Morphological changes in Proteus mirabilis O18 biofilm under the influence of a urease inhibitor and a homoserine lactone derivative

Abstract

Proteus mirabilis is a pathogenic gram-negative bacterium that frequently causes kidney infections, typically established by ascending colonization of the urinary tract. The present study is focused on ureolytic activity and urease inhibition in biofilms generated by P. mirabilis O18 cells. Confocal microscopy revealed morphological alterations in biofilms treated with urea and a urease inhibitor (acetohydroxamic acid, AHA), as some swarmer cells were found to protrude from the biofilm. The presence of a quorum-sensing molecule (N-butanoyl homoserine lactone, BHL) increased biofilm thickness and its ureolytic activity. Laser interferometric determination of diffusion showed that urea easily diffuses through P. mirabilis biofilm, while AHA is blocked. This may suggest that the use of urease inhibitors in CAUTIs may by less effective than in other urease-associated infections. Spectroscopic studies revealed differences between biofilm and planktonic cells indicating that polysaccharides and nucleic acids are involved in extracellular matrix and biofilm formation.

Fig. 1

Proteus mirabilis O18 pDsRed2 biofilm stained with live/dead BacLight stain (Invitrogen), 72-h culture. a Stacked images, b live cells, c dead cells

Fig. 2

Morphological changes in Proteus mirabilis O18 pDsRed2 biofilm after treatment with AHA (a), urea (b), and both (c). Images acquired with a Leica confocal microscope. Scale bar 5 μm

Fig. 3

Swarming motility of Proteus mirabilis O18 pDsRed in a test plate with LB agar (1 %, w/v) supplemented with AHA (200 μg/mL)

Fig. 4

Determination of planktonic cells (absorbance at 550 nm) and biofilm mass (crystal violet method, absorbance at 595 nm) for Proteus mirabilis O18 pDsRed strain in increasing concentration of AHA

Fig. 5

Kinetics of ureolytic activity of Proteus mirabilis O18 biofilms treated with 10 nM BHL and control

Fig. 6

A nucleopore membrane covered with Proteus mirabilis O18 biofilm. a Membrane surface—light passing through a membrane with round pores, b live Proteus mirabilis cells on the membrane surface, c biofilm stained for dead cells. Live/dead staining was performed with FilmTracer™ dyes (Invitrogen). The images were obtained using a Carl Zeiss Axio Scope.A1 epifluorescent microscope

Fig. 7

Biofilm area coverage of CV-stained membranes. Gray levels from 1 (black) to 256 (white) represent the levels of coverage, where high numbers correspond to low coverage, and low numbers to high coverage

Fig. 8

Interferometric analysis of the acetohydroxamic acid (AHA) and urea released from the lower cuvette through a Proteus mirabilis O18 biofilm attached to nucleopore membranes at room temperature measured with a laser interferometric system. Representative results are presented. The initial concentration of both substances was 250 μg/mL

Fig. 9

Scheme visualizing the obtained data. The addition of 10 nM BHL increased both biofilm thickness and its ureolytic activity. Diffusion of the urease inhibitor is blocked, while urea diffuses freely. Starvation conditions may induce the presence of protruding swarmer cells. Biofilm mass tightly covers the nucleopore membrane

Fig. 10

Representative FT-IR spectra (4,000–800 cm⁻¹) of Proteus mirabilis planktonic cells (a), Proteus mirabilis biofilm, (b) and a superposition of the above-mentioned cultures (c)