The BvgAS signal transduction system regulates biofilm development in Bordetella

Abstract

The majority of Bordetella sp. virulence determinants are regulated by the BvgAS signal transduction system. BvgAS mediates the control of multiple phenotypic phases and a spectrum of gene expression profiles specific to each phase in response to incremental changes in the concentrations of environmental signals. Studies highlighting the critical role of this signaling circuitry in the Bordetella infectious cycle have focused on planktonically growing bacterial cells. It is becoming increasingly clear that the major mode of bacterial existence in the environment and within the body is a surface-attached state known as a biofilm. Biofilms are defined as consortia of sessile microorganisms that are embedded in a matrix. During routine growth of Bordetella under agitating conditions, we noticed the formation of a bacterial ring at the air-liquid interface of the culture tubes. We show here that this surface adherence property reflects the ability of these organisms to form biofilms. Our data demonstrate that the BvgAS locus regulates biofilm development in Bordetella. The results reported in this study suggest that the Bvg-mediated control in biofilm development is exerted at later time points after the initial attachment of bacteria to the different surfaces. Additionally, we show that these biofilms are highly tolerant of a number of antimicrobials, including the ones that are currently recommended for treatment of veterinary and human infections caused by Bordetella spp. Finally, we discuss the significance of the biofilm lifestyle mode as a potential contributor to persistent infections.

FIG. 1.

(A) Formation of a bacterial ring (indicated by the arrow) at the air-liquid interface of polystyrene culture tubes rotating in a roller drum by the wt strain and the Bvg⁻ phase-locked strain of B. bronchiseptica. (B) Microtiter assay of biofilm formation at 24 h by wt and mutant strains of B. bronchiseptica. (C) Kinetics of biofilm formation for B. bronchiseptica strains showing the OD₅₄₀s of solubilized crystal violet from surface-attached cells grown in microtiter plates and assayed at specified time intervals. Each data point is the average for six wells, and error bars indicate the standard errors. Representative data from one of at least five independent experiments are shown. The wt and different mutant strains were grown in SS medium under Bvg⁺ phase conditions. (D) Complementation of the BvgAS locus restores biofilm formation. Microtiter assays were performed as described for panel C. (E) Conditions were the same as for panel C, except that the wt strain was grown in SS medium in the presence of modulator MgSO₄ or nicotinic acid (Nic).

FIG. 1.

(A) Formation of a bacterial ring (indicated by the arrow) at the air-liquid interface of polystyrene culture tubes rotating in a roller drum by the wt strain and the Bvg⁻ phase-locked strain of B. bronchiseptica. (B) Microtiter assay of biofilm formation at 24 h by wt and mutant strains of B. bronchiseptica. (C) Kinetics of biofilm formation for B. bronchiseptica strains showing the OD₅₄₀s of solubilized crystal violet from surface-attached cells grown in microtiter plates and assayed at specified time intervals. Each data point is the average for six wells, and error bars indicate the standard errors. Representative data from one of at least five independent experiments are shown. The wt and different mutant strains were grown in SS medium under Bvg⁺ phase conditions. (D) Complementation of the BvgAS locus restores biofilm formation. Microtiter assays were performed as described for panel C. (E) Conditions were the same as for panel C, except that the wt strain was grown in SS medium in the presence of modulator MgSO₄ or nicotinic acid (Nic).

FIG. 2.

Quantitative analysis of biofilm populations of the wt and phase-locked mutant strains in silicone tubing continuous flow devices. Log phase cultures of each strain (OD₆₀₀, ∼0.5) were inoculated into a section of silicone tubing that had first been primed with medium. The cells were allowed to attach to the tubing for the designated time points, followed by the continuous passage of growth medium through the tubing for 48 h. The tubings were then longitudinally sectioned, and the biofilm-grown cells were harvested, resuspended, and enumerated by plate counts (see Materials and Methods). The number of bacteria obtained for each strain was normalized to that recovered from the wt strain, which is represented as 100%.

FIG. 3.

Scanning electron micrographs of the B. bronchiseptica wt strain or the Bvg⁻ phase-locked strain grown for 6, 24, and 72 h on glass coverslips as described in Materials and Methods. Bar = 1 μm.

FIG. 4.

The kinetics of biofilm formation for B. parapertussis. The wt strain 12822 was grown in SS medium either in the absence (black bars) or presence (white bars) of the modulator MgSO₄. The OD₅₄₀ of solubilized crystal violet from surface-attached cells grown in microtiter plates and assayed at specified time-intervals is shown. Each data point is the average for six wells, and error bars indicate the standard errors. Representative data from one of at least three independent experiments are shown.

FIG. 5.

Scanning electron micrographs comparing biofilm formation for B. parapertussis wt strain 12822 grown in the absence (A) or presence (B) of the modulator MgSO₄. The cultures were grown for 96 h on the surfaces of glass coverslips. Bars = 1 μm.

FIG. 6.

Scanning electron micrographs comparing biofilm formation for B. pertussis wt strain Bp 536 (A) and Bvg⁻ phase-locked strain Bp537 (B) grown for 96 h on the surfaces of glass coverslips. Bars = 1 μm.