Distinguishing between metabolically active and dormant bacteria on paper

Area of Science:

• Microbiology
• Biotechnology
• Analytical Chemistry

Background:

• Bacteria exist in metabolically active and dormant states, impacting environmental and industrial settings.
• Rapid shifts in bacterial metabolic states can lead to detrimental outcomes, such as pathogen proliferation or industrial process disruption.
• Understanding bacterial metabolic states is crucial for managing risks associated with pathogens and optimizing industrial processes.

Purpose of the Study:

• To develop rapid, cost-effective, paper-based colorimetric assays for distinguishing between metabolically active and dormant bacteria.
• To assess the applicability of these assays for common bacteria such as Escherichia coli and Salmonella Typhimurium.
• To enable quick determination of the ratio of dormant to metabolically active bacteria in various settings.

Main Methods:

• Development of two distinct paper-based colorimetric assays utilizing enzymatic reactions.
• Assay 1: Detection of oxidoreductase activity in active bacteria using iodophenyl-nitrophenyl-phenyl tetrazolium salt (INT) and methylphenazinium methyl sulfate.
• Assay 2: Detection of alkaline phosphatase activity in dormant bacteria using INT and para-nitrophenyl phosphate salt.
• Incorporation of bacteriophage immobilization (P22 for Salmonella, T4 for E. coli) for specific bacterial capture.

Main Results:

• Colorimetric changes observed within 60 minutes, visible at 10^3 CFU and quantifiable at 10^6 CFU.
• Assays successfully detected both Escherichia coli and Salmonella Typhimurium due to common bacterial enzymes.
• Specific bacterial capture enhanced detection limits to 10^2 CFU for Salmonella and 10^4 CFU for E. coli.

Conclusions:

• The developed paper-based assays provide a rapid and low-cost method for determining bacterial metabolic states.
• The methodology is versatile for various applications in environmental monitoring, industrial process control, and healthcare.
• Specific capture using bacteriophages allows for targeted detection of individual bacterial species, addressing limitations of broad-spectrum enzyme detection.