Antibiotic Susceptibility Testing of the Gram-Negative Bacteria Based on Flow Cytometry

Abstract

Rapidly treating infections with adequate antibiotics is of major importance. This requires a fast and accurate determination of the antibiotic susceptibility of bacterial pathogens. The most frequently used methods are slow because they are based on the measurement of growth inhibition. Faster methods, such as PCR-based detection of determinants of antibiotic resistance, do not always provide relevant information on susceptibility, particularly that which is not genetically based. Consequently, new methods, such as the detection of changes in bacterial physiology caused by antibiotics using flow cytometry and fluorescent viability markers, are being explored. In this study, we assessed whether Alexa Fluor® 633 Hydrazide (AFH), which targets carbonyl groups, can be used for antibiotic susceptibility testing. Carbonylation of cellular macromolecules, which increases in antibiotic-treated cells, is a particularly appropriate to assess for this purpose because it is irreversible. We tested the susceptibility of clinical isolates of Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, to antibiotics from the three classes: β-lactams, aminoglycosides, and fluoroquinolones. In addition to AFH, we used TO-PRO®-3, which enters cells with damaged membranes and binds to DNA, and DiBAC4 (3), which enters cells with depolarized membranes. We also monitored antibiotic-induced morphological alterations of bacterial cells by analyzing light scattering signals. Although all tested dyes and light scattering signals allowed for the detection of antibiotic-sensitive cells, AFH proved to be the most suitable for the fast and reliable detection of antibiotic susceptibility.

Strong increase in the autofluorescence of cells signals struggle for survival

“…We also evaluated the viability of cells treated with tetracycline using the viability markers, Alexa Fluor™ 633 Hydrazide (AFH) which targets carbonyl groups, and TO-PRO™-3 which binds to DNA in cells with damaged membranes 26 , 27 . Both viability markers confirmed that tetracycline did not kill the cells (Fig.…”

A New Series of Pyrrole-Based Chalcones: Synthesis and Evaluation of Antimicrobial Activity, Cytotoxicity, and Genotoxicity

“…The cell suspensions were analysed in a BD Accuri C6 flow cytometer (Becton-Dickinson, Mansfield, MA, USA). The cell scattergram (forward scatter: FS, side scatter: SS), the autofluorescence (without fluorocrome), and the intensity of fluorescence at FL1 (green fluorescence, 525 nm) and FL2 (red fluorescence, 620 nm) were recorded using a logarithmic scale [ 32 , 33 ].…”

3D microfluidic gradient generator for combination antimicrobial susceptibility testing

“…Conventional MIC testing 7 (Supplementary Materials Fig. S1a) involves overnight incubation of a patient-collected bacterial sample in the presence of growth media and dilute antibiotic solutions 8 to determine the lowest dose of a single antibiotic required to inhibit the proliferation of bacteria and increase bacterial colony density, which is known as the MIC value 9,10 .…”

“…Conventional AST techniques, while well-established, generally require multiple independent manual laborintensive fluidic handling procedures, involving a minimum of~16-24 h for sample enrichment and dilution, followed by~24-72 h for complete AST analysis 8,21 . As a result, the duration from sample collection to delivery of definitive AST results in clinical settings can take anywhere from 2 days to 1 week 10 . A standard clinical procedure while a clinician awaits AST evaluation, therefore, is to prescribe a large dose of a broad-spectrum antibiotic to stop the infection from worsening, which often contributes to the emergence and propagation of AMR in the first place [22][23][24] .…”