichia coli. J Bacteriol, 2009, 191(21): 6592-6601.
17.	Klemm P, Schembri MA. Fimbrial surface display systems in bacteria: from vaccines to random libraries. Microbiology (Reading), 2000, 146(Pt 12): 3025-3032.
18.	Struve CB, Krogfelt KA. Characterization of Klebsiella pneumoniae type 1 fimbriae by detection of phase variation during colonization and infection and impact on virulence. Infect Immun, 2008, 76(9): 4055-4065.
19.	Stahlhut SG, Struve C, Krogfelt KA, et al. Biofilm formation of Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae. FEMS Immunol Med Microbiol, 2012, 65(2): 350-359.
20.	Schroll C, Barken KB, Krogfelt KA, et al. Role of type 1 and type 3 fimbriae in Klebsiella pneumoniae biofilm formation. BMC Microbiol, 2010, 10: 179.
21.	Jagnow J, Clegg S. Klebsiella pneumoniae MrkD-mediated biofilm formation on extracellular matrix- and collagen-coated surfaces. Microbiology (Reading), 2003, 149(Pt 9): 2397-2405.
22.	Russo TA, Olson R, Macdonald U, et al. Aerobactin mediates virulence and accounts for increased siderophore production under iron-limiting conditions by hypervirulent (hypermucoviscous) Klebsiella pneumoniae. Infect Immun, 2014, 82(6): 2356-2367.
23.	Russo TA, Olson R, Macdonald U, et al. Aerobactin, but not yersiniabactin, salmochelin, or enterobactin, enables the growth/survival of hypervirulent (hypermucoviscous) Klebsiella pneumoniae ex vivo and in vivo. Infect Immun, 2015, 83(8): 3325-3333.
24.	Qu TT, Zhou JC, Jiang Y, et al. Clinical and microbiological characteristics of Klebsiella pneumoniae liver abscess in East China. BMC Infect Dis, 2015, 15: 161.
25.	Bulger J, Macdonald U, Olson R, et al. Metabolite transporter PEG344 is required for full virulence of hypervirulent Klebsiella pneumoniae strain hvKP1 after pulmonary but not subcutaneous challenge. Infect Immun, 2017, 85(10): e00017-e00093.
26.	Li H, Zhang J, Liu Y, et al. Molecular characteristics of carbapenemase-producing Enterobacteriaceae in China from 2008 to 2011: predominance of KPC-2 enzyme. Diagn Microbiol Infect Dis, 2014, 78(1): 63-65.
27.	Yao B, Xiao X, Wang F, et al. Clinical and molecular characteristics of multi-clone carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae isolates in a tertiary hospital in Beijing, China. Int J Infect Dis, 2015, 37: 107-112.

1. Paczosa MK, Mecsas J. Klebsiella pneumoniae: going on the offense with a strong defense. Microbiol Mol Biol Rev, 2016, 80(3): 629-661.
2. Lee CR, Lee JH, Park KS, et al. Global dissemination of carbapenemase-producing Klebsiella pneumoniae: epidemiology, genetic context, treatment options, and detection methods. Front Microbiol, 2016, 7: 895.
3. Zhang Y, Zeng J, Liu W, et al. Emergence of a hypervirulent carbapenem-resistant Klebsiella pneumoniae isolate from clinical infections in China. J Infect, 2015, 71(5): 553-560.
4. Fu P, Tang Y, Li G, et al. Pandemic spread of bla_KPC-2 among Klebsiella pneumoniae ST11 in China is associated with horizontal transfer mediated by IncFII-like plasmids. Int J Antimicrob Agents, 2019, 54(2): 117-124.
5. Russo TA, Marr CM. Hypervirulent Klebsiella pneumoniae. Clin Microbiol Rev, 2019, 32(3): e00001-19.
6. Zhang S, Zhang X, Wu Q, et al. Clinical, microbiological, and molecular epidemiological characteristics of Klebsiella pneumoniae-induced pyogenic liver abscess in southeastern China. Antimicrob Resist Infect Control, 2019, 8: 166.
7. Wang JL, Chen KY, Fang CT, et al. Changing bacteriology of adult community-acquired lung abscess in Taiwan: Klebsiella pneumoniae versus anaerobes. Clin Infect Dis, 2005, 40(7): 915-922.
8. Chang WN, Huang CR, Lu CH, et al. Adult Klebsiella pneumoniae meningitis in Taiwan: an overview. Acta Neurol Taiwan, 2012, 21(2): 87-96.
9. Gu D, Dong N, Zheng Z, 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.
10. Siu LK, Huang DB, Chiang T. Plasmid transferability of KPC into a virulent K2 serotype Klebsiella pneumoniae. BMC Infect Dis, 2014, 14: 176.
11. Lee CR, Lee JH, Park KS, et al. Antimicrobial resistance of hypervirulent Klebsiella pneumoniae: epidemiology, hypervirulence-associated determinants, and resistance mechanisms. Front Cell Infect Microbiol, 2017, 7: 483.
12. Russo T, Olson R, Fang CT, et al. Identification of biomarkers for differentiation of hypervirulent Klebsiella pneumoniae from classical K. pneumoniae. J Clin Microbiol, 2018, 56(9): e00718-e00776.
13. Compain F, Babosan A, Brisse S, et al. Multiplex PCR for detection of seven virulence factors and K1/K2 capsular serotypes of Klebsiella pneumoniae. J Clin Microbiol, 2014, 52(12): 4377-4380.
14. Lam MMC, Wick RR, Wyres KL, et al. Genetic diversity, mobilisation and spread of the yersiniabactin-encoding mobile element ICEKp in Klebsiella pneumoniae populations. Microb Genom, 2018, 4(9): e000196.
15. Yu VL, Hansen DS, Ko WC, et al. Virulence characteristics of Klebsiella and clinical manifestations of K. pneumoniae bloodstream infections. Emerg Infect Dis, 2007, 13(7): 986-993.
16. Stahlhut SG, Tchesnokova V, Struve C, et al. Comparative structure-function analysis of mannose-specific FimH adhesins from Klebsiella pneumoniae and Escherichia coli. J Bacteriol, 2009, 191(21): 6592-6601.
17. Klemm P, Schembri MA. Fimbrial surface display systems in bacteria: from vaccines to random libraries. Microbiology (Reading), 2000, 146(Pt 12): 3025-3032.
18. Struve CB, Krogfelt KA. Characterization of Klebsiella pneumoniae type 1 fimbriae by detection of phase variation during colonization and infection and impact on virulence. Infect Immun, 2008, 76(9): 4055-4065.
19. Stahlhut SG, Struve C, Krogfelt KA, et al. Biofilm formation of Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae. FEMS Immunol Med Microbiol, 2012, 65(2): 350-359.
20. Schroll C, Barken KB, Krogfelt KA, et al. Role of type 1 and type 3 fimbriae in Klebsiella pneumoniae biofilm formation. BMC Microbiol, 2010, 10: 179.
21. Jagnow J, Clegg S. Klebsiella pneumoniae MrkD-mediated biofilm formation on extracellular matrix- and collagen-coated surfaces. Microbiology (Reading), 2003, 149(Pt 9): 2397-2405.
22. Russo TA, Olson R, Macdonald U, et al. Aerobactin mediates virulence and accounts for increased siderophore production under iron-limiting conditions by hypervirulent (hypermucoviscous) Klebsiella pneumoniae. Infect Immun, 2014, 82(6): 2356-2367.
23. Russo TA, Olson R, Macdonald U, et al. Aerobactin, but not yersiniabactin, salmochelin, or enterobactin, enables the growth/survival of hypervirulent (hypermucoviscous) Klebsiella pneumoniae ex vivo and in vivo. Infect Immun, 2015, 83(8): 3325-3333.
24. Qu TT, Zhou JC, Jiang Y, et al. Clinical and microbiological characteristics of Klebsiella pneumoniae liver abscess in East China. BMC Infect Dis, 2015, 15: 161.
25. Bulger J, Macdonald U, Olson R, et al. Metabolite transporter PEG344 is required for full virulence of hypervirulent Klebsiella pneumoniae strain hvKP1 after pulmonary but not subcutaneous challenge. Infect Immun, 2017, 85(10): e00017-e00093.
26. Li H, Zhang J, Liu Y, et al. Molecular characteristics of carbapenemase-producing Enterobacteriaceae in China from 2008 to 2011: predominance of KPC-2 enzyme. Diagn Microbiol Infect Dis, 2014, 78(1): 63-65.
27. Yao B, Xiao X, Wang F, et al. Clinical and molecular characteristics of multi-clone carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae isolates in a tertiary hospital in Beijing, China. Int J Infect Dis, 2015, 37: 107-112.

West China Medical Journal

Analysis of virulence genes of 376 Klebsiella pneumoniae strains

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