Multiparameter Flow Cytometric Analysis of Antibiotic Effects on Membrane Potential, Membrane Permeability, and Bacterial Counts of Staphylococcus aureus and Micrococcus luteus

Abstract

Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts.Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated Staphylococcus aureus and Micrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC 2 (3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC 2 (3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min. At concentrations of <1 g/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 g/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose-and time-dependent decreases in MP. Tetracycline concentrations of <1 g/ml did not change permeability, while a tetracycline concentration of 4 g/ml permeabilized 50% of the bacteria; 4 g of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially, with no effect on permeability; chloramphenicol did not change either permeability or MP. Erythromycin pretreatment of bacteria prevented streptomycin and amoxicillin effects. Flow cytometry provides a sensitive means of monitoring the dynamic cellular events that occur in bacteria exposed to antibacterial agents; however, it is probably simplistic to expect that changes in a single cellular parameter will suffice to determine the sensitivities of all species to all drugs.

Anti-methicillin Resistant Staphylococcus aureus Compound Isolation from Halophilic Bacillus amyloliquefaciens MHB1 and Determination of Its Mode of Action Using Electron Microscope and Flow Cytometry Analysis

“…Many investigators have used this method to study the interaction between antibacterial agents and bacteria. The cell viability of antibiotics (amoxicillin, tetracycline and erythromycin) treated S. aureus and Micrococcus luteus were studied by Novo et al [24] using flow cytometric analysis. In the above analysis, the fluorescent probe TO-PRO Ò -3 was used for binding to the damaged cells.…”

“…The cell viability of MRSA treated purified compound was determined by using BD FACScalibur flow cytometry system (BD Biosciences, USA) [24]. The bacterial suspension of 10 7 CFU/mL was treated with 49 MIC of compound for 30 min,1 h and 2 h. For negative control, the untreated (live) bacterial suspension was used.…”

A defensin from clam Venerupis philippinarum: Molecular characterization, localization, antibacterial activity, and mechanism of action

“…TO-PRO-3 is generally excluded by prokaryotic and eukaryotic cells with intact cytoplasmic membranes. Therefore, the ratio of TO-PRO-3 fluorescence to DiOC2(3) green fluorescence produces a normalized indicator of membrane permeability (Novo et al, 2000).…”

“…PBS was used as negative control and the pore-forming lantibiotic nisin Z (1.5 μg/ml corresponds to 1 × the MIC, Sigma-Aldrich) was used as positive control. Flow cytometric studies were performed according to the method described by Novo et al (2000).…”

Viability and physiological state transitions of Rhizopus oligosporus sporangiospores in tempe starter culture

“…This technique has demonstrated its potential as a means to assess the physiological state of damaged cells (Ueckert et al 1997). FCM has been successfully used in several studies to assess the viability of microbial cells in probiotic products and dairy starters (Bunthof and Abee 2002), starved cultures Kaprelyants et al 1996), cells having been exposed to antibiotics (Novo et al 2000) or high hydrostatic pressure (Ritz et al 2001).…”