Rapid microbial viability assay using scanning electron microscopy: a proof-of-concept using Phosphotungstic acid staining

Zmerli, Omar; Bellali, Sara; Haddad, Gabriel; Hisada, Akiko; Ominami, Yusuke; Raoult, Didier; Khalil, Jacques Bou

doi:10.1016/j.csbj.2023.07.010

Cited by 4 publications

(1 citation statement)

References 49 publications

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“…In the past two decades, a number of other methods had been developed for differentiating dead bacteria from live/dead cell mixtures, including electron microscopy, − real-time fluorescent quantitative PCR (qPCR), surface-enhanced Raman scattering, aggregation-induced emission (AIE), and fluorescent techniques combined with fluorescence spectra, , optical microscopy, − or flow cytometry (FCM). , However, none of these techniques meets the requirements of both high sensitivity and fast detection. For instance, fluorescence spectra can hardly determine the accurate death rate of bacteria that are treated by antibiotics .…”

Section: Introductionmentioning

confidence: 99%

Rapid and Sensitive Quantification of Bacterial Viability Using Ratiometric Fluorescence Sensing

He,

Chen,

Wang

et al. 2024

Anal. Chem.

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Section: Introductionmentioning

confidence: 99%

Rapid and Sensitive Quantification of Bacterial Viability Using Ratiometric Fluorescence Sensing

He,

Chen,

Wang

et al. 2024

Anal. Chem.

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Detoxification of ars genotypes by arsenite-oxidizing bacteria through arsenic biotransformation

Chang,

Kim,

Lee

2024

Environ Geochem Health

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Antimicrobial Susceptibility Testing for Colistin: Extended Application of Novel Quantitative and Morphologic Assay Using Scanning Electron Microscopy

Zmerli,

Bellali,

Haddad

et al. 2024

International Journal of Microbiology

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Background. Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram-negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM-AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin. Methods. Forty-four Gram-negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼107 CFU/mL were exposed to Colistin at EUCAST-ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII-Tabletop-SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA-staining and morphologic changes. Results. We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard-based broth microdilution method. Conclusions. We provide an extended application of the proof of concept for the utilization of the SEM-AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.

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Rapid microbial viability assay using scanning electron microscopy: a proof-of-concept using Phosphotungstic acid staining

Cited by 4 publications

References 49 publications

Rapid and Sensitive Quantification of Bacterial Viability Using Ratiometric Fluorescence Sensing

Rapid and Sensitive Quantification of Bacterial Viability Using Ratiometric Fluorescence Sensing

Detoxification of ars genotypes by arsenite-oxidizing bacteria through arsenic biotransformation

Antimicrobial Susceptibility Testing for Colistin: Extended Application of Novel Quantitative and Morphologic Assay Using Scanning Electron Microscopy

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