Klebsiella pneumoniae is recognized as an important opportunistic pathogen commonly associated with nosocomial urinary tract infections (UTIs), including catheter-associated UTIs (CAUTIs), as well as sepsis and pneumonia (Podschun & Ullmann, 1998).
Several virulence factors have been identified in K. pneumoniae (Podschun & Ullmann, 1998; Struve et al., 2003). The prominent polysaccharide capsule expressed by most isolates, together with the lipopolysaccharide layer, protects the bacteria against phagocytosis and the bactericidal activity of serum. Fimbrial adhesins facilitate adherence to specific tissue surfaces as well as abiotic surfaces for example indwelling catheters. It is well known that most K. pneumoniae isolates express two fimbrial adhesins: type 1 fimbriae and type 3 fimbriae; however, sequence analyses suggest that other yet uncharacterized fimbrial variants may also be expressed by K. pneumoniae. We recently established that type 1 fimbriae are essential for the ability of K. pneumoniae to cause UTI (Struve et al., 2008), whereas the expression of type 3 fimbriae has been shown to promote biofilm formation on biotic as well as abiotic surfaces (Langstraat et al., 2001; Di Martino et al., 2003; Jagnow & Clegg, 2003; Struve et al., 2009). Furthermore, recent studies have reported type 3 fimbriae expression in Escherichia coli, mediated by conjugative plasmids and associated with profoundly enhanced biofilm formation (Burmølle et al., 2008; Ong et al., 2008). Interestingly, the transcription factor OxyR has most recently been shown to be involved in K. pneumoniae biofilm formation partly by regulating fimbrial expression (Hennequin & Forestier, 2009).
Urinary catheters are standard medical devices utilized in both hospital and care facilities. The most frequent complication associated with these devices is the development of nosocomial CAUTIs. Indwelling urinary catheters favor biofilm formation of uropathogens such as K. pneumoniae by providing a surface for the attachment of bacterial adhesins, thus enhancing microbial colonization and the development of biofilm. However, the mechanism of K. pneumoniae biofilm formation on abiotic surfaces is not well understood. We have developed a method to identify the genes involved in K. pneumoniae biofilm formation on abiotic surfaces by screening a clone library of K. pneumoniae DNA expressed in E. coli.
Construction of the clone library was carried out using the EpiFOS™ Fosmid Library Production Kit (Epicentre) according to the manufacturer's instructions. Briefly, DNA from C3091, a well-characterized virulent and biofilm-forming K. pneumoniae strain isolated from a patient with urinary tract infection (Oelschlaeger & Tall, 1997; Struve et al., 2003, 2009), was purified, sheared to fragments of approximately 40 kb and ligated into fosmid vector pEpiFOS-5 (Epicenter). The fosmid library was transduced into E. coli EPI100 (Epicentre). A total of 1152 E. coli clones, each carrying a fosmid containing approximately 40 kb of K. pneumoniae DNA, were isolated.
To identify clones containing K. pneumoniae DNA promoting biofilm formation, the clone library was screened for increased biofilm formation compared with the EPI100 E. coli strain carrying the empty fosmid vector. The 1152 clones were screened in a semi-automatic microtiter plate assay and biofilm formation was quantified by crystal violet staining as previously described using a Biomek 2000 automated robot (Beckman Coulter Inc.) (O'Toole & Kolter, 1998). Briefly, clones were inoculated into the wells of a microtiter plate containing ABT minimal medium with 0.5% glucose added and grown for 48 h at 60 r.p.m., 37 °C. Then, the plates were washed with saline and crystal violet was added for 15 min. After washing with saline, the crystal violet was dissolved by ethanol, and A595 nm was measured. All experiments were performed in triplicate.
Nine clones with significantly increased biofilm formation (P values between 0.007 and 0.04 as evaluated by an unpaired t-test with Welch corrections) were identified (Fig. 1a). To exclude that the enhanced biofilm formation was related to enhanced growth, the identified clones' growth rates were evaluated in direct competition with the EPI100 parent strain. All clones exhibited similar growth rates (data not shown).
To identify biofilm-promoting genes, fosmids from the nine clones were subjected to random in vitro mutagenesis using the EZ-Tn5™〈KAN-2〉 Insertion Kit (Epicentre) according to the manufacturer's instructions. Briefly, fosmid vectors were purified and mixed with the EZ-Tn5™〈KAN-2〉 Transposon, transformed into EPI100 electrocompetent E. coli cells and plated on Luria–Bertani (LB) agar containing (50 μg mL−1) kanamycin.
Approximately 100 random transposon mutants of each fosmid clone were isolated and screened for attenuated biofilm formation compared with the parent fosmid clones with no transposon insertion (Fig. 1b). As seen in Fig. 1b, approximately 25% of the mutants have been disrupted in a gene (or genes) important for biofilm formation. To identify the biofilm promoting genes, the fosmids from attenuated mutants were purified and sequenced with primers localized in the inserted transposon.
Of the nine fosmid clones, Clones 1H2, 2E3, 4D6, 4G9 and 10E5 (Fig. 1a) were found to contain the type 3 fimbriae gene cluster (mrk), which has previously been shown to be an important biofilm promoter in K. pneumoniae. Indeed, all the five clones expressed functional type 3 fimbriae as shown by type 3 fimbriae-specific hemagglutinations assays performed as described previously (Struve et al., 2009). Furthermore, after mutagenesis of these clones, the attenuated mutants were found to be disrupted in the mrk gene cluster, verifying that the markedly enhanced biofilm formation of these five clones was due to the expression of type 3 fimbriae.
The three clones 5F4, 5G7 and 8H11 (Fig. 1a) all contained a DNA fragment homologous to a region in the sequenced K. pneumoniae strain MGH 78578 encoding KPN_00060 to KPN_00084 (GenBank: CP000647). Subsequent mutagenesis revealed that several genes in this region influence biofilm formation including genes involved in amino acid synthesis and genes involved in l-arabinose metabolism. We will initiate further studies to clarify the role of this chromosomal region in biofilm formation
Interestingly, the clone 4D8 (Fig. 1a) was found to encode a large putative cell surface protein of unknown function homologous to KPN_00994 (GenBank: ABR76430) in strain MGH 78578. This gene is characterized by a large repeat region spanning approximately 7000 bp, encoding serine-aspartate (SD) repeats, suggesting a close relationship with the SD-repeat protein multigene family found in Staphylococcus spp. This protein family is mainly known for its fibronectin and collagen-binding properties (Josefsson et al., 1998; Sakinc et al., 2006). In future experiments, we will construct a specific SD-repeat protein mutant of K. pneumoniae C3091 to further characterize the role of this gene in biofilm formation.
To investigate whether the identified clones also exhibit enhanced biofilm formation on catheters, a catheter biofilm assay described previously by Burmølle et al. (2008) was applied. Briefly, clones were grown O/N in LB at 37 °C with appropriate antibiotics and 50 μL was transferred to 5 mL M9 minimal media containing three pieces of catheter. The Catheter pieces (BARD, 100% silicone foley catheter, REF 165814CE, Bard Limited, UK) were 1 cm in length and split down the middle in order to increase the surface area. After a 48-h incubation with light shaking at 37 °C, the catheter pieces were washed twice, then stained by 0.5% crystal violet, followed by washing. Finally, the pieces were covered by 2 mL ethanol and the OD600 nm of the solution was measured. Catheter pieces incubated in M9 media, but not exposed to bacteria, were used as negative controls. All experiments were performed in triplicate. Clone 4D8, encoding the SD-repeat protein and the clones 5F4, 5G7 and 8H11, containing the genes involved in amino acid synthesis and arabinose metabolism exhibited no increase in biofilm formation on catheters compared with the EPI100 parent strain (data not shown). However, Clones 1H2, 2E3, 4D6, 4G9 and 10E5, all containing the type 3 fimbriae gene cluster, exhibited a fourfold increase in biofilm formation on catheters when compared with the EPI100 parent strain (data not shown).
In conclusion, a novel approach to identify the genes involved in biofilm formation was successfully applied. Thus, by screening a clone library of a K. pneumoniae genome for enhanced biofilm formation we identified novel K. pneumoniae genes promoting biofilm formation. Our methodology was validated as type 3 fimbriae, previously shown to promote biofilm, were identified in the present study. In future studies, we will perform complementation experiments to exclude polar effects on downstream genes and construct and evaluate specific K. pneumoniae mutants to further characterize the biofilm-promoting genes identified in the present study. We believe that screening of clone libraries encompasses a novel attractive approach for screening in infection models for identification of virulence genes and the method is easily applicable for other bacterial species. To our knowledge, besides type 3 fimbriae, none of the genes identified in this study have previously been associated with biofilm formation in K. pneumoniae.
CAUTIs are a worldwide problem, and one of the leading causative agents of CAUTIs is K. pneumoniae. Little is known about the nature of the mechanisms involved in K. pneumoniae biofilm formation on artificial surfaces. We therefore used this comprehensive novel double-screening method to identify, in a high throughput manner, genes involved in biofilm formation of K. pneumoniae. The novel biofilm-promoting genes identified in the present study will, in future studies, be subjected to further analysis and evaluation to assess the exact role played by the specific gene in the formation of biofilm. Elucidation of the mechanism of biofilm formation by pathogens is the first step towards the development of novel approaches for the treatment and prevention of biofilm-related infections.