David J. Mason and Vanya A. Gant
Division of Infection and Immunity, Department of Microbiology,
United Medical and Dental Schools of Guy's and St.Thomas's Hospitals,
London SE1 7EH, UK

email: d.mason@umds.ac.uk
email: v.gant@umds.ac.uk

Use of flow cytometry to investigate morphological and physiological properties of individual organisms within bacterial populations is becoming increasingly popular. The technique is an excellent tool for analyzing microbial responses to antibiotics. Antibiotic-induced changes in specific physiological functions of the cell of interest can be probed with selected fluorescent dyes. One such dye, bis-(1,3- dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)), a lipophilic anion, is sensitive to changes in membrane potential. It will enter into eukaryotic membranes only if their membrane potential has collapsed (Wilson and Chused, 1985). Mason et al., (1994) extended the use of this dye to prokaryotes and have demonstrated its application to the rapid detection of antibiotic susceptibility. Damage to membrane integrity can be monitored using propidium iodide (PI). This small cationic molecule binds to nucleic acids provided it has access to them through a damaged cell membrane. These properties have been exploited to detect antibiotic-induced membrane damage (Gant et al., 1993; Mason et al., 1995).

In this paper we demonstrate the use of these two fluorescent dyes and flow cytometry in monitoring changes in bacterial membrane potential or integrity induced by the quinolone antibiotic (DNA-gyrase inhibitor), ciprofloxacin.

Bacterial strains, media and antibiotic. The bacterial strains used were Escherichia coli KL16 and a clinical isolate of Haemophilus influenzae. Both species were cultured in Iso- Sensitest broth (filtered through a 0.2um-pore-size filter for flow cytometry) at 37 C. For growth of H. influenzae the broth was supplemented with Filde's extract (1%) and NADH (10mg/l). Ciprofloxacin was a gift from Bayer (U.K.); the compound was initially dissolved in 0.01M NaOH and then diluted to required concentrations in distilled water.

Susceptibility testing. The minimum inhibitory concentration (MIC) of ciprofloxacin was determined by using a standard broth dilution method and Iso-Sensitest broth (Stokes and Ridgway, 1987). The ciprofloxacin MIC for E. coli KL16 and H. influenzae was 0.06ug/ml.

Experimental design. Both species were grown to early logarithmic phase (107CFU/ml) in broth culture. Cultures were divided into three equal volumes (10ml); sparfloxacin was added to two of the cultures at 1 and 100 times the MIC. The third culture was retained as a control, and all cultures were incubated for 120min. A sample (1ml) was removed from the control culture prior to incubation, and further 1ml volumes were removed from all cultures after 30, 60, 90 and 120 min. Each sample was spun for 1min in an Eppendorf centrifuge at 13,000rpm, and the pellet washed in broth once and finally resuspended in fresh broth. Two aliquots (0.2ml) were removed from each sample and stained with DiBAC4(3) and PI.

Bacterial staining. DiBAC4(3) (excitation 493nm, emission 516nm) and PI (excitation 536nm, emission 617nm) were supplied by Molecular Probes Inc., U.S.A and Sigma Chemical Co., U.K. respectively. DiBAC4(3) was dissolved in acetone to give a stock solution of 1mg/l. This was diluted 1:10 in 70% ethanol to give a working solution of 100 ug/ml. PI was dissolved in deionised water to a concentration of 100 ug/ml. DiBAC4(3) or PI were added to 0.2ml of cell suspensions to give a final concentration of 10 ug/ml.

Flow cytometric analysis. Flow cytometric analysis was carried out using a Bryte HS (Bio-Rad, U.K.) dual parameter flow cytometer fitted with a mercury-xenon-arc lamp. The instrument used a jet-over-open-surface flow cell configuration, and was equipped with two light scatter detectors (greater than 15 degrees and less than 15 degrees) and two fluorescence detectors (beam split at 520 nm). Fluorescence detection (gated by light scatter parameters) was carried out using a FITC filter block with the following characteristics: excitation, 470-490 nm; band stop, 510 nm; and emission greater than 520nm. All detectors were used with logarithmic amplification; sample flow and sheath pressure were set to 2 ul/min and 0.7 Bar respectively.

In the following experimental results the proportion of cells in the bacterial population which exhibited dye-associated fluorescence (and therefore had depolarised cell membranes or damaged membrane integrity depending on the dye) is expressed as a percentage.

The effects of ciprofloxacin action on E. coli KL16. Fig. 1 shows dual parameter histograms of forward angle light scatter versus PI fluorescence for a culture of E. coli KL16 exposed to ciprofloxacin at the MIC for 120 min. Forward angle light scattered by organisms is seen to increase over 120 min (similar results were obtained with H. influenzae). At 100xMIC, however, no increase in forward angle light scatter was observed from either bacterial species. Propidium iodide fluorescence at the MIC increased slowly over 120 min, with 15% of organisms being rendered fluorescent after 120min. In contrast, the proportion of cells from a control culture exhibiting propidium iodide- asscociated fluorescence after 120min was 1.5%. Propidium iodide uptake following exposure to100xMIC of ciprofloxacin for 120 min was similar to that seen at the MIC.

The proportion of cells exhibiting DiBAC4(3)-associated fluorescence following exposure to the MIC of ciprofloxacin reached 20% after 120min. This represented a minor increase over the control value (12%), but did not reach statistical significance at the 5% level (Mann-Whitney U test). In contrast, exposure to 100xMIC for 120min resulted in 95% of the population exhibiting DiBAC4(3)-associated fluorescence (Mason et al., 1995).

The effects of ciprofloxacin action on Haemophilus influenzae. In cultures exposed to ciprofloxacin at the MIC the proportion of organisms exhbiting DiBAC4(3)- or PI-associated fluorescence after 120 min was 7% and 1% respectively. Following exposure to 100xMIC for 120min the proportion of organisms stained with DiBAC4(3) or PI increased to 54% and 28% respectively (Fig. 2). The proportion of organisms from control cultures rendered fluorescent by DiBAC4(3) or PI was approximately 10% and 5% respectively.

The results illustrate how flow cytometry can be used to detect heterogeneity wthin a microbial population in terms of susceptibility to an antibiotic. Other techniques for susceptibility testing such as the disc diffusion assay or the broth dilution method, consider the bacterial population as a whole and any heterogeneity in response to an antibiotic within this goes un-detected.

In addition, detection of membrane damage using the fluorescent physiological probes demonstrates how flow cytometry can be used to provide information on the mechanism of antimicrobial activity of antibiotics. The extent of membrane damage induced by ciprofloxacin was shown to increase with antibiotic concentration in both bacterial species. This concentration-dependent effect may be due to ciprofloxacin complexing with membrane stabilizing cations (Chapman and Georgopapadakou, 1988; Smith, 1990). Changes in forward angle light scatter profiles upon exposure to ciprofloxacin at the MIC represent filamentation of the organisms (confirmed by phase contrast microscopy). For a more detailed discussion of how these results relate to the mechanism of action of this antibiotic see Mason et al., (1995).

The authors wish to thank Rhône D. P. C. Europe and the Trustees of St Thomas' Hospital for their financial support.

Chapman, J. S., and N. H. Georgopapadakou. 1988. Routes of quinolone permeation in Escherichia coli. Antimicrob. Agents Chemother. 32:438-442.

Gant, V. A., G. Warnes, I. Phillips, and G. F. Savidge. 1993. The application of flow cytometry to the study of bacterial responses to antibiotics. J. Med. Microbiol. 39:147-154.

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Mason, D. J., E. G. M. Power, H. Talsania, I. Phillips and V. A. Gant. 1995. Antibacterial action of ciprofloxacin. Antimicrob. Agents Chemother. 39:2752-2758.

Smith, J. T. 1990. Effects of physiological cation concentration on 4-Quinolone absorbtion and potency, p.15-21. In G. C. Crumplin (ed), The 4-Quinolones Antibacterial Agents in Vitro. Springer-Verlag, London.

Stokes, E. J., and G. L. Ridgway. 1987. Clinical microbiology. Edward Arnold Publishers Ltd, London.

Wilson, H. A., and T. M. Chused. 1985. Lymphocyte membrane potential and Ca2+ sensitive potassium channels described by oxonol dye fluorescence measurements. J. Cellular Physiol. 125:72- 81.

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