Department of Microbiology, College of Medicine, University of Iowa, Iowa City, IA 52242, USA
Correspondence
Steven Clegg
steven-clegg{at}uiowa.edu
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ABSTRACT |
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A real-time video of biofilm formation by K. pneumoniae IA565 is available as supplementary data with the online version of this paper at http://mic.sgmjournals.org.
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INTRODUCTION |
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We have recently demonstrated that the type 3 fimbriae of Klebsiella pneumoniae influence the development of biofilms in plastic, continuous flow-through chambers (Langstraat et al., 2001). The type 3 fimbriae are expressed by several species of opportunistic pathogens and are characterized by their ability to mediate agglutination, in vitro, of treated erythrocytes (Clegg et al., 1994
; Old & Adegbola, 1985
; Old et al., 1985
). The MrkA polypeptide comprises the major structural component of type 3 fimbriae and is polymerized to form the fimbrial shaft. The MrkD protein is believed to function as the type 3 fimbrial adhesin and mediates binding to extracellular matrix (ECM) proteins such as collagen molecules (Schurtz et al., 1994
; Schurtz Sebghati et al., 1998
; Tarkkanen et al., 1990
, 1992
). The efficient development of biofilms by K. pneumoniae on plastic surfaces was independent of the presence of the MrkD adhesin, but facilitated by the presence of the fimbrial shaft on the bacterial surface. The collagen-binding MrkD molecule appears to play little role in the development of biofilms on these plastic surfaces, whereas the MrkA shaft protein is an important contributing factor. Therefore, bacterial binding leading to colonization and biofilm formation on plastic devices such as endotracheal tubes may be more efficient by type 3 fimbriate bacteria. However, in-dwelling devices such as catheters and tubes are coated over time, in situ, with host-derived material (Donlan, 2001
; Francois et al., 1998
). Consequently, specific receptorligand binding, such as the MrkDcollagen interaction, could also play a role in biofilm development during infection. Also, MrkD-mediated binding to extracellular matrices is likely to have a role in colonization of damaged epithelial surfaces.
During the course of our studies investigating the role of type 3 fimbriae in biofilm development on abiotic plastic surfaces we observed that fimbriate bacteria were limited in their ability to form biofilms on glass surfaces. Consequently, coverslips coated with ECM or collagen should provide a good surface to investigate the role of MrkA and MrkD proteins of the type 3 fimbriae on biofilm formation in continuous-flow chambers coated with these substances.
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METHODS |
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Detection of type 3 fimbriae.
The ability of bacteria to produce type 3 fimbriae was determined using monospecific antisera raised against purified fimbriae as reported previously (Schurtz et al., 1994). Haemagglutinating activity of K. pneumoniae strains was determined using tanned erythrocytes according to the method of Old et al. (1985)
. Bacterial binding to collagen was performed using the ELISA described in detail previously (Schurtz Sebghati et al., 1998
). Purified human placenta collagen (types V and IV) and ECM were purchased from BD Biosciences and coating concentrations were determined by standard techniques (Korhonen et al., 1997
; Kukkonen et al., 1993
; Tarkkanen et al., 1997
).
Biofilm formation on uncoated glass surfaces.
The once flow-through continuous culture system, developed by Parsek & Greenberg (1999), was used to assay biofilm development on glass slides that are affixed to the top of the flow cell. Biofilm development on the glass slides incubated at 37 °C using GCAA broth (diluted 1 : 50) as the medium was examined at 6, 24, 48 and 72 h post-inoculation. Following incubation the glass coverslips were gently removed from the flow cell, ensuring that the correct orientation of the coverslip was maintained, and the coverslips were placed on top of a clean, sterile flow-through chamber. In this way fluorescence from bacteria growing on the plastic surfaces of the biofilm chamber could be eliminated. Subsequently, imaging of the biofilm was performed using a Bio-Rad MRC600 confocal microscope and images are presented as composite sections through the xy planes of these sections as described in detail elsewhere (Langstraat et al., 2001
; Parsek & Greenberg, 1999
). The complete area of the slides exposed to the biofilm chamber was examined by confocal microscopy and representative sections were analysed to calculate the depth of biofilm formed.
Biofilm formation on collagen- and cellular-coated coverslips.
To examine biofilm formation on treated slides the following modifications to the procedure described above were performed. Collagen-coated glass or plastic coverslips were prepared as described previously using optimal coating concentrations of collagen solution or human ECM (Schurtz et al., 1994). Collagen coating (0·005 mg ml-1) was performed overnight at 4 °C in carbonate buffer by incubating the flow cell filled with coating solution, and before inoculation the cell was flushed with PBS to remove excess unbound collagen. When using human ECM the coating concentration was 0·109 mg ml-1.
In a separate series of experiments, semiconfluent monolayers of a human bronchial epithelial (HBE) cell line were also used to coat the coverslips. Chambers were inoculated with 2x105 cells and incubated under 5 % CO2 for 24 h at 37 °C in Dulbecco's Modified Eagle Medium supplemented with 10 % fetal bovine serum and 25 µg chloramphenicol ml-1. Under these conditions the coverslips are coated with a semiconfluent monolayer of HBE cells. Following bacterial inoculation the flow rate of the chamber was set at 130 µl min-1 and the tissue culture fluid plus 10 % fetal bovine serum was used as culture medium that was supplemented with appropriate antibiotics. For the biofilm assays the HBE cells were stained with 1 µM cell tracker orange (Molecular Probes) according to the manufacturer's instructions.
Finally, to examine the ability of K. pneumoniae strains to form biofilms on the ECM produced in vitro by respiratory cells, the HBE cells were grown as a confluent monolayer on the slides of the biofilm chamber. Inoculation of the chamber with the HBE cells was as described above and the cells were grown for 48 h. Under these conditions the HBE cells were observed to form a continuous and confluent monolayer when examined by microscopy. Subsequently, the cells were removed from the slides by treatment in situ with 25 mM EGTA as described in detail elsewhere (Hornick et al., 1995). This treatment results in effective removal of the HBE cells without damaging the matrix laid down by these cells. The ability of GFP-labelled bacteria to form biofilms on this matrix was determined as described above.
The imaging of the biofilms on coated coverslips was performed as described above for untreated surfaces. The biofilm development was monitored at 6, 24 and 48 h post-inoculation for the ECM- and collagen-coated slides, and 24 h for the HBE-coated slides.
Bacterial adherence to HBE cells grown in vitro.
Examination of the binding of K. pneumoniae strains to HBE cells in vitro was performed as described previously (Hornick et al., 1995).
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RESULTS |
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Bacterial growth on human ECM- and collagen-coated surfaces
Since we have previously demonstrated that the MrkD adhesin mediates binding in vitro to extracellular matrices and collagen (Langstraat et al., 2001; Schurtz Sebghati et al., 1998
), we investigated the ability of K. pneumoniae strains to grow on human ECM-coated surfaces in a continuous culture system. Optimal coating concentrations of matrix and collagen on the coverslips have been determined previously (Schurtz et al., 1994
; Tarkkanen et al., 1990
) and these conditions were used to coat the coverslips of the flow-through chambers. Careful removal of the coverslips from the chambers enabled us to examine growth of the GFP-producing bacteria on these surfaces in the absence of fluorescence due to growth on any uncoated plastic of the chambers. After 6 h incubation patchy areas of growth of K. pneumoniae IA565 were observed on the ECM-coated slides and the maximum depth of the biofilm formed in these regions was approximately 12·5 µm (Fig. 1
). No significant growth of K. pneumoniae IApc35 or IA
T3 was observed on the slides after 6 h incubation.
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At 48 h post-inoculation of the chambers, strain IA565 continued to exhibit dense growth on the collagen-coated surfaces, although the maximum depth of the biofilm was approximately 25·5 µm which is less than that observed at 24 h. However, even after 3 days incubation the fimbriate but non-adhesive strain, IApc35, still exhibited only limited growth and a similar reduction in the depth of biofilm was observed. The non-fimbriate and non-adhesive strain was not observed to grow on the collagen-coated slides under any conditions of incubation.
Fig. 2 demonstrates the time-course of biofilm development by K. pneumoniae IA565 on human ECM. Initial bacterial growth was limited during the first 16 h of incubation. Subsequently, extensive growth occurred over the following 16 h with the formation of characteristic pillars of bacterial growth. A real-time video of GFP-labelled K. pneumoniae IA565 growing over a 32 h time period on human ECM is available as supplementary data with the online version of this paper at http://mic.sgmjournals.org
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DISCUSSION |
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Coating of the coverslip surfaces with human ECM or purified collagen allows for the efficient formation of biofilms by fimbriate bacteria bearing the MrkD adhesin. Over a prolonged period of incubation (48 h or longer) we observed that the depth of the biofilm mass did decrease. This may be due to removal of distal layers of bacteria by the constant flushing of medium through the chambers once a maximum depth is formed after approximately 24 h. Also, we found that it was necessary to remove the coverslips from the chambers to view biofilm formation since, as we previously reported, GFP-producing fimbriate bacteria grow and efficiently adhere to the poorly coated plastic surfaces of the chamber and interfere with our observations of the collagen-coated surface. The coverslips were removed to ensure minimal disturbance of the biofilm that was present, but biofilms formed after 48 h incubation may be more fragile than those of earlier time points. It is clear, however, that the fimbriate strain lacking the MrkD adhesin did not form biofilms on the collagen as efficiently as the wild-type strain.
It is known that K. pneumoniae type 3 fimbriae do not mediate adherence to confluent monolayers of HBE cells (Hornick et al., 1992, 1995
). In addition, we have demonstrated that these fimbriae facilitate binding to the basement membrane of human lung tissue sections (Hornick et al., 1992
). Consequently, we decided to investigate whether the MrkD adhesin would enable K. pneumoniae biofilms to be formed on the ECM produced by HBE cells grown in vitro on the coverslips of the chambers. In one series of experiments we ensured that the HBE cells were not grown to confluency so that extracellular material produced by the cells would be exposed on the glass surfaces. The location of the biofilm produced by K. pneumoniae IA565 under these conditions is consistent with the interaction of the fimbrial adhesin with the ECM. Large areas of bacterial growth were associated with clusters of HBE cells where the ECM is most likely to be exposed to the bacteria. As previously reported, adherence assays using HBE cells grown in a similar way indicated poor direct binding to the HBE cells. Consequently, we propose that the large areas of bacterial growth around the cells in the chambers is due to MrkD-mediated binding to the ECM by small numbers of fimbriate bacteria. Subsequently, these bacteria grow and establish biofilm formation in these regions. The non-adhesive strains were not able to grow on the slides under these conditions. Although we did not directly demonstrate that the MrkD protein interacts with any specific type of collagen in the ECM of the HBE cells, it is known that collagen is an integral component of the matrix synthesized by cells in vitro (Fenwick et al., 2001
; Hastie et al., 2002
; Yurchenko & O'Rear, 1994
). However, the composition of the ECM produced by the HBE cells has not been investigated in detail. The ability of the MrkD adhesin to facilitate adherence and subsequent growth on ECMs was confirmed using commercially available, purified human ECM. Extensive and complete biofilm formation on this material was only observed using the strain expressing the MrkD molecule.
As indicated above, we believe that the MrkD adhesin does not mediate direct bacterial binding to the HBE cell membranes in the chambers. However, to confirm that the MrkD adhesin binds to the HBE-generated matrix it was possible to remove a confluent layer of HBE cells from the coverslips to expose any material laid down by the HBE cells. In this case extensive biofilm formation was only observed with MrkD-positive bacteria. The ability of MrkD-possessing fimbriae to grow on the underlying matrix of epithelial cells cultured in vitro strongly suggests that disruption of an intact epithelial surface is necessary for type 3 fimbriate bacteria to grow on host surfaces. This is consistent with the clinical observations associated with nosocomially acquired infections due to K. pneumoniae (Craven et al., 1990; Duma, 1985
; Riser & Noone, 1981
).
The use of the once flow-through chambers coated with matrix proteins or as a support for culturing cells from a relevant host provides an excellent model system to investigate the interaction of K. pneumoniae with human cells and tissues in a dynamic environment. In this respect it has advantages over static and closed systems, but does differ from the natural site of infection by presenting the bacteria to host material in a fluid environment rather than at an air/mucosal surface interface located in the respiratory tract. However, the ability to investigate the stages of adherence, colonization and growth on host-derived tissues and matrices will facilitate a molecular analysis of gene expression in K. pneumoniae.
In humans, susceptibility to infections of the airways by the opportunist K. pneumoniae is most frequently associated with predisposing factors. During secondary infections, exfoliated and denuded epithelial surfaces may expose collagenous receptors that enable MrkD-mediated adherence of K. pneumoniae. Subsequent growth at these sites in the form of biofilms could enable the bacteria to avoid efficient killing by alveolar macrophages. For patients with in-dwelling endotracheal tubes the type 3 fimbriae may have a dual role in the infective process. Immediately after insertion of these devices the hydrophobic nature of the type 3 fimbrial shaft could facilitate attachment to the polymer surfaces of these tubes with subsequent growth on the device. Also, it has been demonstrated that, with time, these tubes are coated, in situ, with host-derived material (Donlan, 2001; Francois et al., 1998
). Therefore, a second role of the type 3 fimbriae in these types of infection may involve a specific receptor ligand interaction involving MrkD-mediated adherence to matrix proteins covering the tubes.
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ACKNOWLEDGEMENTS |
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Received 22 April 2003;
revised 9 June 2003;
accepted 12 June 2003.