Evaluation of rapid colistin susceptibility directly from positive blood cultures using a flow cytometry assay

Silva, Daniela Fonseca e; Silva-Dias, Ana; Gomes, Rui; Martins-Oliveira, Inês; Ramos, Maria da Conceição Pereira; Rodrigues, Acácio G.; Cantón, Rafael; Pina-Vaz, Cidália

doi:10.1016/j.ijantimicag.2019.08.016

Cited by 27 publications

(16 citation statements)

References 10 publications

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“…FCM-based AST methods have been described previously, most recently by Fonseca and colleagues, who presented a patented method for colistin AST. 13 , 28 In contrast to that assay, our method utilizes standard laboratory equipment and widely available reagents. Even though this evaluation was performed using an acoustic flow cytometer, the assay could theoretically be run on other flow cytometers with sufficient resolution for small particle detection.…”

Section: Discussionmentioning

confidence: 99%

Rapid high-resolution detection of colistin resistance in Gram-negative bacteria using flow cytometry: a comparison with broth microdilution, a commercial screening test and WGS

Ekelund

Hetland

Löhr

et al. 2021

Journal of Antimicrobial Chemotherapy

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Background Even though both EUCAST and CLSI consider broth microdilution (BMD) as the reference method for antimicrobial susceptibility testing (AST) of colistin, the method exhibits potential flaws related to properties of the colistin molecule. Objectives To develop a flow cytometry method (FCM) for colistin AST and to validate it against BMD, a commercial screening test and WGS. Methods Colistin-mediated loss of membrane integrity in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. was detected with the fluorescent probe YoPro-1 by FCM. An international collection of 65 resistant and 109 susceptible isolates were analysed and the colistin concentration required to reach the EC50 was compared with the BMD MIC and the presence of genotypic resistance markers. Results The overall FCM sensitivity and specificity for colistin resistance was 89% and 94%, with E. coli > K. pneumoniae > P. aeruginosa, whereas the performance for Acinetobacter spp. was poor. All tested E. coli were correctly categorized. Three K. pneumoniae isolates with genotypic findings consistent with colistin resistance were detected by FCM but not BMD. Compared with BMD, FCM delivered AST results with a 75% reduction of time. Conclusions Here, we present a rapid FCM-based AST assay for qualitative and quantitative testing of colistin resistance in E. coli and K. pneumoniae. The assay revealed probable chromosomal colistin resistance in K. pneumoniae that was not detected by BMD. If confirmed, these results question the reliability of BMD for colistin testing.

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Section: Discussionmentioning

confidence: 99%

Rapid high-resolution detection of colistin resistance in Gram-negative bacteria using flow cytometry: a comparison with broth microdilution, a commercial screening test and WGS

Ekelund

Hetland

Löhr

et al. 2021

Journal of Antimicrobial Chemotherapy

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“…Innovative and rapid AST systems have been described, which can be divided in genomic, proteomic and phenotypic methods [13]. We previously described a qualitative AST based on flow cytometry for main antibiotics including colistin directly from positive blood cultures [14] with excellent results [11]. The assay described here is quantitative and qualitative, simple to perform, accurate and provide a rapid AST report directly from colonies.…”

Section: Discussionmentioning

confidence: 99%

Ultra-rapid flow cytometry assay for colistin MIC determination in Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii

Silva

Andrade

Gomes³

et al. 2020

Clinical Microbiology and Infection

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Objectives: Both EUCAST and CLSI recommend broth microdilution for antimicrobial susceptibility testing of colistin, but this method is cumbersome and takes 16-24 h to give results. Our objective was to evaluate a rapid quantitative colistin MIC susceptibility assay based on flow cytometry analysis (FASTcolistin MIC) in comparison with standard broth microdilution assay. Methods: One hundred and sixteen Gram-negative bacilli (78 Enterobacterales, 28 Pseudomonas aeruginosa and 10 Acinetobacter baumannii) were studied in parallel using standard broth microdilution following EUCAST recommendations and FASTcolistin MIC kit. In the last one, a bacteria suspension (0.5 MacFarland) was prepared, diluted in Muller-Hinton broth, incubated in the susceptibility panel containing different colistin concentrations (range 0.125-64 mg/L) with a fluorescent probe and incubated 1 h at 35ºC. After that, a flow cytometry analysis using CytoFLEX (Beckmam) was performed. Using a dedicated software (BioFAST) an automated MIC result was obtained after 1.5 h. Performance evaluation was performed according to the ISO standard 20776-2. Reproducibility and repeatability, categorical (CA) and essential agreement (EA), and lot-to-lot variation and operator-to-operator variability, as well as time to results were determined. Results: Overall, 100% CA (CI 97-100%) and 95.7% EA (CI 90-98%) was obtained with high repeatability (100%; CI 80-100%)and reproducibility (97%; (CI 83-99%)). Absence of lot-to-lot variations or differences in the operators' performance was observed. Conclusions: FASTcolistin MIC is an accurate, reliable and ultra-rapid method (1 h incubation versus 24 h) for susceptibility testing of colistin of common Gram-negative bacilli recovered in clinical laboratories.

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“…By observing the growth of fewer bacteria, we enter the "microscopic" regime, whereby changes in individual bacteria can be detected. A number of techniques, such as flow cytometry [114,116], forward laser light scattering [26,109] and nanostructureenhanced methods [118,121] can provide such microscopic information and allow us to reduce the assessment of susceptibility from 24-48 h down to 2-6 h (see Table 3), simply because they are able to observe a small number of bacteria rather than large colonies.…”

Section: Discussionmentioning

confidence: 99%

“…In particular, it ignores the issue of heteroresistance and the presence of persister cells [10,12]. Flow cytometry [114,116] and droplet microfluidics [131] are already able to pick up heterogeneity, but only at the microscopic scale. We therefore see exciting opportunities for techniques such as SPRi [51] or GMR imaging [32,79] to add their nanoscale sensing ability to this problem.…”

Section: Discussionmentioning

confidence: 99%

“…Critically, the bacterial suspension is hydrodynamically focussed through a nozzle, so that individual cells pass through the laser beam, as illustrated in the schematic in Figure 5 integrity of the bacterial membrane and its functionality (such as the membrane potential) as well as bacterial viability. Thanks to this richness of information, flow cytometry has been successfully used for AST, both with and without fluorescent tagging, with time to result of 1.5-8 h [114][115][116].…”

Section: Flow Cytometrymentioning

confidence: 99%

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Nanophotonics for bacterial detection and antimicrobial susceptibility testing

2020

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Photonic biosensors are a major topic of research that continues to make exciting advances. Technology has now improved sufficiently for photonics to enter the realm of microbiology and to allow for the detection of individual bacteria. Here, we discuss the different nanophotonic modalities used in this context and highlight the opportunities they offer for studying bacteria. We critically review examples from the recent literature, starting with an overview of photonic devices for the detection of bacteria, followed by a specific analysis of photonic antimicrobial susceptibility tests. We show that the intrinsic advantage of matching the optical probed volume to that of a single, or a few, bacterial cell, affords improved sensitivity while providing additional insight into single-cell properties. We illustrate our argument by comparing traditional culture-based methods, which we term macroscopic, to microscopic free-space optics and nanoscopic guided-wave optics techniques. Particular attention is devoted to this last class by discussing structures such as photonic crystal cavities, plasmonic nanostructures and interferometric configurations. These structures and associated measurement modalities are assessed in terms of limit of detection, response time and ease of implementation. Existing challenges and issues yet to be addressed will be examined and critically discussed.

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Evaluation of rapid colistin susceptibility directly from positive blood cultures using a flow cytometry assay

Cited by 27 publications

References 10 publications

Rapid high-resolution detection of colistin resistance in Gram-negative bacteria using flow cytometry: a comparison with broth microdilution, a commercial screening test and WGS

Rapid high-resolution detection of colistin resistance in Gram-negative bacteria using flow cytometry: a comparison with broth microdilution, a commercial screening test and WGS

Ultra-rapid flow cytometry assay for colistin MIC determination in Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii

Nanophotonics for bacterial detection and antimicrobial susceptibility testing

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