Rapid flow cytometric bacterial detection and determination of susceptibility to amikacin in body fluids and exudates

Abstract

A flow cytometry-based method for rapid and quantitative detection of bacteria in various clinical specimens and for rapid determination of antibiotic effect is described. Achieving such a measurement with high sensitivity required discrimination between bacteria and other particles which were often present in clinical samples in high concentrations. This discrimination was facilitated by detecting the bacterial characteristic light scatter and fluorescence signals following staining, e.g., with the fluorescent nucleic acid-binding dye ethidium bromide, as well as by measuring bacterial proliferation during short time intervals. Antibiotic susceptibility was measured by observing the inhibition of such proliferation. The method was applied to 43 clinical specimens from various sources, such as wound exudates, bile, serous cavity fluids, and bronchial lavage. Bacterial detection, achieved in less than 2 h, agreed with results of conventional methods with a sensitivity of 74% and a specificity of 88%. Susceptibility to amikacin was detected in l h in 92% of 13 positive specimens.

Antimicrobial susceptibility testing in 90 min by bacterial cell count monitoring

“…Nevertheless, these papers support our idea that bacterial cell count monitoring can be used as a very fast procedure to reliably predict antimicrobial susceptibility. The application of cell count monitoring may even go further as Cohen et al [14] showed in 1989 that flow cytometry could reliably predict the presence of antimicrobial susceptibility for amikacin directly in clinical samples containing polyflora. They showed that amikacin susceptibility was reliably detected within 1 h in 92% of 13 clinical samples.…”

Current and future applications of flow cytometry in aquatic microbiology

“…In many cases this function cannot be detected due to irreversible cell damage or fastidious growth conditions. This type of assay can be used to demonstrate antibiotic susceptibility within 2 h from sampling [79]. Another way to detect replication is by cell tracking, where covalently labelled cells halve their £uorescence upon cell division [80].…”

“…Dye extrusion can be overcome either by using dyes that cross-link inside the cell [79] or by using appropriate metabolic inhibitors. Thus, staining procedures such as the Chemchrome kit by Chemunex (Maisons-Alford, France) can achieve a good staining for a large spectrum of pathogens [99].…”

“…Physiological or taxonomic probes are often used in double staining procedures and include a nucleic acid dye for the quanti¢cation of total bacteria. With antibody £uorescence for example, it is essential to ensure that only antibody £uorescent events associated with nucleic acid are analysed [79,129,130].…”

Flow cytometry for rapid assessment of viability after exposure to a quaternary ammonium compound

“…Flow cytometry has been used as a rapid method for testing Diaper and Edwards 1994;Porter et N/. 1995), investigating dormancy and stanation (Kaprelyants and Kell 1993 ;Diaper and Edwards 1994), testing susceptibility to antibiotics (Martinez et al 1982;Steen et al 1982; Cohen and Sahar 1989;Gant et a/. 1993;Mason et al 1994) and for studjing the effect of heat, sonication and electroporation on bacterial vitality (Lopez-Amoros et al.…”