The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms

Abstract

Determination of the MIC, based on the activities of antibiotics against planktonic bacteria, is the standard assay for antibiotic susceptibility testing. Adherent bacterial populations (biofilms) present with an innate lack of antibiotic susceptibility not seen in the same bacteria grown as planktonic populations. The Calgary Biofilm Device (CBD) is described as a new technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. The CBD produces 96 equivalent biofilms for the assay of antibiotic susceptibilities by the standard 96-well technology. Biofilm formation was followed by quantitative microbiology and scanning electron microscopy. Susceptibility to a standard group of antibiotics was determined for National Committee for Clinical Laboratory Standards (NCCLS) reference strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 29213. Growth curves demonstrated that biofilms of a predetermined size could be formed on the CBD at specific time points and, furthermore, that no significant difference (P > 0.1) was seen between biofilms formed on each of the 96 pegs. The antibiotic susceptibilities for planktonic populations obtained by the NCCLS method or from the CBD were similar. Minimal biofilm eradication concentrations, derived by using the CBD, demonstrated that for biofilms of the same organisms, 100 to 1,000 times the concentration of a certain antibiotic were often required for the antibiotic to be effective, while other antibiotics were found to be effective at the MICs. The CBD offers a new technology for the rational selection of antibiotics effective against microbial biofilms and for the screening of new effective antibiotic compounds.

FIG. 1

CBD for biofilm antibiotic susceptibility testing. (A) Tilt table used to create the shear force required for biofilm formation. The table can be placed in incubators to control temperature or oxygen tension. (B) Cutaway view of the device showing the pins sitting in the channels of the incubation tray. (C) Top plate of the device.

FIG. 2

Growth curves of E. coli ATCC 25922 (A), P. aeruginosa ATCC 27853 (B), and S. aureus ATCC 29213 (C) demonstrating that a biofilm of a specific size can be produced over a specific period of growth on the CBD. The number of bacteria per peg was determined by breaking pegs from the lid at appropriate times and determining the bacterial number as described in Materials and Methods.

FIG. 3

Mean number of CFU of P. aeruginosa per peg on the MBEC device determined by plating bacteria sonicated from each peg position for biofilms grown for 4 or 24 h, as described in Materials and Methods. The values obtained were compared by analysis of variance and Bartlett’s test for homogeneity of variance. No significant difference was found between rows (P = 0.9982).

FIG. 4

Scanning electron micrograph of an E. coli biofilm formed on the MBEC device. Pins from the device were broken off and fixed as described in Materials and Methods. Bar, 50 μm.