Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

Summary

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro using a 96-well microtiter plate. Planktonic or biofilm bacteria are exposed to serial dilutions of the antimicrobial agent of choice. Viability is assayed by growth on agar plates.

Abstract

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria are inoculated into the wells of a 96-well microtiter plate. In the case of the planktonic assay, serial dilutions of the antimicrobial agent of choice are added to the bacterial suspensions. In the biofilm assay, once inoculated, the bacteria are left to form a biofilm over a set period of time. Unattached cells are removed from the wells, the media is replenished and serial dilutions of the antimicrobial agent of choice are added. After exposure to the antimicrobial agent, the planktonic cells are assayed for growth. For the biofilm assay, the media is refreshed with fresh media lacking the antimicrobial agent and the biofilm cells are left to recover. Biofilm cell viability is assayed after the recovery period. The MBC-P for the antimicrobial agent is defined as the lowest concentration of drug that kills the cells in the planktonic culture.  In contrast, the MBC-B for a strain is determined by exposing preformed biofilms to increasing concentrations of antimicrobial agent for 24 hr. The MBC-B is defined as the lowest concentration of antimicrobial agent that kills the cells in the biofilm.

Introduction

Antibiotic resistance assays were initially developed to assay resistance of planktonic (free-swimming) cultures of bacteria. Since many bacterial infections involve biofilms (surface-attached cells), we were interested in developing a method to assay biofilm-specific antibiotic resistance. However, most antibiotic resistance assays are poorly suited for measuring the resistance of biofilms. For example, determining the minimal inhibitory concentration (MIC) is the gold standard for determining antibiotic resistance of planktonic bacterial cultures ¹. This assay entails mixing a diluted planktonic culture with a dilution series of antibiotic.  The concentration of antibiotic that inhibits the visible growth of the planktonic cells is the MIC. Since this assay relies on inhibition of growth, by definition, it cannot work with biofilm cultures, which requires examining the antibiotic sensitivity of cells in a pregrown biofilm. Instead of measuring growth inhibition, the MBC-B assay described here determines the concentration of antibiotic that kills cells already existing in a biofilm. Thus, this assay aims to mimic antibiotic treatments of established biofilm infections, and provide a more relevant view of the bacterial antibiotic resistance in vivo.

Since biofilms are generally more antibiotic resistant than planktonic cultures ^2-4 , it was necessary to devise a method that directly relates the antibiotic resistance of a biofilm to that of a planktonic culture. Thus another goal of this method is to be able to directly compare the level of antibiotic resistance between planktonic and biofilm cells. The MBC-P and MBC-B assays described here make this possible because cells are cultured under similar conditions. We have utilized this method to study several genes that are important for biofilm-specific antibiotic resistance ^5-8 .

Protocol

1. MBC-B

1. Growing a biofilm (adapted from O'Toole⁹).
   1. Grow a culture of the wild-type strain of interest and mutant strain for 16 hr in a rich medium at 37 °C.
   2. Dilute the saturated overnight cultures 1:100 into fresh medium for antibiotic resistance assays. A standard medium for P. aeruginosa is M63 minimal medium supplemented with magnesium sulfate and arginine (see Table 1). This medium stimulates the formation of a more robust biofilm.
   3. Add 100 μl of the dilution per well in a 96-well microtiter dish (see Table 1). Since these assays are typically performed in triplicate for each strain, there should be 24 wells of each strain.
   4. Incubate the microtiter plate for 24 hr at 37 °C.
2. Exposing the preformed biofilm to a concentration gradient of antibiotic
   1. Prepare a 10x dilution series of antibiotic for 7 wells. Example: for the antibiotic tobramycin, the final concentrations in the wells are 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.006 mg/ml ^5-8 . From a stock of 25 mg/ml, prepare 10x dilutions of 4, 2, 1, 0.5, 0.25, 0.125, and 0.06 mg/ml. Leave on ice.
   2. Remove the spent supernatant (containing planktonic cells) using a multichannel pipette (see Table 1).
   3. Add 90 μl M63 (Mg/Arg) to all of the wells.
   4. Add 10 μl of each 10x antibiotic concentration in order to achieve the desired final concentrations. Add 10 μl water (no antibiotic control) to the final well for each replicate and strain.
   5. Incubate the microtiter plate for 24 hr at 37 °C.
3. Refreshing the media and allowing for the detachment of live cells.
   1. Remove the spent supernatant (containing planktonic cells) using a multichannel pipette.
   2. Add 115 μl M63 (Mg/Arg) to all of the wells.
   3. Incubate the microtiter plate for 24 hr at 37 °C.
4. Assay for live cells.
   1. Label two LB agar plates per 96-well microtiter plate.
   2. Sterilize the multiprong device (see Table 1) by dipping the prongs in 100% ethanol and passing the prongs across the open flame of a Bunsen burner. Repeat. Let the prongs cool slightly. Using the multiprong device, transfer ~3 μl (amount that is typically retained on the tips of the prongs) of planktonic culture from each well of the microtiter plate to the surface of an LB agar plate.
   3. Incubate the LB agar plates for 16 hr at 37 °C.
   4. Determine minimal bactericidal concentration of the antibiotic by identifying, by eye, the cut-off for bacterial growth (Figure 1).

2. MBC-P 

1. Preparing bacterial strains
   1. Grow a culture of the wild-type strain of interest and mutant strain for 16 hr in a rich medium at 37 °C.
   2. Dilute the saturated overnight cultures 1:100 into fresh medium for antibiotic resistance assays. A standard medium for P. aeruginosa is M63 minimal medium supplemented with magnesium sulfate and arginine (see Table 1).
   3. Add 90 μl of the dilution per well in a 96-well microtiter dish (see Table 1). Since these assays are typically performed in triplicate for each strain, there should be 24 wells of each strain.
2. Exposing planktonic cells to a concentration gradient of antibiotic
   1. Prepare 10x dilution series of antibiotic for 7 wells. Example: for the antibiotic tobramycin, the final concentrations in the wells are 0.032, 0.016, 0.008, 0.004, 0.002, 0.001, and 0.0005 mg/ml. From a stock of 25 mg/ml, prepare dilutions of 0.32, 0.16, 0.08, 0.04, 0.02, 0.01, and 0.005 mg/ml. Leave on ice.
   2. Add 10 μl of each 10x antibiotic concentration in order to achieve the desired final concentrations. Add 10 μl water (no antibiotic control) to the final well for each replicate and strain.
   3. Incubate the microtiter plate for 24 hr at 37 °C.
3. Assay for live cells.
   1. Label two LB agar plates per 96-well microtiter plate.
   2. Sterilize the multiprong device (see Table 1) by dipping the prongs in 100% ethanol and passing the prongs across the open flame of a Bunsen burner. Repeat. Let the prongs cool slightly. Using the multiprong device, transfer ~3 μl (amount that is typically retained on the tips of the prongs) of planktonic culture from each well of the microtiter plate to the surface of an LB agar plate.
   3. Incubate the LB agar plates for 16 hr at 37 °C.
   4. Determine minimal bactericidal concentration of the antibiotic by identifying, by eye, the cut off for bacterial growth (Figure 2).

Results

MBC-P and MBC-B assays were carried out, comparing the sensitivity of PA14 wild type with PA14 ∆ndvB. Tobramycin was used as the antibiotic. Results corresponding to step 1.4.4 (Figure 1) and step 2.3.4 (Figure 2) are presented. PA14 and ∆ndvB were inoculated into the MBC-P and MBC-B assays in triplicate. After completing steps 1.0-1.4 of the MBC-B protocol and steps 2.0-2.3 of the MBC-P protocol, the viable cells were plated onto an LB agar plate. Concentra...

Discussion

Antibiotic resistance in planktonic cells is defined as an increase in the minimum inhibitory concentration (MIC) of an antibiotic due to a permanent change in the cells (e.g. a mutation). The mechanisms of biofilm-specific resistance or tolerance that have been identified to date are the result of the expression of wild type genes within biofilms. Thus, the classical definition of resistance does not apply to biofilms. However, another set of definitions has been presented: resistance mechanisms prevent the ant...

Materials

Name	Company	Catalog Number	Comments
1x M63			Prepare as a 5x M63 stock by dissolving 15g KH2PO4, 35g K2HPO4 and 10g (NH4)2SO4 in 1 L of water. This stock does not need to be autoclaved and can be stored at room temperature.  Dilute 5x stock 1:5, autoclave, cool, then add the desired components.
KH2PO4	Fisher	P285-500
K2HPO4	Fisher	P288-500
(NH4)2SO4	Sigma	A5132
Magnesium sulfate	Fisher	M63-500	Add to 1 mM final concentration.  Prepare as a 1 M stock in water and autoclave.
Tobramycin	Sigma		Prepare 50 mg/m stock. Aliquot and store at -20°C.
Arginine	Sigma	A5131	Add to 0.4% final concentration.  Prepare as a 20% stock in water and filter sterilize.  This alternative carbon/energy source can replace glucose and casamino acids
96-well microtiter plates	Corning	3595	Sterile, flat-bottom, low evaporation
Tranferpette (multichannel pipette)	BrandTech	2703610	8-channel, 20-200 μl
Multiprong device	Dan-Kar	MC48	48 prongs fit into ½ of a 96-well microtiter plate