Flow Cytometric Investigation of Filamentation, Membrane Patency ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2000, p. 682–687 0066-4804/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 44, No. 3

Flow Cytometric Investigation of Filamentation, Membrane Patency, and Membrane Potential in Escherichia coli following Ciprofloxacin Exposure H. J. WICKENS,1 R. J. PINNEY,1 D. J. MASON,2

AND

V. A. GANT3*

Microbiology Section, Department of Pharmaceutics, The School of Pharmacy, University of London, London WC1N 1AX,1 Department of Microbiology, University College Hospitals Trust, London WC1E 6DB,3 and Department of Biological Sciences, Warwick University, Coventry CV4 7AL,2 United Kingdom Received 19 July 1999/Returned for modification 14 October 1999/Accepted 16 December 1999

Ninety-eight percent of the cells in a population of Escherichia coli in log-phase growth lost colony-forming ability after being exposed for 3 h to the quinolone antibiotic ciprofloxacin at four times the MIC in nutrient broth, a concentration easily reached in vivo. Flow cytometric analysis, however, demonstrated that only 68% of this bacterial population had lost membrane potential, as judged by the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)], and only 30% could no longer exclude the nucleic acid-binding dye propidium iodide (PI), reflecting lost membrane integrity, efflux mechanisms, or both. Subsequent removal of ciprofloxacin and resuspension in nutrient broth resulted in renewed cell division after 2 h, with a calculated postantibiotic effect (PAE) time of 57 min. The proportion of DiBAC- and PI-fluorescent cells in this recovering population remained stable for more than 4 h after antibiotic removal. Eighty percent of cells present at drug removal were filamentous. Their number subsequently decreased with time, and the increase in particle count seen at the end of the PAE resulted from the division of short cells. Exposure to ciprofloxacin in the presence of the protein synthesis inhibitor chloramphenicol increased colony-forming ability to 60% of starting population numbers. In contrast to ciprofloxacin alone, this antibiotic combination resulted in insignificant filamentation and no dye uptake. Subsequent drug removal and resuspension in nutrient broth resulted in the appearance of filaments within 1 h, with 69% of the population forming filaments at 3 h. Dye uptake was also seen, with 20% of the population fluorescing with either dye after 4 h. We were unable to relate dye uptake to the viable count. Cell division resumed 240 min after removal of both drugs, yielding a PAE calculated at 186 min. Inhibition of protein synthesis with chloramphenicol prevented ciprofloxacin-induced changes in bacterial morphology, cell membrane potential, and ability to exclude nucleic acid-binding dye. These changes persisted beyond the end of the classically defined PAE and were not a definite indicator of cell death as defined by loss of colony formation, which related at least in part to filamentation. The combination of flow cytometry with fluorescent probes allows several physiological parameters to be measured on individual cells within a bacterial culture. Thus, loss of a bacterial cell’s ability to exclude nucleic acid-binding dyes because of destroyed membrane patency or efflux mechanisms may be detected using propidium iodide (PI) (7, 13, 16), and uptake of the fluorochrome bis-(1,3-dibutylbarbituric acid) trimethine oxonol (referred to below as DiBAC) can be related to loss of membrane potential (17). Flow cytometry yields further information related to cell volume and internal architecture such as filamentation (7, 8, 15, 16). We have used flow cytometry in conjunction with traditional viable-counting techniques to investigate changes in colony-forming ability, filament formation, membrane potential, and barrier function in cells exposed to, and recovering from, treatment with the quinolone antibiotic ciprofloxacin. Exposure to ciprofloxacin, like exposure to other agents that damage DNA, induces SOS response-controlled DNA repair and cell filamentation (6). Whether these SOS functions contribute either to cell survival or to cell death after quinolone treatment remains unclear (4, 5, 11, 14, 21, 22, 24, 26). Filaments remain metabolically active for many hours (16) and persist for prolonged periods after drug removal (8, 9). This led

Guan and Burnham (9) to suggest that filamentation contributes to the period of growth suppression which occurs after quinolone removal, an example of postantibiotic effect (PAE) (3). PAE may be viewed as an expression of the time taken by the damaged cell population to recover and reestablish exponential cell division. Traditionally, this has been quantified using viable-counting techniques (3): samples of recovering cultures are plated onto a suitable medium, and subsequent colony formation is assessed. The possibility remains that filaments generated by quinolone treatment may continue to be metabolically active but unable to form colonies; thus, there is a potential for continuing production of metabolites, with possible clinical consequences (16). We have used flow cytometry in conjunction with fluorescent probes of bacterial function to ask questions of both antibiotic effect, and recovery therefrom, which are not possible to answer using classic determinations of colony-forming ability. Specifically, we investigated the relationship between quinolone-induced filamentation and colony formation, as well as the fate of individual cells in the context of recovery from quinolone-induced damage after drug removal in the presence and absence of chloramphenicol, an established inhibitor of protein synthesis.

* Corresponding author. Mailing address: Department of Microbiology, University College Hospitals Trust, Grafton Way, London WC1E 6DB, United Kingdom. Phone: 44-171-380 9517. Fax: 44-171388 8514. E-mail: [email protected].

MATERIALS AND METHODS Bacterial strain. Escherichia coli AB1157 ara-14 argE3 galK2 hisG4 lacY1 leuB6 mtl-1 proA2 rpsL31 thi-1 supE44 thr-1 tsx-33 xyl-5 F⫺ (12) was used throughout.

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FIG. 1. Dot plots of forward angle light scatter and DiBAC fluorescence for filamentous and nonfilamentous E. coli AB1157. Three separate populations are shown. Untreated, log-phase control cells plot below the horizontal line (nonfluorescent) and to the left of the vertical line (control cell length). Ethanoltreated (fluorescent) cells plot above the horizontal line and to the left of the vertical line (control length). Cells treated with four times the MIC of ciprofloxacin for 3 h plot in the second quadrant, indicating both fluorescence and filamentation.

Media and antimicrobials. Cultures were grown overnight at 37°C in Oxoid no. 2 nutrient broth (NB) (Unipath Ltd., Basingstoke, United Kingdom [U.K.]), which was solidified with 1.5% Lab M (Bury, U.K.) agar to make nutrient agar (NA). Ciprofloxacin was generously donated by Bayer (Newbury, U.K.) and

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chloramphenicol was purchased from Sigma (Poole, U.K.). Stock solutions were freshly prepared in sterile distilled water. Drug exposure and cell recovery. Experiments were performed in triplicate; all data points are the means of three independent determinations. Incubation and exposure were carried out at 37°C. E. coli AB1157 was grown overnight in NB and diluted 1 in 50 (vol/vol) into NB containing 0.02 ␮g of ciprofloxacin/ml (equivalent to four times the MIC, as determined by plate assay under these experimental conditions), giving 1% survivors after a 180-min exposure. Cultures were also diluted into NB containing the same concentration of ciprofloxacin as well as 20 ␮g of chloramphenicol/ml to inhibit protein synthesis (60% survival after 3 h). Cells growing in drug-free NB were used as a control. After 180 min, each culture was filtered through a 0.45-␮m-pore-size HA filter disk (Millipore, Watford, U.K.) and the filter was washed with two 10-ml portions of warm (37°C) NB. The filter was then placed in 5 ml of warm NB, and cells were resuspended for 30 s using a vortex mixer (Whirlimixer; Fisons Ltd., Loughborough, U.K.). Suspensions of drug-exposed and control cells were maintained at 37°C. They were sampled immediately post-washout and at hourly intervals for 7 h. Sampling consisted of the simultaneous removal of aliquots for flow cytometric analysis and plating in triplicate on NA after appropriate dilutions in NB. Viable counts (CFU per milliliter) were determined from these plates after a further 24-h incubation at 37°C. Viable counts of control cultures increased about 100-fold during the 180-min period in drug-free NB post-washout, and appropriate dilutions were made to allow accurate colony counting. Estimation of PAE. Plots of log viable count against time were used to determine PAE as described by Craig and Gudmundsson (3), applying the formula PAE ⫽ T ⫺ C, where T is the time in minutes taken for the viable count of a drug-exposed culture to increase by 1 log cycle and C is the equivalent period for the untreated control. Flow cytometric determination of fluorescence, light scatter, and particle concentration. PI was obtained from Sigma; DiBAC was purchased from Molecular Probes Inc. (Eugene, Oreg.). PI was dissolved in deionized water to a concentration of 100 ␮g/ml. DiBAC was dissolved in 70% ethanol at 1 mg/ml and further diluted in deionized water to a final concentration of 100 ␮g/ml. Portions (20 ␮l) of these solutions were added to 180-␮l aliquots of the recovering cultures to give final dye concentrations of 10 ␮g/ml. The mixtures were held for 5 min in the dark at room temperature before analysis in fluorochrome-containing medium with a Bryte HS Flow Cytometer (Bio-Rad, Hemel Hempstead, U.K.) fitted with a mercury-xenon arc lamp. The instrument is equipped with two light scatter detectors (⬍15° and ⬎15°, measuring forward and side scatter, respectively) and three fluorescence detectors detecting appropriately filtered

FIG. 2. Viable counts (vc) and total particle counts (tpc) (A) and numbers of filamentous (“long” particle counts [lpc]) and control length (“short” particle counts [spc]) cells (B) during the PAE following 3 h of exposure of E. coli AB1157 to four times the MIC of ciprofloxacin (Cip) in NB. Zero on the x axes represents the point at which the drug was washed out and cells were resuspended in drug-free NB. Counts of control cultures grown in NB are shown in panel A.

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FIG. 3. Viable counts (vc) and total particle counts (tpc) (A) and numbers of filamentous and nonfilamentous cells (lpc and spc, respectively) (B) during the PAE following 3 h of exposure of E. coli AB1157 to four times the MIC of ciprofloxacin (Cip) in combination with 20 ␮g of chloramphenicol (Cm)/ml in NB. Counts of control cells exposed to Cm in NB are also shown in panel A.

light at green (515 to 565 nm; to detect diBAC fluorescence), orange (565 to 605 nm), and red (⬎605 nm; to detect PI fluorescence) wavelengths. Dye excitation was achieved at 470 to 490 nm. The beam splitter was designed to allow detection of light emission at 520 nm and above. At least 5,000 events were recorded for each sample, and all experiments were conducted in triplicate on separate days. Instrument performance was monitored and adjusted as appropriate using fluorescent 1.5-␮m-diameter calibration beads obtained from the manufacturer (Bio-Rad). Data analysis. Instrument noise was excluded by setting electronic gates to both light scatter parameters; remaining events were then analyzed for fluorescence intensity. Total particle counts (equivalent to the concentration of viable plus nonviable cells) could then be recorded, as the cytometer employs accurate volumetric sample injection. The flow cytometer measures fluorescence on a logarithmic scale spanning 4 decades, with gain settings adjusted to place nonfluorescent cells within the 1st decade (below the horizontal dotted line in Fig. 1). Forward light scatter, approximately relating to cell volume (1, 23), was measured on a similar 4-decade logarithmic scale. Samples of log-phase cells, treated with cold 70% ethanol to destroy both membrane potential and barrier function, were used as positive controls for dye uptake (seen in the first quadrant of Fig. 1), with untreated log-phase cells serving as the negative controls (fourth quadrant, Fig. 1). A third population of cells (second quadrant, Fig. 1) was treated with four times the MIC of ciprofloxacin for 3 h to produce a filamentous population, confirmed by light microscopy to contain 96% of cells at least twice as long as the untreated controls (data not shown). The vertical line in Fig. 1 has been positioned such that more than 95% of the antibiotic-treated population falls to the right of the line, while more than 95% of untreated cells fall to the left. Using plots of forward scatter against side scatter (data not shown), these two populations, designated “long” and “short,” respectively, were isolated by separate computer-generated gates into populations that included more than 95% of each group and excluded at least 95% of the other.

RESULTS Effect of ciprofloxacin on bacterial number, morphology, and viable count. Figure 2A shows a plot of the concentrations of individual bacterial particles detected by flow cytometry as well as viable counts determined by classical methods. The apparent 10-fold difference in particle count at time zero is due

to the fact that the control cultures increased about 10-fold during incubation and were then diluted 100-fold so that the inoculum size would be equivalent to the CFU in the antibiotic-treated cultures at the time of the second antibiotic addition. These dilutions allowed the cells to undergo a complete growth curve post-washout as opposed to entering stationary phase within 2 to 3 h. The bacterial particle count from control cultures not exposed to antibiotic closely followed the viable count up to a particle count of 4 ⫻ 107/ml, beyond which the cytometer becomes inaccurate because of particle coincidence within the illumination window. In contrast, particle counts of ciprofloxacin-exposed cultures were initially 102-fold higher than their viable counts, reflecting the 99% loss of colonyforming ability observed after 3 h of exposure to four times the MIC of ciprofloxacin in NB. Viable counts remained unchanged for 2 h before resumption of exponential growth. Total particle counts remained constant for 4 h, however, before beginning to increase in line with the viable count. These data led to a calculated PAE for ciprofloxacin of 57 min. The total particle concentration and viable count following recovery from ciprofloxacin are once again shown in Fig. 2B, this time in addition to particle counts for those cells which the cytometer classified as either unchanged in morphology or filamented. At the end of 3 h of exposure to ciprofloxacin, 79% of the bacterial particles had formed filaments; filament numbers then decreased slightly over the subsequent 7 h after antibiotic washout. By contrast, the number of bacteria which had not formed filaments remained constant for 2 h and then increased in line with the viable count. Effect of chloramphenicol on ciprofloxacin-induced changes. Figure 3A shows that, as previously described (10, 24), 60% of the bacterial population is capable of forming colonies after

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FIG. 4. Cell size and permeability to bioreactive dyes during the PAE after exposure of E. coli AB1157 to four times the MIC of ciprofloxacin alone (A) or in combination with 20 ␮g of chloramphenicol/ml (B). Open triangles (‚) and diamonds ({) represent filamentous and nonfilamentous particle counts, respectively, as percentages of total particle counts. Solid circles (F) and squares (■) indicate the percentages of the total cell population that fluoresced when stained with PI and DiBAC, respectively.

exposure to ciprofloxacin in the presence of chloramphenicol, which antagonizes its bacterial activity. As expected, this leads to a much-reduced difference between particle count and viable count immediately after drug removal. Figure 3A shows that in control experiments, no loss of viability occurred during exposure to chloramphenicol; total particle counts and viable counts post-washout were therefore indistinguishable. The figure further shows that total particle counts of the chloramphenicol-exposed cultures then increase in line with the viable count in the recovering population. Cultures exposed to ciprofloxacin in the presence of chloramphenicol gave a calculated PAE of 186 min, which is approximately the sum of the PAEs of 57 and 120 min calculated after exposure to ciprofloxacin and chloramphenicol, respectively. Figure 3B shows that only 9% of the bacterial population had formed filaments immediately after washout of the ciprofloxacin-plus-chloramphenicol combination, and approximately half of the total population formed colonies. The proportion of cells forming filaments rose rapidly immediately post-washout to approximately 50% within 1 h and then rose more gradually for another 3 h before eventually declining after 5 h. The nonfilamented proportion of cells declined between 2 and 3 h post-washout; interestingly, the viable count remained unchanged throughout this period. After 4 h postwashout the nonfilamented particle count began to increase in line with the viable count. Morphology and dye permeability of cell populations during the PAE of ciprofloxacin. Ninety-six percent of the bacterial population in control log-phase cultures was classified as nonfilamentous according to the criteria defined in Materials and Methods (data not shown). As previously stated, the ratio of

filaments to morphologically “normal” cells immediately following ciprofloxacin exposure was found to be 4:1. Figure 4A shows that this ratio began to decline steadily afterwards, such that only 14% of the population remained as filaments 7 h after drug removal. This proportional reduction was attributed to renewed proliferation of nonfilamented cells after approximately 3 h rather than to lysis of filaments, since the filament count remained relatively constant throughout the 7-h postwashout period (Fig. 2B). Control experiments and experimental parameters had defined approximately 2% of cells in logarithmic growth as being sufficiently unable to exclude PI to be classed as fluorescent (7, 18). Figure 4A shows that 30% of the population fluoresced with PI immediately following ciprofloxacin washout and that this percentage increased to 43% after 2 h, before falling to 12% after 7 h. By the same criteria, approximately 4% of cells in control cultures could be defined as fluorescent by uptake of DiBAC (data not shown). In contrast, ciprofloxacin exposure promoted DiBAC uptake in 68% of the population immediately after drug washout; again, this proportion slowly increased to more than 80% during the next 3 h, before falling to 19% at 7 h (Fig. 4A). Finally, we were careful to document any relationship between fluorescence intensity and cell volume (as determined by light scattering), since increased fluorescence of a cell could simply reflect increased mass rather than increased cell labeling by the dye. For both dyes, we found only minor shifts in fluorescence intensity according to cell volume, which were far outweighed by antibiotic-induced effects in some cells. Chloramphenicol significantly inhibited changes in filamentation and dye uptake until its removal. Thus, simultaneous exposure to ciprofloxacin and chloramphenicol resulted in only

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FIG. 5. Permeability to DiBAC and PI of long and short cell populations during the PAE of E. coli AB1157 after exposure to four times the MIC of ciprofloxacin in NB (A) and four times the MIC of ciprofloxacin in combination with 20 ␮g of chloramphenicol/ml in NB (B). Open triangles (‚) and diamonds ({) represent uptake of PI by long and short cell populations, respectively (as defined in Materials and Methods). Solid triangles (Œ) and diamonds (}) represent uptake of DiBAC by long and short populations, respectively.

9% filamentation immediately post-washout (Fig. 4B). This proportion increased to 70% after a further 3 h but then declined to 11% after 7 h. There was, again, poor correlation between the uptake of either dye and growth potential: while 40% of cells had lost colony-forming ability, only 10% of the population was significantly permeable to DiBAC or PI at drug washout (Fig. 4B). DiBAC permeability increased gradually with time in drug-free NB, peaking at 22% after 4 h. The proportion of cells permeable to PI decreased for the first 2 h, before also peaking at 22%, 4 h post-washout (Fig. 4B). Figure 5A shows the differential permeability of long and short cells to both dyes after exposure to ciprofloxacin alone; Fig. 5B demonstrates the additional effect of chloramphenicol on these cells. The figure shows the considerable inhibition of ciprofloxacin-induced permeability to both DiBAC and PI consequent upon chloramphenicol exposure. DISCUSSION One of the objectives of this work was to determine the relationship between SOS-controlled cell filamentation and both quinolone-induced loss of colony formation and postexposure recovery. While 80% of the bacterial population formed filaments after exposure for 3 h to four times the MIC of ciprofloxacin in NB, only 1% of cells retained colony-forming ability. Filamentation is therefore not a necessary prerequisite for loss of colony-forming ability. Furthermore, not all nonfilamented cells can grow and divide to form colonies (Fig. 2B). The data rather demonstrate that only 4% of control length cells retain colony-forming ability. Guan and Burnham (9) suggested that filaments may divide during and after quinolone treatment to form further filaments. We were unable to confirm this suggestion, as our data demonstrate that, if anything, the number of filaments declines throughout the PAE of

ciprofloxacin (Fig. 2B). Rather, we suggest that the increase in viable cell count at the end of the PAE results from an increase in the number of control length cells rather than from the division of filaments, although we cannot exclude the alternative but unlikely possibility that filaments may both divide and then once again grow to a length identical to that of their parent cells. Chloramphenicol significantly reduces ciprofloxacin-induced kill, which led Smith and coworkers (10, 24) to propose that inhibition of protein synthesis blocks mechanism B, one of their three putative quinolone lethality mechanisms. Chloramphenicol also blocks ciprofloxacin-induced SOS repair (19, 20), filamentation (Fig. 3B) (22, 25), and ability to exclude potential-sensitive and DNA-binding dyes (Fig. 5). We consider that the appearance of filamentation after the removal of both drugs represents resumed protein synthesis in quinolonedamaged cells, allowing growth but not division. During the period of filament formation there was no decrease in viable count, implying that those cells which had sustained damage sufficient to induce filamentation were not at any stage capable of colony formation, despite retaining metabolic activity (16). Another objective of the work was to investigate the relationship between bioreactive dye uptake and the ability of ciprofloxacin-treated populations of E. coli to form colonies. We and others (7, 18) have suggested a relationship between dye uptake and loss of colony-forming ability. Our observation of 98% loss of colony-forming ability in the presence of only 30% PI fluorescence immediately post-washout suggests that these two physiological parameters are not intimately related; not surprisingly, many other factors may inhibit colony formation independently of changes in membrane permeability, drug efflux mechanisms, or both. A similar lack of correlation between viability and membrane integrity was seen in those experiments incorporating chloramphenicol. In this case, 40% of cells had

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lost colony-forming ability whereas only 9% were permeable to PI. While incorporation of PI, therefore, may be a “lethal” event, other antibiotic-induced mechanisms of loss of colony formation must exist to explain our viable-count results. Jepras et al. (13) suggested that permeability to DiBAC, which has been equated to loss of membrane potential, relates more closely to loss of colony-forming ability. Our data support their conclusions in that our results relating to DiBAC permeability mirrored more closely the proportion of non-colony-forming cells (99%) than did those relating to PI permeability immediately after ciprofloxacin treatment. Once again, however, a significant number of bacteria capable of excluding DiBAC, and therefore assumed to be maintaining an intact membrane potential through energy-dependent mechanisms, were not capable of colony formation. The proportion of cells taking up DiBAC after ciprofloxacin exposure was approximately equal to the proportion of filaments present. Further analysis revealed, however, that of the 67% of cells filamenting following recovery from exposure to ciprofloxacin and chloramphenicol, only 17% took up DiBAC. Posttreatment filamentation was less extensive in cultures exposed to ciprofloxacin in the presence of chloramphenicol (67 compared to 89% with ciprofloxacin alone [Fig. 4]). Similarly, chloramphenicol considerably inhibited the percentage of filaments taking up DiBAC after ciprofloxacin exposure (35 versus 85% [Fig. 5]). This may relate to differences in the extent of damage associated with the SOS response caused by ciprofloxacin in cells competent for protein synthesis, compared to damage resulting from quinolone treatment in the absence of protein synthesis (2). In summary, we have investigated the PAE of ciprofloxacin on E. coli at the single-cell level using a combination of flow cytometric and viable-counting methods, and we have found that, although the calculated PAE for the quinolone was 57 min, we could detect drug effects on cell function, as assessed by two different molecular probes and morphology, for at least 4 h for a significant proportion of cells after drug removal. Filamentation was not a prerequisite for cell death; neither did it necessarily correlate with membrane damage. Chloramphenicol limits both ciprofloxacin-induced filamentation and changes in dye permeability; these changes relate to protein synthesis as determined by washout experiments, where such changes once again appear when protein synthesis resumes. This supports the theories of Smith (10, 24) and Chen et al. (2), namely, that quinolone lethality can proceed via either a chloramphenicol-sensitive or a chloramphenicol-insensitive mechanism. Finally, flow cytometric techniques once again demonstrate the heterogeneous nature of antibiotic-induced effects, and such effects may be highly relevant to the dynamics of the mechanisms underpinning antibiotic resistance. Such techniques may be useful in future antibacterial drug development.

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ACKNOWLEDGMENTS We thank the University of London, The University of London School of Pharmacy, and the Newby Trust for their generous financial support. REFERENCES 1. Boye, E., H. B. Steen, and K. Skarstad. 1983. Flow cytometry of bacteria: a promising tool in experimental and clinical microbiology. J. Gen. Microbiol. 129:973–980.

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