Optimization of a High-Throughput 384-Well Plate-Based Screening Platform with Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 15442 Biofilms

Abstract

In recent years, bacterial infections have become a main concern following the spread of antimicrobial resistance. In addition, bacterial biofilms are known for their high tolerance to antimicrobials and they are regarded as a main cause of recalcitrant infections in humans. Many efforts have been deployed in order to find new antibacterial therapeutic options and the high-throughput screening (HTS) of large libraries of compounds is one of the utilized strategies. However, HTS efforts for anti-biofilm discovery remain uncommon. Here, we miniaturized a 96-well plate (96WP) screening platform, into a 384-well plate (384WP) format, based on a sequential viability and biomass measurements for the assessment of anti-biofilm activity. During the assay optimization process, different parameters were evaluated while using Staphylococcus aureus and Pseudomonas aeruginosa as the bacterial models. We compared the performance of the optimized 384WP platform to our previously established 96WP-based platform by carrying out a pilot screening of 100 compounds, followed by the screening of a library of 2000 compounds to identify new repurposed anti-biofilm agents. Our results show that the optimized 384WP platform is well-suited for screening purposes, allowing for the rapid screening of a higher number of compounds in a run in a reliable manner.

Coupling Additive Manufacturing with Hot Melt Extrusion Technologies to Validate a Ventilator-Associated Pneumonia Mouse Model

“…S. aureus is the most common cause of VAP among Gram-positive bacteria [ 7 , 43 ] and the PRINT-AID consortium has identified antibiofilm leads against S. aureus that could be incorporated into endotracheal tubes [ 44 ]. The challenge to establish a VAP mouse model lies not only in the technical, but also in the biological aspects.…”

Screening of FDA-Approved Drugs Using a 384-Well Plate-Based Biofilm Platform: The Case of Fingolimod

“…For the compound library screening, resazurin staining was performed in 384-well plates according to the protocol previously optimized in our group [ 21 ] with following modifications. Briefly, the planktonic solution was first transferred into a new 384-well plate and the optical density (OD) of the planktonic solution was measured using a Varioskan LUX Multimode microplate reader (Thermo Scientific, Vantaa, Finland).…”

“…In the present study, we screened a collection of FDA-approved drugs using a previously optimized 384-well plate-based platform that allows screening the effect of the compounds on both the viability and biomass of the biofilm [ 21 ]. The screening was performed against Staphylococcus aureus , an important pathogen responsible for a large variety of healthcare-associated infections such as device-related infections, wound infections and sepsis [ 22 ].…”

Strategies and Approaches for Discovery of Small Molecule Disruptors of Biofilm Physiology

“…A widely utilized method for unbiased screening is biofilm staining, using crystal violet, to measure biofilm biomass [ 111 , 112 ]. In one such example, a library of 9831 marine microbial-derived extracts were screened to identify compounds that were able to inhibit A. baumannii biofilm formation.…”