Department of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong
Received 20 May 2004; returned 9 July 2004; revised 18 August 2004; accepted 18 August 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: In triplicate, baseline PAEs were evaluated by exposing E. coli to rifampicin and tobramycin individually and simultaneously for 1 h. PAEs were further assessed in a second study, with the organism exposed first to rifampicin for 1 h, followed by a second 1 h tobramycin exposure, commencing at the beginning, middle and end of the PAE phase induced by rifampicin. The third study was similar to the above, but with the sequence of the two antibiotics reversed, i.e. tobramycin then rifampicin.
Results: The PAE produced by simultaneous exposure of the combination showed an apparent additive interaction (PAE: 5.0±0.3 h) when compared with the PAE of individual antibiotics (rifampicin alone: 3.0±0.1 h; tobramycin alone: 1.5±0.1 h). However, an antagonistic interaction was observed in the second study, with a more pronounced degree of antagonism at the beginning, dissipating towards the end of the previous rifampicin PAE (PAE at the beginning: 2.6±0.3 h; the middle: 1.5±0.2 h; and at the end: 1.7±0.3 h). By subtracting the residual contribution from the first rifampicin exposure, the net average PAEs attributed to the second tobramycin exposure actually increased, from 0.4 to 1.7 h from the beginning to the end of the rifampicin PAE. For the third study, an additive interaction was again observed when the organism was exposed to tobramycin first (PAE at the beginning: 4.7±0.4 h; the middle: 3.7±0.7 h; and at the end: 3.1±0.4 h). The timing of the second rifampicin exposure had no impact to the interaction; after correction, the net mean PAEs attributed to the second rifampicin exposure were maintained at 3.2, 3.2 and 3.1 h.
Conclusions: The present data suggest that the expression of interaction type on PAE by an antibiotic combination was dependent on the mode, sequence and interval of exposure. The impact of these variables should not be overlooked when clinical dosing regimens are optimized.
Keywords: PAE , rifampicin , tobramycin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When antibiotics in combination are not administered simultaneously, it is typical that one antibiotic is administered prior to the other. Therefore, optimal antimicrobial responses should also consider the sequence and interval of dosing. At the time of this study, the authors were not aware of any published reports examining the impact of sequential dosing of antibiotic combinations on PAE. To gain further insights in this area of research, tobramycin and rifampicin were utilized in this study as a test combination against a standard strain of Escherichia coli. The selection of this antibiotic combination was based on its known mechanisms of action rather than its clinical relevance; tobramycin acts primarily by disrupting protein synthesisleading to altered cell membrane permeability and cell deathwhereas rifampicin inhibits DNA-dependent RNA polymerase activity in susceptible cells. As a result of the distinct mechanisms of action, the likelihood of a pharmacological interaction between the two antibiotics is high. In this study, particular attention was given to the three variables, e.g. mode, sequence and interval of exposure of the two antibiotics, and the relevance of their interactions to PAE.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lyophilized E. coli ATCC 25922, purchased from Difco Laboratories (Detroit, MI, USA), was used. After initial isolation, an organism from a single colony was maintained on agar slants at 4°C.
Culture media
MuellerHinton broth supplemented with 25 mg of Ca2 + and 12.5 mg of Mg2 + per litre (MHB-S) was used throughout. Nutrient agar (lot 73271JE) was employed for the colony count assay via a pour plate technique. Both culture media were purchased from Difco Laboratories, Detroit, MI, USA and were sterilized per manufacturer's instructions.
Antimicrobial agents
Rifampicin and tobramycin were purchased from Sigma Chemical Co., St. Louis, MO, USA. Aqueous stock antibiotic solutions were prepared and stored frozen at 20°C before use.
MIC
MICs of the two antibiotics and test organism were measured by the macrodilution technique17 after 18 h of incubation at 37°C.
Bacterial culture
The test organism maintained on an agar slant was transferred to 10 mL of MHB-S and incubated overnight at 37°C. This overnight culture was diluted with MHB-S and further incubated for 23 h at 37°C to enter logarithmic growth. The actively growing culture was subsequently diluted with MHB-S to achieve turbidity matching that of a 0.5 McFarland standard.
PAE assessments
To start the experiment, 0.1 mL of the adjusted culture at 0.5 McFarland was introduced to 9.9 mL of antibiotic-containing MHB-S to yield a total volume of 10 mL. Three sets of experiments, each in triplicate, were carried out; (1) E. coli was exposed to rifampicin and tobramycin individually and simultaneously for 1 h each, (2) the organism was first exposed to rifampicin for 1 h, followed by exposure for 1 h to tobramycin, at the beginning (T0; immediately after antibiotic removal of the first rifampicin exposure), middle (T1 = 2 h) and end (T2 = 4 h) of the first PAE induced by rifampicin and (3) the studies described in (2) were repeated, but with the sequence of the two antibiotics reversed, i.e. tobramycin, then rifampicin, with T0 also started immediately after removal of the first tobramycin exposure, and at the middle (T1 = 1 h) and end (T2 = 2 h) of the first PAE induced by tobramycin. With the anticipation of variability in the PAE measurements, the above time schedules would conform to the intended study conditions, i.e. exposure of the second antibiotic at the beginning, middle and end of the PAE of the first exposure. At the end of every 1 h exposure, the antibiotic(s) was removed by washing three times using sterile 0.9% saline and centrifugation at 1200 g for 10 min. For all studies, the concentrations of rifampicin and tobramycin were 25 mg/L (1.63.2 x MIC) and 2 mg/L (1 x MIC), respectively. These concentrations were established in our preliminary studies to limit the possibility of the viable bacterial densities in samples falling below the 200 cfu/mL limit of quantification over the entire study period for all conditions. Throughout the experiments, cultures were kept at 37°C using a calibrated water bath. Samples (0.1 mL) were withdrawn from the test culture hourly (up to 12 h) and submitted to the pour plate assay until steady growth was observed after removal of the last antibiotic exposure. PAE was determined as the difference between the time required for the viable count in the test culture to increase by one log unit immediately after antibiotic removal and the time required after the same procedure for the control culture. To avoid overgrowth of the control culture, dilution (10100-fold) using pre-warmed MHB-S was performed at the same time points as scheduled antibiotic exposures during the study. For clarity, data pertaining to the control cultures (all in simple logarithmic growth) are not shown in the Figures described in the Results section.
Data interpretation
Interpretation of the interaction expressed by the combination was based on the comparison of PAE ascribed to the antibiotic used in the second exposure and the PAE of that antibiotic alone. The combination was considered to be additive, antagonistic and synergistic when the former PAE was similar to, shorter and longer than the latter, respectively.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The MICs of rifampicin and tobramycin were 816 and 2 mg/L, respectively.
PAE
Following 1 h of antibiotic exposure, the mean (±S.D.) PAE estimates were 1.5 ± 0.1 h for tobramycin alone and 3.0 ± 0.1 h for rifampicin alone. Simultaneous exposure to both antibiotics for 1 h resulted in a PAE of 5.0 ± 0.3 h. Figure 1 shows the changes in bacterial density over time under the three exposure conditions. The combination would, therefore, be considered additive. Both the PAE and extent of bacterial killing were greatest when the organism was exposed to the combination in comparison to the antibiotics alone. In addition, the extent of bacterial density reduction produced by the combination immediately before antibiotic removal was approximately the sum of those by the two antibiotics alone.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The effects of sequential dosing on the pharmacodynamic behaviour of the two study antibiotics have been demonstrated in this study. As the present PAE data reveal, the conclusion on the interaction type can vary depending on the experimental conditions for the same pair of antibiotics. When the test organism was exposed to the test combination simultaneously, the interaction appeared to be additive. However, exposure first to rifampicin antagonized the PAE expressed by the second (tobramycin) exposure, and such antagonism was time-dependent during the PAE of rifampicin. On the other hand, when the organism was exposed first to tobramycin, the second (rifampicin) did not elicit similar antagonism during the PAE of tobramycin, but rather an additive interaction with no time-dependency.
In terms of bacterial count, simultaneous exposure to the antibiotic combination produced the most extensive reduction as compared with each antibiotic alone. The extent of reduction, similar to PAE, appeared to be additive in nature (Figure 1). However, when rifampicin was applied first, the bactericidal effect exhibited by the tobramycin (applied second) increased along with a lower degree of antagonism expressed on PAE when it was introduced towards the end of the rifampicin PAE (Figure 2). In the third set of experiments, the similar and parallel reduction in bacterial count produced by the second rifampicin exposures was consistent with the additive effect observed on PAE when exposure to tobramycin was first (Figure 3).
Interpretation of these data requires a broader understanding of the pharmacological activities of the two antibiotics. Rifampicin and tobramycin act on different bacterial targets; however, the former exhibits a relatively strong bacteriostatic effect against the test organism, whereas the latter is more bactericidal. Previous studies showed that bacteriostatic agents antagonize both bactericidal activity and the PAE of bactericidal agents.18,19 Current data from the second set of studies point in the same direction, i.e. antagonism was observed when exposure to rifampicin preceded that to tobramycin, and it was not apparent when the sequence of exposure was reversed or exposure to the two antibiotics was simultaneous.
In regard to the timing of the second antibiotic exposure, the complete pharmacodynamic profile has to be considered. Data showing the longest delay in bacterial regrowth from each of the three sets of experiments are presented in Figure 4. Although the test organism received a total of 1 h of rifampicin exposure plus 1 h of tobramycin exposure in all cases, the sequence of rifampicin/tobramycin (tobramycin exposure at the end of the rifampicin PAE) delayed bacterial regrowth for the longest time, whereas the delay was shortest when exposure was simultaneous. This suggests that the former was pharmacodynamically the most attractive. In terms of the extent of bacterial density reduction over the study period, the same conclusion can be drawn. The positive relationship observed between the extent of bacterial killing and the length of PAE is in good agreement with the data we reported previously.20 In summary, present data support the need to consider the mode, sequence as well as interval of exposure when an antibiotic combination is used because all these factors contribute to the two pharmacodynamic attributes studied, i.e. bactericidal activity and PAE. More importantly, these factors should not be overlooked when antibiotic combinations are utilized in the clinic.
|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Mandal, S., Mandal, M. D. & Pal, N. K. (2003). Combination effect of ciprofloxacin and gentamicin against clinical isolates of Salmonella enterica serovar Typhi with reduced susceptibility to ciprofloxacin. Japanese Journal of Infectious Diseases 56, 1567.[ISI][Medline]
3
.
Sweeney, M. T. & Zurenko, G. E. (2003). In vitro activities of linezolid combined with other antimicrobial agents against Staphylococci, Enterococci, Pneumococci, and selected gram-negative organisms. Antimicrobial Agents and Chemotherapy 47, 19026.
4 . Critchley, I. A., Sahm, D. F., Kelly, L. J. et al. (2003). In vitro synergy studies using aztreonam and fluoroquinolone combinations against six species of Gram-negative bacilli. Chemotherapy 49, 448.[CrossRef][ISI][Medline]
5
.
Jung, R., Husain, M., Choi, M. K. et al. (2004). Synergistic activities of moxifloxacin combined with piperacillin-tazobactam or cefepime against Klebsiella pneumoniae, Enterobacter cloacae, and Acinetobacter baumannii clinical isolates. Antimicrobial Agents and Chemotherapy 48, 10557.
6 . Erdem, I., Kucukercan, M. & Ceran, N. (2003). In vitro activity of combination therapy with cefepime, piperacillin-tazobactam, or meropenem with ciprofloxacin against multidrug-resistant Pseudomonas aeruginosa strains. Chemotherapy 49, 2947.[CrossRef][ISI][Medline]
7
.
Dawis, M. A., Isenberg, H. D., France, K. A. et al. (2003). In vitro activity of gatifloxacin alone and in combination with cefepime, meropenem, piperacillin and gentamicin against multidrug-resistant organisms. Journal of Antimicrobial Chemotherapy 51, 120311.
8
.
Jacqueline, C., Caillon, J., Le Mabecque, V. et al. (2003). In vitro activity of linezolid alone and in combination with gentamicin, vancomycin or rifampicin against methicillin-resistant Staphylococcus aureus by time-kill curve methods. Journal of Antimicrobial Chemotherapy 51, 85764.
9
.
Gunderson, B. W., Ibrahim, K. H., Hovde, L. B. et al. (2003). Synergistic activity of colistin and ceftazidime against multiantibiotic-resistant Pseudomonas aeruginosa in an in vitro pharmacodynamic model. Antimicrobial Agents and Chemotherapy 47, 9059.
10
.
Fish, D. N., Choi, M. K. & Jung, R. (2002). Synergic activity of cephalosporins plus fluoroquinolones against Pseudomonas aeruginosa with resistance to one or both drugs. Journal of Antimicrobial Chemotherapy 50, 10459.
11 . Kato, K., Iwai, S., Sato, T. et al. (2002). In-vitro activity of ciprofloxacin combined with flomoxef against Bacteroides fragilis, compared with that of ciprofloxacin combined with clindamycin. Journal of Infection and Chemotherapy 8, 1903.[CrossRef][Medline]
12 . Hostacka, A. (1997). Comparison of postantibiotic effects of imipenem and netilmicin alone and in combination against Pseudomonas aeruginosa. Arzneimittelforschung 47, 9657.[ISI][Medline]
13 . Sood, P., Mandal, A. & Mishra, B. (2000). Postantibiotic effect of a combination of antimicrobial agents on Pseudomonas aeruginosa. Chemotherapy 46, 1736.[CrossRef][ISI][Medline]
14
.
Chan, C. Y., Au-Yeang, C., Yew, W. W. et al. (2001). Postantibiotic effects of antituberculosis agents alone and in combination. Antimicrobial Agents and Chemotherapy 45, 36314.
15 . Nyhlen, A., Ljungberg, B., Nilsson-Ehle, I. et al. (2002). Postantibiotic effect of meropenem and ciprofloxacin in the presence of 5-fluorouracil. Chemotherapy 48, 1828.[CrossRef][ISI][Medline]
16 . Gudmundsson, S., Erlendsdottir, H., Gottfredsson, M. et al. (1990). The postantibiotic effect induced by antimicrobial combinations. Scandinavian Journal of Infectious Diseases Supplementum 74, 8093.[Medline]
17 . National Committee for Clinical Laboratory Standards. (1993). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyThird Edition: Approved Standard M7-A3. NCCLS, Villanova, PA, USA.
18 . Gudmundsson, S., Vogelman, B. & Craig, W. A. (1994). Decreased bactericidal activity during the period of the postantibiotic effect. Journal of Antimicrobial Chemotherapy 34, 92130.[Abstract]
19 . Li, R. C., Schentag, J. J. & Nix, D. E. (1993). The fractional maximal effect method: a new way to characterize the effect of antibiotic combinations and other nonlinear pharmacodynamic interactions. Antimicrobial Agents Chemotherapy 37, 52331.[Abstract]
20 . Li, R. C., Lee, S. W. & Kong, C. H. (1997). Correlation between bactericidal activity and postantibiotic effect for five antibiotics with different mechanisms of action. Journal of Antimicrobial Chemotherapy 40, 3945.[Abstract]
|