Catheter-associated urinary tract infection by Pseudomonas aeruginosa is mediated by exopolysaccharide-independent biofilms

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen that is especially adept at forming surface-associated biofilms. P. aeruginosa causes catheter-associated urinary tract infections (CAUTIs) through biofilm formation on the surface of indwelling catheters. P. aeruginosa encodes three extracellular polysaccharides, PEL, PSL, and alginate, and utilizes the PEL and PSL polysaccharides to form biofilms in vitro; however, the requirement of these polysaccharides during in vivo infections is not well understood. Here we show in a murine model of CAUTI that PAO1, a strain harboring pel, psl, and alg genes, and PA14, a strain harboring pel and alg genes, form biofilms on the implanted catheters. To determine the requirement of exopolysaccharide during in vivo biofilm infections, we tested isogenic mutants lacking the pel, psl, and alg operons and showed that PA14 mutants lacking these operons can successfully form biofilms on catheters in the CAUTI model. To determine the host factor(s) that induces the ΔpelD mutant to form biofilm, we tested mouse, human, and artificial urine and show that urine can induce biofilm formation by the PA14 ΔpelD mutant. By testing the major constituents of urine, we show that urea can induce a pel-, psl-, and alg-independent biofilm. These pel-, psl-, and alg-independent biofilms are mediated by the release of extracellular DNA. Treatment of biofilms formed in urea with DNase I reduced the biofilm, indicating that extracellular DNA supports biofilm formation. Our results indicate that the opportunistic pathogen P. aeruginosa utilizes a distinct program to form biofilms that are independent of exopolysaccharides during CAUTI.

FIG 1

P. aeruginosa colonization of catheters during CAUTI is independent of PEL, PSL, and alginate exopolysaccharides. The indicated organs from mice infected with PA14, PA14 ΔpelD, PA14 ΔpelA-G, or PA14 ΔpelA-G ΔalgD-A in the murine model of CAUTI were harvested, homogenized, and plated for counting of the numbers of CFU. Each symbol represents an individual mouse. Bars represent means. Points at 100 CFU per macerated organ represent samples with counts below the limit of detection. ns, differences were not significant, as determined by one-way ANOVA.

FIG 2

Scanning electron micrographs of catheters isolated from mouse bladders. Catheters were isolated from mice infected with either PAO1 (A) or PA14 ΔpelD/pMMB-PA3702 (B) and visualized by SEM. Asterisks, divided bacteria; arrowheads, filamentous bacteria.

FIG 3

PA14 and PA14 ΔpelD do not compete in the murine CAUTI model. Mice were inoculated with either PA14 and PA14 ΔpelD-lux or PA14-lux and PA14 ΔpelD. Each symbol represents the ratio of PA14 ΔpelD/PA14 in either the bladder or kidneys of an individual mouse. Bars represent means.

FIG 4

Mouse and human urine induce biofilm formation by PA14 ΔpelD. (A) Quantification of the crystal violet eluted from catheters after staining for colonization by PA14/pMMB-PA3702 or PA14 ΔpelD/pMMB-PA3702 in the presence of PBS-T with or without the indicated amounts of mouse urine. (B and C) Quantification of PA14 ΔpelD/pMMB-PA3702 (B) or PA14/pMMB-PA3702 (C) biofilms in PBS-T with or without the indicated amounts of human urine. (D) PA14 ΔpelD biofilm formation in LB or artificial urine medium (AUM). Statistical significance (*, P < 0.05) was determined by using Student's t test. Abs, absorbance.

FIG 5

Urea induces biofilm formation by PA14 ΔpelD. Quantification of crystal violet biofilm formation assays for colonization of either PA14/pMMB-PA3702 (A) or PA14 ΔpelD/pMMB-PA3702 (B) in the presence of LB or LB supplemented with urea, citric acid, creatinine, glycine, histidine, or glutamic acid. (C) Biofilm formation assays with catheters inoculated with either PA14/pMMB-PA3702 (closed bars) or PA14 ΔpelD/pMMB-PA3702 (open bars) in PBS-T with or without urea at the indicated concentrations. Statistical significance (*, P < 0.05) was determined by using Student's t test.

FIG 6

Scanning electron microscopy reveals biofilm formation by PA14 ΔpelD/pMMB-PA3702 in the presence of urea. PA14/pMMB-PA3702 (A and C) and PA14 ΔpelD/pMMB-PA3702 (B and D) were visualized after incubation for 16 h on catheter tubing in the absence or presence of 0.5 M urea. (Insets) Photos taken at higher magnification.

FIG 7

Urea causes the release of DNA from a subset of P. aeruginosa cells. PA14 (A) or PA14 ΔpelD (B) overnight cultures were subcultured 1:100 and placed under agarose pads containing LB with the indicated concentrations of urea or SDS. Samples were incubated for 8 h at 37°C in a humidified chamber. Cells were imaged by phase-contrast microscopy, and eDNA was detected by propidium iodide staining (red). (Insets) Magnified images of rounded cells (arrowheads) that stain positive with propidium iodide.

FIG 8

DNase I treatment reduces the amount of biofilms formed in the presence of urea. (A) PA14 and PA14 ΔpelD biofilms in borosilicate tubes were treated with 5 μg of DNase I or buffer as a control at 37°C for 90 min and stained with crystal violet. (B) Quantification of eluted crystal violet after DNase I or control treatment. Statistical significance (*, P < 0.05) was determined by using Student's t test.