Critical Assessment of Methods to Quantify Biofilm Growth and Evaluate Antibiofilm Activity of Host Defence Peptides

Abstract

Biofilms are multicellular communities of bacteria that can adhere to virtually any surface. Bacterial biofilms are clinically relevant, as they are responsible for up to two-thirds of hospital acquired infections and contribute to chronic infections. Troublingly, the bacteria within a biofilm are adaptively resistant to antibiotic treatment and it can take up to 1000 times more antibiotic to kill cells within a biofilm when compared to planktonic bacterial cells. Identifying and optimizing compounds that specifically target bacteria growing in biofilms is required to address this growing concern and the reported antibiofilm activity of natural and synthetic host defence peptides has garnered significant interest. However, a standardized assay to assess the activity of antibiofilm agents has not been established. In the present work, we describe two simple assays that can assess the inhibitory and eradication capacities of peptides towards biofilms that are formed by both Gram-positive and negative bacteria. These assays are suitable for high-throughput workflows in 96-well microplates and they use crystal violet staining to quantify adhered biofilm biomass as well as tetrazolium chloride dye to evaluate the metabolic activity of the biofilms. The effect of media composition on the readouts of these biofilm detection methods was assessed against two strains of Pseudomonas aeruginosa (PAO1 and PA14), as well as a methicillin resistant strain of Staphylococcus aureus. Our results demonstrate that media composition dramatically alters the staining patterns that were obtained with these dye-based methods, highlighting the importance of establishing appropriate biofilm growth conditions for each bacterial species to be evaluated. Confocal microscopy imaging of P. aeruginosa biofilms grown in flow cells revealed that this is likely due to altered biofilm architecture under specific growth conditions. The antibiofilm activity of several antibiotics and synthetic peptides were then evaluated under both inhibition and eradication conditions to illustrate the type of data that can be obtained using this experimental setup.

Antibacterial mechanisms of GN‐2 derived peptides and peptoids against Escherichia coli

“…As most clinical bacterial infections are biofilm‐related, it is essential to prove antimicrobial effects in biofilm experiments. It is obviously essential to note that the assay platforms for evaluating anti‐biofilm effects are many and that the smallest change in the protocol might impact significantly on the outcome . The classical repeated motif peptoids introduced by Patch and Barron back in 2003 are one of the most highly studied antimicrobial peptoids and has naturally also been studied for its anti‐biofilm properties.…”

Methods Used for the Eradication of Staphylococcal Biofilms

“…For example, Traba et al used LIVE/DEAD staining for susceptibility monitoring of a S. aureus biofilm to reactive discharge gases [101,102]. Haney et al used this method in their study to investigate the impact of media composition on staining patterns and the activity of antibiotics and antimicrobial peptides (AMPs) against Pseudomonas aeruginosa and S. aureus strains [103]. The CLSM imaging revealed that the MgSO 4 composition can affect the biofilm architecture of P. aeruginosa.…”

“…On the other hand, it should be emphasized that both fluorophores can be used separately, depending on the purpose of their application. For instance, in the study by Sonesson et al on staphylococcal enzymes (staphopains) and their proteolytic activity, the SYTO9 with atetramethylrhodamine (TAMRA)-labelled LL-37 peptide were used to determine the peptide binding to S. aureus cells [103]. Furthermore, LIVE/DEAD staining allowed for the investigation into how particular compounds may interact with a S. aureus biofilm.…”

Investigation on the effect of vitamin C on growth & biofilm-forming potential of Streptococcus mutans isolated from patients with dental caries

“…On clinical grounds, they are produced to allow the bacteria to survive in response to stresses in the form of antibiotics, nutrient limitation, changes in environmental temperature or oxygen, and others [7]. Bio lms have gained much clinical relevance not only in dentistry but also in medical practice, as they are thought to be responsible for up to two-thirds of hospital-acquired infections [8] and to be the source of many persistent and chronic infections [9]. The bio lm mode confers an antibiotic resistance that can be associated with di cult-to-eradicate infections such as those of medical devices [10,11].…”