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

• Published in: European journal of clinical microbiology & infectious diseases 2020-10, Vol.39 (10), p.1907-1914
• Main Authors: Martins-Oliveira, Inês, Pérez-Viso, Blanca, Quintas, Sofia, Silva-Dias, Ana, Gomes, Rosário, Rodrigues, Acácio G, Cantón, Rafael, Pina-Vaz, Cidalia
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - pharmacology; Anti-Bacterial Agents - therapeutic use; Antiinfectives and antibacterials; Antimicrobial agents; Assaying; Bacilli; Biomedical and Life Sciences; Biomedicine; Blood; Blood Culture; Bottles; Cephalosporins - pharmacology; Cephalosporins - therapeutic use; Dyes; Flow Cytometry; Fluorescence; Gram-negative bacilli; Gram-Negative Bacteria - drug effects; Gram-Negative Bacteria - isolation & purification; Gram-Negative Bacterial Infections - diagnosis; Gram-Negative Bacterial Infections - drug therapy; Gram-Negative Bacterial Infections - microbiology; Gram-Positive Bacteria - drug effects; Gram-Positive Bacteria - isolation & purification; Gram-Positive Bacterial Infections - diagnosis; Gram-Positive Bacterial Infections - drug therapy; Gram-Positive Bacterial Infections - microbiology; Humans; Internal Medicine; Medical Microbiology; Membrane permeability; Membrane potential; Membranes; Microbial Sensitivity Tests; Original Article; Portugal; Pseudomonas aeruginosa; Reproducibility; Spain; Susceptibility; Tazobactam; Tazobactam - pharmacology; Tazobactam - therapeutic use
• ISSN: 0934-9723; 1435-4373
• DOI: 10.1007/s10096-020-03926-4

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.