High-throughput metal susceptibility testing of microbial biofilms

Abstract

Background: Microbial biofilms exist all over the natural world, a distribution that is paralleled by metal cations and oxyanions. Despite this reality, very few studies have examined how biofilms withstand exposure to these toxic compounds. This article describes a batch culture technique for biofilm and planktonic cell metal susceptibility testing using the MBEC assay. This device is compatible with standard 96-well microtiter plate technology. As part of this method, a two part, metal specific neutralization protocol is summarized. This procedure minimizes residual biological toxicity arising from the carry-over of metals from challenge to recovery media. Neutralization consists of treating cultures with a chemical compound known to react with or to chelate the metal. Treated cultures are plated onto rich agar to allow metal complexes to diffuse into the recovery medium while bacteria remain on top to recover. Two difficulties associated with metal susceptibility testing were the focus of two applications of this technique. First, assays were calibrated to allow comparisons of the susceptibility of different organisms to metals. Second, the effects of exposure time and growth medium composition on the susceptibility of E. coli JM109 biofilms to metals were investigated.

Results: This high-throughput method generated 96-statistically equivalent biofilms in a single device and thus allowed for comparative and combinatorial experiments of media, microbial strains, exposure times and metals. By adjusting growth conditions, it was possible to examine biofilms of different microorganisms that had similar cell densities. In one example, Pseudomonas aeruginosa ATCC 27853 was up to 80 times more resistant to heavy metalloid oxyanions than Escherichia coli TG1. Further, biofilms were up to 133 times more tolerant to tellurite (TeO3(2-)) than corresponding planktonic cultures. Regardless of the growth medium, the tolerance of biofilm and planktonic cell E. coli JM109 to metals was time-dependent.

Conclusion: This method results in accurate, easily reproducible comparisons between the susceptibility of planktonic cells and biofilms to metals. Further, it was possible to make direct comparisons of the ability of different microbial strains to withstand metal toxicity. The data presented here also indicate that exposure time is an important variable in metal susceptibility testing of bacteria.

Figure 1

Growth of bacterial biofilms in the MBEC assay. (A) Mean cell density of Pseudomonas aeruginosa ATCC 27853 biofilms on the pegs in different rows of the MBEC assay. Each value is expressed as the mean and standard deviation of 4 to 6 trials. There is no significant difference between cell density of biofilms in the different rows (p = 0.842 using one-way ANOVA). (B) SEM photomicrograph of a P. aeruginosa biofilm on the peg surface. (C) Mean cell density of Escherichia coli TG1 on the pegs in different rows of the MBEC assay. Each value is expressed as the mean and standard deviation of 4 to 6 trials. There is no significant difference between cell density of biofilms in the different rows (p = 0.274 using one-way ANOVA). (D) SEM photomicrograph of an E. coli biofilm on the peg surface. The bar represents 5 μm.

Figure 2

An overview of the high-throughput protocol for metal susceptibility testing using the MBEC assay. (A) Frozen stocks of bacteria were streaked out on the appropriate agar medium to obtain a first- and a subsequent second-subculture. (B) Colonies were collected from second-subcultures and suspended in broth medium to a 1.0 McFarland Standard. (C) This suspension was diluted 30-fold in broth, and the 1 in 30 dilution was used to inoculate the MBEC assay. (D) The inoculated device was placed on a rocking table in an incubator. (E) Serial dilutions of metal cations and oxyanions were set up along length of a microtiter plate along (the challenge plate). (F) The biofilms were rinsed to remove loosely adherent planktonic bacteria. (G) The first peg from each row was removed. These pegs were used to verify growth of the biofilms on the pegs. The peg lid was then inserted into the challenge plate. (H) During exposure, metals diffuse into the biofilm while planktonic cells are shed from the surface of the biofilm. Sloughed cells serve as the inoculum for planktonic MIC and MBC determinations. (I) The exposed biofilms were rinsed twice and the peg lid was inserted into fresh recovery medium containing the appropriate neutralizing agent (the recovery plate). The biofilms were disrupted into the recovery medium by sonciation on a water table sonicator. (J) Aliquots of planktonic cultures were transferred from the challenge plate to a microtiter plate containing the appropriate neutralizing agents (the neutralizing plate). (K) An aliquot from the recovery and neutralizing plates were spotted onto rich agar media. (L) MIC values are determined by reading the optical density at 650 nm (OD₆₅₀) of the challenge plate after the desired period of incubation using a microtiter plate reader. Spot plates were qualitatively scored for growth to obtain MBC and MBEC values. MBEC values were redundantly determined by determining the A₆₅₀ of the recovery plates after incubation.