Susceptibility of biofilms to Bdellovibrio bacteriovorus attack

Abstract

Biofilms are communities of microorganisms attached to a surface, and the growth of these surface attached communities is thought to provide microorganisms with protection against a range of biotic and abiotic agents. The capability of the gram-negative predatory bacterium Bdellovibrio bacteriovorus to control and reduce an existing Escherichia coli biofilm was evaluated in a static assay. A reduction in biofilm biomass was observed as early as 3 h after exposure to the predator, and an 87% reduction in crystal violet staining corresponding to a 4-log reduction in biofilm cell viability was seen after a 24-h exposure period. We observed that an initial titer of Bdellovibrio as low as 10(2) PFU/well or an exposure to the predator as short as 30 min is sufficient to reduce a preformed biofilm. The ability of B. bacteriovorus to reduce an existing biofilm was confirmed by scanning electron microscopy. The reduction in biofilm biomass obtained after the first 24 h of exposure to the predator remained unchanged even after longer exposure periods and reinoculation of the samples with fresh Bdellovibrio; however, no genetically stable resistant population of the host bacteria could be detected. Our data suggest that growth in a biofilm does not prevent predation by Bdellovibrio but allows a level of survival from attack greater than that observed for planktonic cells. In flow cell experiments B. bacteriovorus was able to decrease the biomass of both E. coli and Pseudomonas fluorescens biofilms as determined by phase-contrast and epifluorescence microscopy.

FIG. 1.

Predation on E. coli biofilms by B. bacteriovorus. (A) E. coli biofilms were developed for 18 h in 96-well microtiter plates (pretreatment), followed by a 24-h exposure to B. bacteriovorus lysate (+B.b.) or a sterile lysate solution (−B.b.), and then rinsed and stained with CV. (B) Quantification of biofilm biomass over time. B. bacteriovorus lysate (▪) or a sterile lysate solution control (□) was added to a developed E. coli biofilm, the dishes were rinsed and stained with CV, and the amount of CV staining was quantified at OD₅₅₀ for each time point. Each value represents the mean of 24 wells from one representative experiment; error bars indicate the standard errors. Each experiment was carried out three times, yielding similar results each time. The difference in OD₅₅₀ at each time point from 6 h to 48 h was statistically significant (P < 0.001) (C) Quantification of biofilm cell viability. E. coli biofilms were developed ∼18 h in a 96-well microtiter plate, followed by exposure to B. bacteriovorus or a sterile lysate. Biofilm cell viability assays of B. bacteriovorus-treated (▪) and control, sterile lysate-treated (□) samples were performed after the wells were rinsed and sonicated. Each value represents the mean of four wells from one representative experiment; error bars indicate the standard errors. Each experiment was carried out three times yielding similar results. The difference in viability between these treatments at each time point was statistically significant (P < 0.001). (D) SEM images taken after E. coli biofilms were developed for 18 h on a polyvinyl chloride plastic coverslip (pretreatment) and exposed for 24 h to a sterile lysate solution (−Bdellovibrio) or a B. bacteriovorus lysate (+Bdellovibrio). The arrow indicates attached B. bacteriovorus. Scale bar, 2 μm. Magnification, ×10,000. Each experiment was carried out three times, yielding similar results. Images were viewed at the air-liquid interface.

FIG. 2.

Cell viability counts of biofilm and planktonic E. coli. Wild-type E. coli biofilms were formed for 18 h in a 96-well microtiter plate, followed by 48 h of exposure to B. bacteriovorus (▪) or sterile lysate (□), and viability counts were determined after the wells were rinsed and sonicated. Planktonic E. coli were mixed with B. bacteriovorus (▴) or with sterile lysate (▵), and the bacterial viability counts were determined. Each value represents the mean of four wells from one representative experiment, and error bars indicate the standard errors. The difference in viability at 24 and 48 h between the biofilm versus planktonic cells was statistically significant (P < 0.001). Each experiment was carried out three times, yielding similar results.

FIG. 3.

Monitoring Bdellovibrio attack in flow cells. E. coli (A) and P. fluorescens (B) biofilms were developed in a flow cell system after inoculation with sterile lysate solution (I to III) or B. bacteriovorus lysate (IV to VI). After 48 h the chambers were analyzed by phase-contrast microscopy (I and IV) (dark areas are adherent bacteria) or stained with the BacLight viability stain for 45 min and then rinsed for 45 min to remove excess dye. Syto-9 panels (II and V) indicate viable cells (green, intact membranes), and PI panels (III and VI) indicate dead or compromised cells (red, damaged membranes). Scale bar, 20 μm. Magnification, ×650. Each experiment was carried out three times, with two replicates for each treatment, yielding similar results. At least 10 different areas of each sample were examined; representative images are shown here.