A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Summary

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Abstract

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Introduction

Bacteria can switch between two very different lifestyles, planktonic and sessile, of which a biofilm is the most common example. In biofilms, bacteria form structured communities embedded in a self-produced matrix¹. This self-produced matrix is a barrier between the bacteria and their external environment, and it protects the microbial cells, keeping them in close proximity. The composition of the biofilm matrix varies between and even within species, but it mostly consists of a tight network of lipopolysaccharides, extracellular DNA, and proteins. The matrix serves as a physical barrier inhibiting the entrance of harmful agents, but it also protects the b....

Protocol

1. Growing the Bacteria

1. Pre-culture the bacteria overnight in a tryptic soy broth (TSB) at 37 °C with 220 rpm shaking (16-18 hr).
2. Dilute the pre-culture 100-1,000 times (depending on the growth rate of the bacteria; here, 1,000 times is used for S. aureus) in fresh TSB and let it grow at 37 °C and 200 rpm to reach exponential growth (optical density at 595 nm (OD₅₉₅) between 0.2 and 0.6).
   NOTE: This step requires strain-specific optimization.

2. Biofilm Formation: Pre- and Post-exposure

1. Dilute the exponentially grown culture 100 times (this equals approximately 10....

Results

In the proposed platform, the effects on the viability, biomass, and biofilm matrix are quantified. In the working sequence (Figure 1), one sample plate is stained with resazurin and subsequently with crystal violet to simultaneously evaluate the effects on bacterial biofilm viability and on total biofilm biomass. Both assays can be performed consecutively in the same plate because it was demonstrated earlier that a first staining with resazurin had no statistically signi.......

Discussion

There is no single method that can simultaneously measure the effect of a compound on the viability, biomass, and biofilm matrix. Therefore, there is a need for combining assays in order to detect an effect on the three endpoints, preferably at a primary screening stage.

Resazurin is a very simple staining protocol consisting only of the addition of the redox probe. However, establishing the optimal incubation time of the biofilms with the resazurin is crucial to the success of this assay. In .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Resazurin	Sigma Aldrich	R7017
Crystal violet	Sigma Aldrich	HT90132
Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate	Thermo Fisher Scientific	W11261
LIVE/DEAD BacLight	Molecular Probes	L7012	SYTO 9 for staining viable cells green and propidium iodide for staining dead cells red
Phosphate Buffered Saline
Tryptone soy agar	Lab M, Neogen	LAB011
Tryptine soy broth	Lab M, Neogen	LAB004
F96 Well Plate Polystyrene Sterile Clear Flat bottom	Thermo Fisher Scientific	161093
BRAND caps, strips of 8	Sigma Aldrich	BR781413-300EA
Branson CPX series ultrasonic bath	Sigma Aldrich	Z769428-1EA
Multipipette	Thermo Fisher Scientific
Multidrop dispenser	Thermo Fisher Scientific
Biomek 3000	Beckman Coulter
Varioskan Flash Multiplate reader	Thermo Fisher Scientific
Staphylococcus aureus	ATCC	25923