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Archives of microbiology
Jul 05, 2024;206 (8): 344.

High-throughput combination assay for studying biofilm formation of uropathogenic Escherichia coli.

M Li, C D Cruz, P Ilina, P Tammela
Author Information
  1. M Li: Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, Helsinki, FI-00014, Finland. ORCID
  2. C D Cruz: Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, Helsinki, FI-00014, Finland. ORCID
  3. P Ilina: Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, Helsinki, FI-00014, Finland. ORCID
  4. P Tammela: Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, Helsinki, FI-00014, Finland. paivi.tammela@helsinki.fi. ORCID
PMID: 38967798 DOI: 10.1007/s00203-024-04029-w

Abstract

Uropathogenic Escherichia coli, the most common cause for urinary tract infections, forms biofilm enhancing its antibiotic resistance. To assess the effects of compounds on biofilm formation of uropathogenic Escherichia coli UMN026 strain, a high-throughput combination assay using resazurin followed by crystal violet staining was optimized for 384-well microplate. Optimized assay parameters included, for example, resazurin and crystal violet concentrations, and incubation time for readouts. For the assay validation, quality parameters Z' factor, coefficient of variation, signal-to-noise, and signal-to-background were calculated. Microplate uniformity, signal variability, edge well effects, and fold shift were also assessed. Finally, a screening with known antibacterial compounds was conducted to evaluate the assay performance. The best conditions found were achieved by using 12 µg/mL resazurin for 150 min and 0.023% crystal violet. This assay was able to detect compounds displaying antibiofilm activity against UMN026 strain at sub-inhibitory concentrations, in terms of metabolic activity and/or biomass.

Keywords

Assay optimization Biofilm Crystal violet Resazurin Screening UPEC

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MeSH Term

Biofilms
Uropathogenic Escherichia coli
High-Throughput Screening Assays
Xanthenes
Anti-Bacterial Agents
Gentian Violet
Oxazines
Microbial Sensitivity Tests
Urinary Tract Infections
Humans

Chemicals

Xanthenes
resazurin
Anti-Bacterial Agents
Gentian Violet
Oxazines
Journal Article

Word Cloud

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