Hypervirulent Klebsiella pneumoniae serotype K1 clinical isolates form robust biofilms at the air-liquid interface

Autoři: Meritxell Cubero aff001;  Sara Marti aff001;  Mª Ángeles Domínguez aff001;  Aida González-Díaz aff001;  Dàmaris Berbel aff001;  Carmen Ardanuy aff001
Působiště autorů: Department of Microbiology, Hospital Universitari de Bellvitge, Instituto de Investigación Biomédica de Bellvitge (IDIBELL), Barcelona, Spain aff001;  Research Network for Respiratory Diseases (CIBERES), ISCIII, Madrid, Spain aff002;  Spanish Network for Research in Infectious Diseases (REIPI), Barcelona, Spain aff003;  Departamento de Patología y terapéutica experimental, Universitat de Barcelona, Barcelona, Spain aff004
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222628


The prevalence of a new hypervirulent and hypermucoviscous K. pneumoniae phenotype (Hmv) is increasing worldwide, mainly linked to serotypes K1 and K2. Since capsular thickness can directly affect the capability to form biofilms, we aimed to evaluate the association between the Hmv phenotype with adhesion and biofilm formation in a collection of clinical K. pneumoniae isolates.

We selected 38 Hmv clinical isolates [15 serotype K1; 9 serotype K2; 3 non-K1/K2 (rmpA+); 11 non-K1/K2 (rmpA-)] and 7 non-Hmv clinical isolates. The Hmv phenotype was assessed through the mucoviscosity test. Serum resistance was determined by bacterial viability tests in pooled human serum. Adhesion was evaluated with the Biofilm Ring Test®, and biofilm formation was identified by crystal violet staining (Solid-Liquid, SLI-biofilm) or visual examination (Air-Liquid, ALI-biofilm).

This study linked for the first time the formation of robust ALI-biofilm plugs by K. pneumoniae to the capsular serotype K1, a group of hypervirulent strains which are generally highly susceptible to the antimicrobial agents. Among all the studied isolates, the capsular serotype K1 presented lower initial adhesion despite having the adhesins mrkD and fimH but higher ALI-biofilm formation than isolates with other capsular serotypes (K2 or non-K1/K2). This structure might confer increased resistance to a group of hypervirulent K. pneumoniae serotype K1.

Klíčová slova:

Biology and life sciences – Organisms – Bacteria – Klebsiella – Microbiology – Medical microbiology – Microbial pathogens – Bacterial pathogens – Bacteriology – Bacterial biofilms – Bacterial physiology – Adhesins – Biofilms – Microbial physiology – Cell biology – Cellular structures and organelles – Pili and fimbriae – Medicine and health sciences – Pathology and laboratory medicine – Pathogens – Virulence factors – Pathogen motility – Research and analysis methods – Specimen preparation and treatment – Staining – Crystal violet staining


1. Kanj S, Kanafani Z. Current Concepts in Antimicrobial Therapy Against Resistant Gram-Negative Organisms: Extended-Spectrum β-Lactamase–Producing Enterobacteriaceae, Carbapenem-Resistant Enterobacteriaceae, and Multidrug-Resistant Pseudomonas aeruginosa. Mayo Clin Proc. 2011;86: 250–259. doi: 10.4065/mcp.2010.0674

2. Podschun R, Ullmann U. Klebsiella spp. as Nosocomial Pathogens: Epidemiology, Taxonomy, Typing Methods, and Pathogenicity Factors. Clin Microbiol Rev. 1998;11: 589–603. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC88898/pdf/cm000589.pdf 9767057

3. Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol. 2004;2: 95–108. doi: 10.1038/nrmicro821 15040259

4. Patel G, Shah N, Sharma R. Pyogenic Liver Abscess, Bacteremia, and Meningitis with Hypermucoviscous Klebsiella pneumoniae: An Unusual Case Report in a Human T-Cell Lymphotropic Virus Positive Patient of Caribbean Origin in the United States. Case Rep Infect Dis. Hindawi Publishing Corporation; 2013;2013: 676340. doi: 10.1155/2013/676340 24490092

5. Cubero M, Grau I, Tubau F, Pallarés R, Dominguez MA, Liñares J, et al. Hypervirulent Klebsiella pneumoniae clones causing bacteraemia in adults in a teaching hospital in Barcelona, Spain (2007–2013). Clin Microbiol Infect. 2016;22: 154–160. doi: 10.1016/j.cmi.2015.09.025

6. Struve C, Roe CC, Stegger M, Stahlhut SG, Hansen DS, Engelthaler DM, et al. Mapping the Evolution of Hypervirulent Klebsiella pneumoniae. MBio. 2015;6: e00630–15. doi: 10.1128/mBio.00630-15 26199326

7. Wu KM, Li NH, Yan JJ, Tsao N, Liao TL, Tsai HC, et al. Genome sequencing and comparative analysis of Klebsiella pneumoniae NTUH-K2044, a strain causing liver abscess and meningitis. J Bacteriol. 2009;191: 4492–4501. doi: 10.1128/JB.00315-09 19447910

8. Schembri MA, Dalsgaard D, Klemm P. Capsule Shields the Function of Short Bacterial Adhesins. J Bacteriol. 2004;186: 1249–1257. doi: 10.1128/JB.186.5.1249-1257.2004 14973035

9. Struve C, Krogfelt KA. Role of capsule in Klebsiella pneumoniae virulence: lack of correlation between in vitro and in vivo studies. FEMS Microbiol Lett. 2003;218(1): 149–154. Available: www.fems-microbiology.org 12583911

10. Fang CT, Chuang YP, Shun CT, Chang SC, Wang JT. A Novel Virulence Gene in Klebsiella pneumoniae Strains Causing Primary Liver Abscess and Septic Metastatic Complications. J Exp Med. Rockefeller University Press; 2004;199: 697–705. doi: 10.1084/jem.20030857 14993253

11. Wu MC, Lin TL, Hsieh PF, Yang HC, Wang JT. Isolation of Genes Involved in Biofilm Formation of a Klebsiella pneumoniae Strain Causing Pyogenic Liver Abscess. PLoS One. 2011;6: e23500. doi: 10.1371/journal.pone.0023500 21858144

12. Lai Y-C, Peng H-L, Chang H-Y. RmpA2, an Activator of Capsule Biosynthesis in Klebsiella pneumoniae CG43, Regulates K2 cps Gene Expression at the Transcriptional Level. J Bacteriol. 2003;185: 788–800. doi: 10.1128/JB.185.3.788-800.2003 12533454

13. Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA, Dance D, et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci. 2015;112: e3574–e3581. doi: 10.1073/pnas.1501049112 26100894

14. Brisse S, Fevre C, Passet V, Issenhuth-Jeanjean S, Gis Tournebize R, Diancourt L, et al. Virulent Clones of Klebsiella pneumoniae: Identification and Evolutionary Scenario Based on Genomic and Phenotypic Characterization. PLoS One. 2009;4: e4982. doi: 10.1371/journal.pone.0004982 19319196

15. Bachman MA, Oyler JE, Burns SH, Caza M, Lépine F, Dozois CM, et al. Klebsiella pneumoniae Yersiniabactin Promotes Respiratory Tract Infection through Evasion of Lipocalin 2. Infect Immun. 2011;79: 3309–3316. doi: 10.1128/IAI.05114-11 21576334

16. Diago-Navarro E, Chen L, Passet V, Burack S, Ulacia-Hernando A, Kodiyanplakkal RP, et al. Carbapenem-Resistant Klebsiella pneumoniae Exhibit Variability in Capsular Polysaccharide and Capsule Associated Virulence Traits. J Infect Dis. 2014;210: 803–813. doi: 10.1093/infdis/jiu157 24634498

17. Puig C, Marti S, Hermans PWM, de Jonge MI, Ardanuy C, Liñares J, et al. Incorporation of phosphorylcholine into the lipooligosaccharide of nontypeable Haemophilus influenzae does not correlate with the level of biofilm formation in vitro. Infect Immun. 2014;82: 1591–1599. doi: 10.1128/IAI.01445-13 24452688

18. Puig C, Domenech A, Garmendia J, Langereis JD, Mayer P, Calatayud L, et al. Increased Biofilm Formation by Nontypeable Haemophilus influenzae Isolates from Patients with Invasive Disease or Otitis Media versus Strains Recovered from Cases of Respiratory Infections. Appl Environ Microbiol. 2014;80: 7088–7095. doi: 10.1128/AEM.02544-14 25192997

19. Chavant P, Gaillard-Martinie B, Talon R, Hébraud M, Bernardi T. A new device for rapid evaluation of biofilm formation potential by bacteria. J Microbiol Methods. 2007;68: 605–612. doi: 10.1016/j.mimet.2006.11.010 17218029

20. Nait Chabane Y, Marti S, Rihouey C, Alexandre S, Hardouin J. Characterisation of Pellicles Formed by Acinetobacter baumannii at the Air-Liquid Interface. PLoS One. 2014;9: e111660. doi: 10.1371/journal.pone.0111660 25360550

21. Shon AS, Bajwa RP, Russo TA. Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: a new and dangerous breed. Virulence. 2013;4: 107–118. doi: 10.4161/viru.22718 23302790

22. Merino S, Camprubi S, Alberti S, Benedi V-J, Tomas JM. Mechanisms of Klebsiella pneumoniae Resistance to Complement-Mediated Killing. Infect Immun. 1992;60: 2529–2535. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC257192/pdf/iai00030-0395.pdf 1587619

23. June HW, Wu AM, Cheng GT, Chang XY, Tsai SF, Wu TS. Contribution of fucose-containing capsules in Klebsiella pneumoniae to bacterial virulence in mice. Exp Biol Med. 2008;233: 64–70. doi: 10.3181/0706-RM-170 18156307

24. Fang C-T, Lai S-Y, Yi W-C, Hsueh P-R, Liu K-L, Chang S-C. Klebsiella pneumoniae Genotype K1: An Emerging Pathogen That Causes Septic Ocular or Central Nervous System Complications from Pyogenic Liver Abscess. Clin Infect Dis. 2007;45: 284–293. doi: 10.1086/519262 17599305

25. Nassif X, Sansonetti PJ. Correlation of the Virulence of Klebsiella pneumoniae KI and K2 with the Presence of a Plasmid Encoding Aerobactin. Infect Immun. 1986;54: 603–608. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC260211/pdf/iai00099-0009.pdf

26. Hentzien M, Rosman J, Decré D, Brenkle K, Mendes-Martins L, Mateu P. Seven hypervirulent ST380 Klebsiella pneumoniae septic localizations Sept localisations. Médecine Mal Infect. 2017;47: 171–173. doi: 10.1016/j.medmal.2016.10.002

27. Stahlhut SG, Struve C, Krogfelt KA, Reisner A. Biofilm formation of Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae. FEMS immunol med microbiol. 2012;65: 350–359. doi: 10.1111/j.1574-695X.2012.00965.x 22448614

28. Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol. 2009;11: 1034–1043. doi: 10.1111/j.1462-5822.2009.01323.x 19374653

29. Alcántar-Curiel MD, Blackburn D, Saldaña Z, Gayosso-Vázquez C, Iovine N, De La Cruz MA, et al. Multi-functional analysis of Klebsiella pneumoniae fimbrial types in adherence and biofilm formation. Virulence. 2013;129: 129–138. doi: 10.4161/viru.22974 23302788

30. Guo Y, Wang S, Zhan L, Jin Y, Duan J, Hao Z, et al. Microbiological and Clinical Characteristics of Hypermucoviscous Klebsiella pneumoniae Isolates Associated with Invasive Infections in China. Front Cell Infect Microbiol. 2017;7: 24. doi: 10.3389/fcimb.2017.00024 28203549

31. Wang H, Wilksch JJ, Strugnell RA, Gee ML. Role of Capsular Polysaccharides in Biofilm Formation: An AFM Nanomechanics Study. ACS Appl Mater Interfaces. 2015;7: 13007–13013. doi: 10.1021/acsami.5b03041 26034816

32. Armitano J, Méjean V, Jourlin-Castelli C. Gram-negative bacteria can also form pellicles. Environ Microbiol Rep. 2014;6: 534–544. doi: 10.1111/1758-2229.12171 25756106

33. Hadjifrangiskou M, Gu AP, Pinkner JS, Kostakioti M, Zhang EW, Greene SE, et al. Transposon Mutagenesis Identifies Uropathogenic Escherichia coli Biofilm Factors. J Bacteriol. 2012;194: 6195–6205. doi: 10.1128/JB.01012-12 22984258

34. Carabarin-Lima A, León-Izurieta L, Del R, Rocha-Gracia C, Castañeda-Lucio M, Torres C, et al. First evidence of polar flagella in Klebsiella pneumoniae isolated from a patient with neonatal sepsis. J Med Microbiol. 2016;65: 729–737. doi: 10.1099/jmm.0.000291 27283194

Článek vyšel v časopise


2019 Číslo 9

Nejčtenější v tomto čísle

Tomuto tématu se dále věnují…


Zvyšte si kvalifikaci online z pohodlí domova

Ulcerative colitis_muž_břicho_střeva
Ulcerózní kolitida
nový kurz

Blokátory angiotenzinových receptorů (sartany)
Autoři: MUDr. Jiří Krupička, Ph.D.

Antiseptika a prevence ve stomatologii
Autoři: MUDr. Ladislav Korábek, CSc., MBA

Citikolin v neuroprotekci a neuroregeneraci: od výzkumu do klinické praxe nejen očních lékařů
Autoři: MUDr. Petr Výborný, CSc., FEBO

Zánětlivá bolest zad a axiální spondylartritida – Diagnostika a referenční strategie
Autoři: MUDr. Monika Gregová, Ph.D., MUDr. Kristýna Bubová

Všechny kurzy