scholarly journals Rapid assessment of antibiotic effects on Escherichia coli by bis-(1,3-dibutylbarbituric acid) trimethine oxonol and flow cytometry.

1997 ◽  
Vol 41 (9) ◽  
pp. 2001-2005 ◽  
Author(s):  
R I Jepras ◽  
F E Paul ◽  
S C Pearson ◽  
M J Wilkinson
Keyword(s):  
Escherichia Coli ◽  
Flow Cytometry ◽  
Membrane Potential ◽  
Rapid Assessment ◽  
Physiological Changes ◽  
Analytical Technique ◽  
E Coli ◽  
Flow Cytometric ◽  
Bacterial Cell Viability ◽  
Sensitive Analytical Technique

The effects of selected antibiotics on Escherichia coli were studied by flow cytometry with the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. The actions of azithromycin, cefuroxime, and ciprofloxacin at five times the MIC on E. coli were compared by the traditional CFU assay and flow cytometry. Changes in viable counts of bacteria determined with DiBAC4(3) and by flow cytometry following treatment with the antibiotics showed trends similar to those found by the CFU assays. However, viable counts determined by flow cytometry following antibiotic treatment were 1 to 2 logs higher than those determined by the corresponding CFU assays. All the results obtained by flow cytometry were provided within 10 min after sampling, whereas the conventional CFU assay results took at least 18 h. The results indicated that flow cytometry is a sensitive analytical technique that can rapidly monitor the physiological changes of individual microorganisms following antibiotic action and can provide information on the mode of action of a drug. The membrane potential probe DiBAC4(3) provides a robust flow cytometric indicator for bacterial cell viability.

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

2000 ◽  
Vol 44 (4) ◽  
pp. 827-834 ◽  
Author(s):  
David J. Novo ◽  
Nancy G. Perlmutter ◽  
Richard H. Hunt ◽  
Howard M. Shapiro
Keyword(s):  
Staphylococcus Aureus ◽  
Flow Cytometry ◽  
Membrane Potential ◽  
Membrane Permeability ◽  
Micrococcus Luteus ◽  
Flow Cytometric Analysis ◽  
Bacterial Counts ◽  
Flow Cytometric ◽  
Permeability Parameter ◽  
Particle Counts

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 andMicrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC2(3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC2(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 μg/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 μg/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 μg/ml did not change permeability, while a tetracycline concentration of 4 μg/ml permeabilized 50% of the bacteria; 4 μg 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.

scholarly journals Evaluation of Physiological Effects Induced by Manuka Honey Upon Staphylococcus aureus and Escherichia coli

2019 ◽  
Vol 7 (8) ◽  
pp. 258 ◽  
Author(s):  
Patricia Combarros-Fuertes ◽  
Leticia M. Estevinho ◽  
Rita Teixeira-Santos ◽  
Acácio G. Rodrigues ◽  
Cidália Pina-Vaz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Membrane Potential ◽  
Membrane Integrity ◽  
Efflux Pumps ◽  
Coli Strain ◽  
Antibacterial Action ◽  
Manuka Honey ◽  
E Coli ◽  
Action Mechanisms

Several studies have explored the antimicrobial properties of manuka honey (MkH). However, the data available regarding antibacterial action mechanisms are scarcer. The aim of this study was to scrutinize and characterize primary effects of manuka honey (MkH) upon the physiological status of Staphylococcus aureus and Escherichia coli (as Gram-positive and Gram-negative bacteria models, respectively), using flow cytometry (FC) to reveal its antibacterial action mechanisms. Effects of MkH on membrane potential, membrane integrity and metabolic activity were assessed using different fluorochromes in a 180 min time course assay. Time-kill experiments were carried out under the same conditions. Additionally, MkH effect on efflux pumps was also studied in an E. coli strain with an over-expression of several efflux pumps. Exposure of bacteria to MkH resulted in physiological changes related to membrane potential and membrane integrity; these effects displayed slight differences among bacteria. MkH induced a remarkable metabolic disruption as primary physiological effect upon S. aureus and was able to block efflux pump activity in a dose-dependent fashion in the E. coli strain.

In Vitro Antibacterial Activity and Mechanism of Monocaprylin against Escherichia coli and Staphylococcus aureus

2018 ◽  
Vol 81 (12) ◽  
pp. 1988-1996 ◽  
Author(s):  
JIANYU WANG ◽  
MAOMAO MA ◽  
JUN YANG ◽  
LONG CHEN ◽  
PING YU ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Membrane Potential ◽  
Cell Membrane ◽  
Sodium Benzoate ◽  
Rapid Change ◽  
Potassium Sorbate ◽  
E Coli

ABSTRACT In the present study, the antibacterial activity of monocaprylin in comparison with sodium benzoate and potassium sorbate against Staphylococcus aureus and Escherichia coli was assessed by measuring MIC, MBC, effect of pH on MIC, and incubation temperature on bactericidal efficacy. Results showed that monocaprylin exhibited an excellent antibacterial activity against both strains, with the lowest MIC and MBC of 1.28 mg/mL. A MIC of monocaprylin remained unchanged despite the pH values of culture medium, ranging from 5 to 9, unlike that of potassium sorbate or sodium benzoate. Furthermore, monocaprylin at MBC effectively reduced the population of E. coli and S. aureus by &gt;5.5 log CFU/mL at 25°C within 6 h and decreased E. coli by approximately 5.0 log CFU/mL and S. aureus by 2.9 log CFU/mL at 12 h. The underlying mechanism of monocaprylin was then investigated by measuring β-galactosidase activity, membrane potential, release of cellular contents, scanning electron microscopy, and transmission electron microscopy observations. Results indicated that monocaprylin killed E. coli by the rapid change in permeability and integrity of cell membrane, leading to decline of membrane potential, leakage of nucleic acids and proteins, and ultimately cell membrane disintegration and lysis. On the other hand, monocaprylin might exert its antibacterial activity against S. aureus mainly by diffusing across the cell wall, collapsing the cell membrane, and disturbing the order of intracellular contents. These findings indicated that monocaprylin had better antibacterial ability compared with traditional synthetic preservatives and might be a potential antibacterial additive independent of pH.

scholarly journals Development and Characterization of a Fluorescent-Bacteriophage Assay for Detection of Escherichia coli O157:H7

1999 ◽  
Vol 65 (4) ◽  
pp. 1397-1404 ◽  
Author(s):  
Lawrence Goodridge ◽  
Jinru Chen ◽  
Mansel Griffiths
Keyword(s):  
Escherichia Coli ◽  
Flow Cytometry ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Lower Detection ◽  
Unreliable Method ◽  
Direct Epifluorescent Filter Technique ◽  
Bacterial Concentrations

ABSTRACT In this paper we describe evaluation and characterization of a novel assay that combines immunomagnetic separation and a fluorescently stained bacteriophage for detection of Escherichia coliO157:H7 in broth. When it was combined with flow cytometry, the fluorescent-bacteriophage assay (FBA) was capable of detecting 104 cells/ml. A modified direct epifluorescent-filter technique (DEFT) was employed in an attempt to estimate bacterial concentrations. Using regression analysis, we calculated that the lower detection limit was between 102 and 103cells/ml; however, the modified DEFT was found to be an unreliable method for determining bacterial concentrations. The results of this study show that the FBA, when combined with flow cytometry, is a sensitive technique for presumptive detection of E. coliO157:H7 in broth cultures.

scholarly journals Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS)

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1719-1729 ◽  
Author(s):  
Michael Berney ◽  
Hans-Ulrich Weilenmann ◽  
Thomas Egli
Keyword(s):  
Escherichia Coli ◽  
Membrane Potential ◽  
Glucose Uptake ◽  
Efflux Pump ◽  
Cellular Functions ◽  
Solar Disinfection ◽  
E Coli ◽  
Pump Activity ◽  
Uva Light ◽  
Uptake Activity

The effectiveness of solar disinfection (SODIS), a low-cost household water treatment method for developing countries, was investigated with flow cytometry and viability stains for the enteric bacterium Escherichia coli. A better understanding of the process of injury or death of E. coli during SODIS could be gained by investigating six different cellular functions, namely: efflux pump activity (Syto 9 plus ethidium bromide), membrane potential [bis-(1,3-dibutylbarbituric acid)trimethine oxonol; DiBAC4(3)], membrane integrity (LIVE/DEAD BacLight), glucose uptake activity (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose; 2-NBDG), total ATP concentration (BacTiter-Glo) and culturability (pour-plate method). These variables were measured in E. coli K-12 MG1655 cells that were exposed to either sunlight or artificial UVA light. The inactivation pattern of cellular functions was very similar for both light sources. A UVA light dose (fluence) of <500 kJ m−2 was enough to lower the proton motive force, such that efflux pump activity and ATP synthesis decreased significantly. The loss of membrane potential, glucose uptake activity and culturability of >80 % of the cells was observed at a fluence of ∼1500 kJ m−2, and the cytoplasmic membrane of bacterial cells became permeable at a fluence of >2500 kJ m−2. Culturable counts of stressed bacteria after anaerobic incubation on sodium pyruvate-supplemented tryptic soy agar closely correlated with the loss of membrane potential. The results strongly suggest that cells exposed to >1500 kJ m−2 solar UVA (corresponding to 530 W m−2 global sunlight intensity for 6 h) were no longer able to repair the damage and recover. Our study confirms the lethal effect of SODIS with cultivation-independent methods and gives a detailed picture of the ‘agony’ of E. coli when it is stressed with sunlight.

scholarly journals Novel Method for Rapid Assessment of Antibiotic Resistance in Escherichia coli Isolates from Environmental Waters by Use of a Modified Chromogenic Agar

2007 ◽  
Vol 73 (7) ◽  
pp. 2224-2229 ◽  
Author(s):  
A. J. Watkinson ◽  
G. R. Micalizzi ◽  
J. R. Bates ◽  
S. D. Costanzo
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Environmental Samples ◽  
Rapid Assessment ◽  
Treatment Plant ◽  
Wastewater Effluent ◽  
Susceptibility Test ◽  
E Coli ◽  
Novel Method ◽  
Disk Susceptibility

ABSTRACT We validated a novel method for screening Escherichia coli resistance to antibiotics in environmental samples using modified Difco MI agar (Becton Dickinson) impregnated with selected antibiotics (tetracycline, ampicillin, cephalexin, and sulfamethoxazole), termed MI-R. This method combines an existing rapid assessment technique for E. coli enumeration with clinical reference data for breakpoint analysis of antibiotic resistance and was developed to address issues encountered when clinical methods are used with environmental samples. Initial trials conducted using strains of E. coli with resistance to the selected antibiotics showed that this method was reproducible and accurate with respect to antibiotic resistance. Trials using wastewater effluent demonstrated the precision of the method, and the levels of resistance found in effluent were directly comparable to the levels of antibiotic resistance determined using the more traditional CLSI (formerly NCCLS) disk susceptibility test. All wastewater isolates growing on MI-R plates were confirmed to be resistant using the CLSI disk susceptibility test. Bacterial resistance to ampicillin (38% ± 4% overall), sulfamethoxazole, tetracycline (21% ± 3% overall), and ciprofloxacin (6% ± 1%) were found in wastewater effluent. A successful trial was also conducted with water collected from the Brisbane River, Australia. The levels of antibiotic resistance in E. coli ranged from 0 to 47% for ampicillin, from 0 to 24% for tetracycline, from 0 to 63% for sulfamethoxazole, and from 0 to 1% for ciprofloxacin, with the highest incidence of resistance associated with wastewater treatment plant discharges. This method has great potential for rapid and representative assessment of antibiotic resistance in E. coli and could allow increased sample analysis, resulting in greater confidence in spatial analysis in environmental studies.

scholarly journals On The Mechanisms of Nitrite Utilization by Escherichia coli Cells during Stationary Growth

2015 ◽  
Vol 10 (1) ◽  
pp. 193-205 ◽  
Author(s):  
Н.А. Ри ◽  
N.A. Ree
Keyword(s):  
Escherichia Coli ◽  
Membrane Potential ◽  
Model Analysis ◽  
Enzyme Concentration ◽  
Utilization Rate ◽  
Nitrite Accumulation ◽  
Stationary Growth ◽  
E Coli ◽  
Physiological Investigation ◽  
Operon Expression

Periplasmic Nrf nitrite reductase is the main nitrite utilizing enzyme in Escherichia coli cells when concentration of substrate in the medium is low. The model of nitrite utilization by E. coli cells, cultivated during stationary growth in the chemostat, was earlier created. The model took into account the dynamic of the nrf and nir operons, coding enzymes that metabolize and transport nitrite. The model analysis revealed that to describe the kinetic of nitrite accumulation in the chemostat at micromolar nitrite concentrations, additional assumption about higher nitrite utilization rate in the cell then that was estimated from genetic investigations is to be incorporated in the model. In this study it was shown that the discrepancy between genetic and physiological investigation results under low nitrite concentrations may be explained by properties of Nrf enzyme, which catalytic activity and stability depends on its localization in the periplasm, and its secretion to the periplasm is determined on the presence of membrane potential. The model analysis revealed that at low nitrite concentration local change of enzyme concentration during transition from cellular cytoplasm to periplasm under membrane potential influence is sufficient for higher periplasmic Nrf activity achievement, than it can be expected according to nrf operon expression level.

scholarly journals A medium for the rapid enumeration of Escherichia coli in the presence of other faecal coliforms in tropical waters

1982 ◽  
Vol 88 (2) ◽  
pp. 265-273 ◽  
Author(s):  
R. C. Wright
Keyword(s):  
Escherichia Coli ◽  
Water Quality ◽  
Membrane Filtration ◽  
Rapid Assessment ◽  
Faecal Coliforms ◽  
E Coli ◽  
Rapid Enumeration ◽  
Selective Membrane ◽  
Filtration Medium ◽  
Tube Dilution

SummaryA selective membrane filtration medium is described for use in the rapid assessment of water quality in tropical countries where the incidence of faecal coliforms other than E. coli presents problems in the interpretation of results. The medium gives comparable results to MPN values obtained in the multiple tube dilution test using modified Gray's glutamate medium, and to membrane filtration counts obtained using M-FC broth and membrane-enriched Teepol broth, whilst differentiation of E. coli is enhanced.

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