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Evaluation of ultra-rapid susceptibility testing of ceftolozane-tazobactam by a flow cytometry assay directly from positive blood cultures

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Abstract

The urgent need for rapid antimicrobial susceptibility is broadly apparent from government reports to the lay press. Accordingly, we developed a flow-cytometry assay (FCM) for evaluating ceftolozane-tazobactam (C/T) susceptibility directly on blood cultures (BC) requiring < 2 h from flag positivity to report. The protocol was optimized with C/T-susceptible and C/T-resistant gram-negative bacilli inoculated in BC aerobic bottles (Becton-Dickinson, USA), and afterward optimized for different C/T concentrations (1/4, 2/4, 4/4, and 8/4 mg/L) for 1 h incubation (37 °C), followed by FCM and software analysis. Fluorescent membrane permeability and membrane potential dyes were comparatively used to detect early cell lesions using the CytoFLEX cytometer (Beckman-Coulter, USA). Repeatability, reproducibility, and stability of the assay up to 48 h after BC positivity were determined. Internal validation was performed in spiked BC bottles with 130 Enterobacterales and 32 Pseudomonas aeruginosa isolates from Porto University (Portugal), including 13 ATCC isolates. Additionally, 64 gram-negative bacilli recovered from positive BC at Ramon y Cajal Hospital (Madrid, Spain) were tested. Categorical agreement (CA) and analytical errors were calculated comparing FCM with broth microdilution results. Only the membrane potential dyes clearly distinguished CT-susceptible and CT-resistant isolates. Excellent repeatability, reproducibility, and inter-method concordance was observed. Overall, CA was 99.1% using EUCAST criteria with 2 major errors and 98.7% with CLSI criteria with 2 major and 1 minor errors. A new, accurate, and ultra-rapid FCM (< 2 h) for testing C/T susceptibility gave accurate results and would expand current FCM antimicrobial susceptibility assay.

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Acknowledgments

We thank Merck Sharp & Dohme, LDA, who kindly provided ceftolozane-tazobactam for these studies.

Funding

This work received funding from H2020 FTIPilot 2016 project no. 730713 “FAST-bact A novel fast and automated test for antibiotic susceptibility testing for Gram positive and negative bacteria.”

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Authors and Affiliations

  1. FASTinov, S.A., Matosinhos, Portugal

    Inês Martins-Oliveira, Ana Silva-Dias, Rosário Gomes, Acácio G Rodrigues & Cidalia Pina-Vaz

  2. Division of Microbiology, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal

    Inês Martins-Oliveira, Sofia Quintas, Acácio G Rodrigues & Cidalia Pina-Vaz

  3. Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain

    Blanca Pérez-Viso & Rafael Cantón

  4. Faculty of Medicine, University of Porto, CINTESIS - Center for Health Technology and Services Research, Porto, Portugal

    Ana Silva-Dias, Acácio G Rodrigues & Cidalia Pina-Vaz

Authors
  1. Inês Martins-Oliveira
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  2. Blanca Pérez-Viso
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  3. Sofia Quintas
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  4. Ana Silva-Dias
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  5. Rosário Gomes
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  6. Acácio G Rodrigues
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  7. Rafael Cantón
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  8. Cidalia Pina-Vaz
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Corresponding author

Correspondence to Cidalia Pina-Vaz.

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Conflict of interest

Rafael Cantón has participated in educational programs sponsored by MSD. FASTinov is a spin-off of Porto University founded by some of the authors.

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The study was approved by the ethical committee of this center (Ref. 161-17).

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Martins-Oliveira, I., Pérez-Viso, B., Quintas, S. et al. Evaluation of ultra-rapid susceptibility testing of ceftolozane-tazobactam by a flow cytometry assay directly from positive blood cultures. Eur J Clin Microbiol Infect Dis 39, 1907–1914 (2020). https://doi.org/10.1007/s10096-020-03926-4

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  • DOI: https://doi.org/10.1007/s10096-020-03926-4

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