Figure 1

Predation by B. bacteriovorus wt and HI mutants. (A) Plaque predation assays. Wild-type B. bacteriovorus lysates (B.b wt) or HI mutants (HI-A, B, C) were grown and transferred to a thick lawn of K. pneumoniae host cells (pre-treatment). DDNB and heat killed (30 min at 90°C) HI mutant A were used as negative controls. Forty-eight hours after inoculation a clear lytic halo formed at the point of inoculation. Each experiment was carried out three times with three replicates for each treatment, yielding similar results- representative images are shown here. (B) Biofilm predation assays. E. coli biofilms were developed for 18 hrs in 96 well microtiter plates (pre-treatment), followed by 48 hr exposure to B. bacteriovorus lysate, HI mutants (HI-A, B, C), DDNB or heat killed HI mutant A, then rinsed and stained with CV. Each experiment was carried out three times, with 24 wells for each treatment, yielding similar results- representative images are shown here. (C) Quantification of biofilm biomass. B. bacteriovorus lysate, HI mutants, DDNB or heat killed HI mutant A, were added to a developed E. coli biofilm. Forty-eight hours later the dishes were rinsed, stained with CV and the amount of CV staining was quantified at OD₆₀₀ for each time point. Each value represents the mean of 12 wells from one representative experiment. Error bars indicate standard errors. Each experiment was carried out three times yielding similar results. The difference in biofilm reduction between B. bacteriovorus lysate, HI-A, B, C and the negative controls (DDNB and the heat killed HI-A) was statistically significant (P < 0.001). (D) Cell viability counts of planktonic E. coli. Planktonic E. coli cells were mixed with B. bacteriovorus lysate, HI mutants (HI-A, B, C), DDNB or heat killed HI mutant A, and the bacterial viability counts determined. Each experiment was carried out three times yielding similar results. Each value represents the mean of 3 lysates from one representative experiment. Error bars indicate standard errors. The difference in host viability at 24 hr between B. bacteriovorus lysate, HI-A, B, C and the negative controls (DDNB and the heat killed HI-A) was statistically significant (P < 0.001). The difference in host viability at 24 hr between B. bacteriovorus lysate and HI-A was statistically significant (P = 0.05).