ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1992, p. 1852-1858 0066-4804/92/091852-07$02.00/0
Vol. 36, No. 9
Copyright X) 1992, American Society for Microbiology
Postantibiotic Sub-MIC Effects of Vancomycin, Roxithromycin, Sparfloxacin, and Amikacin INGA ODENHOLT-TORNQVIST,* ELISABETH LOWDIN, AND OTTO CARS Department of Infectious Diseases, Uppsala University, Uppsala, Sweden Received 14 January 1992/Accepted 15 June 1992
The sub-MIC effects (SMEs) and the postantibiotic sub-MIC effects (PA SMEs) of vancomycin, roxithromycin, and sparfloxacin for Streptococcus pyogenes and Streptococcus pneumoniae and of amikacin for Escherichia coli and Pseudomonas aeruginosa were investigated. A postantibiotic effect was induced by exposing strains to 10x the MIC of the antibiotic for 2 h in vitro. After the induction, the exposed cultures were washed to eliminate the antibiotics. Unexposed controls were treated similarly. Thereafter, the exposed cultures (PA SME) and the controls (SME) were exposed to different subinhibitory concentrations (0.1, 0.2, and 0.3 x the MIC) of the same drug and growth curves for a period of 24 h were compared. In general, the PA SMEs were much more pronounced than the SMEs. However, for amikacin and E. coli the SME of 0.2 and 0.3 x the MIC also had an initial bactericidal effect. The longest PA SMEs were demonstrated for the combinations with the most pronounced killing during the induction and for the combinations which exhibited the longest PAEs. (This material was presented in part at the 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 29 September to 2 October 1991.)
In the early 1940s, the dosage schedules devised for penicillin therapy were based on the assumption that drug concentrations must be maintained above the MIC. This assumption was based on previous experiments with the sulfonamides (1, 26). However, a few years after the introduction of penicillin, it was shown both in animal experiments and in clinical studies that a discontinuous penicillin therapy was as effective as a continuous one, even if the concentrations in serum had fallen under the MIC (4, 26, 28). At that time, Eagle et al. showed, both in vitro and in a thigh infection model in mice, that there was a lag phase before regrowth of bacteria after a first challenge of penicillin (5, 6). This concept aroused new interest in the 1970s and was named the postantibiotic effect (PAE) (3, 14, 33). The PAE has been extensively studied both in vitro and in vivo and has been cited as one explanation for the success of intermittent dosing regimens (3, 32). However, in most antibioticbacterium combinations the sum of the time that the drug concentration is above the MIC and the PAE does not cover the whole dosing interval. Also, in the in vivo situation, a suprainhibitory concentration of a drug will always be followed by subinhibitory concentrations (sub-MICs). We have shown earlier that subinhibitory antibiotic concentrations may have different effects on bacteria exposed previously to suprainhibitory antibiotic concentrations (postantibiotic sub-MIC effect [PA SME]), compared with the effects on bacteria not previously exposed to antibiotics (sub-MIC effect [SME]) (18, 20). In most of the investigated combinations with P-lactam antibiotics, we found a pronounced difference in time between the PA SME and the SME, with two exceptions. In the combinations with no PAE, neither a PA SME nor an SME was found, and in the combination of imipenem with Pseudomonas aeruginosa, both a long PA SME and an SME were seen (20). The aim of this study was to investigate the occurrence of PA SMEs and SMEs for compounds other than ,B-lactam antibiotics, such as vancomycin, roxithromycin, sparfloxacin, and amikacin. *
MATERIALS AND METHODS Cultures. The strains used in this study were as follows: Streptococcus pyogenes, group A, M12, NCTC P 1800, Streptococcus pneumoniae ATCC 6306, P. aeruginosa ATCC 27853, and Escherichia coli ATCC 25922. The grampositive strains were grown in Todd-Hewitt broth, and the gram-negative strains were grown in Mueller-Hinton broth supplemented with 50 mg of Ca2" and 25 mg of Mg2+ per liter. The gram-positive strains were cultured for 6 h at 37°C in 5% CO2 in air, resulting in approximately 5 x 10' CFU/ml, and the gram-negative strains were cultured for 6 h at 37°C, resulting in approximately 109 CFU/ml. Antibiotics. The antibiotics were obtained as reference powders with known potencies from the following pharmaceutical companies: vancomycin from Eli Lilly Sweden AB, Stockholm, Sweden; roxithromycin from Roussel Nordiska AB, Stockholm, Sweden; sparfloxacin from Rhone-Poulenc Rorer, Helsingborg, Sweden; and amikacin from BristolMyers Squibb, Bromma, Sweden. Dilutions were made in distilled water on the day the experiments were performed. Determination of MICs. MICs were determined in fluid media by using a twofold dilution with an inoculum of approximately 105 CFU of the test strain per ml and were read after 24 h. The MIC was defined as the lowest concentration of the antibiotic allowing no visible growth. Determinations of MICs were performed in triplicate on separate occasions. Since the MIC of amikacin for E. coli ATCC 25922 was higher than that reported by other authors (see Results), the MIC for this combination was determined five times on different occasions. Induction of the postantibiotic phase and determination of the PAE. The following antibiotic-bacterium combinations were studied: vancomycin with S. pyogenes group A, M12, NCTC P 1800 and with S. pneumoniae ATCC 6306; roxithromycin with S. pyogenes group A, M12, NCTC P 1800 and with S. pneumoniae ATCC 6306; sparfloxacin with S. pyo-
Corresponding author. 1852
POSTANTIBIOTIC SUB-MIC EFFECTS
VOL. 36, 1992
1853
TABLE 1. PAE, PA SME, and SME for different antibiotic-bacterium combinations. Antibiotic-bacterium combination
O.1x
the MIC
Duration (h) of effect' 0.2x the MIC
SME
PA SME
SME
3.5 (2.9-3.8) 0 (0-0.1)
5.1 (3.6-6.0)
0 (0-0.2)
Vancomycin-S. pneumo-
3.0 (1.8-4.5) 0 (-0.5-0.2)
8.8 (8.5-8.8)
1.6 (1.2-2.2)
niae ATCC 6306 RoXithromycin-S. pyogenes M12, NCTC P 1800
5.0 (4.4-6.0) 0.1 (0-0.3)
6.3 (5.4-10.2)
0.1 (0-0.2)
Vancomycin-S. pyogenes
PA SME
6.8 (5.4-10.6)
M12, NCTC P 1800
RoXithromycin-S. pneumoniae ATCC 6306
Sparfloxacin-S. pyogenes M12, NCTC P 1800 Sparfloxacin-S. pneumo-
0.3x the MIC
PAE
>7.8-22.3) 0.5 (-0.4-0.6) 2.2 (1.5-2.4)
SME
' PA SME
22.7) >9.7-22.6) 2.0 (1.9-2.3) >21.3 (>21.3) 0.1 (0-0.5)
>9.7-9.7-7.8-7.8-22.3 (>22.3) >9.3-
Vol. 36, No. 9
Copyright X) 1992, American Society for Microbiology
Postantibiotic Sub-MIC Effects of Vancomycin, Roxithromycin, Sparfloxacin, and Amikacin INGA ODENHOLT-TORNQVIST,* ELISABETH LOWDIN, AND OTTO CARS Department of Infectious Diseases, Uppsala University, Uppsala, Sweden Received 14 January 1992/Accepted 15 June 1992
The sub-MIC effects (SMEs) and the postantibiotic sub-MIC effects (PA SMEs) of vancomycin, roxithromycin, and sparfloxacin for Streptococcus pyogenes and Streptococcus pneumoniae and of amikacin for Escherichia coli and Pseudomonas aeruginosa were investigated. A postantibiotic effect was induced by exposing strains to 10x the MIC of the antibiotic for 2 h in vitro. After the induction, the exposed cultures were washed to eliminate the antibiotics. Unexposed controls were treated similarly. Thereafter, the exposed cultures (PA SME) and the controls (SME) were exposed to different subinhibitory concentrations (0.1, 0.2, and 0.3 x the MIC) of the same drug and growth curves for a period of 24 h were compared. In general, the PA SMEs were much more pronounced than the SMEs. However, for amikacin and E. coli the SME of 0.2 and 0.3 x the MIC also had an initial bactericidal effect. The longest PA SMEs were demonstrated for the combinations with the most pronounced killing during the induction and for the combinations which exhibited the longest PAEs. (This material was presented in part at the 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 29 September to 2 October 1991.)
In the early 1940s, the dosage schedules devised for penicillin therapy were based on the assumption that drug concentrations must be maintained above the MIC. This assumption was based on previous experiments with the sulfonamides (1, 26). However, a few years after the introduction of penicillin, it was shown both in animal experiments and in clinical studies that a discontinuous penicillin therapy was as effective as a continuous one, even if the concentrations in serum had fallen under the MIC (4, 26, 28). At that time, Eagle et al. showed, both in vitro and in a thigh infection model in mice, that there was a lag phase before regrowth of bacteria after a first challenge of penicillin (5, 6). This concept aroused new interest in the 1970s and was named the postantibiotic effect (PAE) (3, 14, 33). The PAE has been extensively studied both in vitro and in vivo and has been cited as one explanation for the success of intermittent dosing regimens (3, 32). However, in most antibioticbacterium combinations the sum of the time that the drug concentration is above the MIC and the PAE does not cover the whole dosing interval. Also, in the in vivo situation, a suprainhibitory concentration of a drug will always be followed by subinhibitory concentrations (sub-MICs). We have shown earlier that subinhibitory antibiotic concentrations may have different effects on bacteria exposed previously to suprainhibitory antibiotic concentrations (postantibiotic sub-MIC effect [PA SME]), compared with the effects on bacteria not previously exposed to antibiotics (sub-MIC effect [SME]) (18, 20). In most of the investigated combinations with P-lactam antibiotics, we found a pronounced difference in time between the PA SME and the SME, with two exceptions. In the combinations with no PAE, neither a PA SME nor an SME was found, and in the combination of imipenem with Pseudomonas aeruginosa, both a long PA SME and an SME were seen (20). The aim of this study was to investigate the occurrence of PA SMEs and SMEs for compounds other than ,B-lactam antibiotics, such as vancomycin, roxithromycin, sparfloxacin, and amikacin. *
MATERIALS AND METHODS Cultures. The strains used in this study were as follows: Streptococcus pyogenes, group A, M12, NCTC P 1800, Streptococcus pneumoniae ATCC 6306, P. aeruginosa ATCC 27853, and Escherichia coli ATCC 25922. The grampositive strains were grown in Todd-Hewitt broth, and the gram-negative strains were grown in Mueller-Hinton broth supplemented with 50 mg of Ca2" and 25 mg of Mg2+ per liter. The gram-positive strains were cultured for 6 h at 37°C in 5% CO2 in air, resulting in approximately 5 x 10' CFU/ml, and the gram-negative strains were cultured for 6 h at 37°C, resulting in approximately 109 CFU/ml. Antibiotics. The antibiotics were obtained as reference powders with known potencies from the following pharmaceutical companies: vancomycin from Eli Lilly Sweden AB, Stockholm, Sweden; roxithromycin from Roussel Nordiska AB, Stockholm, Sweden; sparfloxacin from Rhone-Poulenc Rorer, Helsingborg, Sweden; and amikacin from BristolMyers Squibb, Bromma, Sweden. Dilutions were made in distilled water on the day the experiments were performed. Determination of MICs. MICs were determined in fluid media by using a twofold dilution with an inoculum of approximately 105 CFU of the test strain per ml and were read after 24 h. The MIC was defined as the lowest concentration of the antibiotic allowing no visible growth. Determinations of MICs were performed in triplicate on separate occasions. Since the MIC of amikacin for E. coli ATCC 25922 was higher than that reported by other authors (see Results), the MIC for this combination was determined five times on different occasions. Induction of the postantibiotic phase and determination of the PAE. The following antibiotic-bacterium combinations were studied: vancomycin with S. pyogenes group A, M12, NCTC P 1800 and with S. pneumoniae ATCC 6306; roxithromycin with S. pyogenes group A, M12, NCTC P 1800 and with S. pneumoniae ATCC 6306; sparfloxacin with S. pyo-
Corresponding author. 1852
POSTANTIBIOTIC SUB-MIC EFFECTS
VOL. 36, 1992
1853
TABLE 1. PAE, PA SME, and SME for different antibiotic-bacterium combinations. Antibiotic-bacterium combination
O.1x
the MIC
Duration (h) of effect' 0.2x the MIC
SME
PA SME
SME
3.5 (2.9-3.8) 0 (0-0.1)
5.1 (3.6-6.0)
0 (0-0.2)
Vancomycin-S. pneumo-
3.0 (1.8-4.5) 0 (-0.5-0.2)
8.8 (8.5-8.8)
1.6 (1.2-2.2)
niae ATCC 6306 RoXithromycin-S. pyogenes M12, NCTC P 1800
5.0 (4.4-6.0) 0.1 (0-0.3)
6.3 (5.4-10.2)
0.1 (0-0.2)
Vancomycin-S. pyogenes
PA SME
6.8 (5.4-10.6)
M12, NCTC P 1800
RoXithromycin-S. pneumoniae ATCC 6306
Sparfloxacin-S. pyogenes M12, NCTC P 1800 Sparfloxacin-S. pneumo-
0.3x the MIC
PAE
>7.8-22.3) 0.5 (-0.4-0.6) 2.2 (1.5-2.4)
SME
' PA SME
22.7) >9.7-22.6) 2.0 (1.9-2.3) >21.3 (>21.3) 0.1 (0-0.5)
>9.7-9.7-7.8-7.8-22.3 (>22.3) >9.3-
