Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Jeff A. Shaw, Travis J. Bourret

Research output: Contribution to journalArticle

Abstract

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number147
DOIs
StatePublished - May 13 2019

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Salmonella
Chromosomes
Throughput
Bacterial Chromosomes
Polymerase Chain Reaction
Microorganisms
Genetic Markers
Base Pairing
Virulence
Research Design
Bacteria
Costs and Cost Analysis
DNA
Genetic engineering
Experiments
Design of experiments
Personnel
Costs
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

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