Journal of Antimicrobial Chemotherapy (1979) 5, 365-373
Synergjsm between amikacin and cefazolin against Staphylococcus aureus: a comparative study of oxacillin-sensitive and oxacillin-resistant strains
Jack Levy and Jean Klastersky
Synergism between amikacin and cefazolin was studied in vitro on 9 oxacillinresistant and 10 oxacillin-sensitive isolates of Staphylococcus aureus. Minimal bactericidal concentrations of amikacin and cefazolin alone or combined in a fixed proportion were determined. The results were compared with those obtained from the study of bacterial killing curves using the same drugs alone or in combination and in concentrations similar to those achieved in sera of patients treated with usual doses of these antibiotics. These two techniques demonstrated a synergistic activity of amikacin and cefazolin against most oxacillin-resistant strains. As far as oxacillin-sensitive strains are concerned, a synergistic effect was seen only during the early part of the bactericidal process. The bactericidal activity of the sera of 5 patients who were given usual doses of amikacin and cefazolin alone or in combination was also measured against the 19 strains. For the oxacillin-resistant strains, the bactericidal activity of sera containing both drugs was significantly higher than that of sera containing cefazolin alone. In contrast, the oxacillin-sensitive strains were equally susceptible to sera containing cefazolin alone or in combination with amikacin. These in vitro data suggest that a combination of a cephalosporin with an aminoglycoside might be useful for the treatment of infections caused by oxacilUnresistant staphylococci. Introduction Previous in vitro studies have shown that combinations of P-lactamase resistant penicillins or cephalosporins with aminoglycosides were as effective as vancomycin in killing oxacillin-resistant staphylococci (Bulger, 1967; Klastersky, 1972). These combinations have been recommended as a treatment for infections due to these resistant cocci. Recently, their use has been proposed as a therapeutic regimen for severe staphylococcal infections even when the offending pathogens were sensitive to oxacillin. An increased early bactericidal activity against oxacillin-sensitive strains has been demonstrated in vitro Please address requests for reprints to Dr J. Klastereky, Dept. of Medicine, Institut Jutes Bordet, 1, rue Heger-Bordet, 1000 Bruxdles, Belgium. 365 O3O5-7453/79/O4O365+O9 $01.00/0 © 1979 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Karolinska Institutet on May 27, 2015
Service de Midecine Interne {Laboratoire d'Investigation Clinique H. J. Tagnon) Institut Jules Bordet et Clinique Pidiatrique de PHSpital Saint-Pierre, University Libre de Bruxelles, Bruxelles, Belgique
366
J. Levy and J. Klastersky
using such combinations (Watanakunakorn & Glotzbecker, 1974). In animal experiments, such in vitro results have been correlated with an increased eradication of the bacteria from infected sites (Sande & Johnson, 1975; Sande & Courtney, 1976) and with higher survival rates (Steigbigel, Greenman & Remington, 1975; Campos, Rabinovich & Smith, 1974). The present study was undertaken to investigate a possible synergistic activity of cefazolin and amikacin against oxacillin-sensitive and oxacillin-resistant staphylococci. Results of in vitro tests were correlated with the antimicrobial activity of sera of subjects receiving amikacin, cefazolin or a combination of both drugs. Materials and methods
Antibiotics Cefazolin as standard powder and amikacin sulfate solution were provided by Bristol Laboratories (Syracuse N.Y.). Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC) Overnight cultures of the 19 strains of Staphylococcus aureus in Trypticase Soy Broth (TSB) (Difco) were diluted to give 10* colony forming units (cfu)/ml. Using plastic microtitre plates, serial twofold dilutions of the antibiotics were made in a volume of 25 ul of TSB: the range of concentrations varied from 120 mg/1 to 0-1 mg/1 for cefazolin and from 40 mg/1 to 003 mg/1 for amikacin. As far as cefazolin plus amikacin is concerned, proportions similar to those achieved in the blood of patients treated with usual doses of these drugs were employed, i.e. cefazolin : amikacin=3 : 1. The concentrations ranged thus from 120 mg/1 of cefazolin+40 mg/1 of amikacin to 01 mg/1 of cefazolin+0-03 mg/1 of amikacin. The bacterial suspension, in a volume of 25 ul, was then added to each well and the plates were incubated for 18 h at 37°C. The MIC was denned as the lowest concentration of antibiotic that prevented gross turbidity after incubation at 37°C for 18 h; one microlitre of each well in which no growth occurred was removed via a calibrated loop, plated on blood agar plates and incubated at 37°C for 18 h. The highest dilution that did not contain cultivable bacteria was considered to represent the minimal bactericidal concentration (bactericidal end point : killing of ^99-9% of the initial inoculum). Significant enhancement of the antistaphylococcal activity (synergjsm) was denned as at least a fourfold decrease in MBC of both antibiotics when used in the combination as compared to the MBC of each antibiotic used alone.
Downloaded from http://jac.oxfordjournals.org/ at Karolinska Institutet on May 27, 2015
Micro-organisms Nineteen strains of Staphylococcus aureus were included in this study; they were obtained from various clinical materials (blood, pus, sputum, urine). Nine were resistant to oxacillin and 10 were sensitive to oxacillin according to a classical disc method (Churcher, 1968). Oxacillin impregnated discs (2 ug; B.D. Mdrieux; manufactured under BBLlicense) were used on 5 % NaCl Mueller Hinton agar (Difco). An inhibition zone of 11 mm or larger was considered as indicating sensitivity to the drug. All oxacillin-resistant strains grew up to the edge of the oxacillin disc. Bacteriophage typing (performed by Prof. J. Beumer, Institut Pasteur du Brabant, Brussels, Belgium) indicated that all 19 strains were different.
AmfknHn plus cefazolin against oxadHin-resistant S. aarau
367
Bactericidal activity of the serum Five male patients who were hospitalized at the Institut Jules Bordet and who gave informed consent for that part of the study were given on three consecutive days, cefazolin (21 mg/kg i.m.), amikacin (7 mg/kg i.m.) and cefazolin (21 mg/kg i.m.) plus amikacin (7 mg/kg i.m.). All had normal renal function and none of them showed any signs of infection at the time of the study. Venous blood samples were drawn one hour after the injection of the drugs and the sera obtained were stored at — 20°C until used. The concentrations of antibiotics in the sera containing amikacin and cefazolin alone were determined using the technique of Bennet (Bennet, Brodie, Benner & Kirby, 1966) with Bacillus subtilis as the test organism. The antibacterial activities of the 15 sera were measured for the 19 strains of Staphylococcus aureus with the same technique as the one used for the determination of the MBC; twofold dilutions of the sera performed with TBS as diluent were used instead of antibiotics solutions. Results MIC—MBC determinations MBC of amikacin and cefazolin alone and in combination for the 19 strains are shown in Table I. For the 9 oxacillin-resistant strains and for 1 oxacillin-sensitive strain (no. 14) the addition of amikacin to cefazolin resulted in a fourfold or more decrease of the MBC as compared to the MBC of cefazolin alone. For 7 oxacillin-resistant strains (nos 1, 2, 3, 5, 6, 7, 8) a fourfold decrease of the MBC of amikacin occurred when amikacin and cefazolin were used together. For these 7 strains, the combination can thus be considered as fully synergistic. The same consideration applies to strain 14 which was oxacillin-sensitive. For the 9 other oxacillin-sensitive strains, the MBC of cefazolin in the combination was not significantly different from the MBC of cefazolin when used alone. Thus, synergism, as defined earlier, was found, by the present technique, for 7 out of 9 oxacillin-resistant
Downloaded from http://jac.oxfordjournals.org/ at Karolinska Institutet on May 27, 2015
Bacterial killing curves The concentration of antibiotics used in this part of the study was the mean of the concentrations observed in the serum 1 h afteT administration of amikacin (7 mg/kg, i.m.), cefazolin (21 mg/kg i.m.), and cefazolin (21 mg/kg i.m.) plus amikacin (7 mg/kg i.m.) to five patients. Flasks containing these antibiotics, in a volume of 100 ml of TSB, were incubated with 1 ml of cultures of the various strains adjusted to a concentration of 108 cfu/ml in order to obtain a final concentration of 10* cfu/ml. After 0, 1,2, 4, 6, 20 and 44 h of incubation at 37°C, 001 ml from each flask were removed and plated on blood agar plates in order to perform colony counts. The samples were plated undiluted and in 100-fold dilutions in TSB. Because only 001 ml was sampled, 100 cfu/ml was the lowest number of organisms which could be detected. Synergism with this method was defined as a decrease of at least 2 log in the colony count obtained with the combination of antibiotics as compared with the colony count obtained with the single most active agent. Serial colony counts of control flasks (without antibiotics) were performed for all strains simultaneously to the experiments with antibiotics. Killing curves studies were performed on selected strains (nos 5, 6, 12, 14, 17) using for the combination of cefazolin with amikacin one fourth of the concentrations used when the drugs were tested alone in the same experiment.
J. Levy and J. Klastersky
368
Table I. Minimal bactericidal concentration of amikacin, cefazoJin and their combination for 19 strains of Staphylococcus aureus and mean bactericidal activities of sera in 5 patients after administration of cefazolin (21 mg/kg), amikacin (7 mg/kg) and cefazolin plus amikacin Strains
Oxacillin R*
Amikacin Cefazolin 1-2 2-5 5 5 10 10 2-5 5 1-2
7-5 3-7 120 60 60 120 60 60 7-5
5 5 5 5 1-2 5 10 10 10 10
0-4 0-4 0-9 0-4 0-9 0-2 0-4 0-2 0-4 0-4
Bactericidal dilution of serum (reciprocal)
Amikacin
Amikacin
cefazolin
Amikacin Cefazolin cefazolin
0-3 0-3 1-2 2-5 1-2 1-2 0-6 0-6 0-6
+0-9 +0-9 +3-7 +7-5 +3-7 +3-7 +1-8 +1-8 +1-8
4 4 8 8 4 4 8 4 8
2 4 4 4 2 4 4 4 16
16 8 16 16 8 8 32 8 32
01 +0-4 0-07 + 0-2 0-3 +0-9 0-1 +0-4 0-07+0-2 0-1 +0-4 0-1 +0-4 007 + 0-2 01 +0-4 0-1 +0-4
8 4 8 8 8 4 4 4 4 4
64 64 64 64 128 128 128 256 256 128
128 128 64 128 64 128 128 128 128 128
Oxacillin S* 10 11 12 13 14 15 16 17 18 19
*R, resistant; S, sensitive.
strains and for 1 out of 10 oxacillin-sensitive strains. It is also clear from Table I that oxacillin-sensitive and oxacillin-resistant strains were equally sensitive to amikacin. Bactericidal activity of the sera The mean bactericidal activities of the sera of the 5 patients are also shown in Table I. For each of the oxacillin-resistant strains (nos 1 to 9), the sera containing both amikacin and cefazolin were bactericidal at a higher dilution than sera containing amikacin or cefazolin alone (sign test / >
Synergjsm between amikacin and cefazolin against Staphylococcus aureus: a comparative study of oxacillin-sensitive and oxacillin-resistant strains
Jack Levy and Jean Klastersky
Synergism between amikacin and cefazolin was studied in vitro on 9 oxacillinresistant and 10 oxacillin-sensitive isolates of Staphylococcus aureus. Minimal bactericidal concentrations of amikacin and cefazolin alone or combined in a fixed proportion were determined. The results were compared with those obtained from the study of bacterial killing curves using the same drugs alone or in combination and in concentrations similar to those achieved in sera of patients treated with usual doses of these antibiotics. These two techniques demonstrated a synergistic activity of amikacin and cefazolin against most oxacillin-resistant strains. As far as oxacillin-sensitive strains are concerned, a synergistic effect was seen only during the early part of the bactericidal process. The bactericidal activity of the sera of 5 patients who were given usual doses of amikacin and cefazolin alone or in combination was also measured against the 19 strains. For the oxacillin-resistant strains, the bactericidal activity of sera containing both drugs was significantly higher than that of sera containing cefazolin alone. In contrast, the oxacillin-sensitive strains were equally susceptible to sera containing cefazolin alone or in combination with amikacin. These in vitro data suggest that a combination of a cephalosporin with an aminoglycoside might be useful for the treatment of infections caused by oxacilUnresistant staphylococci. Introduction Previous in vitro studies have shown that combinations of P-lactamase resistant penicillins or cephalosporins with aminoglycosides were as effective as vancomycin in killing oxacillin-resistant staphylococci (Bulger, 1967; Klastersky, 1972). These combinations have been recommended as a treatment for infections due to these resistant cocci. Recently, their use has been proposed as a therapeutic regimen for severe staphylococcal infections even when the offending pathogens were sensitive to oxacillin. An increased early bactericidal activity against oxacillin-sensitive strains has been demonstrated in vitro Please address requests for reprints to Dr J. Klastereky, Dept. of Medicine, Institut Jutes Bordet, 1, rue Heger-Bordet, 1000 Bruxdles, Belgium. 365 O3O5-7453/79/O4O365+O9 $01.00/0 © 1979 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Karolinska Institutet on May 27, 2015
Service de Midecine Interne {Laboratoire d'Investigation Clinique H. J. Tagnon) Institut Jules Bordet et Clinique Pidiatrique de PHSpital Saint-Pierre, University Libre de Bruxelles, Bruxelles, Belgique
366
J. Levy and J. Klastersky
using such combinations (Watanakunakorn & Glotzbecker, 1974). In animal experiments, such in vitro results have been correlated with an increased eradication of the bacteria from infected sites (Sande & Johnson, 1975; Sande & Courtney, 1976) and with higher survival rates (Steigbigel, Greenman & Remington, 1975; Campos, Rabinovich & Smith, 1974). The present study was undertaken to investigate a possible synergistic activity of cefazolin and amikacin against oxacillin-sensitive and oxacillin-resistant staphylococci. Results of in vitro tests were correlated with the antimicrobial activity of sera of subjects receiving amikacin, cefazolin or a combination of both drugs. Materials and methods
Antibiotics Cefazolin as standard powder and amikacin sulfate solution were provided by Bristol Laboratories (Syracuse N.Y.). Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC) Overnight cultures of the 19 strains of Staphylococcus aureus in Trypticase Soy Broth (TSB) (Difco) were diluted to give 10* colony forming units (cfu)/ml. Using plastic microtitre plates, serial twofold dilutions of the antibiotics were made in a volume of 25 ul of TSB: the range of concentrations varied from 120 mg/1 to 0-1 mg/1 for cefazolin and from 40 mg/1 to 003 mg/1 for amikacin. As far as cefazolin plus amikacin is concerned, proportions similar to those achieved in the blood of patients treated with usual doses of these drugs were employed, i.e. cefazolin : amikacin=3 : 1. The concentrations ranged thus from 120 mg/1 of cefazolin+40 mg/1 of amikacin to 01 mg/1 of cefazolin+0-03 mg/1 of amikacin. The bacterial suspension, in a volume of 25 ul, was then added to each well and the plates were incubated for 18 h at 37°C. The MIC was denned as the lowest concentration of antibiotic that prevented gross turbidity after incubation at 37°C for 18 h; one microlitre of each well in which no growth occurred was removed via a calibrated loop, plated on blood agar plates and incubated at 37°C for 18 h. The highest dilution that did not contain cultivable bacteria was considered to represent the minimal bactericidal concentration (bactericidal end point : killing of ^99-9% of the initial inoculum). Significant enhancement of the antistaphylococcal activity (synergjsm) was denned as at least a fourfold decrease in MBC of both antibiotics when used in the combination as compared to the MBC of each antibiotic used alone.
Downloaded from http://jac.oxfordjournals.org/ at Karolinska Institutet on May 27, 2015
Micro-organisms Nineteen strains of Staphylococcus aureus were included in this study; they were obtained from various clinical materials (blood, pus, sputum, urine). Nine were resistant to oxacillin and 10 were sensitive to oxacillin according to a classical disc method (Churcher, 1968). Oxacillin impregnated discs (2 ug; B.D. Mdrieux; manufactured under BBLlicense) were used on 5 % NaCl Mueller Hinton agar (Difco). An inhibition zone of 11 mm or larger was considered as indicating sensitivity to the drug. All oxacillin-resistant strains grew up to the edge of the oxacillin disc. Bacteriophage typing (performed by Prof. J. Beumer, Institut Pasteur du Brabant, Brussels, Belgium) indicated that all 19 strains were different.
AmfknHn plus cefazolin against oxadHin-resistant S. aarau
367
Bactericidal activity of the serum Five male patients who were hospitalized at the Institut Jules Bordet and who gave informed consent for that part of the study were given on three consecutive days, cefazolin (21 mg/kg i.m.), amikacin (7 mg/kg i.m.) and cefazolin (21 mg/kg i.m.) plus amikacin (7 mg/kg i.m.). All had normal renal function and none of them showed any signs of infection at the time of the study. Venous blood samples were drawn one hour after the injection of the drugs and the sera obtained were stored at — 20°C until used. The concentrations of antibiotics in the sera containing amikacin and cefazolin alone were determined using the technique of Bennet (Bennet, Brodie, Benner & Kirby, 1966) with Bacillus subtilis as the test organism. The antibacterial activities of the 15 sera were measured for the 19 strains of Staphylococcus aureus with the same technique as the one used for the determination of the MBC; twofold dilutions of the sera performed with TBS as diluent were used instead of antibiotics solutions. Results MIC—MBC determinations MBC of amikacin and cefazolin alone and in combination for the 19 strains are shown in Table I. For the 9 oxacillin-resistant strains and for 1 oxacillin-sensitive strain (no. 14) the addition of amikacin to cefazolin resulted in a fourfold or more decrease of the MBC as compared to the MBC of cefazolin alone. For 7 oxacillin-resistant strains (nos 1, 2, 3, 5, 6, 7, 8) a fourfold decrease of the MBC of amikacin occurred when amikacin and cefazolin were used together. For these 7 strains, the combination can thus be considered as fully synergistic. The same consideration applies to strain 14 which was oxacillin-sensitive. For the 9 other oxacillin-sensitive strains, the MBC of cefazolin in the combination was not significantly different from the MBC of cefazolin when used alone. Thus, synergism, as defined earlier, was found, by the present technique, for 7 out of 9 oxacillin-resistant
Downloaded from http://jac.oxfordjournals.org/ at Karolinska Institutet on May 27, 2015
Bacterial killing curves The concentration of antibiotics used in this part of the study was the mean of the concentrations observed in the serum 1 h afteT administration of amikacin (7 mg/kg, i.m.), cefazolin (21 mg/kg i.m.), and cefazolin (21 mg/kg i.m.) plus amikacin (7 mg/kg i.m.) to five patients. Flasks containing these antibiotics, in a volume of 100 ml of TSB, were incubated with 1 ml of cultures of the various strains adjusted to a concentration of 108 cfu/ml in order to obtain a final concentration of 10* cfu/ml. After 0, 1,2, 4, 6, 20 and 44 h of incubation at 37°C, 001 ml from each flask were removed and plated on blood agar plates in order to perform colony counts. The samples were plated undiluted and in 100-fold dilutions in TSB. Because only 001 ml was sampled, 100 cfu/ml was the lowest number of organisms which could be detected. Synergism with this method was defined as a decrease of at least 2 log in the colony count obtained with the combination of antibiotics as compared with the colony count obtained with the single most active agent. Serial colony counts of control flasks (without antibiotics) were performed for all strains simultaneously to the experiments with antibiotics. Killing curves studies were performed on selected strains (nos 5, 6, 12, 14, 17) using for the combination of cefazolin with amikacin one fourth of the concentrations used when the drugs were tested alone in the same experiment.
J. Levy and J. Klastersky
368
Table I. Minimal bactericidal concentration of amikacin, cefazoJin and their combination for 19 strains of Staphylococcus aureus and mean bactericidal activities of sera in 5 patients after administration of cefazolin (21 mg/kg), amikacin (7 mg/kg) and cefazolin plus amikacin Strains
Oxacillin R*
Amikacin Cefazolin 1-2 2-5 5 5 10 10 2-5 5 1-2
7-5 3-7 120 60 60 120 60 60 7-5
5 5 5 5 1-2 5 10 10 10 10
0-4 0-4 0-9 0-4 0-9 0-2 0-4 0-2 0-4 0-4
Bactericidal dilution of serum (reciprocal)
Amikacin
Amikacin
cefazolin
Amikacin Cefazolin cefazolin
0-3 0-3 1-2 2-5 1-2 1-2 0-6 0-6 0-6
+0-9 +0-9 +3-7 +7-5 +3-7 +3-7 +1-8 +1-8 +1-8
4 4 8 8 4 4 8 4 8
2 4 4 4 2 4 4 4 16
16 8 16 16 8 8 32 8 32
01 +0-4 0-07 + 0-2 0-3 +0-9 0-1 +0-4 0-07+0-2 0-1 +0-4 0-1 +0-4 007 + 0-2 01 +0-4 0-1 +0-4
8 4 8 8 8 4 4 4 4 4
64 64 64 64 128 128 128 256 256 128
128 128 64 128 64 128 128 128 128 128
Oxacillin S* 10 11 12 13 14 15 16 17 18 19
*R, resistant; S, sensitive.
strains and for 1 out of 10 oxacillin-sensitive strains. It is also clear from Table I that oxacillin-sensitive and oxacillin-resistant strains were equally sensitive to amikacin. Bactericidal activity of the sera The mean bactericidal activities of the sera of the 5 patients are also shown in Table I. For each of the oxacillin-resistant strains (nos 1 to 9), the sera containing both amikacin and cefazolin were bactericidal at a higher dilution than sera containing amikacin or cefazolin alone (sign test / >
