Isolation of genes involved in biofilm formation of a Klebsiella pneumoniae strain causing pyogenic liver abscess

Abstract

Background: Community-acquired pyogenic liver abscess (PLA) complicated with meningitis and endophthalmitis caused by Klebsiella pneumoniae is an emerging infectious disease. To investigate the mechanisms and effects of biofilm formation of K. pneumoniae causing PLA, microtiter plate assays were used to determine the levels of biofilm formed by K. pneumoniae clinical isolates and to screen for biofilm-altered mutants from a transposon mutant library of a K. pneumoniae PLA-associated strain.

Methodology/principal findings: The biofilm formation of K. pneumoniae was examined by microtiter plate assay. Higher levels of biofilm formation were demonstrated by K. pneumoniae strains associated with PLA. A total of 23 biofilm-decreased mutants and 4 biofilm-increased mutants were identified. Among these mutants, a biofilm-decreased treC mutant displayed less mucoviscosity and produced less capsular polysaccharide (CPS), whereas a biofilm-increased sugE mutant displayed higher mucoviscosity and produced more CPS. The biofilm phenotypes of treC and sugE mutants also were confirmed by glass slide culture. Deletion of treC, which encodes trehalose-6-phosphate hydrolase, impaired bacterial trehalose utilization. Addition of glucose to the culture medium restored the capsule production and biofilm formation in the treC mutant. Transcriptional profile analysis suggested that the increase of CPS production in ΔsugE may reflect elevated cps gene expression (upregulated through rmpA) in combination with increased treC expression. In vivo competition assays demonstrated that the treC mutant strain was attenuated in competitiveness during intragastric infection in mice.

Conclusions/significance: Genes important for biofilm formation by K. pneumoniae PLA strain were identified using an in vitro assay. Among the identified genes, treC and sugE affect biofilm formation by modulating CPS production. The importance of treC in gastrointestinal tract colonization suggests that biofilm formation contributes to the establishment and persistence of K. pneumoniae infection.

Figure 1. Biofilm formation of K. pneumoniae clinical isolates.

Overnight cultures of K. pneumoniae PLA and non-tissue-invasive strains were grown in fresh Luria-Bertani broth at a ratio of 1∶100 in polystyrene plates at 37°C for 5 hr. The sessile bacteria were stained with crystal violet, washed to remove unbound cells, eluted with 95% ethanol, and monitored the absorbance at 550 nm. Data shown are the means of 3 independent experiments (duplicate in each experiment), and error bars shows the standard deviation (SDs). p = 2.599×10⁻⁶, by Student's t-test. Arrow head, the absorbance values (A₅₅₀) of NTUH-K2044.

Figure 2. Biofilm formation of transposon mutants of K. pneumoniae NTUH-K2044.

Overnight cultures of K. pneumoniae PLA and non-tissue-invasive strains were grown in fresh Luria-Bertani broth at a ratio of 1∶100 in polystyrene plates at 37°C for 5 hr. The sessile bacteria were stained with crystal violet, washed to remove unbound cells, eluted with 95% ethanol, and monitored the absorbance at 550 nm. Data are representative of three independent experiments (duplicate in each experiment).

Figure 3. Mucoviscosity, capsular polysaccharide (CPS) production and biofilm formation of K. pneumoniae NTUH-K2044 and isogenic mutants.

A, K. pneumoniae NTUH-K2044 and the transposon mutants Tn3-2 (wza), Tn19-27 (wzc), Tn16-6 (treC), and Tn10-23 (sugE) grown overnight were pelleted at 12,000 g for 10 min. B, The amount of CPS production nof the wild-type strain NTUH-K2044, treC, sugE, and wza mutants were determined by the sulfuric acid assay. *, p<.05. Data are representative of three independent experiments (triplicate in each experiment).

Figure 4. Schemas of transposon-insertion site and biofilm formation of K. pneumoniae NTUH-K2044 and isogenic mutants.

A, Schemas of transposon-insertion site of treC mutant (Tn16-6, Tn18-57, and Tn19-70) and sugE mutant (Tn10-23). Broad arrows, the locations and orientations of the open-reading frames. R, the ribosomal binding site. B, Biofilm formation of K. pneumoniae NUTH-K2044, the deletion mutants, and the complementation strains. Bacteria were grown at 37°C in polystyrene plates containing fresh Luria-Bertani broth for 5 h. The sessile bacteria were stained with crystal violet, washed to remove unbound cells, eluted with 95% ethanol, and monitored the absorbance at 550 nm. *, p<.05. Data are representative of three independent experiments (triplicate in each experiment).

Figure 5. Biofilm formation by K. pneumoniae NTUH-K2044 and its isogenic mutants.

Cover slides were placed into 24-well plates, which had been paved with glass beads. GFP-expressing K. pneumoniae strains were cultured overnight, inoculated into fresh LB for 18 h. The cover slides were washed with 1×PBS twice while shaking. The fluorescent signals were examined with a confocal microscope under 400×. The photographs depicted mimic images through the x–y plot (central), the y–z plot (right), and the x–z plot (below) of the biofilms. Data represent one of three independent experiments.

Figure 6. Biofilm development of K. pneumoniae ΔtreC on glass slides and the comparison of the treBC expression differences between biofilm and planktonic cells.

A, K. pneumoniae NTUH-K2044 (WT), ΔtreC, ΔtreC/ΔtreC+ were grown at 37°C on glass slides, harvested at 4, 8, 16, 24, 48, and 72 h, and observed with a confocal microscope under 400× oil lens. The 177 sections obtained at 0.2 µm intervals were processed using Bitplane Imaris. B, The wild-type strains which were grown under planktonic culture condition or under biofilm slide culture condition were harvested at 4, 8, 16, 24, 48, and 72 h. The gene expression was determined by real-time RT-PCR. The data showed that the fold change in the expression of treB and treC gene in the biofilm cells with respect to that of in the planktonic cells. The expression of licC was taken as an internal control. Data represent one of two independent experiments.

Figure 7. Biofilm and capsular polysaccharide production of ΔtreC in Luria-Bertani broth supplemented with glucose.

A, Biofilm formation by K. pneumoniae NTUH-K2044 and ΔtreC on glass surface. GFP-expressing K. pneumoniae strain was grown on glass cover slide and harvest at 16 h. The cover slides were washed with PBS twice while shaking. The fluorescent signals were observed with a confocal microscope using oil immersion under 400×. The 177 sections obtained at 0.2 µm intervals were processed using Bitplane Imaris. Data represent one of two independent experiments. B, The capsular polysaccharide of wild-type strain (WT) and ΔtreC grown in Luria-Bertani (LB) broth, or LB broth supplemented with 0.4% of glucose were extracted and determined by sulfuric acid assay. *, p<0.05. Data shown are mean from three independent experiments.

Figure 8. The transcriptional levels of treC and capsule-associated genes in the sugE mutant.

Total RNA was extracted from K. pneumoniae NTUH-K2044 wild-type strain (WT) and ΔsugE cultivated in LB broth. The expression level of each gene in wild-type strain was taken as basal levels to obtain the value of relative level in ΔsugE strain. Data shown are the means of 3 independent experiments, and error bars shows the SDs.

Figure 9. The in vivo competitiveness of ΔtreC.

Groups of 6 BALB/c mice were fed with 2×10⁶ K. pneumoniae. (ΔplacZ strain mixed with the wild-type strain or the ΔtreC strain, the ratio was 1∶ 1). The bacteria were harvested from colon when mice were euthanized on the 7^th day after infection and plated onto LB plates. The ratio of lacZ-negative colonies to lacZ-positive colonized was determined. Competition index of ΔtreC group vs ΔplacZ mutant group, 0.083±0.25, *p = 0.0039.