Comparison of Two Methods for Determining In Vitro Postantibiotic ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1988, 0066-4804/88/040450-04$02.00/0 Copyright © 1988, American Society for Microbiology

p. 450-453

Vol. 32, No. 4

Comparison of Two Methods for Determining In Vitro Postantibiotic Effects of Three Antibiotics on Escherichia coli DENISE L. RESCOTT,1 DAVID E. NIX,12* PATRICIA HOLDEN,' AND JEROME J. SCHENTAG'2 School of Pharmacy, State University of New York at Buffalo,2 and The Clinical Pharmacokinetics Laboratory, Millard Fillmore Hospital, 3 Gates Circle,l* Buffalo, New York 14209 Received 28 August 1987/Accepted 12 January 1988

The postantibiotic effect (PAE) for 10 isolates of Escherichia coli was measured by two methods after 1 h of to ampicillin, ciprofloxacin, or tobramycin. The reference method involved serial colony counting to determine growth after antibiotic exposure in relation to control growth. A spectrophotometric procedure was developed with the Abbott MS-2 research system. This method measured the time to detection of growth after exposure and compared this with the time for growth detection in control chambers having the same initial colony count. A reference curve of time to growth versus log initial CFU per milliliter was used to standardize control growth. PAE was determined after exposure to antibiotic at two and six times the MIC and with inocula ranging from 103 to 109 CFU/ml. There was a statistically significant correlation between PAE measured by the spectrophotometric and the reference methods, and the residuals about the regression line were normally distributed. The mean PAE determined by both methods was statistically different for tobramycin-exposed, but not for ampicillin- or ciprofloxacin-exposed, organisms. There was a concentration-dependent PAE for ciprofloxacin and tobramycin. The PAEs for ciprofloxacin (151 min) and tobramycin (108 min) at concentrations six times the MIC were prolonged compared with those measured at two times the MIC (69 and 66 min, respectively). PAE was inversely related to the exposed inoculum for ciprofloxacin and tobramycin. The PAE for E. coli exposed to ampicillin was minimal and was not affected by either concentration or inoculum. The MS-2 method for determining PAE yields similar results, but is less laborious than the reference method. exposure

Postantibiotic effect (PAE) is the lag phase or recovery period of bacterial growth after brief exposure to an antibiotic. The presence of PAE may be an important consideration in designing antibiotic dosage regimens (4, 7, 9). A prolonged PAE should allow extension of antibiotic dosing intervals beyond the time that antibiotic concentrations fall below the MIC (8). Theoretically, antimicrobial agents with minimal PAEs may require serum concentrations above MICs for an entire dosing interval. The reference method for PAE measurement is the broth technique, involving serial colony counting and broth subculture of growth (4). Consequently, the technique is very time consuming and tedious, because numerous colony counts must be performed after exposure to the antibiotic. A more rapid and less tedious method would greatly facilitate the clinical study of PAE as a factor in design of antibiotic dosage regimens. A procedure was developed to measure PAE with the Abbott MS-2 research system (MS-2). The MS-2 method was considerably less time consuming than the broth technique, but required validation because it utilizes optical endpoints rather than survival. The MS-2 method and the broth technique were compared by using three antibiotics against 10 Escherichia coli isolates. Variables known to alter the duration of the PAE, including antimicrobial agent concentration and inoculum size, were also evaluated utilizing the MS-2.

ATCC 25922 (American Type Culture Collection, Rockville, Md.) was used as a reference strain for all procedures. The isolates were stored on nutrient agar slants, transferred weekly, and streaked for purity. MIC. MICs were determined for each isolate in duplicate against ampicillin (Sigma Chemical Co., St. Louis, Mo.), ciprofloxacin (Miles Inc., Pharmaceutical Division, West Haven, Conn.), and tobramycin (Eli Lilly & Co., Indianapolis, Ind.), using the broth microdilution test as outlined by the National Committee for Clinical Laboratory Standards (12). The antibiotics were serially diluted in cation-supplemented Mueller-Hinton broth (MHB-S). An inoculum of approximately 5 x 105 CFU/ml was used. MICs were recorded after 16 to 18 h of incubation at 35°C. Inoculum. The inocula for all procedures were prepared by incubating each organism overnight at 35°C in MHB-S. The turbidity was adjusted to a 0.5 McFarland standard, and the sample was diluted into fresh MHB-S to achieve the desired inoculum. For the control curves, the organisms were incubated overnight; then 1 ml was added to fresh MHB-S and incubated at 35°C until growth was observed. The turbidity was adjusted to a 0.5 McFarland standard, and the sample was diluted to obtain the desired inoculum. Inoculum size was verified with nutrient agar pour plates. PAE. Comparison of the two methods for PAE determination was performed at twice the MIC at an inoculum of 5 x 105 CFU/ml by the following methods. (i) Broth technique. The reference tube macrodilution method was performed as described by Craig and Gudmundsson (4). For each organism, an inoculum of approximately 5 x 105 CFU/ml was prepared. The organisms were then exposed in duplicate to each of the antibiotics-ampicillin, ciprofloxacin, and tobramycin-at twice the MIC for 1 h at 35°C. After antibiotic exposure, a 1:100 dilution was made into fresh MHB-S, which was maintained at 35°C for

MATERIALS AND METHODS Organisms. Ten clinical isolates of E. coli were chosen based on their ampicillin susceptibility by standard disk diffusion methods (11). An organism with an inhibitory zone of greater than 14 mm was considered susceptible. E. coli *

Corresponding author. 450

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the duration of the test. This dilution step effectively negates antibiotic activity (2). The organisms were exposed to twice the MIC versus four to five times the MIC to avoid concentration effects on viable organisms. This is particularly apparent with tobramycin as a consequence of its rapid bactericidal effects (2). When the lower concentration was used, viable organisms remained after a 1:100 dilution into drug-free medium. Colony counts were determined with nutrient agar pour plates in the initial inoculum, upon dilution to remove the antibiotic, and every 15 min thereafter for 2 h with ampicillin-exposed organisms. For tobramycin and ciprofloxacin, colony counts were performed every 0.5 h after exposure to the antibiotic for 4 h to take into account the anticipated longer suppression of growth. The control growth rate for the same organism was determined under identical conditions without antibiotic exposure. Control colony counts were determined at 30-min intervals for 3 h. PAEs were determined as the time required for the CFU per milliliter in the antibiotic-exposed culture to increase by 1 log1o above the count immediately after removal of the antibiotic, minus the time required for the control culture to increase by 1 log1o. (H-) MS-2. A spectrophotometric method to measure PAEs was developed utilizing the Abbott MS-2 research system. The procedure involves an inoculum of approximately 5 x 105 CFU/ml exposed in duplicate to twice the MIC of ampicillin, tobramycin, and ciprofloxacin for 1 h at 35°C. A 1:100 dilution was made in MHB-S, and a portion of this diluted sample was used to determine viable colony counts immediately after antibiotic exposure. The diluted sample postexposure was added to a research cartridge in duplicate and incubated at 35°C with constant agitation in the MS-2. Transmittance readings were recorded for each well at 5-min intervals. An Apple Ile computer (Apple Computer, Inc., Cupertino, Calif.) was interfaced to the MS-2 system to measure the time required for a 5% decrease in transmittance for each well. A 5% decrease in transmittance was used to define the point at which detection of growth occurred. Any growth delays or abnormal growth rate resulting from PAE will alter the time required to reach the threshold concentration, or a 5% decrease in transmittance. To determine the PAE, the time for a given unexposed inoculum to reach threshold must be subtracted. Control growth was determined by placing an inoculum of approximately 5 x 105 CFU/ml into well 1 of the research cartridge and performing serial twofold dilutions in wells 2 through 10. The time required for a 5% decrease in transmittance was then measured for each well. A plot of time versus initial CFU per milliliter in each well was linear. Perpendicular least-squares regression was used to determine the best-fit line, where the y-intercept represented the approximate detection limit of the MS-2 and the slope represented the instantaneous growth rate constant. The PAE was calculated as the time required for a 5% decrease in transmittance for the antibiotic-exposed organisms minus the time required for the same inoculum in the control to decrease 5% in transmittance. Effect of inoculum size. An inoculum of 5 x 103, 5 x 105, 5 x i07, and 1 x 109 CFU of each clinical isolate per ml was exposed to each antibiotic in duplicate in the same manner as described for the MS-2 method. All inocula were verified by using nutrient agar pour plates. To obtain a 1 x 109 inoculum, we allowed a fresh culture to reach a turbidity equal to that of a 1.0 McFarland standard. The resulting PAEs were compared at each of the inoculum levels. Concentration effect. Each isolate was exposed to two and

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six times the MIC of each antibiotic for 1 h in duplicate to determine the effect of varying exposure concentration. PAEs were determined in the same manner as described above for the MS-2 method. The PAEs were compared at each concentration. Statistics. The correlation of PAE results by each method was evaluated by the t test comparing the regression slope with a line with a slope of 1 at the 0.05 level of significance. Regression analysis of the two methods was performed by perpendicular least-squares estimation. Residuals from the regression line were evaluated by the chi-square goodnessof-fit test. An unpaired t test was used to evaluate differences between methods. Differences in PAE for different inoculum sizes were evaluated by analysis of variance at the 0.05 level of significance. Tukey's test was employed for individual differences within groups. A paired t test was performed on the PAEs resulting from exposure to two antimicrobial agent concentrations at the 0.05 level of significance. RESULTS The range of MICs of ampicillin for the clinical isolates was 1.56 to 3.13 ,ug/ml; of ciprofloxacin, 0.006 to 0.025 jig/ml; and of tobramycin, 0.39 to 1.56 ,ug/ml. PAE durations for both the reference and MS-2 method are shown in Table 1. The average inoculum for both methods was (5.1 + 2.6) x 105 CFU/ml (range, 2.8 x 105 to 1.7 x 106 CFU/ml). Mean PAEs were: for ampicillin, 5.4 ± 7.7 and 5.5 + 8.2 min; for ciprofloxacin, 69.7 ± 21.1 and 75.6 ± 30.6 min; and for tobramycin, 54.0 ± 19.6 and 85.3 ± 26.8 min for the reference and MS-2 methods, respectively. The methods did not differ for mean ampicillin and ciprofloxacin PAEs, while the differences in the mean PAE for tobramycin-exposed organisms were significant (P < 0.05). The average deviation from the mean for duplicate testing was 20% for the reference method and 10% for the MS-2 method. Within-day coefficient of variation was 13% for the reference method and 6% for the MS-2 method. Between-day variability (percent relative standard deviation) for both methods was large and ranged from 30 to 50%. A PAE of 20 min or less was considered insignificant owing to the limitations of the testing procedure. Negative numbers, sometimes encountered, having a value of 20 min or less were assigned a value of zero. The regression line of PAE by MS-2 and reference methods, brought through the origin, had a slope of 1.32 (Fig. 1). The slope, evaluated statistically, was not different from a slope of 1. If the two methods are equally able to predict PAE, the slope would theoretically be 1.0 with an intercept of 0. The residuals about the regression line were normally distributed, showing no systematic deviation from the line, suggesting both methods to be equally variable. Concentration effects on PAE. The PAE was determined after exposure to each antibiotic at two and six times the MIC to determine concentration effect utilizing the MS-2. The PAE was significantly prolonged at six times the MIC of ciprofloxacin and tobramycin (151 ± 78 and 108 ± 85 min, respectively) compared with twice the MIC (69 ± 38 and 66 ± 29 min, respectively; P < 0.025). One organism (EC1) was unevaluable because there was rio growth after exposure to tobramycin at six times the MIC. There was no significant effect of concentration on the PAE with ampicillin (4.18 ± 13 and 3.0 ± 10 min at two and six times the MIC, respectively). Inoculum effects on PAE. The PAE was inversely related to the exposed inoculum for ciprofloxacin and tobramycin.

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TABLE 1. Mean PAE

duration determined by the reference method and the MS-2 method for ampicillin ciprofloxacin, and tobramycin PAE (min)

Ampicillin

Ciprofloxacin

16.0 0.0 4.4 0.0 6.0 23.0 0.0 0.0 2.0 8.6 0.0

57.0 66.5

EC1 EC2 EC3 EC4 ECS EC6 EC7 EC8 EC9 EC10 ATCC 25922

Tobramycin

42.0 50.5 33.4 62.0 32.0 61.0 67.0 32.0 55.0 60.6 98.0

35.0 57.0 72.0 107.6 90.0 86.0 53.0 86.0 56.0

5.45 7.73

Mean SD a

MS-2 method

Reference method

Organism

53.95a 19.62

69.65 21.05

Ampicillin

Ciprofloxacin

0.0 5.7 14.7 7.0 4.7 0.0 0.0 0.0 0.0 2.0 26.0

98.5 37.3 65.0 54.3 52.7 80.0 153.3 66.7 72.0 81.3 70.3

5.46 8.23

Tobramycin

121.0 118.0 97.7 89.7 73.0 77.0 87.0 31.7 72.0 59.7 111.4

85.29a 26.83

75.58 30.63

Significant difference between mean values (P < 0.05).

Each organism was exposed to the antibiotics at various inocula (Fig. 2) ranging from 5 x 103 to 1 x 109 CFU/ml. The actual inoculum for the exposure of 1 x 109 CFU/ml was 3.3 x 10 CFU/ml. The difference in PAE for ciprofloxacin and tobramycin was significant for the range of inocula used when tested by analysis of variance by repeated measures design (P < 0.05). When the Tukey test was applied, a significant difference was observed for ciprofloxacin between inocula of 103 and 109 CFU/ml, and a significant difference was observed for tobramycin between inocula of 103 and 107 CFU/ml (P < 0.05). There was no effect of inoculum size on the ampicillin PAE. Three organisms were not included in the analysis of the 103 inoculum for tobramycin because they all failed to grow postexposure. DISCUSSION PAE describes the recovery period or the suppression of growth after short exposure to antimicrobial agents. Most

previous work for gram-negative organisms has been in vitro determination of the PAE. In general, beta-lactam antibiotics produce little or no PAE with gram-negative organisms, except in high concentrations and with imipenem (3, 4). Inhibitors of protein synthesis such as aminoglycosides and the quinolones which act on DNA gyrase generally produce the longest PAEs for gram-negative organisms (2, 4). The reference methods for measurement of in vitro PAE, the broth and membrane-agar techniques (4), are well accepted but tedious to perform. The broth technique (4) was used in this experiment. This involves sampling at 15- to 30-min intervals to determine growth kinetics compared with control or unexposed growth. To perform this method on one organism exposed to one antibiotic in duplicate with a control, the procedure requires approximately 140 plates, 1.7 liters of media, 50 serial dilutions, 6 h to perform, and an additional 2 h to read the plates after overnight incubation. The procedure may be shortened by using automated dilution and plating procedures. 200

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140

150

-120 * WI

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IC

00 20 0

103

105

1O07

109

Inocuhm (CFU/mi) 0

20

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60

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100

120

140

160

Reference Method PAE (minutes)

FIG. 1. Regression of PAE by the MS-2 method reference method (y = 1.319x; r = 0.741).

versus

the

FIG. 2. Effect of inoculum size on the duration of PAE. Data are represented as mean + standard deviation. Actual mean inocula for the 103, 10i, 107, and 109 ranges were 3.7 x 103, 3.7 x 105, 3.7 x 107, and 3.3 x 108 CFU/ml, respectively. , ampicillin; , ciprofloxacin; i1 , tobramycin.

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The MS-2 method was developed to minimize the effort expended to determine the PAE, to minimize the need for serial colony counting and its potential for variability, and to maximize the number of microorganisms and antibiotics that can be screened in a day. The method requires approximately 2 h of laboratory time to perform, 25 plates, 0.5 liter of media, eight serial dilutions, and 0.5 h to read the plates after overnight incubation, per organism and antibiotic in duplicate. Only initial colony counts after removal of the antibiotic and initial counts of the accompanying control are needed. Several investigators have described PAE measurement with the use of spectrophotometry (1, 13, 14). Previous work by Bergan et al. (1) described detection of the PAE with a model utilizing a photometric tube apparatus. In their report, there was a discrepancy between turbidity and colony counts. In our preliminary work for the MS-2 method, the 5% decrease in transmittance was found by parallel colony counts that range from 5 x 106 to 4 x 107 CFU/ml, the detection threshold of the MS-2. This was in agreement with our measured threshold in this study of 1.6 x 106 to 6.4 x 106 CFU/ml based on the control regression line. When the initial colony count was significantly below this threshold concentration, there was an apparent growth lag measured by the MS-2. During this apparent growth lag, log-phase growth may be present. It is particularly important to stress that with the utilization of this spectrophotometric method, the observed lag in growth is dependent on the initial CFU per milliliter as well as the growth rate of the organism during the monitoring period. One must measure and correct for a low initial CFU per milliliter postexposure, particularly with potent bactericidal drugs such as tobramycin, by using a control curve. The MS-2 method was used in this study to determine concentration effects on the PAE. The effect of concentration on PAE was first investigated by Eagle (5). He found that a PAE was only observed at concentrations of penicillin that were lethal to gram-positive microorganisms. An effect of concentration on the PAE has been reported for E. coli (2, 4) for tobramycin at most multiples of the MIC and for ampicillin at eight times the MIC. In the present experiment, there was a significant increase in the duration of the PAE at six times the MIC of tobramycin and ciprofloxacin. There was no effect on PAE with an ampicillin concentration six times the MIC. The effect of the inoculum size on the duration of PAE has been examined by Eagle and Musselman (6) with Staphylococcus aureus. Higher inocula were associated with slight decreases in the duration of PAE. McDonald et al. (10) also found a slight decrease in the duration of PAE with increasing inocula. Similar results were found in this study utilizing the MS-2. For ciprofloxacin and tobramycin, there was a significant decrease in PAE with increasing inocula. This was particularly apparent when evaluating an inoculum of 5 x 103 versus 5 x 107 and 1 x 109 CFU/ml (Fig. 2). Ampicillin lacked a PAE, so inoculum effects were not observed. There was considerable variability in both the reference and MS-2 methods. Factors accounting for this variability include the inherent error in serial colony-counting procedures, a between-day difference in inoculum, and true differences in growth rate or recovery period of the organism. When a 1 log1o increase in viable organisms was measured, ciprofloxacin- and ampicillin-exposed organisms generally resumed a logarithmic growth phase after the suppressive period resulting from antibiotic exposure. With tobramycin, however, the organisms did not resume the same growth as the control, but resumed a stunted or slower growth phase after the removal of the antibiotic throughout the study. In a

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separate experiment, there was no effect on growth rate when the organism was incubated with tobramycin concentrations that were 50- to 200-fold lower than the MIC. Thus, PAE in the reference method for tobramycin-exposed organisms may be underestimated because a 1 log1o increase in viable organisms may have occurred before resumption of the normal logarithmic growth rate. In the MS-2 method, having threshold growth detection at approximately 106 to 10' CFU/ml, the organisms resumed a normal growth rate. Therefore, PAE as predicted by resumption of normal growth may be more accurately estimated in the MS-2 method. These experiments defined an alternative, less laborious technique for the measurement of PAE. For the three antibiotics chosen, the MS-2 and the reference method are similar. There is considerable between-day variability found among the measurements for both methods, indicating a need for more refinement in this area. The MS-2 method allows a rapid screening for the PAE of several microorganisms and antibiotics at one time. This new technique can foster an expanded utilization of in vitro PAE in the clinical research environment. Further in vivo studies are needed to better define the clinical impact of PAE and the applicability of in vitro testing to design of antibiotic dosing regimens. LITERATURE CITED

1. Bergan, T., I. B. Carlson, and J. E. Fuglesang. 1980. An in vitro model for monitoring bacterial responses to antibiotic agents under simulated in vivo conditions. Infection 8:S96-5102. 2. Bundtzen, R. W., A. U. Gerber, D. L. Cohn, and W. A. Craig. 1981. Postantibiotic suppression of bacterial growth. Rev. In-

fect. Dis. 3:28-37. 3. Bustamante, C. I., G. L. Drusano, B. A. Tatem, and H. C. Standiford. 1984. Postantibiotic effect of imipenem on Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 26:678682. 4. Craig, W. A., and S. Gudmundsson. 1986. The postantibiotic effect, p. 515-536. In V. Lorian (ed.), Antibiotics in laboratory medicine, 2nd ed. The Williams & Wilkins Co., Baltimore. 5. Eagle, H. 1949. The recovery of bacteria from the toxic effects of penicillin. J. Clin. Invest. 28:832-836. 6. Eagle, H., and A. D. Musselman. 1949. The slow recovery of bacteria from the toxic effects of penicillin. J. Bacteriol. 58:475-490. 7. Gengo, F. M., T. W. Mannion, C. H. Nightingale, and J. J. Schentag. 1984. Integration of pharmacokinetics and pharmacodynamics of methicillin in curative treatment of experimental endocarditis. J. Antimicrob. Chemother. 14:619-631. 8. Gerber, A. U., P. Wiprachtiger, U. Stettler-Spichiger, and G. Lebek. 1982. Constant infusions vs. intermittent doses of gentamicin against Pseudomonas aeruginosa in vitro. J. Infect. Dis. 145:554-560. 9. McCormack, J. P., and J. J. Schentag. 1987. Potential impact of quantitative susceptibility tests on the design of aminoglycoside dosing regimens. Drug Intell. Clin. Pharm. 21:187-192. 10. McDonald, P. J., W. A. Craig, and C. M. Kunin. 1977. Persistent effect of antibiotics on Staphylococcus aureus after exposure for limited periods of time. J. Infect. Dis. 135:217-223. 11. National Committee for Clinical Laboratory Standards. 1984. Performance standards for antimicrobial disk susceptibility tests, 3rd ed. National Committee for Clinical Laboratory Standards, Villanova, Pa. 12. National Committee for Clinical Laboratory Standards. 1983. Methods for dilution susceptibility tests for bacteria that grow aerobically. National Committee for Clinical Laboratory Standards, Villanova, Pa. 13. Ravizzola, G., A. Caruso, N. Manca, E. Savoldi, and A. Turano. 1983. In vitro activity of cefotetan and other cephalosporins on Klebsiella and resistance to inactivating bacterial enzymes. J. Antimicrob. Chemother. ll(Suppl. A):133-138. 14. Shah, P. M. 1981. Bactericidal activity of ampicillin and amoxicillin. J. Antimicrob. Chemother. 8(Suppl. C):93-99.