Type 3 fimbrial shaft (MrkA) of Klebsiella pneumoniae, but not the fimbrial adhesin (MrkD), facilitates biofilm formation

Abstract

Isolates of Klebsiella pneumoniae are responsible for opportunistic infections, particularly of the urinary tract and respiratory tract, in humans. These bacteria express type 3 fimbriae that have been implicated in binding to eucaryotic cells and matrix proteins. The type 3 fimbriae mediate binding to target tissue using the MrkD adhesin that is associated with the fimbrial shaft comprised of the MrkA protein. The formation of biofilms in vitro by strains of K. pneumoniae was shown to be affected by the production of fimbriae on the bacterial surface. However, a functional MrkD adhesin was not necessary for efficient biofilm formation. Nonfimbriate strains were impaired in their ability to form biofilms. Using isogenic fimbriate and nonfimbriate strains of K. pneumoniae expressing green fluorescent protein it was possible to demonstrate that the presence of type 3 fimbriae facilitated the formation of dense biofilms in a continuous-flowthrough chamber. Transformation of nonfimbriate mutants with a plasmid possessing an intact mrk gene cluster restored the fimbrial phenotype and the rapid ability to form biofilms.

FIG. 1

Production of a biofilm on the surfaces of microtiter plates by K. pneumoniae 43816, the nonfimbriate mutant MBM100, and MBM100 transformed with pFK12. Bacteria were incubated in plates containing GCAA broth for 8 h to optimize type 3 fimbrial expression. Crystal violet was used to quantitate biofilm formation as described in Materials and Methods. The bars indicate means ± standard errors of the means (error bars) from three experiments.

FIG. 2

Biofilm formation in microtiter plates by K. pneumoniae IA565 and its derivatives. Biofilm formation was determined after 10 h of incubation for all strains and the results (means ± standard errors of the means [error bars]) of three independent experiments are shown for each strain.

FIG. 3

Formation of biofilms by K. pneumoniae IA565 and its derivatives over a 24-h incubation period. Optimal biofilm formation was observed after approximately 10 h for each strain.

FIG. 4

Epifluorescence and confocal microscopy of K. pneumoniae IA565, IApc35, and IAΔT3 biofilms produced by bacteria expressing GFP. (Top) Composite sections obtained by imaging through the x-y plane of the biofilms. Images for an 8 μm depth are presented for strains IA565 and IApc35, whereas the depth of image for strain IAΔT3 is 40 μm. (Bottom) Saggital views of a z series of bacterial biofilms. The scale indicates the depth of z-series sections obtained.

FIG. 5

Scanning confocal laser microscopy of biofilms formed by K. pneumoniae 43816 and MBM100. (A) Images of the fimbriate strain were produced by performing a composite analysis of an x-y series of images through a 20-μm depth. (B) The nonfimbriate mutant, MBM100, analyzed by identical procedures to those used in panel A.

FIG. 6

Biofilm formation, using the microtiter plate assay, by K. pneumoniae isolates exhibiting phenotypic fimbrial phase variation. Biofilm formation was determined after 8 h of incubation following inoculation of the plates using the appropriate phase variant. The strains were obtained from the University of Iowa Hospitals and Clinics microbiology laboratory or the Iowa Sate Hygienic Laboratory. The sites of isolation for the following are as indicated: IA172, sputum; IA927, water; IA904, blood; IA912, tissue biopsy specimen. Bars represent means ± standard errors of the means (error bars) from three experiments.