Implementation of Microfluidics for Antimicrobial Susceptibility Assays: Issues and Optimization Requirements

Abstract

Despite the continuous emergence of multi-drug resistant pathogens, the number of new antimicrobials reaching the market is critically low. Natural product peptides are a rich source of bioactive compounds, and advances in mass spectrometry have achieved unprecedented capabilities for the discovery and characterization of novel molecular species. However, traditional bioactivity assay formats hinder the discovery and biochemical characterization of natural product antimicrobial peptides (AMPs), necessitating large sample quantities and significant optimization of experimental parameters to achieve accurate/consistent activity measurements. Microfluidic devices offer a promising alternative to bulk assay systems. Herein, a microfluidics-based bioassay was compared to the traditional 96-well plate format in respective commercially-available hardware. Bioactivity in each assay type was compared using a Viola inconspicua peptide library screened against E. coli ATCC 25922. Brightfield microcopy was used to determine bioactivity in microfluidic channels while both common optical and fluorescence-based measurements of cell viability were critically assessed in plate-based assays. Exhibiting some variation in optical density and fluorescence-based measurements, all plate-based assays conferred bioactivity in late eluting V. inconspicua library fractions. However, significant differences in the bioactivity profiles of plate-based and microfluidic assays were found, and may be derived from the materials comprising each assay device or the growth/assay conditions utilized in each format. While new technologies are necessary to overcome the limitations of traditional bioactivity assays, we demonstrate that off-the-shelf implementation of microfluidic devices is non-trivial and significant method development/optimization is required before conventional use can be realized for sensitive and rapid detection of AMPs in natural product matrices.

Challenges to antimicrobial susceptibility testing of plant-derived polyphenolic compounds

“…Recently, Parsley et al ( 83 ) found significant differences in the bioactivity profiles of natural product peptides against E. coli between a microfluidic assay and a plate-based method. They suggested that the differences were owed to different bacterial growth conditions and different plastic materials involved in each assay and pointed out that microfluidic-based methods required significant optimisation to be reliably applied for AST.…”

The Present and Future Role of Microfluidics for Protein and Peptide-Based Therapeutics and Diagnostics

“…However, when compared to the traditional format of measuring via 96-well plate analysis, the results obtained were dissimilar, owing to differing oxygenation levels during incubation subjected to each system. The 96-well plate was exposed to a greater level of oxygen than the MFs assays, causing incomparability of the systems and hence the results of this study were inconclusive [57]. However, there is strong potential for future research in this area to explore different approaches for obtaining AMP activity using MFs.…”

“…Owing to increased pathogen drug resistance, the production of novel antimicrobials is limited, which is why a new area of antimicrobials in the form of peptide therapeutics should be explored. Despite the categorization of over 3000 AMPs, currently, only 7 antimicrobial peptides (AMPs) are approved for clinical use by the FDA [57], owing mainly to a lack of clinical efficacy. Bioassays performed using an MF design allow detecting antimicrobial activity.…”

Microfluidics for antibiotic susceptibility testing

“…A simple 8-channel device was used to compare the antimicrobial activity of a library of peptides, showing difference to the activity of same compounds when tested in a well plate, and suggesting that the choice of materials for a microfluidic device is critical. 99 A separate approach in which the resistance or susceptibility was determined by counting the number of dead bacterial cells was shown in a simple microchannel device. 100 2.3.2.…”