ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1991, p. 1261-1263 0066-4804/91/061261-03$02.00/0 Copyright C 1991, American Society for Microbiology
Vol. 35, No. 6
Human Serum Enhances the Postantibiotic Effect of Fluoroquinolones against Staphylococcus aureus ROSS J. DAVIDSON,'* GEORGE G. ZHANEL,12 ROBERT PHILLIPS,3 AND DARYL J. HOBAN' Department of Medical Microbiology' and Faculty of Pharmacy,2 University of Manitoba, and Health Sciences Centre, Winnipeg, Manitoba R3A JR9, and Rhone Poulenc Pharma Inc., Montreal, Quebec,3 Canada Received 24 April 1990/Accepted 28 March 1991
The postantibiotic effect (PAE) of fluoroquinolones against Staphylococcus aureus was determined in Mueller-Hinton broth and normal human serum. At both 4x and 1Ox the MIC, serum significantly increased the duration of the PAE in all strains tested (P < 0.05). Reducing the pH of the serum from 7.9 to 7.2 had no effect on the PAE. Heat treating the serum (56°C, 30 min) reduced the PAE of ciprofloxacin at 1Ox the MIC approximately 25% (P < 0.05). The PAE of cloxacillin was reduced approximately 80% in serum, and PAE experiments with gentamicin and cephalexin produced findings similar to those obtained with the fluoroquinolones. Serum increased the MICs of ciprofloxacin and norfloxacin less than twofold and increased the MIC of pefloxacin a'pproximately fourfold. We conclude that normal human serum considerably increases the PAE of fluoroquinolones against S. aureus.
Cation (50 mg of CaCl2 per liter, 25 mg of MgSO4 per liter)-supplemented MHB (pH 7.2 to 7.4) (Difco Laboratories, Detroit, Mich.) was used for both MIC determinations and PAE experiments. Colony counts were performed using Trypticase soy agar (Scott Laboratories, Fiskeville, R.I.) supplemented with 5% defibrinated sheep blood. Normal human serum samples were obtained from healthy adult volunteers with no history of antimicrobial therapy within the previous 2 months. The serum was pooled and stored at -70°C. The pH of the pooled serum was between 7.8 and 7.9. For heat-inactivation studies, serum was heated for 30 min in a 56°C water bath. Serum derived from recalcified frozen plasma (2 ml of 1 M CaCl2 per 100 ml of plasma) was also used in some of the PAE determinations. Studies performed with serum derived from plasma provided results very similar to those of studies performed with fresh human serum. MICs were determined by using arithmetic dilutions in both MHB and normal human serum by using a broth microdilution technique, as described by the National Committee for Clinical Laboratory Standards (7). The MIC was defined as the lowest concentration of antimicrobial agent that prevented visible growth after 18 h of incubation at 350C. A culture in the logarithmic phase of growth was obtained by diluting an overnight culture 1:10 into MHB or normal human serum. These were allowed to grow for several hours until the optical density at 580 nm reached 0.3. One milliliter of log-phase culture was then added to 9.0 ml of antimicrobial agent-containing MHB or serum, resulting in a final inoculum of approximately 106 to 107 CFU/ml. All antimicrobial agents were evaluated at 4 and lOx the MIC, and all experiments were performed in a nonshaking water bath at 37°C. After 2 h of exposure, antimicrobial agents were removed by a 1:100 dilution of the test medium into prewarmed MHB or serum. The following controls were included with each experiment: (i) a growth control prepared and treated in a fashion similar to that for the test solution but without exposure to antimicrobial agents and (ii) a residual antibiotic control containing 1/100 of the test antimicrobial concentration. Counts of CFU for the test cultures
Suppression of bacterial growth, or the postantibiotic effect (PAE), following brief exposure of microorganisms to antimicrobial agents has been observed for decades. This effect has been demonstrated both in vitro and in vivo and is exhibited by most antimicrobial agents (3, 11). The majority of in vitro work to date has concentrated on this effect in standard bacteriological media. Some investigators, however, have studied this effect in certain biological fluids including human serum and urine. Bundtzen et al. (1) reported that broth containing 90% heat-inactivated human serum abolished the PAE of rifampin and reduced the PAE of tetracycline by 54% in Escherichia coli. When the MICs in serum were determined and these new MICs were evaluated, the PAE was reestablished. Chin and Neu (2) reported that ciprofloxacin produced a 4-h PAE with Pseudomonas aeruginosa in both Mueller-Hinton broth (MHB) and MHBnormal human serum (50:50). Van der Auwera and Klastersky (9) measured the PAE against Staphylococcus aureus, Listeria monocytogenes, and Mycobacterium fortuitum in serum samples drawn from patients 1 and 6 h posttreatment with high doses of amikacin. Significant PAEs were demonstrated with S. aureus and L. monocytogenes. This study was designed to investigate the PAE of fluoroquinolones against S. aureus and to determine the effect of normal human serum on the PAE. Three methicillin-susceptible S. aureus strains were selected for study: two clinical isolates (F651 and F988) and a reference strain (ATCC 29213). The organisms were stored in skim milk at -70°C. Ciprofloxacin (Miles Pharmaceuticals, Rexdale, Ontario, Canada), norfloxacin (Merck Frosst, Rayway, N.J.), pefloxacin (Rhone Poulenc, Montreal, Quebec, Canada), gentamicin (Schering Corp. Ltd., Pointe Claire, Quebec, Canada), cloxacillin (Bristol-Myers, Syracuse, N.Y.), and cephalexin (Sigma Chemical Co., St. Louis, Mo.) were used in the following experiments. Stock solutions of all antimicrobial agents were prepared from standard powders. Antimicrobial concentrates were diluted in appropriate media and used the same day. *
Corresponding author. 1261
1262
ANTIMICROB. AGENTS CHEMOTHER.
NOTES TABLE 1. MICs of antimicrobial agents in MHB and normal human serum MIC (kLg/ml) of:
Organism
F651 F988 ATCC 29213
Ciprofloxacin
Norfloxacin
Pefloxacin
MHB
Serum
MHB
Serum
MHB
Serum
0.34 0.25 0.32
0.50 0.45 0.70
0.62 0.45 0.82
0.85 0.80 1.1
0.40 0.38 0.50
1.3 1.6 3.6
and the growth controls were determined prior to exposure to antimicrobial agents, after 2 h of exposure, and after dilution. Thereafter, all cultures, including the residual antibiotic control, were assessed for growth every 30 min until marked turbidity was noticed. Serial 10-fold dilutions were prepared by using sterile cold 0.85% NaCl. Aliquots of 0.01 ml and/or 0.1 ml of the appropriate dilutions were plated by using a spread plate technique. Plates were incubated at 37°C and read after 18 to 24 h. The PAE was determined as previously described (1, 6). All MIC determinations were performed in triplicate, and the mean values were calculated. MIC results are displayed in Table 1. Growth kinetics of the organism measured in MHB and serum controls were very similar (Fig. 1). In each case, the growth controls increased 1 log approximately 80 to 90 min after dilution. In addition, the residual antibiotic controls, included in both MHB and serum experiments, grew at the same rate as that of the growth controls, indicating that a 1:100 dilution was effective in removing the activity of the antimicrobial agents. Results of all fluoroquinolone PAE determinations are displayed in Table 2. All experiments were repeated a minimum of two times on separate days. All three fluoroquinolones produced prolonged PAEs in all organisms and at
108
both concentrations tested. Increasing the drug concentration from 4 to 1Ox the MIC resulted in an increased PAE in both MHB and serum. Experiments performed with human serum had a profound effect on the duration of the PAE. In the majority of experiments, the PAE in serun was double that of corresponding experiments in MHB. Of interest is the fact that while the PAE was enhanced in serum, bactericidal activity was reduced approximately 1 log (Fig. 1). Other investigators have reported that serun appeared to reduce the activity of fluoroquinolones against gram-positive cocci or had little effect (4, 8, 10). After resumption of growth, treated cells in both MHB and serum eventually reached the same growth rate as that of the controls. In order to provide insight into this phenomenon, MICs were determined using human serum. The MICs of ciprofloxacin and norfloxacin increased less than twofold; however, a four-fold increase was noted in the MIC of pefloxacin.
Gram stains of cells during the PAE were made in both broth and serum to exclude the possibility that serum promotes clumping of the cells after resumption of growth and thereby artificially reduces the number of CFU. Examination of the stains showed no considerable increase in clumping when experiments were performed with serum. Because ciprofloxacin consistently produced the longest PAE in MHB and serum, it was selected for pH and heat-inactivation studies (data not shown). Decreasing the pH of serum from between 7.8 and 7.9 to 7.2 (similar to that of MHB) had no effect on the duration of the PAE. At this reduced pH, ciprofloxacin at lOx the MIC produced a PAE of 2.9 h. Heat treating the serum, however, reduced the PAE of ciprofloxacin at lOx the MIC from 3.2 to 2.4 h (standard deviation, 0.2 h), a 25% decrease (P < 0.05). Similar experiments with MHB and serum were performed using an aminoglycoside (gentamicin) and two betalactams (cloxacillin [high protein binding] and cephalexin
DNutlon
105
106 dF 0
-J
Time (min) FIG. 1. PAE of fluoroquinolones at lOx the MIC for S. aureus in MHB and normal human serum.
1263
NOTES
VOL. 35, 1991
TABLE 2. PAE of fluoroquinolones against S. aureus in MHB and normal human serum Ciprofloxacin
Organism
Serum
MHB
4x
lox
4x
lox
Mean PAE (h) (SD)W Norfloxacin Serum MHB lox lox 4x 4x
Pefloxacin
MHB 4x
Serum
lox
0.9 (0.1) 1.5 (0.05) 2.3 (0.2) 3.2 (0.5) 0.6 (0.1) 1.4 (0.05) 1.6 (0.2) 2.4 (0.1) 0.7 (0.1) 1.3 (0.05) F651 1.1 (0.1) 1.8 (0.1) 2.4 (0.2) 3.7 (0.3) 1.1 (0.1) 1.5 (0.1) 2.2 (0.2) 2.9 (0.2) 1.0 (0.1) 1.4 (0.1) F988 1.8 (0.1) 2.5 (0.1) 2.8 (0.2) 3.8 (0.6) 1.7 (0.2) 2.0 (0.1) 2.6 (0.3) 3.3 (0.2) 1.2 (0.1) 1.5 (0.1) ATCC 29213 a The PAE of each fluoroquinolone was tested in MHB and serum at 4 and lOx the MIC.
[low protein binding]) to determine whether this phenomenon was restricted to the fluoroquinolones. The PAE of cloxacillin in serum was considerably reduced, from 1.2 to 0.35 h, approximately 80%. Cloxacillin MICs in serum were approximately 16-fold higher than those in MHB, and therefore increased protein binding appears to be responsible for the reduced PAE (1). When the organisms were exposed to cloxacillin at a concentration that produced the same antimicrobial activity in serum as in MHB, the PAE was increased from 1.2 to 2.3 h, approximately twofold. Unlike cloxacillin MICs, cephalexin MICs in serum were similar to those obtained in MHB (within one well). The PAE of cephalexin in serum, however, was approximately 2.5-fold higher than that in similar experiments performed with MHB. The PAE increased from 0.8 h in MHB to 2.2 h in serum. Experiments performed with gentamicin exhibited results similar to those with the fluoroquinolones. The PAE of gentamicin in serum was increased from 2.2 h in MHB to 3.8 h, approximately 70%. This study demonstrates that normal human serum has a profound effect on the PAE of fluoroquinolones against S. aureus. Despite the reduced bactericidal activity in serum, the duration of the PAE in serum was increased for all three fluoroquinolones. Considering the significant increase of the PAE in serum and the results of MIC determinations in serum, we must assume that some component of serum is acting in combinattion with the antimicrobial agents (5). Reducing the pH of serum to match that of MHB (pH 7.2) had little to no effect on the duration of the PAE. Thus, we conclude that the pH of serum does not significantly affect the PAE within this range. In addition, we have demonstrated that heat-inactivated serum (56°C, 30 min) reduced the PAE of ciprofloxacin at lOx the MIC from 3.2 to 2.4 h. This represents a difference of over 25%. The decrease of the PAE in heat-inactivated serum suggests that complement or some other heat-labile component may have an important role in the PAE. It appears, however, that other, more stable component(s) may also have a role in the PAE. More work in this area is required to fully elucidate the role of serum in the PAE.
4x
lox
2.1 (0.2) 1.8 (0.3) 1.9 (0.2)
2.6 (0.2) 2.9 (0.3) 2.5 (0.3)
This study was supported in part by a grant from Rhone-Poulenc Pharma Inc., Montreal, Quebec, Canada. G.G.Z. was sponsored by a Postdoctoral Research Fellowship from the Pharmaceutical Manufacturers Association of the Canada Health Research Foundation (PMAC-HRF) and the Medical Research Council of Canada (MRC). We thank Mary Wegrzyn for her expert secretarial assistance. REFERENCES 1. Bundtzen, R. W., A. U. Gerber, D. L. Cohn, and W. A. Craig. 1981. Postantibiotic suppression of bacterial growth. Rev. Infect. Dis. 3:28-37. 2. Chin, N., and H. C. Neu. 1987. Postantibiotic suppressive effect of ciprofloxacin against gram-positive and gram-negative bacteria. Am. J. Med. 82:58-62. 3. Craig, W. A., and S. Gudmundsson. 1984. The postantibiotic effect, p. 515-536. In V. Lorian (ed.), Antibiotics in laboratory medicine, 2nd ed. The Williams & Wilkins Co., Baltimore. 4. Dutcher, B. S., A. M. Reynard, M. E. Beck, and R. K. Cunningham. 1978. Potentiation of antibiotic bactericidal activity by normal human serum. Antimicrob. Agents Chemother. 13:820826. 5. Leggett, J. E., and W. A. Craig. 1989. Enhancing effect of serum ultrafiltrate on the activity of cephalosporins against gramnegative bacilli. Antimicrob. Agents Chemother. 33:35-40. 6. 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. 7. National Committee for Clinical Laboratory Standards. 1988. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Publication no. M7-T2. National Committee for Clinical Laboratory Standards, Villanova, Pa. 8. Reeves, D. S., M. J. Bywater, and H. A. Holt. 1984. The activity of enoxacin against clinical bacterial isolates in comparison with that of five other agents, and factors affecting that activity. J. Antimicrob. Chemother. 14:(Suppl. C):7-17. 9. Van der Auwera, P., and J. Klastersky. 1987. Serum bactericidal activity and postantibiotic effect in serum of patients with urinary tract infection receiving high-dose amikacin. Antimicrob. Agents Chemother. 31:1061-1068. 10. Wise, R., J. M. Andrews, J. P. Ashby, and R. S. Matthews. 1988. In vitro activity of lomefloxacin, a new quinolone antimicrobial agent, in comparison with those of other agents. Antimicrob. Agents Chemother. 32:617-622. 11. Zhanel, G. G., D. J. Hoban, and G. K. M. Harding. 1991. The postantibiotic effect: a review of in vitro and in vivo data. DICP Ann. Pharmacother. 25:153-163.
Vol. 35, No. 6
Human Serum Enhances the Postantibiotic Effect of Fluoroquinolones against Staphylococcus aureus ROSS J. DAVIDSON,'* GEORGE G. ZHANEL,12 ROBERT PHILLIPS,3 AND DARYL J. HOBAN' Department of Medical Microbiology' and Faculty of Pharmacy,2 University of Manitoba, and Health Sciences Centre, Winnipeg, Manitoba R3A JR9, and Rhone Poulenc Pharma Inc., Montreal, Quebec,3 Canada Received 24 April 1990/Accepted 28 March 1991
The postantibiotic effect (PAE) of fluoroquinolones against Staphylococcus aureus was determined in Mueller-Hinton broth and normal human serum. At both 4x and 1Ox the MIC, serum significantly increased the duration of the PAE in all strains tested (P < 0.05). Reducing the pH of the serum from 7.9 to 7.2 had no effect on the PAE. Heat treating the serum (56°C, 30 min) reduced the PAE of ciprofloxacin at 1Ox the MIC approximately 25% (P < 0.05). The PAE of cloxacillin was reduced approximately 80% in serum, and PAE experiments with gentamicin and cephalexin produced findings similar to those obtained with the fluoroquinolones. Serum increased the MICs of ciprofloxacin and norfloxacin less than twofold and increased the MIC of pefloxacin a'pproximately fourfold. We conclude that normal human serum considerably increases the PAE of fluoroquinolones against S. aureus.
Cation (50 mg of CaCl2 per liter, 25 mg of MgSO4 per liter)-supplemented MHB (pH 7.2 to 7.4) (Difco Laboratories, Detroit, Mich.) was used for both MIC determinations and PAE experiments. Colony counts were performed using Trypticase soy agar (Scott Laboratories, Fiskeville, R.I.) supplemented with 5% defibrinated sheep blood. Normal human serum samples were obtained from healthy adult volunteers with no history of antimicrobial therapy within the previous 2 months. The serum was pooled and stored at -70°C. The pH of the pooled serum was between 7.8 and 7.9. For heat-inactivation studies, serum was heated for 30 min in a 56°C water bath. Serum derived from recalcified frozen plasma (2 ml of 1 M CaCl2 per 100 ml of plasma) was also used in some of the PAE determinations. Studies performed with serum derived from plasma provided results very similar to those of studies performed with fresh human serum. MICs were determined by using arithmetic dilutions in both MHB and normal human serum by using a broth microdilution technique, as described by the National Committee for Clinical Laboratory Standards (7). The MIC was defined as the lowest concentration of antimicrobial agent that prevented visible growth after 18 h of incubation at 350C. A culture in the logarithmic phase of growth was obtained by diluting an overnight culture 1:10 into MHB or normal human serum. These were allowed to grow for several hours until the optical density at 580 nm reached 0.3. One milliliter of log-phase culture was then added to 9.0 ml of antimicrobial agent-containing MHB or serum, resulting in a final inoculum of approximately 106 to 107 CFU/ml. All antimicrobial agents were evaluated at 4 and lOx the MIC, and all experiments were performed in a nonshaking water bath at 37°C. After 2 h of exposure, antimicrobial agents were removed by a 1:100 dilution of the test medium into prewarmed MHB or serum. The following controls were included with each experiment: (i) a growth control prepared and treated in a fashion similar to that for the test solution but without exposure to antimicrobial agents and (ii) a residual antibiotic control containing 1/100 of the test antimicrobial concentration. Counts of CFU for the test cultures
Suppression of bacterial growth, or the postantibiotic effect (PAE), following brief exposure of microorganisms to antimicrobial agents has been observed for decades. This effect has been demonstrated both in vitro and in vivo and is exhibited by most antimicrobial agents (3, 11). The majority of in vitro work to date has concentrated on this effect in standard bacteriological media. Some investigators, however, have studied this effect in certain biological fluids including human serum and urine. Bundtzen et al. (1) reported that broth containing 90% heat-inactivated human serum abolished the PAE of rifampin and reduced the PAE of tetracycline by 54% in Escherichia coli. When the MICs in serum were determined and these new MICs were evaluated, the PAE was reestablished. Chin and Neu (2) reported that ciprofloxacin produced a 4-h PAE with Pseudomonas aeruginosa in both Mueller-Hinton broth (MHB) and MHBnormal human serum (50:50). Van der Auwera and Klastersky (9) measured the PAE against Staphylococcus aureus, Listeria monocytogenes, and Mycobacterium fortuitum in serum samples drawn from patients 1 and 6 h posttreatment with high doses of amikacin. Significant PAEs were demonstrated with S. aureus and L. monocytogenes. This study was designed to investigate the PAE of fluoroquinolones against S. aureus and to determine the effect of normal human serum on the PAE. Three methicillin-susceptible S. aureus strains were selected for study: two clinical isolates (F651 and F988) and a reference strain (ATCC 29213). The organisms were stored in skim milk at -70°C. Ciprofloxacin (Miles Pharmaceuticals, Rexdale, Ontario, Canada), norfloxacin (Merck Frosst, Rayway, N.J.), pefloxacin (Rhone Poulenc, Montreal, Quebec, Canada), gentamicin (Schering Corp. Ltd., Pointe Claire, Quebec, Canada), cloxacillin (Bristol-Myers, Syracuse, N.Y.), and cephalexin (Sigma Chemical Co., St. Louis, Mo.) were used in the following experiments. Stock solutions of all antimicrobial agents were prepared from standard powders. Antimicrobial concentrates were diluted in appropriate media and used the same day. *
Corresponding author. 1261
1262
ANTIMICROB. AGENTS CHEMOTHER.
NOTES TABLE 1. MICs of antimicrobial agents in MHB and normal human serum MIC (kLg/ml) of:
Organism
F651 F988 ATCC 29213
Ciprofloxacin
Norfloxacin
Pefloxacin
MHB
Serum
MHB
Serum
MHB
Serum
0.34 0.25 0.32
0.50 0.45 0.70
0.62 0.45 0.82
0.85 0.80 1.1
0.40 0.38 0.50
1.3 1.6 3.6
and the growth controls were determined prior to exposure to antimicrobial agents, after 2 h of exposure, and after dilution. Thereafter, all cultures, including the residual antibiotic control, were assessed for growth every 30 min until marked turbidity was noticed. Serial 10-fold dilutions were prepared by using sterile cold 0.85% NaCl. Aliquots of 0.01 ml and/or 0.1 ml of the appropriate dilutions were plated by using a spread plate technique. Plates were incubated at 37°C and read after 18 to 24 h. The PAE was determined as previously described (1, 6). All MIC determinations were performed in triplicate, and the mean values were calculated. MIC results are displayed in Table 1. Growth kinetics of the organism measured in MHB and serum controls were very similar (Fig. 1). In each case, the growth controls increased 1 log approximately 80 to 90 min after dilution. In addition, the residual antibiotic controls, included in both MHB and serum experiments, grew at the same rate as that of the growth controls, indicating that a 1:100 dilution was effective in removing the activity of the antimicrobial agents. Results of all fluoroquinolone PAE determinations are displayed in Table 2. All experiments were repeated a minimum of two times on separate days. All three fluoroquinolones produced prolonged PAEs in all organisms and at
108
both concentrations tested. Increasing the drug concentration from 4 to 1Ox the MIC resulted in an increased PAE in both MHB and serum. Experiments performed with human serum had a profound effect on the duration of the PAE. In the majority of experiments, the PAE in serun was double that of corresponding experiments in MHB. Of interest is the fact that while the PAE was enhanced in serum, bactericidal activity was reduced approximately 1 log (Fig. 1). Other investigators have reported that serun appeared to reduce the activity of fluoroquinolones against gram-positive cocci or had little effect (4, 8, 10). After resumption of growth, treated cells in both MHB and serum eventually reached the same growth rate as that of the controls. In order to provide insight into this phenomenon, MICs were determined using human serum. The MICs of ciprofloxacin and norfloxacin increased less than twofold; however, a four-fold increase was noted in the MIC of pefloxacin.
Gram stains of cells during the PAE were made in both broth and serum to exclude the possibility that serum promotes clumping of the cells after resumption of growth and thereby artificially reduces the number of CFU. Examination of the stains showed no considerable increase in clumping when experiments were performed with serum. Because ciprofloxacin consistently produced the longest PAE in MHB and serum, it was selected for pH and heat-inactivation studies (data not shown). Decreasing the pH of serum from between 7.8 and 7.9 to 7.2 (similar to that of MHB) had no effect on the duration of the PAE. At this reduced pH, ciprofloxacin at lOx the MIC produced a PAE of 2.9 h. Heat treating the serum, however, reduced the PAE of ciprofloxacin at lOx the MIC from 3.2 to 2.4 h (standard deviation, 0.2 h), a 25% decrease (P < 0.05). Similar experiments with MHB and serum were performed using an aminoglycoside (gentamicin) and two betalactams (cloxacillin [high protein binding] and cephalexin
DNutlon
105
106 dF 0
-J
Time (min) FIG. 1. PAE of fluoroquinolones at lOx the MIC for S. aureus in MHB and normal human serum.
1263
NOTES
VOL. 35, 1991
TABLE 2. PAE of fluoroquinolones against S. aureus in MHB and normal human serum Ciprofloxacin
Organism
Serum
MHB
4x
lox
4x
lox
Mean PAE (h) (SD)W Norfloxacin Serum MHB lox lox 4x 4x
Pefloxacin
MHB 4x
Serum
lox
0.9 (0.1) 1.5 (0.05) 2.3 (0.2) 3.2 (0.5) 0.6 (0.1) 1.4 (0.05) 1.6 (0.2) 2.4 (0.1) 0.7 (0.1) 1.3 (0.05) F651 1.1 (0.1) 1.8 (0.1) 2.4 (0.2) 3.7 (0.3) 1.1 (0.1) 1.5 (0.1) 2.2 (0.2) 2.9 (0.2) 1.0 (0.1) 1.4 (0.1) F988 1.8 (0.1) 2.5 (0.1) 2.8 (0.2) 3.8 (0.6) 1.7 (0.2) 2.0 (0.1) 2.6 (0.3) 3.3 (0.2) 1.2 (0.1) 1.5 (0.1) ATCC 29213 a The PAE of each fluoroquinolone was tested in MHB and serum at 4 and lOx the MIC.
[low protein binding]) to determine whether this phenomenon was restricted to the fluoroquinolones. The PAE of cloxacillin in serum was considerably reduced, from 1.2 to 0.35 h, approximately 80%. Cloxacillin MICs in serum were approximately 16-fold higher than those in MHB, and therefore increased protein binding appears to be responsible for the reduced PAE (1). When the organisms were exposed to cloxacillin at a concentration that produced the same antimicrobial activity in serum as in MHB, the PAE was increased from 1.2 to 2.3 h, approximately twofold. Unlike cloxacillin MICs, cephalexin MICs in serum were similar to those obtained in MHB (within one well). The PAE of cephalexin in serum, however, was approximately 2.5-fold higher than that in similar experiments performed with MHB. The PAE increased from 0.8 h in MHB to 2.2 h in serum. Experiments performed with gentamicin exhibited results similar to those with the fluoroquinolones. The PAE of gentamicin in serum was increased from 2.2 h in MHB to 3.8 h, approximately 70%. This study demonstrates that normal human serum has a profound effect on the PAE of fluoroquinolones against S. aureus. Despite the reduced bactericidal activity in serum, the duration of the PAE in serum was increased for all three fluoroquinolones. Considering the significant increase of the PAE in serum and the results of MIC determinations in serum, we must assume that some component of serum is acting in combinattion with the antimicrobial agents (5). Reducing the pH of serum to match that of MHB (pH 7.2) had little to no effect on the duration of the PAE. Thus, we conclude that the pH of serum does not significantly affect the PAE within this range. In addition, we have demonstrated that heat-inactivated serum (56°C, 30 min) reduced the PAE of ciprofloxacin at lOx the MIC from 3.2 to 2.4 h. This represents a difference of over 25%. The decrease of the PAE in heat-inactivated serum suggests that complement or some other heat-labile component may have an important role in the PAE. It appears, however, that other, more stable component(s) may also have a role in the PAE. More work in this area is required to fully elucidate the role of serum in the PAE.
4x
lox
2.1 (0.2) 1.8 (0.3) 1.9 (0.2)
2.6 (0.2) 2.9 (0.3) 2.5 (0.3)
This study was supported in part by a grant from Rhone-Poulenc Pharma Inc., Montreal, Quebec, Canada. G.G.Z. was sponsored by a Postdoctoral Research Fellowship from the Pharmaceutical Manufacturers Association of the Canada Health Research Foundation (PMAC-HRF) and the Medical Research Council of Canada (MRC). We thank Mary Wegrzyn for her expert secretarial assistance. REFERENCES 1. Bundtzen, R. W., A. U. Gerber, D. L. Cohn, and W. A. Craig. 1981. Postantibiotic suppression of bacterial growth. Rev. Infect. Dis. 3:28-37. 2. Chin, N., and H. C. Neu. 1987. Postantibiotic suppressive effect of ciprofloxacin against gram-positive and gram-negative bacteria. Am. J. Med. 82:58-62. 3. Craig, W. A., and S. Gudmundsson. 1984. The postantibiotic effect, p. 515-536. In V. Lorian (ed.), Antibiotics in laboratory medicine, 2nd ed. The Williams & Wilkins Co., Baltimore. 4. Dutcher, B. S., A. M. Reynard, M. E. Beck, and R. K. Cunningham. 1978. Potentiation of antibiotic bactericidal activity by normal human serum. Antimicrob. Agents Chemother. 13:820826. 5. Leggett, J. E., and W. A. Craig. 1989. Enhancing effect of serum ultrafiltrate on the activity of cephalosporins against gramnegative bacilli. Antimicrob. Agents Chemother. 33:35-40. 6. 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. 7. National Committee for Clinical Laboratory Standards. 1988. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Publication no. M7-T2. National Committee for Clinical Laboratory Standards, Villanova, Pa. 8. Reeves, D. S., M. J. Bywater, and H. A. Holt. 1984. The activity of enoxacin against clinical bacterial isolates in comparison with that of five other agents, and factors affecting that activity. J. Antimicrob. Chemother. 14:(Suppl. C):7-17. 9. Van der Auwera, P., and J. Klastersky. 1987. Serum bactericidal activity and postantibiotic effect in serum of patients with urinary tract infection receiving high-dose amikacin. Antimicrob. Agents Chemother. 31:1061-1068. 10. Wise, R., J. M. Andrews, J. P. Ashby, and R. S. Matthews. 1988. In vitro activity of lomefloxacin, a new quinolone antimicrobial agent, in comparison with those of other agents. Antimicrob. Agents Chemother. 32:617-622. 11. Zhanel, G. G., D. J. Hoban, and G. K. M. Harding. 1991. The postantibiotic effect: a review of in vitro and in vivo data. DICP Ann. Pharmacother. 25:153-163.
