Differential protection from tobramycin by extracellular polymeric substances from Acinetobacter baumannii and Staphylococcus aureus biofilms

Abstract

We investigated biofilms of two pathogens, Acinetobacter baumannii and Staphylococcus aureus, to characterize mechanisms by which the extracellular polymeric substance (EPS) found in biofilms can protect bacteria against tobramycin exposure. To do so, it is critical to study EPS-antibiotic interactions in a homogeneous environment without mass transfer limitations. Consequently, we developed a method to grow biofilms, harvest EPS, and then augment planktonic cultures with isolated EPS and tobramycin. We demonstrated that planktonic cultures respond differently to being treated with different types of EPS (A. baumannii versus S. aureus) in the presence of tobramycin. By harvesting EPS from the biofilms, we found that A. baumannii EPS acts as a "universal protector" by inhibiting tobramycin activity against bacterial cells regardless of species; S. aureus EPS did not show any protective ability in cell cultures. Adding Mg(2+) or Ca(2+) reduced the protective effect of A. baumannii EPS. Finally, when we selectively digested the proteins or DNA of the EPS, we found that the protective ability did not change, suggesting that neither has a significant role in protection. To the best of our knowledge, this is the first study that demonstrates how EPS protects pathogens against antibiotics in a homogeneous system without mass transfer limitations. Our results suggest that EPS protects biofilm communities, in part, by adsorbing antibiotics near the surface. This may limit antibiotic diffusion to the bottom of the biofilms but is not likely to be the only mechanism of protection.

FIG 1

Biofilm membrane reactor. A pump is used to pull medium through the membrane. The medium exits the bottom of the funnel. It is recycled using a drip inlet through the top of the reactor. An air filter is used to maintain constant pressure. The closeup of the membrane shows the biofilm forming on the surface.

FIG 2

Addition of EPS from Acinetobacter baumannii, but not of EPS from Staphylococcus aureus, protects bacteria from tobramycin treatment. (A) Representative growth curves for A. baumannii that was cultured in 0.33× TSB alone (●), with 1 μg/ml tobramycin (○), with 50% (vol/vol) A. baumannii EPS (▼), or with both tobramycin and EPS (△). (B) Representative growth curves for S. aureus that was cultured in 0.33× TSB alone (●), with 1 μg/ml tobramycin (○), with 50% (vol/vol) A. baumannii EPS (▼), or with both tobramycin and EPS (△). (C) Normalized growth rates of cultures challenged with tobramycin, with EPS, or with both tobramycin and EPS. EPSs from both A. baumannii and S. aureus were used, but only EPS from A. baumannii protected against a tobramycin challenge (*, P < 0.001, n = 22). Error bars represent 1 standard deviation.

FIG 3

The growth rate of A. baumannii culture with 50% (vol/vol) extracted A. baumannii EPS responded in a dose-dependent manner to tobramycin (gray bars). Normalized growth rates are reported in OD/h. The controls (black bars) did not include EPS. The results show the average values for eight independent replicates. Error bars represent 1 standard deviation.

FIG 4

Average growth rates of A. baumannii cultures with extracted A. baumannii EPS and 1 μg/ml tobramycin with added Mg²⁺ or Ca²⁺. The decrease in the protective ability of EPS can be seen by comparing the growth rates for the EPS with those of tobramycin cultures. When 1.25 mM Mg²⁺ or Ca²⁺ was added, the growth was inhibited. Two-way analysis of variance confirms that Mg²⁺ and Ca²⁺ are statistically significant in the presence of A. baumannii EPS and 1 μg/ml tobramycin (*, P < 0.001). Error bars represent 1 standard deviation.