Postantibiotic effect of aminoglycosides on Gram-negative bacteria evaluated by a new method

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1 Journal of Antimicrobial Chemotherapy (1988) 22, Postantibiotic effect of aminoglycosides on Gram-negative bacteria evaluated by a new method Barforo Isaksson'*, Lennart Nibson*, Rolf Mailer' and Lars Serin* Departments of Infectious Diseases' and Clinical Bacteriology, University Hospital, S Linkdping, Sweden The in-vitro postantibiotic effect (PAE) of amikacin, gentamicin, netilmitin and tobramycin was investigated by a bioluminescent assay of bacterial ATP. Two strains each of Escherichia coli and Pseudomonas aeruginosa were exposed for 1 h to different concentrations of the aminoglycosides. The aminoglycoside was removed by a 1 ~ 3 dilution, and regrowth of bacteria was followed at hourly intervals by monitoring bacterial ATP. This method simplified the PAE studies and made such studies possible at high aminoglycoside concentrations. The length of the PAE was dose-dependent for all the aminoglycosides studied. The PAEs ranged between three and seven hours for all four strains at the aminoglycoside concentrations normally reached in serum during standard dosing. The long PAE of aminoglycosides, especially after exposure to high drug concentrations, constitutes an argument in favour of administering aminoglycosides in higber-than-usual doses with longer intervals between doses. This proposal is also supported by recent pharmacokinetic, bacteriological and toxicity data. Introduction The postantibiotic effect (PAE) is the phenomenon of suppression of bacterial growth after a short exposure of bacteria to antimicrobials. The term emphasizes that the effect is due to prior antimicrobial exposure rather than to the action of persisting subinhibitory concentrations of a drug (McDonald, Craig & Kunin, 1977). The PAE has been consistently observed for many antibiotics with Gram-positive cocci, but major differences in the PAE are observed for the various classes of antimicrobials with Gram-negative bacteria. Several reports have shown that aminoglycosides and other antibiotics have a PAE on Gram-negative bacteria (Bigger, 19; Eagle, 199; Eagle & Musselman, 199; Bundtzen et al., 1981; McDonald, Craig & Kunin, 1976; Shah, Hubener & Stille, 1978; Bergan & Carlsen, 198; Gerber & Craig, 1981). In investigating the PAE, researchers have generally used viable counts to follow regrowth after drug removal. This method is laborious and, in the case of aminoglycosides, not practical because of the extensive decrease in viability, especially after exposure to high concentrations of the drug. We have developed a new method for determination of the PAE, based on the bioluminescent assay of bacterial ATP (Nilsson, 1978). This is a rapid and sensitive method which can be used for PAE determination of aminoglycosides as well as of other antibiotics. * Corresponding author /88/ $2./ 1988 The British Society for Antimicrobial Chemotherapy Downloaded from at Pennsylvania State University on February 2, 216

2 2 B. Isaksson et al Bacterial strains Materials and methods Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, E. coli LU 2 and P. aeruginosa LU 538 were used. The last two strains were clinical isolates from blood cultures. Antibiotics Aqueous stock solutions of active aminoglycosides (1 mg/1) were prepared from gentamicin sulphate (656/ig/mg; Schering Corp., Blomfield, NJ, USA), netilmicin sulphate (66 /ig/mg; Schering Corp.), tobramycin sulphate (92 /ig/mg; Eli Lilly and Co., Indianapolis, IN, USA) and amikacin base (91 /ig/mg; Bristol Laboratories, Syracuse, NY, USA). These stock solutions were stored frozen. Growth medium Mueller-Hinton Broth (Difco Laboratories, Detroit, Michigan, USA) supplemented with 25 mg/1 Mg 2+ and 5 mg/1 Ca 2+ (Washington & Sutter, 198) was used as the growth medium. MIC determinations Serial two-fold dilutions of aminoglycosides were prepared in the growth medium. Samples of these dilutions (-5 ml) were added to series of test tubes. Bacterial strains in logarithmic phase were diluted to approximately 1 5 cfu/ml and -5 ml samples of these cultures were added to the tubes, which were then incubated at 37 C. Visible growth was recorded after 2 h. Determination of bacterial viability Bacterial numbers were determined as cfu per millilitre by plating after serial dilution (Nilsson & Soren, 1986). Bioluminescence assay of intracellular bacterial A TP Analytical equipment. Light emission from the bioluminescent assay was measured in a 125 Luminometer (LKB-Wallac, Turku, Finland) and recorded on a 125 Display (LKB-Wallac). The extraction of bacterial ATP was performed in a LKB-Biocal 273 incubator (LKB Products, Bromma, Sweden). Analytical reagents. ATP-monitoring reagent (LKB-Wallac) was used in the assay of ATP. Apyrase (purified grade I) (Sigma Chemical Co., St. Louis, Mo, USA) was used to eliminate extracellular ATP before the extraction of intracellular ATP. Other reagents were of analytical grade. Elimination of extracellular ATP. A 5-/il sample from the culture was incubated for 1 min at 37 C with 5 /il of an apyrase solution consisting of % apyrase in supplemented Mueller-Hinton Broth (Difco). Extraction of intracellular ATP. After elimination of extracellular ATP, 5 (A of the apyrase-treated sample was pipetted into 5/il of boiling 1 M Tris buffer, ph 7-75, Downloaded from at Pennsylvania State University on February 2, 216

3 PAE of aminoglycosides 25 containing 2 mm EDTA. After heating for 9 sec, the extracts were cooled before the assay of ATP was performed. This procedure inactivated the apyrase and disrupted the bacterial cells, causing them to release their ATP. Luciferase assay of ATP. Luciferase reagent (1/xl) was added to 55/zl of each extract and the light intensity was recorded. Calculation of assay results. Sample ATP levels were calculated by using assays of standard amounts of ATP as references. Correction was made for background luminescence. Known amounts of ATP added to the extracts were used as internal standards in order to correct for inhibition of the luciferase reaction by the extracts. Determination of PAE with bioluminescence Logarithmic phase bacteria were obtained by incubating the bacterial strains in growth medium for 8-1 h without shaking. The bacterial numbers were monitored by bioluminescent assay of bacterial ATP (1" 6 M ATP corresponding to approximately 1 9 cfu/ml) and the cultures were diluted to approximately 1 7 cfu/ml. Samples of these cultures (-5 ml) were added to tubes containing different concentrations of aminoglycoside in -5 ml of growth medium and the tubes were incubated at 37 C for 1 h without shaking. After removing samples for viable count and ATP determination, the cultures were diluted 1" 3 with prewarmed growth medium. Each experiment included an unexposed control culture prepared and treated as above. Regrowth of bacteria was followed once per hour in the diluted cultures and was monitored by bioluminescent assay of bacterial ATP. Each experiment was repeated three to six times. The PAE was calculated from the regrowth curves with the equation PAE = T C, where T was the time required for the bacterial population in the test culture to increase by 2 log 1 after dilution of the drug, and C was the time required for the bacterial population in the control culture to increase by 2 logj. The ATP levels in aminoglycoside-exposed cultures immediately after 1" 3 dilution were below the detection limit. Therefore the ATP was measured before dilution, and the values obtained, divided by 1 3, were used as the starting values in these calculations. Normally the PAE is calculated on the basis of the time taken for the bacterial numbers to increase by 1 log 1. However, the ATP values after 1 log 1 growth were just above the detection limit. We therefore chose to use the time taken for bacterial numbers, as measured by ATP, to increase by 2 log 1, which we considered adequate since the growth curves for drug-treated and untreated bacteria were parallel once the growth had started. MIC values Results The MIC values of amikacin, gentamicin, netilmicin and tobramycin for the E. coli and P. aeruginosa strains are shown in Table I. Downloaded from at Pennsylvania State University on February 2, 216 Effect of residual antibiotic When studying PAE it is important to ascertain that the dilution after drug exposure is sufficient to prevent the residual drug from affecting bacterial growth. The effects of

4 26 B. Isaksson et al Table I. MIC's of the aminoglycosidcs for the bacterial strains MIC (mg/1) Organism Amikacin Gentamicin Netihnicin Tobramycin E. coli ATCC E. coli LU 2 P. aeruginosa ATCC P. aeruginosa LU the residual aminoglycosides, after 1 2 and 1~ 3 dilutions, on the growth of P. aeruginosa LU 538 and E. coli LU 2, are shown in Figure 1A and B, respectively. After 1 " 2 dilution the residual aminoglycoside still substantially suppressed growth of P. aeruginosa LU 538, even though the aminoglycoside concentration was far below Downloaded from at Pennsylvania State University on February 2, 216 Figure 1 (a) Regrowth or P. aeruginosa LU 538 and its suppression by residual drug, monitored by assay of bacterial ATP after KT 1 dilution of cultures exposed to 6 (A), ( ) 16 ( ), 8 (V), (O), 2 (*) and ( ) mg/1 umiltbrin for I h. (b) Regrowth of E coli LU 2, and its suppression by residual drug, monitored by assay of bacterial ATP after 1"' dilution of cultures exposed to 6 (A), (D). 16 (-it), 8 (V), (O)> 2 (%) and (A) mg/1 amikacin for 1 h.

5 PAE of aminoglycosides 27 TaWe II. Decrease in viability (cfu/ml) in cultures (initially 5 x 1* to 1 7 cfu/ml) of E. coli ATCC 25922, E. coli LU 2, P. aeruginosa ATCC 27852, P. aeruginosa LU 538 exposed to amikacin, gentamicin, netilmicin and tobramycin for 1 h. Cfu/ml remaining after 1 h exposure to antibiotic Concentration E. coli E. coli P. aeruginosa P. aeruginosa Antibiotic (mg/1) ATCC LU 2 ATCC LU 538 Amikacin Gentamicin Netilmicin Tobramycin x1" 7-7 x OxlO 2 1-x1" 1-x1" 1-8 xlo 2 -x1" -3 x 1* 1- x 1 2 * 2- x 1 2 * 6 x Ox 1 2 * 2- x 1 2 * 1-Ox x Ox 1 2 * 1-2 x 1 2 ' 2-5 x 1 2 * 6-2 x 1 2 * 7 x 1 2 * 3-2 x x x x x x 1 2 * 1-3 x 1* 7 x 1 x 1" 3 x 1" 31 x 1 2 * 5-5 x xlo 2 - xlo x OxlO x xlo 3 1-5x1* 3- x 1' 3-OxlO 3 1-6x1* 1- xlo 3 1-2x1* -x1" 5-OxlO x xlo 3 'No viable bacteria was found when the sample from the bacterial culture was dropped on the agar plate. The indicated bacterial numbers were only obtained when the sample was spread over the whole recovery plate surface. the MIC value for the strain (- mg/1) (Figure l(a)). No suppression of bacterial growth was observed after a 1" 3 dilution, even with the more-amikacin-sensitive E. coli LU 2 strain, for which the MIC value was 1 mg/1 (Figure l(b)). Decrease in viability An extensive decrease in viability after a one-hour exposure to the drug was observed for all the aminoglycosides and bacterial strains investigated (Table II). The bactericidal effect increased with the aminoglycoside concentration. When samples from the cultures were dropped on the agar plate, no viable bacteria were found at high concentrations of the aminoglycosides. However, when the sample volume was spread over the whole agar surface (thereby diluting the carried-over antibiotic, viable bacteria were recovered from some of the cultures (Table II). Downloaded from at Pennsylvania State University on February 2, 216 Bacterial regrowth When the bacterial numbers were determined by ATP measurements, the initial decrease of intracellular ATP was not as extensive as that of viability. Regrowth of the E. coli and P. aeruginosa strains was monitored by assay of intracellular ATP after a one-hour exposure to the aminoglycosides, followed by drug

6 28 B. Isakssoo et al Downloaded from at Pennsylvania State University on February 2, Tim* (h) Figure 2. Cultures of. coli ATCC were exposed for one hour to amikacin (a), gcntamicin (b), netilmirin (c), and tobramycin (d) (6 ( ), ( ), 16 ( ), 8 (O), (A), 2 (A), 1 (*). -5 (^r) and (V) mg/1 aminoglycoside). Subsequently the antibiotic activity was eliminated by a 1* } dilution. The regrowth in these cultures were monitored by assay of bactenal ATP. elimination by means of a 1 3 dilution. The regrowth of E. coli ATCC is shown in Figure 2. The time interval before regrowth was initiated increased with aminoglycoside concentration in a dose-dependent manner but the growth rates were similar once regrowth had started.

7 Postantibiotic effect PAE of aminoglycosides 29 The PAEs of the four aminoglycosides for the four strains, after exposure for one hour, were calculated from the regrowth curves and are shown in Table III. Each value is based on three to six experiments. The duration of the PAE was dose-dependent for all the aminoglycosides studied. The figures in boxes in Table III correspond to those concentrations of aminoglycoside usually reached in serum with standard dosing, i.e. mg/1 amikacin, 8 mg/1 gentamicin, netilmicin and tobramycin. The PAEs for E. coli LU 2 at these concentrations was similar ( h) for all the aminoglycosides except netilmicin, which had a longer PAE (6-6 h). All the aminoglycosides produced similar PAEs on E. coli ATCC ( h) at these concentrations (Table III). At 16 mg/1 and mg/1, no regrowth occurred with gentamicin, netilmicin and tobramycin after incubation for 2 h, and this was confirmed by viable counts (Table III). The PAEs for gentamicin and netilmicin (8 mg/1) on the P. aeruginosa strains were h and the PAEs for amikacin ( mg/1) and tobramycin (8 mg/1) were --5- h and h, respectively (Table III). Regrowth in one of six cultures occurred after exposure to and 6 mg/1 amikacin, mg/1 netilmicin, and 16 mg/1 tobramycin for P. aeruginosa ATCC (Table III). No regrowth occurred for this strain after exposure to mg/1 gentamicin or tobramycin. Regrowth in one or two of six cultures occurred for P. aeruginosa LU 538 after exposure to mg/1 gentamicin or 8 mg/1 tobramycin (Table III). No regrowth occurred for this strain after exposure to 16 or mg/1 tobramycin (Table III). Discussion A postantibiotic effect (PAE) has been demonstrated for most antibiotics. The exact mechanism(s) for these effects are not known. In the case of aminoglycosides it may be due to binding of sub-lethal amounts of drug to the ribosomes leading to a subsequent disruption of protein synthesis. The PAE might represent the time needed for resynthesis of ribosomal proteins (Craig & Gudmundsson, 1986). The length of the PAE varies with the duration of antibiotic exposure and the antibiotic concentration. A maximal PAE is normally reached at a concentration of 1 x MIC of the antibiotic (Eagle & Musselman, 199; McDonald, Craig & Kunin, 1977; Shah, Hubener & Stille, 1978; Wilson & Rolinson, 1979; Bundtzen et al., 1981). In the majority of these earlier investigations the length of the PAE was determined by monitoring regrowth of the antibiotic-exposed bacteria by viable counts. This, however, is a laborious method and, in the case of aminoglycosides, it is difficult to assess the PAE by viable counts because of the extensive bactericidal effect of these antibiotics. After dilution of the antibiotic the number of surviving bacteria becomes so low that meaningful viable counts cannot be performed. The great decrease in viability after exposure to aminoglycosides was also seen in the present experiments (Table II). It is likely, however, that viable counts will exaggerate the bactericidal effect of antibiotics, since 'viability' depends not only on the numbers of surviving bacteria in the broth culture, but also on the ability of these surviving bacteria to form colonies on the agar plates. The growth of the bacteria may be suppressed by the aminoglycoside carried over with the dilutent to the agar plates. Furthermore, it is possible that bacteria that have aminoglycosides intracellularly, and on their cell surfaces, may die later, on the agar plates, or may need a period of Downloaded from at Pennsylvania State University on February 2, 216

8 3 B. Isaksson et al Table III. Post-antibiotic effect of aminoglycosides for two strains each of E. colt and P. aeruginosa. Concentration (mg/1) Post-antibiotic effect PAE (h) Mean (Range) Amikacin Gentamicin Netilmicin Tobramycin E. coli ATCC (-1--3) 2-5 (-1--7) 1- (-9-1-9) ( ) 16 1 ( ) 6 E. coli LU (-6-7-1) 7-8 ( ) -2 (-1--3) -3 (-2--) -8 (-7--8) 1- ( ) 2-2 (2-2) [TT ( ) 6-5 (5--8-5) P. aeruginosa ATCC (-2) -7 (-5-1) ( ) 16-5 ( ) 1-* 6 5-5* P. aeruginosa LU (1--6) 1- ( ) 8 31 ( ) 16-5 (3-8-5-) [5] (-2-6-5) 6 61 (5--7-) -5 (---5) 1-9 ( ) 3-3 (31-3-5).7 (-7--8) l&al ( ) -3 (--7) -8 (-6-1-1) 1- ( ) 2-2 (2--2-6) 3-lj (2-9-3-) (-1--) 1-7 (1-2-2-) [3] ( ) 5-8 ( ) 1- (-9-1-1) 1-9 ( ) [To] (3-9--3) 5-7 (5-1-6-) 5-5* 1 (-8-1-3) 2- ( ) 3-9 (3-5--5) 61 ( ) fmjl ( ) -9 (-7-1-) 1- (1--1-7) 2-2 (2-1-2-) 3 (2-9-3-) f^] ( ) -8 (-7-O-9) 1-7 (1--2-) 3-8] (2-8--6) -7 (-1-5-) 61 (-7-7-8) 1-2 (-9-1-5) 1- ( ) 2-9 ( ) -2 (3-8--8) [63] ( ) -3 (-2--5) -8 (-6-1-1) 1-5 ( ) 2- (1-7-2-) T9 (3-7--) - (-2--7) -9 (-8-1-) 2-5 (2--2-6) -7 (-6--8) i-2-5-5) 1-8 (-9-2-7) 5 ( ) 5-8 ( ) 6-9»* ( ) Downloaded from at Pennsylvania State University on February 2, 216, Not determined,, no regrowth,, regrowth in only 1 of 6 tubes, *, regrowth in 2 of 6 tubes. Boxed figures indicate peak serum levels. recovery before they can form colonies (i.e. there may be a PAE on the agar plates). This recovery is probably achieved more easily in broth than on the agar surface. This hypothesis is supported by findings from several experiments with high aminoglycoside concentrations where no colonies were seen on agar plates inoculated with samples

9 PAE of aminogtycosides 31 taken immediately after antibiotic exposure. However, when the samples were taken from the broth culture at later time intervals, increasing numbers of bacterial colonies were recovered. This means that there must have been some viable bacteria present immediately after antibiotic exposure but these were not readily detectable until after a period of recovery in antibiotic-free broth. This recovery phenomenon gives a false impression that there has been cell multiplication, and so can lead to an underestimation of the PAE. In the bioluminescent method the bacterial numbers were assayed from the intracellular ATP content of the sample, and it was assumed that the ATP content of the bacteria is relatively constant. This method constitutes a rapid and sensitive method for enumerating bacteria, and in growing cultures there is a good correlation between viability and intraccllular ATP (Molin, Nilsson & Ansehn, 1983). The bactericidal effect of the aminoglycosides, as measured by a decrease in bacterial ATP, was much less pronounced than that estimated by viable counts. This difference may be accounted for in part by the exaggerated bactericidal effect estimated by viability studies, and also by the possibility that both the ATP of surviving cells and the ATP of some dead, but still intact, bacteria are registered by the bioluminescent method. This implies that the post-exposure estimates of surviving bacteria will tend to be too high and that the PAEs recorded may be over-estimates. In spite of these methodological differences our PAE results at low concentrations of the aminoglycosides were consistent with those reported by other authors (Bundtzen et al., 1981; Craig & Gudmundsson, 1986). In addition, the bioluminescent method offered the possibility of determining the PAEs at high aminoglycoside concentrations, which cannot be achieved by viable counts. In the present experiments a dose-dependent PAE was seen at up to to 6 x MIC, but the effect varied with the different aminoglycosides and bacterial strains. At antibiotic levels attainable in serum after ordinary dosing, the PAE varied between three and seven hours for both E. coli and P. aeruginosa. This period is probably prolonged in vivo by the effect of the low concentrations of aminoglycosides remaining during the dosing interval. Our results (Figure l(a), (b)) showed that aminoglycosides at concentrations far below MIC suppressed the bacterial growth substantially. We have preliminary results showing that the PAE in vitro for amikacin, gentamicin, netilmicin and tobramycin is prolonged when small amounts of antibiotic, i.e. concentrations far below trough levels, are left after an incubation of one hour. Direct evidence that aminoglycosides induce longer PAEs in vivo than in vitro was presented earlier (Gudmundsson et al., 1983; Craig & Gudmundsson, 1986). The long PAE of aminoglycosides, especially after exposure to high antibiotic concentrations, supports alternative dosing schedules, i.e. larger doses with longer dosing intervals. This idea is also supported by the finding that organisms in the postantibiotic phase are more susceptible to the antibacterial activity of human leucocytes (McDonald, Wetherall & Pruul, 1981). Furthermore, dosing of aminoglycosides which is too frequent might even be less effective, since organisms seem to be less susceptible to aminoglycosides during the PAE-phase (Gerber & Craig, 1981). The time required to kill E. coli and Klebsiella pneumoniae with the aminoglycoside antibiotics was prolonged by several hours during the PAE-phase, and closely paralleled the duration of the PAE (Vogelman, Gudmundsson & Craig, 1983). An increasing number of in vitro studies, animal studies, and clinical trials suggest that, in the treatment of serious infections caused by Gram-negative pathogens, one Downloaded from at Pennsylvania State University on February 2, 216

10 B. Isiksson et al daily dose of aminoglycosides may be both equally effective and less toxic than traditional multiple dose regimens (Blaser, Stone & Zinner, 1985; Kapusnik & Sande, 1986; Gerber et al., 1983; Pechere & Bernard, 198; Powell et al., 1983; Thompson & Powell, 198). A once-daily dose regimen of aminoglycosides results in a high serum peak value (Mailer et al., 1988), a long PAE, possibly complete killing, and no regrowth of resistant variants (Soren & Nilsson, 198; Nilsson & Soren, 1986; Nilsson, Soren & Radberg, 1987). On the basis of these data it would seem important to confirm the efficacy of these dosing schedules in multicentrc studies on patients with systemic bacterial infections. References Bcrgan, T. & Carlsen, I. B. (198). Bacterial kill rates of amoxycillin and ampicillin at exponentially diminishing concentrations simulating in vivo conditions. Infection 8, Suppl. 1. S13-8. Bigger, J. W. (19). The bactericidal action of penicillin on Staphylococcus pyogenes. Irish Journal of Medical Science, No. 227, Blaser, J., Stone, B. B. & Zinner, S. H. (1985). Efficacy of intermittent versus continuous administration of netilmicin in a two-compartment in vitro model. Antimicrobial Agents and Chemotherapy 27, Bundtzen, R. W., Gerber, A. U., Conn, D. L. & Craig, W. A. (1981). Postantibiotic suppression of bacterial growth. Reviews of Infectious Diseases 3, Craig, W. A. & Gudmundsson, S. (1986). The postantibiotic effect. In Antibiotics in Laboratory Medicine, 2nd edn (Lorian, V. Ed.), pp Williams & Wilkins, New York. Eagle, H. (199). The recovery of bacteria from the toxic effects of penicillin. Journal of Clinical Investigation 28, 8-6. Eagle, H. & Musselman, A. D. (199). The slow recovery of bacteria from the toxic effects of penicillin. Journal of Bacteriology 58, Gerber, A. U. & Craig, W. A. (1981). Growth kinetics of respiratory pathogens after short exposures to ampicillin and erythromycin in vitro. Journal of Antimicrobial Chemotherapy 8, Suppl. C, Gerber, A. U., Craig, W. A., Brugger, H.-P., Feller, C, Vastola, A. P. & Brandel, J. (1983). Impact of dosing intervals on activity of gentamicin and ticarcillin against Pseudomonas aeruginosa in granulocytopenic mice. Journal of Infectious Diseases 17, Gudmundsson, S., Turnidge, J., Vogelman, B. & Craig, W. A. (1983). The post-antibiotic effect in vivo. In Proceedings of the 13th International Congress of Chemotherapy (K. H. Spitzy & K. Karrer, Eds), Part 117, pp Verlag H. Egerman, Vienna, Austria. Kapusnik, J. E. & Sande, M. A. (1986). Challenging conventional aminoglycoside dosing regimens. The value of experimental models. American Journal of Medicine 8, Suppl. 6B, Mailer, R., Isaksson, R., Nilsson, L. & Soren, L. (1988). A study of amikacin given once versus twice daily in serious infections. Journal of Antimicrobial Chemotherapy 22, McDonald, P. J., Craig, W. A. & Kunin, C. M. (1976) Brief antibiotic exposure and effect on bacterial growth. In Chemotherapy. Volume 2: Laboratory Aspects of Infections (Williams, J. D. & Geddes, A. M., Eds), pp Plenum, New York. McDonald, P. J., Craig, W. A. & Kunin, C. M. (1977). Persistent effect of antibiotics on Staphylococcus aureus after exposure for limited periods of time. Journal of Infectious Diseases 135, McDonald, P. J., Wetherall, B. L. & Pruul, H. (1981). Postantibiotic leukocyte enhancement: increased susceptibility of bacteria pretreated with antibiotics to activity of leukocytes. Reviews of Infectious Diseases 3, 38. Molin,., Nilsson, L. & Ansehn, S. (1983). Rapid detection of bacterial growth in blood cultures by bioluminescent assay of bacterial ATP. Journal of Clinical Microbiology 18, Downloaded from at Pennsylvania State University on February 2, 216

11 PAE of aminoglycosides 33 Nilsson, L. (1978). New rapid bioassay of gentamicin based on luciferase assay of extracellular ATP in bacterial cultures. Antimicrobial Agents and Chemotherapy 1, Nilsson, L. & Soren, L. (1986). Selective growth of resistant variants during incubation of Enterobacteriaceae with four aminoglycosides. Journal of Antimicrobial Chemotherapy 18, Nilsson, L., Soren, L. & Radberg, G. (1987). Frequencies of variants resistant to different aminoglycosides in Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 2, Pechere, J. C. & Bernard, P. A. (198). Gentamicin ototoxicity can be avoided if a new therapeutic regimen is used. An experimental animal model. In Proceedings of the Twenty- Fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 198. Abstract 8, p American Society for Microbiology, Washington, DC. Powell, S. H., Thompson, W. L., Luthe, M. A., Stern, R. C, Grossniklaus, D. A., Bloxham, D. D. et al. (1983). Once-daily vs. continuous aminoglycoside dosing: efficacy and toxicity in animal and clinical studies of gentamicin, netilmicin, and tobramycin. Journal of Infectious Diseases 17, Shah, P. M., Hubener, K.-G. & Stille, W. (1978). In-vitro-Untersuchungen zur intermittierenden Therapie mit Penicillin G und Ampicillin. Medizinische Welt 29, Soren, L. & Nilsson, L. (198). Regrowth of aminoglycoside-resistant variants and its possible implication for determination of MICs. Antimicrobial Agents and Chemotherapy 26, Thompson, W. L. & Powell, S. (198). Aminoglycoside nephrotoxicity in dogs: dependence on drug, dose and regimen. Scandinavian Journal of Infectious Diseases, Suppl. 23, Vogelman, B., Gudmundsson, S., Craig, W. & Middleton, W. S. (1983). Reduced susceptibility of bacteria during the postantibiotic effect (PAE) to tidal antimicrobials. In Program and Abstracts of the Twenty-Third Interscience Conference on Antimicrobial Agents and Chemotherapy, Las Vegas, Abstract 895, p. 29. American Society for Microbiology, Washington, DC. Washington, J. A. & Sutter, V. L. (198). Dilution susceptibility test: agar and macro-broth dilution procedures. In Manual of Clinical Microbiology, 3rd edn, (Lennette, E. H., Balows, A., Hausler, W. J. & Truant, J. P., Eds), pp American Society for Microbiology, Washington, DC. Wilson, D. A. & Rolinson, G. N. (1979). The recovery period following exposure of bacteria to penicillins. Chemotherapy 25, (Received 3 November 1987; revised version accepted 18 February 1988) Downloaded from at Pennsylvania State University on February 2, 216