Multi-functional analysis of Klebsiella pneumoniae fimbrial types in adherence and biofilm formation

Abstract

Klebsiella pneumoniae is an opportunistic pathogen frequently associated with nosocomially acquired infections. Host cell adherence and biofilm formation of K. pneumoniae isolates is mediated by type 1 (T1P) and type 3 (MR/K) pili whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. The E. coli common pilus (ECP) is an adhesive structure produced by all E. coli pathogroups and a homolog of the ecpABCDE operon is present in the K. pneumoniae genome. In this study, we aimed to determine the prevalence of these three fimbrial genes among a collection of 69 clinical and environmental K. pneumoniae strains and to establish a correlation with fimbrial production during cell adherence and biofilm formation. The PCR-based survey demonstrated that 96% of the K. pneumoniae strains contained ecpA and 94% of these strains produced ECP during adhesion to cultured epithelial cells. Eighty percent of the strains forming biofilms on glass produced ECP, suggesting that ECP is required, at least in vitro, for expression of these phenotypes. The fim operon was found in 100% of the strains and T1P was detected in 96% of these strains. While all the strains examined contained mrkA, only 57% of them produced MR/K fimbriae, alone or together with ECP. In summary, this study highlights the ability of K. pneumoniae strains to produce ECP, which may represent a new important adhesive structure of this organism. Further, it defines the multi-fimbrial nature of the interaction of this nosocomial pathogen with host epithelial cells and inert surfaces.

Keywords: E. coli common pilus; K. oxytoca; Klebsiella pneumoniae; adherence; biofilms.

Figure 1. Ultrastructural analysis of K. pneumoniae strains. Bacteria recovered from HeLa cells infection assays were negatively stained (A–C and G) or immuno-gold labeled (D–F, H and I) and visualized by transmission EM. (A and D) Kpn 3; (B and E) Kpn Leo15; (C and F) Kpn 1; (G) Kpn 40 showing pili, which did not reacted with anti-ECP antibodies (H). (I) K. oxytoca MXa showing ECP.

Figure 2. Detection of EcpA in selected K. pneumoniae strains. Immunobloting of whole cells extracts of Klebsiella strains treated with HCl and reacted with anti-ECP antibody. Purified ECP was used as a positive control. Strains Kpn 191–2, Kpn 292 and Kpn 11, which lack ecpA, were negative for EcpA.

Figure 3. High-resolution SEM analysis of selected K. pneumoniae strains adhering to HeLa cells. (A, C and E) Kpn 1; (B, D and F) Kpn Leo15. (C and D) Images at high magnification showing numerous filaments connecting bacteria to each other in aggregates (arrows). (E and F) SIEM showing gold-labeled ECP on adhering bacteria.

Figure 4. High resolution SEM analysis of K. oxytoca 336 associated with the surface of HeLa cells. (A–C) Aggregates of strain 336 gold-labeled ECP connecting bacteria to each other and anchoring bacteria tightly to host cell filopodia structures (arrows). (D) Mock-infected HeLa cells were used as negative control.

Figure 5. Demonstration of ECP and/or MR/K on K. pneumoniae strains adhering to HeLa cells by immunofluorescence. (A, D and G) Kpn 3; (B, E and H) Kpn Leo15; (C, F and I) Kpn 40. ECP are shown in green and MR/K in red. Bacterial and cellular DNA was stained with DAPI (blue). Note the presence of both pili in Kpn 3. Images were taken at 60×.

Figure 6. Immunoblotting of whole cells extracts of selected K. pneumoniae strains. Bacterial extracts treated or not with HCl were reacted with the appropriate anti-pili antibodies to demonstrate the simultaneous production of 3 pili types.

Figure 7. Biofilm formation and ECP production in selected Klebsiella strains. (A) Kpn 1; (B) Kpn Leo15; (C) Kpn 25; (D) Kpn 3; (E) Kpn 13; (F) Klebsiella oxytoca 373; (G) Kpn 5; (H) Kpn JC. Biofilms obtained after 24 h of incubation at 37°C in DMEM were immuno-stained with anti-ECP antibodies (green) and bacteria were stained with propidium iodide (red). Images were taken at 60×.