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. 2007 Jan;73(2):605-14.
doi: 10.1128/AEM.01893-06. Epub 2006 Nov 10.

Vulnerability of pathogenic biofilms to Micavibrio aeruginosavorus

Daniel Kadouri  1 , Nel C Venzon, George A O'Toole
Affiliations

Vulnerability of pathogenic biofilms to Micavibrio aeruginosavorus

Daniel Kadouri et al. Appl Environ Microbiol. 2007 Jan.

Abstract

The host specificity of the gram-negative exoparasitic predatory bacterium Micavibrio aeruginosavorus was examined. M. aeruginosavorus preyed on Pseudomonas aeruginosa, as previously reported, as well as Burkholderia cepacia, Klebsiella pneumoniae, and numerous clinical isolates of these species. In a static assay, a reduction in biofilm biomass was observed as early as 3 hours after exposure to M. aeruginosavorus, and an approximately 100-fold reduction in biofilm cell viability was detected following a 24-h exposure to the predator. We observed that an initial titer of Micavibrio as low as 10 PFU/well or a time of exposure to the predator as short as 30 min was sufficient to reduce a P. aeruginosa biofilm. The ability of Micavibrio to reduce an existing biofilm was confirmed by scanning electron microscopy. In static and flow cell experiments, M. aeruginosavorus was able to modify the overall P. aeruginosa biofilm structure and markedly decreased the viability of P. aeruginosa. The altered biofilm structure was likely caused by an increase in cell-cell interactions brought about by the presence of the predator or active predation. We also conducted a screen to identify genes important for P. aeruginosa-Micavibrio interaction, but no candidates were isolated among the approximately 10,000 mutants tested.

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Figures

FIG. 1.
FIG. 1.
Predation of P. aeruginosa PA14 biofilms by M. aeruginosavorus ARL-13. (A) P. aeruginosa biofilms were developed for 18 h in 96-well microtiter plates (pretreatment), followed by 24 h of exposure to a Micavibrio lysate (+M.a.) or a sterile lysate (−M.a.), and then rinsed and stained with CV. (B) Quantification of biofilm biomass over time. A Micavibrio (▪) or sterile (□) lysate was added to a preformed P. aeruginosa biofilm, the dishes were rinsed and stained with CV, and the amount of CV staining was quantified as the OD550 for each time point. Each value represents the mean for 24 wells from one representative experiment. Error bars show 1 standard deviation. Each experiment was carried out five times, yielding similar results. The difference in OD550 at each time point from 12 h to 96 h was statistically significant (P < 0.001). (C) Quantification of biofilm cell viability. P. aeruginosa biofilms were developed for ∼18 h in a 96-well microtiter plate, followed by exposure to a Micavibrio (▪) or sterile (□) lysate. Samples were obtained after the wells were rinsed and sonicated. Each value represents the mean for three wells from one representative experiment, and error bars indicate standard errors. Each experiment was carried out three times, yielding similar results. The difference in viability between the treatments at each time point was statistically significant (P < 0.001). (D) Scanning electron micrographs after P. aeruginosa biofilms were developed for 18 h on polyvinyl chloride plastic coverslips (pretreatment) and exposed for 24 h to a Micavibrio lysate (+Micavibrio) or a sterile lysate (−Micavibrio). Magnification, ×10,000. Each experiment was performed three times, yielding similar results. Images were viewed at the air-liquid interface.
FIG. 2.
FIG. 2.
Monitoring Micavibrio attack in flow cells. P. aeruginosa PA14 biofilms were developed in a flow cell system for 24 h following inoculation with a Micavibrio (I to III) or sterile (IV to VI) lysate. Seventy-two hours after treatment, 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). Bar, 20 μm; magnification, ×650. Each experiment was performed three times, with two replicates for each treatment, yielding similar results. At least 10 different areas of each sample were examined, and representative images are shown.
FIG. 3.
FIG. 3.
Predation promotes early biofilm formation and cell aggregation. (A) P. aeruginosa biofilms were simultaneously mixed with a Micavibrio (+M.a.) or sterile (−M.a.) lysate, and the wells were rinsed and stained with CV after 24 and 48 h of incubation. (B) Quantification of biofilm biomass over time. A Micavibrio (▪) or sterile (□) lysate was simultaneously mixed with P. aeruginosa cells, the dishes were rinsed and stained with CV, and the amount of CV staining was quantified as the OD550 for each time point. Each value represents the mean for 24 wells from one representative experiment, and error bars indicate 1 standard deviation. Each experiment was carried out five times, yielding similar results. The difference in OD550 at each time point from 24 h to 96 h was statistically significant (P < 0.001). (C) Phase-contrast microscopy images taken 12 (I and II) and 24 (III and IV) h after P. aeruginosa was simultaneously mixed with a Micavibrio lysate (+M.a.) or sterile lysate (−M.a.). Bar, 4 μm; magnification, ×650. This experiment was performed three times, yielding similar results.
FIG. 4.
FIG. 4.
Predation on K. pneumoniae and B. cepacia biofilms by M. aeruginosavorus ARL-13. (A) K. pneumoniae and (B) B. cepacia biofilms were developed for 18 h in 96-well microtiter plates (pretreatment), followed by 24 h of exposure to a Micavibrio lysate (+M.a.) or a sterile lysate (−M.a.), and then rinsed and stained with CV. (C and D) Quantification of biofilm cell viability. K. pneumoniae (C) and B. cepacia (D) biofilms were developed for ∼18 h in 96-well microtiter plates, followed by exposure to a Micavibrio lysate (▪) or a sterile lysate (□), and biofilm cell viability was assessed as described in the legend for Fig. 1. The difference in viability between the treatments at each time point was statistically significant (P < 0.01). (E) Scanning electron micrographs taken after K. pneumoniae (I to III) and B. cepacia (IV to VI) biofilms were developed for 18 h on polyvinyl chloride plastic coverslips (I and IV, pretreatment) and exposed for 24 h to a Micavibrio lysate (II and V, + Micavibrio) or a sterile lysate (III and VI, − Micavibrio). Bar, 4 μm; magnification, ×10,000. Each experiment was performed three times, yielding similar results. Images were viewed at the air-liquid interface.
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