Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials

Abstract

Biofilms are considered to be highly resistant to antimicrobial agents. Strictly speaking, this is not the case-biofilms do not grow in the presence of antimicrobials any better than do planktonic cells. Biofilms are indeed highly resistant to killing by bactericidal antimicrobials, compared to logarithmic-phase planktonic cells, and therefore exhibit tolerance. It is assumed that biofilms are also significantly more tolerant than stationary-phase planktonic cells. A detailed comparative examination of tolerance of biofilms versus stationary- and logarithmic-phase planktonic cells with four different antimicrobial agents was performed in this study. Carbenicillin appeared to be completely ineffective against both stationary-phase cells and biofilms. Killing by this beta-lactam antibiotic depends on rapid growth, and this result confirms the notion of slow-growing biofilms resembling the stationary state. Ofloxacin is a fluoroquinolone antibiotic that kills nongrowing cells, and biofilms and stationary-phase cells were comparably tolerant to this antibiotic. The majority of cells in both populations were eradicated at low levels of ofloxacin, leaving a fraction of essentially invulnerable persisters. The bulk of the population in both biofilm and stationary-phase cultures was tolerant to tobramycin. At very high tobramycin concentrations, a fraction of persister cells became apparent in stationary-phase culture. Stationary-phase cells were more tolerant to the biocide peracetic acid than were biofilms. In general, stationary-phase cells were somewhat more tolerant than biofilms in all of the cases examined. We concluded that, at least for Pseudomonas aeruginosa, one of the model organisms for biofilm studies, the notion that biofilms have greater resistance than do planktonic cells is unwarranted. We further suggest that tolerance to antibiotics in stationary-phase or biofilm cultures is largely dependent on the presence of persister cells.

FIG. 1

Killing of P. aeruginosa cells by carbenicillin. (A) Cells of logarithmic-phase, stationary-phase, and biofilm cultures were treated with carbenicillin for 6 h and then plated for colony counting. The data for biofilm cultures are plotted as CFU per peg of the biofilm device. The limit of detection is indicated by the solid horizontal line. The standard deviation for each point was calculated with n = 3. Biofilm, diamonds; stationary-phase cells, circles; logarithmic-phase cells, squares. (B) Data of panel A replotted as percent survival.

FIG. 2

Killing of P. aeruginosa cells by ofloxacin. (A) Cells of logarithmic-phase, stationary-phase, and biofilm cultures were treated with ofloxacin for 6 h and then plated for colony counting. The conditions were as described in the legend to Fig. 1. Biofilm, diamonds; stationary-phase cells, circles; logarithmic-phase cells, squares. (B) Data of panel A replotted as percent survival.

FIG. 3

Killing of P. aeruginosa cells by tobramycin. (A) Cells of logarithmic-phase, stationary-phase, and biofilm cultures were treated with tobramycin for 6 h and then plated for colony counting. The conditions were as described in the legend to Fig. 1. Note that the experimental points for logarithmic-phase cells essentially align at the y axis due to the scale of this graph. Complete killing of logarithmic-phase cells was achieved with tobramycin at 4 μg/ml. The open square shows that the data point was at or below the limit of detection. Biofilm, diamonds; stationary-phase cells, circles; logarithmic-phase cells, squares. (B) Data of panel A replotted as percent survival.

FIG. 4

Killing of P. aeruginosa cells by peracetic acid. (A) Cells of logarithmic-phase, stationary-phase, and biofilm cultures were treated with peracetic acid for 6 h and then plated for colony counting. The conditions were as described in the legend to Fig. 1. The open squares show that the data points were at or below the limit of detection. Biofilm, diamonds; stationary-phase cells, circles; logarithmic-phase cells, squares. (B) Data of panel A replotted as percent survival.