Vol. 36, No. 10

ANTIMICROBIAL AGENTs AND CHEMOTHERAPY, Oct. 1992, p. 2118-2124

0066-4804/921102118-07$02.00/0 Copyright C 1992, American Society for Microbiology

Analysis of Macromolecular Biosynthesis To Define the Quinolone-Induced Postantibiotic Effect in Eschenichia coli LINONG GUAN,I ROBERT M. BLUMENTHAL, AND JEFFREY C. BURNHAM* Department of Microbiology, Medical College of Ohio, Toledo, Ohio 43699 Received 17 March 1992/Accepted 21 July 1992

Quinolones inhibit DNA gyrase, and the major effects of this inhibition are on replication and trnscription of DNA. The postantibietic effect (PAE) refers to continued inhibition of cell division, In terms of the viable count, following trasient exposure to an antibic. Previous work has shown that quinolone-treated cells have not filly recovered by the time the classicalBy defined PAE has ended. We describe the PAE of the qunolones CI-960, enoxacin, and ciprofloxacin on macromolecular bioyntbesis in the clinal isolate EMckickla coi l96 in an attempt to relate the PAE to the time that it actually takes for the cells to recover fuly. DNA synthesis was inhibited immediately upon exposure to these quinolons at 0.5x or 0.75X the MIC. Thi inhibition continued for several hours following quinolone removal. The effects of these quinolones on RNA and protein synthesis varied; enoxacin treatment at 0.5x the MIC resulted in increase of over 60% in both RNA and t effects on protein synthesis per unit of cell mass, while ciprofloxacin and CI-.0 at that level had no s either RNA or protein synthesis. The effects of enoxacin and ciprofioxacin on bacterial protein profiles were also distinguishable, and these changes coresponded to their PAE on DNA syndteis. Throughout the study, all measures of the physiological status of the cells e ned to normal by the time DNA syntheis per unit of cell mass did so. These results suggest that DNA synthesis per unit of cell mass provides an accurate measure of the time required for quinolone-treated cells to recover flly. an

Suppression of bacterial growth can persist after a short exposure to antimicrobial agents (6). In 1944, Bigger (2) demonstrated that staphylococci do not resume growth for several hours following transient exposure to an inhibitory concentration of penicillin G. More recently, Craig and colleagues (4, 6, 20, 31) have performed extensive investigations of this postantibiotic effect (PAE) with both gram-positive and gram-negative bacteria. In general, grampositive bacteria such as Streptococcus pyogenes, Streptococcus pneumoniae, and Staphylococcus aureus showed a PAE of 1 to 3 h after transient exposure to ,B-lactam antibiotics near the MICs for the strains, while for gramnegative bacteria such as Escherichia coli and Pseudomonas aeruginosa, concentrations much greater than the MICs for the organisms were required to elicit a significant PAE. Inhibitors of protein and RNA synthesis tend to produce the longest PAEs, which have comparable durations in both gram-positive and gram-negative bacteria (4). The major clinical relevance of the PAE is in designing antibiotic dosage regimens, because long PAEs provide the potential for longer intervals between doses (10). Furthermore, a close relationship between the length of the PAE and bacteriocidal activity has been observed (21). Although the phenomenon of PAE has been observed and studied for almost half a century, the precise mechanisms of PAE are still unclear. The observed differences in PAEs of various drug-organism combinations suggest that multiple mechanisms are involved (6). It is possible that bacteria

in amounts sufficient for the continuation of its bacteriostatic effect (4). Quinolone antibiotics are actively taken up (8), act on bacterial DNA gyrase (topoisomerase II) and inhibit DNA replication, and can produce a significant PAE in both gram-positive and gram-negative bacteria at or above the MICs for the bacteria (6, 12, 14, 21, 23). Sub-MICs of quinolones can also induce a PAE on E. coli (14) but not P. aeruginosa (12). The PAE is classically defined as the time that it takes for a treated culture to increase 10-fold in viable cells after drug removal minus the time that it takes for an untreated culture to increase 10-fold in viable cells (6), with no requirement that the two cultures be growing at the same rate following the increase. In other words, even at the end of the classically defined PAE, the treated cells may still be physiologically abnormal. We have previously found that the effects of quinolones on E. coli cell division, septation, and hemolysin activity can last considerably longer than the classicaLly defined PAE (14). The present study had two goals: first, to investigate the quinolone-induced PAE in E. coli through studies on bacterial DNA, RNA, and protein metabolism and, second, to see whether these measurements can complement the classically defined PAE by indicating how long it actually takes for quinolone-treated cells to recover fully. MATERIALS AND METHODS Materials. Enoxacin (CI-919, PD 107779), 14C-enoxacin (15.9 ,uCi/mg), and CI-960 (PD 127391) were provided by Parke-Davis Pharmaceutical Research Division, WarnerLambert Company (Ann Arbor, Mich.). Ciprofloxacin, trimethoprim, and sulfamethoxazole were obtained from Sigma Chemical Co. (St. Louis, Mo.). [methyl-3H]thymidine (specific activity, 85 Ci/mmol), [5,6-3Huridine (specific activity, 46 Ci/mmol), and L-[4,5-3HJleucine (specific activity, 56 Ci/mmol) were from Amersham (Arlington Heights, Ill.);

which have suffered nonlethal damage from an antibiotic simply need time to revert to their normal metabolic state. An alternative possibility is that the drug binds to target sites

*

Corresponding author.

t Present address: Neose Pharmaceuticals, Inc., Horsham, PA

19044.

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VOL. 36, 1992

L-[35SJmethionine (specific activity, 1,139.9 Ci/mmol) was from NEN-Dupont (Wilmington, Del.). Lysozyme, sodium dodecyl sulfate (SDS), DL-methionine, and thiamine were from Sigma Chemical Co.; Liquifluor scintillant was from Du Pont (Boston, Mass.). M9 medium was made with 42 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 7.6 mM NH4C1, 2 mM MgSO4, 0.2% glucose, and 0.1 mM CaCl2 (28). Bacterial strains and culture conditions. E. coli ATCC 25922 was used as a control strain for MIC determinations. E. coli J96, which was generously provided by Sheila Hull (Baylor College of Medicine, Houston, Tex.), was isolated from a patient with acute pyelonephritis (25). The PAEs of quinolones on this strain have been described previously (14). The strains were routinely grown in M9 minimal medium supplemented with 2 ,ug of thiamine per ml at 37°C in a shaking water bath. Bacterial growth. Bacterial growth in the presence or absence of various quinolones was determined by monitoring the optical density of the culture at 520 nm (OD520). Cultures of 30 ml were grown in 125-ml flasks at 37°C with shaking, and the OD520 was determined at 30-min intervals in a Milton Roy Spectronic 501. Since the changes in concentration of nutrients and waste products in media can affect the rates of bacterial DNA, RNA, and protein synthesis (22), dilution with prewarmed medium was done hourly to keep the bacteria in balanced growth over the 5-h course of the experiments. MIC determination. MICs were determined by broth microdilution in M9 medium supplemented with 2 p,g of thiamine per ml as described previously (14). After determination of the approximate MIC, a more accurate MIC was determined by dividing the range between 0.5 x the MIC and 2x the MIC into 10 concentrations for determining the lowest concentration at which no visible growth occurred after incubation at 37°C overnight. The MICs of CI-960, enoxacin, ciprofloxacin, and trimethoprim-sulfamethoxazole for E. coli J96 were 0.004, 0.19, 0.03, and 0.60 p,g/ml, respectively. Measurement of DNA, RNA, and protein synthesis. (i) Antibiotic exposure and drug removal. The inocula were prepared by incubating the bacteria overnight at 37°C in M9 supplemented with 2 p,g of thiamine per ml. The culture was then diluted 1:100 in fresh prewarmed medium and incubated in a shaking water bath until the OD520 reached 0.1 (after 0.5 h in the log phase of growth). Cultures were then exposed to the drug at sub-MICs for 1 h. In order to remove residual antibiotic, the cultures were centrifuged at 2,500 x g and 25°C for 5 min, the supernatants were discarded, and the cell pellets were suspended and diluted in the same prewarmed medium. This procedure was used in parallel on the untreated control culture. The efficiency of this pellet resuspension step for drug removal was measured by using 4C-enoxacin. The same antibiotic treatment and drug removal procedures were followed. After the supernatant was removed, the concentration of residual antibiotic was determined by measuring the radioactivity (in counts per minute) of 14C-enoxacin in the pellets.

(ii) [methyl-3Hlthymidine, [5,6-3Hluridine, and L-[4,5-3H] leucine pulse-labeling. The pulse-labeling assay was modified from the method of Crumplin and Smith (7). At the indicated times, 0.49-ml samples of culture were added to tubes containing 10 pl of [methyl-3H]thymidine (20 p,Ci/ml) for DNA synthesis, 10 pl of [5,6-3Hluridine (50 p,Ci/ml) for RNA synthesis, or 10 p,l of L-[4,5- Hlleucine (50 p,Ci/ml) for protein synthesis. After incubation at 37°C with shaking for 5 min, triplicate 0.1-ml samples from each tube were added

QUINOLONE-INDUCED PAE IN E. COLI

2119

to wells containing 0.1 ml of 50% trichloroacetic acid (TCA) in a 96-well microtiter plate. All of the TCA precipitates were stored at 4°C for at least 1 h and were then filtered through Filtermats on a 12-well cell harvester (Skatron, Sterling, Va.) and rinsed for 1 min with distilled water. Filters were dried overnight at 45°C and put into 4-ml Skatron scintillation vials. Two milliliters of scintillation fluid (1 liter of toluene containing 160 ml of Liquifluor) was added, and the radioactivity was determined in a Beckman LS3810 (Fullerton, Calif.). (iii) Analysis of 35S-labeled proteins in polyacrylamide gels by autoradiography. First, cells were labeled with L-[35S]methionine (30). At the times indicated in the legend to Fig. 4, 0.99-ml samples of culture were added to tubes containing 10 IlI of L-[35 ]methionine (500 ,uCi/ml). After incubation at 37°C with shaking for 5 min, each sample was chased with 100 pl of methionine (5 mg/ml) for 2 min at 25°C. Samples were precipitated for determination of incorporated radioactivity as described above. The remaining portion of each sample was stored at -70°C for gel electrophoresis. Second, samples were prepared and electrophoresis was carried out. The labeled samples were thawed at 20°C for 1 h. Samples of 40 to 60 pl were added to 1.5-ml Eppendorf tubes containing lysis buffer (4 mg of lysozyme per ml, 50 mM glucose, 50 mM Tris-HCl, 10 mM EDTA [pH 8.0]) for a final volume of 100 pl, and the solution was incubated at 20°C for 5 min. One-fourth volume of sample buffer (0.125 M Tris-HCl [pH 6.8], 3% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromophenol blue) was added to each tube, and then the tubes were heated to 100°C for 5 min. Proteins were resolved on 7.5% polyacrylamide gels containing 0.1% SDS as described by Laemmli (18). After electrophoresis, the gels were dried and exposed to Kodak XAR5 X-ray film at -70°C for 72 h.

RESULTS Maintenance of balanced growth ofE. coli J96 and effects of enoxacin. We began by defining the growth conditions necessary to follow macromolecular synthesis over an extended time following quinolone treatment. To ensure that our untreated control cultures remained in balanced growth over the course of these experiments, we periodically diluted them. The doubling time of the logarithmic-phase culture was 59 min in M9 glucose medium at 37°C. Thereafter, a twofold dilution with fresh prewarmed medium was made once every hour to keep the culture in balanced growth. Figure 1A shows that exponential growth could be maintained indefinitely under these conditions, so our data would not be influenced by nutrient limitation or other problems associated with entry into the stationary phase. Cultures such as the ones described above were exposed to enoxacin for 1 h at 0.5 x or 0.75 x the MIC and were then pelleted and resuspended to remove the drug (see below); and the OD520 was followed (Fig. 1B). There was no detectable effect during the 1-h drug treatment itself, but the results indicate a 2-h-long reduction in the rate of mass accumulation relative to that in the untreated control culture. In contrast, by using either M9 glucose or Mueller-Hinton medium and measuring growth by either OD520 determination or viable counts, treatments with enoxacin at the indicated MICs gave fairly constant PAE values of about 0.7 h (data not shown). Lower concentrations of enoxacin (0.25 x or 0.125 x the MIC) had no effect on growth (data not shown). Higher drug concentrations were not used because they are bacteriocidal and lead to the interference of dead

ANTIMICROB. AGENTS CHEMOTHER.

GUAN ET AL.

2120

0;

0.2

o

2

3

4

Tlme (b) 40. B

20

A

A

-1

-os

o

os 1 2 1.5 Hours after removal of enoxcacin

2.5

3

FIG. 1. (A) Maintenance of exponential growth of E. coli J96 by periodic dilution. Cultures were diluted every hour. The coffected

OD was calculated as the OD250 times the cumulative dilution factor. A straight line would indicate that the cultures remained in balanced growth over the time of the experiment, after the cells entered the exponential phase of growth. Effor bars indicate the standard deviation. (B) The effect of a transient sub-MIC of enoxacin on the growth of E. coli J96. An exponential-growth-phase culture was split and treated for 1 h with enoxacin at Ox (O), 0.5x (A), or 0. 75 x (A) the MIC. At time zero, the drug was removed as described in the text. The length of the effects on rate of mass accumulation (ODZO) are shown by plotting the slope of the growth curve, between each successive pair of time points, after subtracting the slope of the untreated control. The units are thus OD520 per hour.

cells in cell mass measurements (14); this was also true for the other quinolones used in this study. Efficiency of drug removal by pelleting of cells. To minimize disturbance of the cultures, drug removal involved just one centrifugation step; this was followed by resuspension in prewarmed medium. Accordingly, we examined the possibility that sigifcant residual amounts of antibiotic remained in the resuspended culture. Since the specific activity of 14C-enoxacin (15.9 ,uCi/mg) was not high enough to test this drug at sub-MICs, lOx and l x the MIs of 14C-enoxacin were used in this test. By using lOx the MIC of 14C_ enoxacin, following centrifugation of triplicate samples after 1 h of exposure, about 95% of the label was in the supernatant that was removed and 5% was in the pellets. By using 1lx the MIC of 14C-enoxacin, all radioactivity was recovered in the supernatant; the actual counts per minute per 0.1-ml sample, corrected for background, were 184 -+- 21 for the medium, 192 +t 22 for the supernatant, and undetectable for the cell pellet. The highest antimicrobial concentration which we used for

the macromolecular biosynthesis assays described below was 0.75x the MIC. If 95% of the drug was removed (the result from using 10x the MIC of 14C-enoxacin), the residual drug concentration in the resuspended pellets should be, at most, 0.04x the MIC (5% x 0.75x the MIC). This value should be compared with the fact that 0.125x the MIC of enoxacin had no measurable effect on DNA synthesis (see Fig. 2D). Measurement of DNA synthesis during and after enoxacin treatment. To compare DNA synthesis in antibiotic-treated and untreated cells, data were expressed per unit of cell mass (OD520). There are several reasons for our use of cell mass instead of viable count. First, although morphological changes in cells treated with sub-MICs of quinolones were observed, bacterial killing did not occur during or after the treatment (14), so there was no interference from dead cells in the measurement of cell mass. Second, the normal length of E. coli cells is about 3 ,um, but the length of filaments induced by treatment with sub-MICs of quinolones can be as much as 65 ,m (9, 14), and a cell with the mass of 20 bacteria would represent 1 CFU when assessed by viable counts. Third, the OD520 was used as a reference unit in studies of the quinolone-induced SOS response (26, 27). A defined cell mass was pulse-labeled for 5 min with [methyl-3HJthymidine at the indicated times during and after enoxacin treatment, and the results are shown in Fig. 2. The control culture remained in balanced growth over the course of the experiment, as indicated by the constant DNA synthesis per OD520. As expected, the effect of enoxacin was concentration dependent. During the 1-h treatment, 0.75x, 0.5x, and 0.25x the MICs of enoxacin caused rapid inhibition of DNA synthesis, while 0.125x the MIC of enoxacin did not show a significant effect. As expected, the inhibition of DNA synthesis did not stop immediately after enoxacin removal, but continued about another 3 h for 0.75 x the MIC (Fig. 2A), 2 h for 0.5 x the MIC (Fig. 2B), and 1 h for 0.25 x the MIC (Fig. 2C). Note that these times are for complete recovery to the untreated control level of DNA synthesis. Effects of quinolone treatment on RNA and protein synthesis. We next compared DNA, RNA, and protein synthesis in cells treated with four different antibiotics at 0.5 x the MICs. The results are given as the incorporation per OD520 of the treated cultures as a percentage of that of the untreated control culture (Fig. 3). After 1 h of exposure, DNA synthesis was inhibited up to 79% by enoxacin (Fig. 3A), 37% by CI-960 (Fig. 3B), and 80% by ciprofloxacin (Fig. 3C). As was seen for enoxacin (Fig. 2), the inhibition of DNA synthesis continued after drug removal: 1 h for CI-960 (Fig. 3B) and more than 4 h for ciprofloxacin (Fig. 3C). On the other hand, recovery of DNA synthesis in the CI-960-treated culture began as soon as the drug was removed (Fig. 3B), but only after 0.5 to 1.0 h in the cases of enoxacin and ciprofloxacin (Fig. 3A and C). The effects of these quinolones on RNA and protein synthesis also varied. Surprisingly, enoxacin caused a transient increase of up to 60% in the rates of protein and RNA synthesis (Fig. 3A), while ciprofloxacin and CI-960 had no such effect. Note that the stimulation of RNA and protein synthesis by enoxacin ended by the time that DNA synthesis had returned to normal levels. Trimethoprim-sulfamethoxazole did not show significant effects on DNA, RNA, or protein synthesis during or after the 1-h treatment (Fig. 3D). Effects of quinolone treatment on the pattern of protein synthesis. The profile of the proteins being made by a cell is a sensitive indicator of its physiological status, even when the identity of the altered bands or spots is unknown. A

QUINOLONE-INDUCED PAE IN E. COLI

VOL. 36, 1992

2121

0-,O7sk11m ... 0

1 - 04 50 40

20 10

0.125xMGC

0.25 x MIc

-2

-1

0

i

24

5 -2

-1

0

1

4

5

Tlme after drug removal (h) FIG. 2. Incorporation of [methyl-3H]thymidine during and after a 1-h exposure to enoxacin. E. coli J96 was grown in M9 glucose medium, and the exponential-phase culture was split to give five cultures treated with Ox (control), 0.75x (A), 0.5x (B), 0.25x (C), or 0.125x (D) the MIC of enoxacin. In each panel the untreated control culture is represented by open circles. At time zero (arrows), the drug was removed as described in the text. At the indicated times, 0.49-ml samples of culture were added to tubes containing 10 pl of [methyl-3H]thymidine for 5 min. Triplicate 0.1-ml samples from each tube were precipitated with TCA. The resulting counts per minute per OD520 are shown, corrected for background, with standard error bars.

comparison of [35S]methionine-pulsed proteins from control cultures with cultures treated with 0.5 x the MIC of enoxacin or ciprofloxacin is shown in Fig. 4. These are the same cultures for which data were shown in Fig. 3. Several alterations in the relative quantities of individual protein bands can be seen in both the enoxacin-treated (Fig. 4A) and ciprofloxacin-treated (Fig. 4B) cultures. The effects of enoxacin and ciprofloxacin on bacterial protein profiles were distinguishable from one another. For example, a new band at 37.5 kDa appeared only in the enoxacin-treated culture and disappeared by 1.5 h after drug removal. Trimethoprimsulfamethoxazole and CI-960 at 0.5 x the MIC did not show significant effects on bacterial protein profiles. Most significantly, the changes in protein bands induced by enoxacin and ciprofloxacin corresponded to the pattern of DNA synthesis; after drug removal for 2 h, both cultures showed full reversion of the protein profile to the pattern of the control culture. DISCUSSION

The phenomena of quinolone-induced PAEs which we previously observed include (i) growth suppression, (ii) altered morphology because of inhibition of cell division and septation, and (iii) reduction of hemolysin activity (14). We examined the effects of transient exposure to sub-MICs of the quinolones CI-960, enoxacin, and ciprofloxacin or to trimethoprim-sulfamethoxazole on macromolecular synthesis in the clinical isolate E. coli J96. This investigation was carried out to study the relationship between the molecular mechanism of action of quinolones and the observed PAE

phenomenon and to relate the classically defined PAE to the time that it actually takes the treated cells to recover fully. The effects of various quinolones on RNA and protein synthesis differ. RNA and protein synthesis are progressively inhibited by quinolones as the quinolone concentration is increased above the most bacteriocidal concentration (9, 19, 29). In this study we found that 0.5 x the MIC of enoxacin increased RNA and protein synthesis during the 1-h treatment and for 1 h after drug removal (Fig. 3A). Ciprofloxacin and CI-960 did not show these effects (Fig. 3B and C). Lewin and colleagues (19, 29) found that enoxacin uses only mechanism A, the bacteriocidal mechanism common to all tested quinolone antibiotics and which depends on RNA and protein synthesis as well as bacteria capable of division in order to be killed (19, 29). The transcription inhibitor rifampin or the translation inhibitor chloramphenicol completely abolishes the bacteriocidal activity of enoxacin (19). In contrast, ciprofloxacin uses both mechanism A and mechanism B, the second of which is not antagonized by rifampin (29). For the purposes of this study, there are two significant conclusions. First, neither total RNA synthesis per unit of cell mass nor total protein synthesis mass is a useful indicator of the physiological status of quinolone-treated cells. Second, both of these parameters are normal by the time that the DNA synthesis per unit of cell mass has returned to the normal level. Effects of quinolones on bacterial protein profiles. Krueger and Walker (17) reported that two heat shock proteins with molecular masses of 73 kDa (dnaK) and 61 kDa (groEL) were induced by nalidixic acid (17). Gudas and Pardee (15) also reported that nalidixic acid treatment of E. coli B/r

2122

ANTIMICROB. AGENTS CHEMOTHER.

GUAN ET AL. B

CI-960

.0.Sx MC.

D

t~~~~~

Trimehprimsulfamethoxazole

-.5x -2

-1

2

3

4

s-2

-

Time after drug removal

0i2

3

s

(h)

FIG. 3. Effects of transient drug treatment on the synthesis of DNA, RNA, and protein. E. coli J96 was grown in M9 glucose medium, and the exponential-phase culture was split to give five cultures treated for 1 h with no drug (mock-treated control) or 0.5 x the MIC of enoxacin (A), CI-960 (B), ciprofloxacin (C), or trimethoprim-sulfamethoxazole (D). At time zero (arrows), the drug was removed as described in the text. At the indicated times, 0.49-ml samples of culture were added to tubes containing 10 p1 of [methyl-3H]thymidine (0), [5,6-3H]uridine (A), or L-[4,5-3H]leucine (5) for 5 min. Trplicate 0.1-ml samples from each tube were precipitated with TCA. The resulting counts per minute per OD520 are shown, corrected for background, as a percentage of the level in the control culture.

caused the induction of a 60-kDa protein that separated with a membrane fraction (15). The quinolone-induced proteins with molecular masses of 70.0 and 58.0 kDa shown in Fig. 4 may be these same heat shock proteins. Distinguishable changes were also seen in the enoxacin- and ciprofloxacintreated cultures (Fig. 4). This result may support the hypothesis of Lewin and colleagues (19, 29) that there are differences in the mechanisms of antibiotic action between enoxacin and ciprofloxacin. It is not clear why the band at 37.5 kDa, which may be the RecA protein (40 kDa), appeared only after drug removal in the culture treated with 0.5 x the MIC of enoxacin. Ciprofloxacin has been shown to induce synthesis of the RecA protein (27), although that work involved slightly higher drug levels, a different host strain, and the use of a recA::lacZ fusion. Regardless of the identities of the induced proteins, all of the changes in protein bands induced by enoxacin or ciprofloxacin corresponded to the pattern of DNA synthesis, with both parameters returning to normal in parallel. Quinolone-induced postantibiotic growth suppression is associated with inhibition of DNA synthesis. Quinolones appear to inhibit DNA synthesis by inhibiting the activity of DNA gyrase, an enzyme responsible for ATP-dependent negative supercoiling (1, 16). Inhibitory effects on nucleoid segregation (13) and DNA synthesis (5) by sub-MICs of quinolones have been reported. Normally, the rate of DNA synthesis is expressed per unit of culture mass, because this value remains constant as long as the culture is in steady-state growth (3). In the experiments reported here, this approach is problematic, because quinolones affect chromosome replication, repair synthesis of DNA, and cell size. In theory, one could obtain the

-800

-4R0

FIG. 4. Effects of transient sub-MICs of quinolones on the pattern of protein synthesis in E. coli J96. Cells were pulse-labeled with 35S-methionine and chased as described in the text. Equal amounts of TCA-precipitable counts were applied to each lane. Molecular weights (in thousands) and positions of standards are indicated on the right. Zero time represents the time at which quinolones were removed from the growth medium. Odd-numbered lanes represent antibiotic-untreated cultures at the following times: -0.5, 0, 0.25, 0.5, 1.0, 1.5, and 2.0 h for both panels A and B. Even-numbered lanes represent cultures treated for 1 h with 0.5x the MIC of enoxacin (A) or ciprofloxacin (B) at the same times given above for the odd-numbered lanes. The most pronounced changes in the protein profiles are indicated by arrows at the left and correspond to polypeptides with molecular masses of 72.0, 70.0, 58.0, 53.5, and 37.5 kDa (A) or 78.0, 70.0, 58.0, and 40.0 kDa (B).

VOL. 36, 1992

QUINOLONE-INDUCED PAE IN E. COLI

TABLE 1. Overview of effects of transient exposure to various antibioticS on E. coli J96 Time (h)

Antibiotic (MIC)

Cell mass accumu-

lationa

lain

DNA

RNA and

srynthesis/potein

Classical

aytess 0D520-

synthesis/ OD:5

PAEb

-

Enoxacin 0.75x 0.50x 0.25x 0.125x

2.0 2.0 0.0 0.0

3.0 2.0 1.0 0.0

NDY 1.0 0.0 0.0

ND 0.7 0.1 ND

Ciprofloxacin (0.50)

2.5

>4.0

0.0

0.9

CI-960 (0.50)

0.0

1.0

0.0

0.1

Trimethoprim-sulfamethoxazole (0.50)

0.0

0.0

0.0

0.0

a The time before the value returns to the level of the untreated control culture. b Data are from a previous study (14), using the definition of PAE given previously (6). c ND, not determined.

2123

and these data yield a correlation coefficient of 0.915. This strong positive correlation exists despite a range of effects from the different quinolones. For example, CI-960 did not cause a significant growth suppression at 0.5x the MIC, although the DNA synthesis of the treated culture was transiently inhibited by about 40%. Since DNA synthesis in cultures treated with 0.5x the MIC of enoxacin or ciprofloxacin was only about 30% of the normal level at the end of the classically defined PAEs, results of our study suggest that the classical definition of PAE gives a value proportional to but shorter than the actual recovery time of quinolone-treated E. coli. The PAEs associated with various quinolones are being taken into account in optimizing quinolone dosage regimens (10). The results presented here suggest that the PAEs provide a very conservative basis for judging the allowable interval between quinolone treatments and that this conservatism is compounded by the profound effects of some quinolones at levels well below the MIC.

ACKNOWLEDGMENTS We thank the Parke-Davis Pharmaceutical Research Division for supporting this research and for providing radiolabeled enoxacin. R.M.B. is supported by the National Science Foundation under grants DMB-8818673 and MCB-9205248.

normal DNA synthesis per unit of cell mass value in an abnormal cell with compensating changes in these three parameters. Nonetheless, it is clear that a culture cannot be considered as having returned to normal growth if it has not attained the normal DNA synthesis per unit of cell mass value. In the event, both DNA synthesis and cell size (and DNA synthesis per unit of cell mass) return to untreated levels in parallel. We found that treatment with enoxacin, CI-960, or ciprofloxacin at 0.5x to 0.75x the MIC led to an immediate reduction in DNA synthesis. Because quinolone treatment increases repair synthesis of DNA (11, 26, 27), the inhibition of chromosome replication may have been even more pronounced than our thymidine incorporation results suggest. This inhibition, parallel to the growth effects, did not stop immediately following drug removal and possibly reflects the time needed to repair the accumulated damage to the DNA or for residual intracellular drug-bound target to be diluted by target synthesis. It should be noted that incorporation of labeled thymidine may also have been affected to some extent by changes in the intracellular pool size of thymidine or the activity of any of several genes involved in thymidine uptake, phosphorylation, or turnover (24). It is nevertheless true that all measures of physiological status return to normal levels by the time that DNA synthesis per unit of cell mass does (Table 1) and that any possible systematic errors in measuring DNA synthesis would affect the apparent extent of inhibition, not the length of the inhibition. A total of seven inhibition time courses were determined: for trimethoprim-sulfamethoxazole, CI-960, and ciprofloxacin, each at 0.5 x their MIC, and for enoxacin at four concentrations (Fig. 2 and 3). The times for recovery from all quinolones to control rates of DNA synthesis and cell mass accumulation are correlated to one another and to the classically defined PAE (the time required for a 10-fold increase in viable count following drug removal minus the time for the same increase in the untreated control culture [6]). A summary of this information is presented in Table 1. Four of the seven conditions have nonzero data for both DNA synthesis per unit of cell mass and the classical PAE,

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