Methicillin-resistant Staphylococcus aureus (MRSA) and MSSA strains were treated with: (a) grapefruit oil (GFO) components, isolated by chromatography and characterised by NMR and mass spectroscopy; (b) antimicrobial agents, or (c) a combination of both to evaluate (MIC determination) intrinsic antibacterial activity and to determine whether GFO components could modulate bacterial sensitivity to the anti-bacterial agents. Preliminary data suggested that the grapefruit component 4-[[(E)-5-(3,3-dimethyl-2-oxiranyl)-3-methyl-2-pentenyl]oxy]-7H-furo[3,2-g]chromen-7-one (2) enhances the susceptibility of test MRSA strains to agents, e.g., ethidium bromide and norfloxacin, to which these micro-organisms are normally resistant.

A continuous culture of Saccharomyces cerevisiae IFO 0233, growing with glucose as the major carbon and energy source, shows oscillations of respiration with a period of 48 min. Samples taken at maxima and minima indicate that (i) periodic changes do not occur as a result of carbon depletion, (ii) intrinsic differences in respiratory activity occur in washed organisms and (iii) a respiratory inhibitor accumulates during respiratory oscillations. Plasma membrane and inner mitochondrial membranes generate transmembrane electrochemical potentials; changes in these can be respectively assessed using anionic or cationic fluorophores. Thus flow cytometric analyses indicated that an oxonol dye [DiBAC(4)(3); bis(1,3-dibutylbarbituric acid)trimethine oxonol] was excluded from yeasts to a similar extent (in >98% of the population) at all stages, showing that the plasma membrane potential was maintained at a steady value. However, uptake of Rhodamine 123 was greatest at that phase characterized by a low respiratory rate. Addition of uncouplers of energy conservation [CCCP (m-chlorocarbonylcyanide phenylhydrazone) or S-13(5-chloro-3-t-butyl-2-chloro-4(1)-nitrosalicylanilide)] to the continuous cultures increased the respiration, but had only a transient effect on the period of the oscillation. Electron microscopy showed changes in mitochondrial ultrastructure during the respiratory oscillation. At low respiration the cristae were more clearly defined due to swelling of the matrix; this corresponds to the 'orthodox' conformation. When respiration was high the mitochondrial configuration was 'condensed'. It has been shown previously that a temperature-compensated ultradian clock operates in S. cerevisiae. It is proposed that mitochondria undergo cycles of energization in response to energetic demands driven by this ultradian clock output.

Aims: To investigate the antibacterial efficacy of vancomycin towards Staphylococcus aureus under aerobic and anaerobic conditions, and to assess the influence of oxygen on the duration of the post-antibiotic effect (PAE) after exposure to vancomycin.Methods and Results: Culture-based techniques and flow cytometric measurements of 5-cyano-2,3-ditolyl tetrazolium chloride (an indicator of redox activity) and the membrane potential-sensitive fluorophore Sytox Green, were used to test four staphylococcal strains. The MICs for all strains, and the duration of PAE, were similar whether tested with or without oxygen. However, a fivefold logarithmic reduction in cell counts was observed in 10-15 h aerobically, depending on strain, compared with longer than 60 h in an anaerobic environment. Flow cytometric data correlated well with counts of colony-forming units under both aerobic and anaerobic conditions.Conclusions: The death rate of Staph. aureus exposed to vancomycin was greater in the presence of oxygen, although MIC values and PAE durations were similar whether tested aerobically or anaerobically. Also, flow cytometry provided a rapid and sensitive alternative to plate counts for the assessment of antibiotics in oxygen-free conditions.Significance and Impact of the Study: This study underlines the need for further anaerobic testing using different strain/antibiotic combinations, the results of which will have clinical significance due to the anaerobic nature of some sites of infection.

Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) is an antimicrobial agent used in hygiene products, plastics and kitchenware, and for treating methicillin-resistant Staphylococcus aureus (MRSA) outbreaks. S. aureus strains with low-level resistance to triclosan have emerged. It has been claimed that strains with decreased susceptibility to biocides may also be less susceptible to antibiotics. We tested the susceptibility of S. aureus clinical isolates to triclosan and several antibiotics. Triclosan MICs ranged between 0.025 and 1 mg/L. Some, but not all, strains were resistant to several antibiotics and showed low-level triclosan resistance. S. aureus mutants with enhanced resistance to triclosan (< or =1 mg/L) were isolated. In several cases this resistance was stably inherited in the absence of triclosan. These mutants were not more resistant than the parent strain to several antibiotics. Changes in triclosan MICs associated with the acquisition of a plasmid encoding mupirocin resistance were not observed, suggesting that the triclosan/mupirocin co-resistance seen in a previous study was not the result of a single resistance gene or separate genes on the same plasmid. The continuous exposure of a triclosan-sensitive S. aureus strain to sub-MIC concentrations of triclosan for 1 month did not result in decreased susceptibility to triclosan or to several antibiotics tested. Triclosan-induced potassium leakage and bactericidal effects on a triclosan-sensitive strain, a resistant strain and a strain selected for increased resistance were compared with those of non-growing organisms, exponentially growing organisms and organisms in the stationary phase. No significant differences between the strains were observed under these conditions despite their different MICs. Biocides have multiple target sites and so MICs often do not correlate with bactericidal activities. The ability of S. aureus to develop resistance to triclosan and the current view that triclosan may have a specific target in Escherichia coli, namely enoyl reductase, underline the need for more research on the mechanisms of action and resistance.

BACKGROUND:Conventional techniques used to assess bactericidal activities of antibodies are time-consuming; flow cytometry has been used as a rapid alternative. In this study, the membrane potential-sensitive fluorescent probes bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) and Sytox Green, the redox dye cyano-2,3-ditolyl tetrazolium chloride (CTC), and the Baclite viability test kit were used to assess the effects of ceftazidime, ampicillin, and vancomycin on clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, respectively.METHODS:Bacterial cultures were grown to early exponential phase, at which point the antibiotics were added at their breakpoint values, and incubation was allowed to continue. At timed intervals, samples were stained and flow cytometric analysis was performed on a Skatron Argus 100 arc-lamp based dual-parameter flow cytometer.RESULTS:All the dyes successfully identified antibiotic-induced damage in the three strains, although different fluorescence responses between the dyes were observed. DiBAC4(3) and Sytox Green overestimated numbers of nonviable bacteria relative to loss of viability as judged by plate counts. CTC, a measure of respiratory activity, revealed antibiotic-induced population heterogeneity illus trated by the development of several subpopulations. The "live" component of the viability kit identified two populations corresponding to viable and nonviable organisms, whereas the "dead" component only revealed single populations, the fluorescence intensity of which increased with antibiotic exposure.CONCLUSIONS:Flow cytometry provides a rapid and sensitive technique for the evaluation of the antibacterial activities of antibiotics. The use of a range of fluorophores specific for different cellular characteristics may be beneficial, bearing in mind the different fluorescence responses observed among the dyes used here.

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Concern has been growing regarding the potential of antibiotic and disinfectant co-resistance in clinically important bacteria. In this study, the susceptibilities of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) to chlorhexidine (CHX), the quaternary ammonium compounds cetylpyridinium chloride (CPC) and benzalkonium chloride (BC), triclosan, dibromopropamidine isethionate (DBPI) and triclocarban were compared. MRSA exhibited low-level resistance to CHX and the QACs, with MICs of 1.5 to 3-fold (CHX), and 2 to 4-fold (QACs) higher than MSSA. However, the MIC values for MRSA ranged between 0.025 (the MIC of MSSA) and 1 microg/mL with triclosan, and between <5 (the MIC of MSSA) and 75 microg/mL with DPBI. Nevertheless, these strains remain relatively sensitive to most of these antimicrobial agents. The bactericidal efficacy of CHX, CPC and DBPI (with the exception of one strain) correlated with their MIC value. This was not observed using triclosan; MRSA and MSSA strains were equally susceptible to its killing effect, regardless of MIC. The permeabilizing agent, ethylenediamine tetraacetic acid (EDTA) was unable to potentiate the antibacterial activities of the biocides against any of the strains tested. Attempts to select for staphylococcal strains with increased resistance to triclosan, CPC or CHX, using disc diffusion, step-wise broth, or repeated exposure/recovery technique, were only partially successful, and resistance was found to be unstable. The susceptibilities of vancomycin-resistant enterococcus (VRE) and vancomycin-sensitive enterococcus (VSE) to the biocides were also compared and found to be similar both in terms of MIC testing and time-kill studies.

ABSTRACT The postantibiotic effect (PAE) following a 2-h exposure of Staphylococcus aureus NCTC 6571 to methicillin (5× the MIC) was investigated with fluorescent probes, 5-cyano-2,3-di-4-tolyl tetrazolium chloride (CTC), an indicator of respiratory activity, and the membrane potential-sensitive compound bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC 4 (3)]. Counts of the numbers of CFU on solid agar correlated well with information gained from the CTC and DiBAC 4 (3) fluorescence intensity distributions obtained by flow cytometry and revealed that the postantibiotic effect was 3.1 h. Due to the capacity of flow cytometry to provide information on the heterogeneity of a bacterial population, both fluorescent probes identified the emergence of an active subpopulation 4 h after removal of the methicillin, indicating the recovery of a small percentage of the population. After removal of the methicillin and resuspension of the cells in methicillin-free medium, a further decrease in the respiratory activity and the membrane integrity of the population was observed, although the CFU counts hardly varied, indicating continued antibiotic-induced damage. Also, CTC fluorescence measurements identified numerous subpopulations during the PAE period; this suggests that the PAE is complex, with individual organisms exhibiting various degrees of recovery. Flow cytometry thus provides a rapid and sensitive alternative to traditional techniques that have been used to study PAE, with the added advantage that physiological changes can be detected as they arise.

Vitality and viability of an alcohol-tolerant wine yeast, used in cider production, were assessed after exposure to alkanol(s) during growth, Criteria employed were: methylene blue reduction, ability to form colonies on yeast extract-peptone-glucose agar medium, glucose driven proton efflux rates (''acidification power''), fermentative (CO2 output) rates and adenylate energy charge values. We also monitored the maintenance of transmembrane electrochemical potential across the plasma membrane as measured by flow cytometry and by scanning confocal laser microscopy of oxonol dye exclusion.Growth rates were diminished by a third by 7.5% (v/v) added ethanol, 1% butan-1-ol or 1.4% isobutanol. Exposure to 19% (v/v) ethanol gave 16% loss of ''viability'', as measured by methylene blue reduction, during the first 29 h of growth, For 1% butan-1-ol, 50% loss of ''viability'' occurred over 40 h, whereas a similar effect of iso-butanol took 55 h. Adenylate charge values were high (<0.8) in growing cultures, remained high in early stationary phase but declined to 0.4 after 115 h, These values were hardly affected by 5 or 7.5% (v/v) ethanol whereas 10% or 15% (v/v) ethanol gave values of 0.58 and 0.16 after only 5 h exposure, 1% butan-1-ol or iso-butanol decreased adenylate charge values to a greater extent than 10% (v/v) ethanol, with the straight chain alcohol the more potent.Oxonol exclusion indicated that the vast majority of cells with greatly diminished vitality have maintained the plasma membrane potential values required to retain viability, despite extensive exposure to alkanol(s). Thus loss of ability to reduce methylene blue indicates diminished vitality but is not a reliable index of loss of viability, ''Acidification power'' was a more sensitive indicator of vitality than adenylate charge values, When mixtures of C-2 + C-4 alcohols were employed effects were generally additive rather than synergistic.

ABSTRACT. The free‐living anaerobic flagellate Hexamita sp. was observed to actively consume O 2 with a K m O 2 of 13 μM. Oxygen consumption increased lineraly with O 2 tension up to a threshold level of 100 μM, above which it was inhibited. Oxygen uptake was supported by a number of substrates but probably not coupled to energy conservation as cytochromes could not be detected spectro‐photometrically. In addition, inhibitors specific for respiratory chain components did not significantly affect O 2 uptake. Respiration was however, partially inhibited by flavoprotein and iron‐sulfur protein inhibitors. NAD(P)H supported O 2 consumption was measured in both particulate and soluble fractions; this activity was partially inhibited by quinacrine. A chemosensory response was observed in cells exposed to air, however no response was observed in the presence of superoxide dismutase plus catalase. Catalase and nonspecific peroxidase activity could not be detected, but superoxide dismutase activity was present. Superoxide dismutase was sensitive to NaN 3 and H 2 O 2 but not KCN, suggesting a Fe prosthetic group. Flow cytometric analysis revealed that thiol levels in live cells were depleted in the presence of t‐butyl H 2 O 2 . The observed NADPH‐driven glutathione reductase activity is believed to recycle oxidized thiols in order to re‐establish reduced thiol levels in the cell. The corresponding thiol cycling enzyme glutathione peroxidase could not be detected. The ability to withstand high O 2 tensions (100 μM) would enable Hexamita to spend short periods in a wider range of habitats. Prologed exposure to O 2 tensions higher than 100 μM leads to irreversible damage and cell death.

Flow cytometry using the anionic membrane potential-sensitive fluorescent probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)), enabled assessment of antibiotic-induced membrane perturbation in five clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and two antibiotic-sensitive reference strains, NCTC 6571 and 8325-4, after establishment of steady-state growth in liquid cultures inoculated from single colonies. Flow cytometric indications of the enhanced DiBAC(4)(3) uptake after treatment with vancomycin at 0.1, 1, 4 and 10 x MIC showed excellent comparison with viability losses quantified as cfu on solid agar in MRSA isolate QC. The antibiotic susceptibility patterns to benzylpenicillin, methicillin and vancomycin for all isolates used in this study could be determined in 2-4 h from an overnight plate culture. This technique thus provides a rapid and reproducible antibiotic sensitivity test which may be applicable in routine clinical practice.

The anaerobic free-living ciliated protozoon Metopus contortus is a grazer in anoxic marine sediments. It does not possess mitochondria, but it does have specialized organelles termed hydrogenosomes which release hydrogen gas. The cationic lipophilic cyanine dye DiOC7(3) is an indicator of transmembrane electrochemical potential. With the aid of confocal laser scanning microscopy (CLSM), the association of this dye with hydrogenosomes in situ was followed. Flow cytometric measurements showed that fluorescence of the membrane potential dye decreased in response to an elevated pH2 in the cell. CLSM also revealed localization of fluorescence of the calcium probe Fluo 3-AM, and of the transmembrane pH gradient probe BCECF-AM, within the lumen of the hydrogenosomes. In addition, hydrogenosomal inclusions were detected. X-ray microanalysis of these electron-dense granules revealed high levels of calcium, phosphate and magnesium. It is concluded that M. contortus hydrogenosomes are calcium stores, have a membrane potential, and an alkaline lumen. These physiological features resemble those of mitochondria in aerobic protozoa.

Flow cytometry of rhodamine 123- or cyanine-stained cider yeast shows that the capacity for mitochondrial uptake of these cationic dyes is lost early in the fermentation. Survival of prolonged anaerobiosis (for at least 22 days) at high ethanol concentrations (at least 11% v/v) during cider fermentation does not require the maintenance of a measurable inner mitochondrial transmembrane electrochemical potential. Confocal laser scanning microscopy of yeasts after exposure to either of the cationic dyes confirms the lack of mitochondrial development and inability of the fermentative organisms to take up the fluorophores. Aeration of samples taken from the fermentation vessels restores the ultrastructure and the dye uptake capacity of the yeasts. This indicates that the changes are reversible, and that the organisms have retained their viability.