A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms

Abstract

Bacterial biofilms show enormous levels of antibiotic resistance, but little is known about the underlying molecular mechanisms. Multidrug resistance pumps (MDRs) are responsible for the extrusion of chemically unrelated antimicrobials from the bacterial cell. Contribution of the MDR-mediated efflux to antibiotic resistance of Pseudomonas aeruginosa biofilms was examined by using strains overexpressing and lacking the MexAB-OprM pump. Resistance of P. aeruginosa biofilms to ofloxacin was dependent on the expression of MexAB-OprM but only in the low concentration range. Unexpectedly, biofilm resistance to ciprofloxacin, another substrate of MexAB-OprM, did not depend on the presence of this pump. Dose-dependent killing indicated the presence of a small "superresistant" cell fraction. This fraction was primarily responsible for very high resistance of P. aeruginosa biofilms to quinolones. Bacterial cells recovered from a biofilm and tested under nongrowing conditions with tobramycin exhibited higher resistance levels than planktonic cells but lower levels than cells of an intact biofilm.

FIG. 1

Role of the MexAB-OprM pump in resistance of biofilms and planktonic cells to killing by antibiotics. (A through C) Biofilms, stationary-phase cells, and nongrowing cells, respectively, treated with ofloxacin (Oflox). (D through F) Biofilms, stationary-phase cells, and nongrowing cells, respectively, treated with ciprofloxacin (Cipro). (G through I) Biofilms, stationary-phase cells, and nongrowing cells, respectively, treated with tetracycline (Tet). (J through L) Biofilms, stationary-phase cells, and nongrowing cells, respectively, treated with tobramycin (Tobra).

FIG. 1

Role of the MexAB-OprM pump in resistance of biofilms and planktonic cells to killing by antibiotics. (A through C) Biofilms, stationary-phase cells, and nongrowing cells, respectively, treated with ofloxacin (Oflox). (D through F) Biofilms, stationary-phase cells, and nongrowing cells, respectively, treated with ciprofloxacin (Cipro). (G through I) Biofilms, stationary-phase cells, and nongrowing cells, respectively, treated with tetracycline (Tet). (J through L) Biofilms, stationary-phase cells, and nongrowing cells, respectively, treated with tobramycin (Tobra).

FIG. 2

Dose-response killing of P. aeruginosa wild-type cells liberated from a biofilm. Cells were dislodged from a biofilm by sonication, placed in Mueller-Hinton growth medium, and treated with tobramycin (Tobra) (diamonds). Similarly prepared cells were diluted prior to tobramycin treatment 10-fold (squares) and 150-fold (triangles).

FIG. 3

Killing of P. aeruginosa biofilms by ofloxacin (Oflx) and tobramycin (Tobra). Conditions were as described for Fig. 1. PAO-JP1 is a lasI mutant defective in the production of HSL, and PAO1 is the wild-type parent strain.