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Quantifying variation in bacterial reproductive fitness: A high-throughput method

Lookup NU author(s): Dr Conor LawlessORCiD



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Copyright © 2021 Frey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. To evaluate changes in reproductive fitness of bacteria, e.g., after acquisition of antimicrobial resistance, a low-cost high-throughput method to analyze bacterial growth on agar is desirable for broad usability. In our bacterial quantitative fitness analysis (BaQFA), arrayed cultures are spotted on agar and photographed sequentially while growing. These time-lapse images are analyzed using a purpose-built open-source software to derive normalized image intensity (NI) values for each culture spot. Subsequently, a Gompertz growth model is fitted to NI values, and fitness is calculated from model parameters. To represent a range of clinically important pathogenic bacteria, we used different strains of Enterococcus faecium, Escherichia coli, and Staphylococcus aureus, with and without antimicrobial resistance. Relative competitive fitness (RCF) was defined as the mean fitness ratio of two strains growing competitively on one plate. BaQFA permitted the accurate construction of growth curves from bacteria grown on semisolid agar plates and fitting of Gompertz models. Normalized image intensity values showed a strong association with the total CFU/ml count per spotted culture (P, 0.001) for all strains of the three species. BaQFA showed relevant reproductive fitness differences between individual strains, suggesting substantially higher fitness of methicillin-resistant S. aureus JE2 than Cowan (RCF, 1.58; P, 0.001). Similarly, the vancomycin-resistant E. faecium ST172b showed higher competitive fitness than susceptible E. faecium ST172 (RCF, 1.59; P, 0.001). Our BaQFA method allows detection of fitness differences between bacterial strains and may help to estimate epidemiological antimicrobial persistence or contribute to the prediction of clinical outcomes in severe infections. IMPORTANCE Reproductive fitness of bacteria is a major factor in the evolution and persistence of antimicrobial resistance and may play an important role in severe infections. With a computational approach to quantify fitness in bacteria growing competitively on agar plates, our high-throughput method has been designed to obtain additional phenotypic data for antimicrobial resistance analysis at a low cost. Furthermore, our bacterial quantitative fitness analysis (BaQFA) enables the investigation of a link between bacterial fitness and clinical outcomes in severe invasive bacterial infections. This may allow future use of our method for patient management and risk stratification of clinical outcomes. Our proposed method uses open-source software and a hardware setup that can utilize consumer electronics. This will enable a wider community of researchers, including those from low-resource countries, where the burden of antimicrobial resistance is highest, to obtain valuable information about emerging bacterial strains.

Publication metadata

Author(s): Frey PM, Baer J, Bergada-Pijuan J, Lawless C, Buhler PK, Kouyos RD, Lemon KP, Zinkernagel AS, Brugger SD

Publication type: Article

Publication status: Published

Journal: mSystems

Year: 2021

Volume: 6

Issue: 1

Online publication date: 02/02/2021

Acceptance date: 11/01/2021

Date deposited: 08/04/2021

ISSN (electronic): 2379-5077

Publisher: American Society for Microbiology


DOI: 10.1128/MSYSTEMS.01323-20


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Funder referenceFunder name
R01 GM117174