Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification

Abstract

Colony forming unit (CFU) determination by agar plating is still regarded as the gold standard for biofilm quantification despite being time- and resource-consuming. Here, we propose an adaption of the high-throughput Start-Growth-Time (SGT) method from planktonic to biofilm analysis, which indirectly quantifies CFU/mL numbers by evaluating regrowth curves of detached biofilms. For validation, the effect of dalbavancin, rifampicin and gentamicin against mature biofilms of Staphylococcus aureus and Enterococcus faecium was measured by accessing different features of the viability status of the cell, i.e., the cultivability (conventional agar plating), growth behavior (SGT) and metabolic activity (resazurin assay). SGT correlated well with the resazurin assay for all tested antibiotics, but only for gentamicin and rifampicin with conventional agar plating. Dalbavancin treatment-derived growth curves showed a compared to untreated controls significantly slower increase with reduced cell doubling times and reduced metabolic rate, but no change in CFU numbers was observed by conventional agar plating. Here, unspecific binding of dalbavancin to the biofilm interfered with the SGT methodology since the renewed release of dalbavancin during detachment of the biofilms led to an unintended antimicrobial effect. The application of the SGT method for anti-biofilm testing is therefore not suited for antibiotics which stick to the biofilm and/or to the bacterial cell wall. Importantly, the same applies for the well-established resazurin method for anti-biofilm testing. However, for antibiotics which do not bind to the biofilm as seen for gentamicin and rifampicin, the SGT method presents a much less labor-intensive method suited for high-throughput screening of anti-biofilm compounds.

Structure-Bioactivity Relationship of the Functionalized Polysulfone with Triethylphosphonium Pendant Groups: Perspective for Biomedical Applications

“…At the set times, 1 mL from the test tubes, in which there is polysulfone suspended in the microbial suspensions, was taken and distributed in sterile Petri plates, on top of which was added culture medium Mueller Hinton Agar (standardized bacteriological growth medium [ 35 ], Oxoid) melted and cooled at 45 °C, so that the microbial cells were not destroyed. After solidification, the Petri plates were incubated at 37 °C for 24 h, after which the microbial load was examined and assessed by counting the colony-forming unit (CFU/mL, standard quantification method used to determine the number of viable cells [ 36 ]). The control is represented by the Petri dish in which Mueller-Hinton agar and 1 mL of microbial suspension (1.5 × 10 8 CFU) were distributed.…”

High Throughput Viability Assay for Microbiology

“…The colony forming unit (CFU) assay is the gold standard for enumerating viable cells in microbiology labs across the world [1][2][3][4][5][6]. The CFU assay combines simplicity with readily available reagents to achieve an enormous dynamic range, commonly measuring between 1 and 100,000,000 viable cells in a sample.…”

“…However, measuring viability across numerous conditions using the CFU assay is time-and resource-intensive while generating a significant amount of plastic waste [4,12].…”

“…Viability measurements are critical in numerous contexts spanning food safety [7], functional genomics [8–10], and drug combination discovery against persister cells [2, 11]. However, measuring viability across numerous conditions using the CFU assay is time- and resource-intensive while generating a significant amount of plastic waste [4, 12].…”

“…Prior approaches to increase the scale of viability measurements included 1) increasing the speed with robotic liquid handling and imaging [1, 4, 13]; 2) decreasing the amount of pipetting by using viability stains [14] or microfluidics [15]; or 3) using cell growth to estimate the initial number of viable cells post-treatment [3]. The most commercially successful alternative to the CFU assay is the Spiral Plater method [16] which deposits the sample in an Archimedes spiral on a solid medium plate.…”