ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1978, p. 412-415
0066-4804/78/0013-0412$02.00/0 Copyright ©) 1978 American Society for Microbiology
Vol. 13, No. 3
Printed in U.S.A.
In Vitro and In Vivo Studies with BL-S786, Cefoxitin, and Cefamandole SMITH SHADOMY,t GERALD WAGNER, AND MELINDA CARVER Department of Medicine, Medical Colkge of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 Received for publication 2 November 1977
The in vitro antimicrobial activities of BL-S786, cefoxitin, and cefamandole against 90 isolates of Enterobacteriaceae, including Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae and E. aerogenes, were studied by using an agar dilution procedure. Comparison of geometric mean minimal inhibitory concentrations showed that BL-S786 was half as active as cefamandole against Enterobacter species, 2 to 4 times more active than cefamandole against all other species, and 4 to 25 times more active than cefoxitin against all species. In vivo experiments employed acute protection tests in infected mice, using five isolates each of the five genera. Drugs were administered intramuscularly in two doses 3 h apart at dosages of 2.5, 5, 10, 20, and 40 mg per mouse. In most instances, BL-S786 was the most efficacious drug, being some 1.3 to 9.1 times more active than cefoxitin in all experiments and 1.5 to 8.7 times more active than cefamandole in most experiments. BL-S786 and cefamandole were -comparable in activity in experiments with E. aerogenes, whereas BL-S786 was supenor in experiments with E. cloacae.
Cefamandole and BL-S786 are newly described, semisynthetic cephalosporin derivatives (4, 6), whereas cefoxitin is a semisynthetic cephamycin (5). All three have broad spectra of antibacterial activity against both gram-negative and gram-positive pathogens (4-7) and are partialy to markedly resistant to fl-lactamases (8, 10, 13). Furthermore, BL-S786 is as active in vivo, if not more so, as cephalothin, cephaloridine, or cefazolin in experimentally infected mice (8). Intramuscularly administered BL-S786 results in both higher serum concentrations and a longer biological half-life than comparable dosages of other cephalosporins (8). The studies reported here were performed to further define the in vitro and in vivo properties of BL-S786 as compared with cefoxitin and cefamandole.
MATERIALS AND METHODS Bacteria. In vitro studies were performed with 90 clinical isolates of gram-negative bacteria. These included Escherichia coli (21 isolates), Klebsiellapneumoniae (19 isolates), Proteus mirabilis (18 isolates), Enterobacter cloacae (17 isolates), and Enterobacter aerogenes (15 isolates). Identifications were made by using the API-20E system (Analytab Products, Inc.). Staphylococcus aureus ATCC 25923 was included for quality control purposes. t Reprint requests: MCV Box 85, Richmond, VA 23298.
Antibiotics and in vitro susceptibility testing. In vitro susceptibility tests were performed by using the WHO-ICS agar dilution procedure (3) and Mueller-Hinton agar (Baltimore Biological Laboratory, BioQuest). Test inocula were prepared from overnight broth cultures grown in Mueller-Hinton broth; densities of the inocula were standardized against a turbi-
dimetric standard as previously described (12). Dilutions of drug, prepared in saline from sterile stock solutions and containing 10 times the final desired concentrations, were added to 9 volumes of sterile, molten Mueller-Hinton agar. Final test concentrations ranged from 0.063 to 128 pg of active drug per ml. The drugs included BL-S786 (as the sodium salt, Bristol Laboratories, 75-L 1302), cefoxitin (as the sodium salt, Merck, Sharp & Dohme Research Laboratories, L-620338-01B), and cefamandole (as cefamandole naftate, Lilly Research Laboratories, CT-28836S). Prepared plates, both with drug and drug-free controls, were inoculated with a replicating device (Melrose Machine Shop) and were incubated at 35°C for 24 h. Susceptibility tests with isolates of P. mirabilis employed plates prepared with 4% agar. In vivo studies. Initially, 10 representative isolates of each genus were selected for in vivo studies to determine their infectivity for mice. Inocula were prepared from overnight broth cultures, washed three times in sterile saline, and adjusted to contain 107 cells per ml; these then were diluted to contain 107, lOi, 1i0, and 104 cells per ml. Six 18- to 22-g female ICR mice (Flow Laboratories, Inc.) were injected intraperitoneally with 1 ml of a diluted suspension. Infected mice were observed through 48 h. Five isolates of each
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IN VITRO AND IN VIVO PROPERTIES OF BL-S786
that produced 100% lethality after 48 h in mice when injected at a concentration of 106 cells per ml were selected for further study. Acute mouse protection tests were performed with the five selected isolates of E. coli, K. pneumoniae, E. aerogenes, E. cloacae, and P. mirabilis. All 25 isolates were susceptible to cefamandole and BL-S786, whereas all 5 isolates of E. aerogenes and 1 isolate of E. cloacae were resistant to cefoxitin. Single experiments were performed with mice injected with one isolate and treated with the three drugs under study. Infective inocula were prepared as above and adjusted to contain 10' cells per ml. For each experiment, 180 mice were injected intraperitoneally with 1 ml of infective inoculum. The mice were then randomized into 15 treatment groups of 10 mice each; 30 mice were retained as controls. At 1 and 4 h after infection, mice in the treatment groups were injected intramuscularly with 0.2-ml volumes of drug prepared in saline. Dosages per injection were the same for all three drugs: 2.5, 5, 10, 20, and 40 mg/kg per 20-g mouse. Treated and untreated mice were maintained through 72 h with observation of survivors at 24, 48, and 72 h. Data analyses. In vitro data for each species were evaluated in terms of cumulative percent inhibition by each of the three drugs; geometric mean minimal inhibitory concentrations were determined. The in vitro activity, in terms of total numbers of organisms susceptible, of BL-S786 at 32 ug/ml was compared statistically (chi-square analysis) with those of cefoxigenus
413
tin and cefamandole at the same concentration (9). In vivo data were evaluated in terms of 50% protective dose (PD50) values based upon 48-h survival rates (11). The in vivo activity of BL-8786 was compared with those of cefoxitin and cefamandole in terms of relative potencies or ratios of PD50 values.
RESULTS
Generally, data from the in vitro studies (Table 1) with cefoxitin and cefamandole were in agreement with recently published values (1, 2), as were those for BL-S786 (6, 7, 8). BL-S786 was the most active of the three compounds against E. coli, K. pneumoniae, and P. mirabilis. BLS786 was, by far, the most active compound when tested against E. coli and K. pneumoniae, particularly at lower concentrations; cefoxitin was the least active at lower concentrations. One isolate of E. coli was resistant to cefamandole but not to the other two drugs, whereas one isolate of K. pneumoniae was resistant to both BL-S786 and cefamandole but not to cefoxitin. BL-S786 was the most active compound against P. mirabilis, whereas cefoxitin was the least active; 4 ,Ag of either BL-S786 or cefamandole per ml was inhibitory for 90% or better of isolates tested, whereas 4 jig of cefoxitin per ml was
TABLE 1. In vitro susceptibility of 90 clinical isolates of Enterobacteriaceae to BL-S786, cefoxitin, and cefamandole as determined by the WHO-ICS agar dilution procedure Organism (no. tested) and compound
Cumulative % inhibited isolates, at concentration Q(g/ml):
128
Ga
95
100
0.74 3.87 1.74
95
95
100
89
89
100
32
64
E. coli (21)
BL-S786 Cefoxitin Cefamandole
29
K. pneumoniae (19)
BL-S786 Cefoxitin Cefamandole P. mirabilis (18) BL-S786 Cefoxitin Cefamandole
79
26
0.62 2.7 1.8 0.46 10.9 1.9
100
E. aerogenes (15)
BL-S786 Cefoxitin Cefamandole
7b 7
67
80
80
80
87
87 7
100 100
3.8 80.6 2.61
E. cloacae (17) 82 88 100 3.83 41 71 82 18 29 53 BL-S786 12b 18 100 95.9 Cefoxitin 94 94 94 100 2.35 24 71 88 88 18 Cefamandole a Geometric mean minimal inhibitory concentration in micrograms per milliliter. b Differences between numbers of isolates inhibited by BL-S786 and cefoxitin were highly significant (chisquare, P < 0.001).
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ANTimICROB. AGENTS CHEMOTHER.
SHADOMY, WAGNER, AND CARVER
inhibitory for only 17%. No isolates of P. mirabilis were totally resistant to any of the three drugs. Data with Enterobacter species were varied. Cefoxitin was devoid of any activity against both E. cloacae and E. aerogenes at a concentration of 16 ,ug/ml or less. Both BL-S786 and cefamandole were active against E. aerogenes at lower concentrations with inhibition of 80% of isolates tested at 8 ,ug of either drug per ml. Cefamandole was someiwhat more active than BL-S786 against E. cloacae, but differences were marginal and nonsignificant. Differences between BL-S786 and cefoxitin when tested against both Enterobacter species were highly significant (chi-square analysis, P < 0.001). One isolate of E. aerogenes and two of E. cloacae were resistant to all three drugs. BL-S786 was found to be the most efficacious drug in vivo (Table 2). PD50 values for BL-S786 in mice infected with E. coli ranged from less than 2.5 mg per mouse in two experiments to 27 mg per mouse in one; BL-S786 failed in one experiment with E. coli. In terms of relative potencies, BL-S786 was 1.5 to 3.5 times more active than cefamandole and 1.3 to 3 times more active than cefoxitin. Cefamandole failed in three of five experiments with E. coli, whereas cefoxitin failed in none. BL-S786 also was the most active drug in experiments with K. pneumoniae, being 2 to 6 times more active than cefoxitin, the second most active drug and 4 to 16 times more active than cefamandole, which failed in four experiments. BL-S786 also was the most active drug in experiments with P. mirabilis, whereas cefamandole was the second most active. BL-S786 was 3.4 to 6.3 times more active than either cefamandole or cefoxitin in these latter experiments in which cefoxitin failed twice, whereas cefamandole failed once. In vivo data with Enterobacter appeared to be species specific, as differences in responses to the three drugs were of borderline significance (chi-square analysis, P < 0.10). PD5o values for BL-S786 were higher in experiments with E. aerogenes than in any of the above experiments, ranging from 9.1 to 31.2 mg per hmouse. In contrast, cefamandole, which had failed in 8 of 15 experiments with E. coli, K. pneumoniae and P. mirabilis, was protective in 4 of 5 experiments with E. aerogenes, with PD50 values ranging from 27.8 to 37.5 mg per mouse. Cefoxitin, which had been protective in all 10 experiments with E. coli and K. pneumoniae and in 3 of the 5 experiments with P. mirabilis, failed in 3 experiments with E. aerogenes. Overall, BL-S786 and cefamandole were comparable in activity against E. aerogenes in terms of PD50 values and were vastly superior to cefoxitin.
TABLE 2. In vivo efficacy of BL-S786, cefamandole, and cefoxitin in mice systemically infected with selected gram-negative pathogensa PD5o (doubled [mg/mouseb]) of: Isolate Organism S786
CMDc
CFX
3 4 6 7 9
11.6 27.1 >40 40 >40 15.4 3.5
25.2 33.9 24.3 7.9 7.0
1
2 17 31 32
11.2 6.5 40 27.2 >40 >40 >40
33.8 17.7 7.2 22.1 31.5
22 24 33 30 15
40 11.9
E. aerogenes
17 24 28 29 41
21.8 9.2 27.3 27.7 31.5
24.8 17.8 >40 37.5 34.7
20.0 26.2 >40 >40 >40
E. cloacae
3 12 13 14 25
10.0
24.2 >40 >40 >40 21.7
E. coli
K. pneumoniae
P. mirabilis
31.8 >40 >40 >40 22.8 a Mice infected with intraperitoneal challenges containing approximately 106 cells. All animals received two intramuscular injections of drug 1 and 4 h after >40 >40 >40
0066-4804/78/0013-0412$02.00/0 Copyright ©) 1978 American Society for Microbiology
Vol. 13, No. 3
Printed in U.S.A.
In Vitro and In Vivo Studies with BL-S786, Cefoxitin, and Cefamandole SMITH SHADOMY,t GERALD WAGNER, AND MELINDA CARVER Department of Medicine, Medical Colkge of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 Received for publication 2 November 1977
The in vitro antimicrobial activities of BL-S786, cefoxitin, and cefamandole against 90 isolates of Enterobacteriaceae, including Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae and E. aerogenes, were studied by using an agar dilution procedure. Comparison of geometric mean minimal inhibitory concentrations showed that BL-S786 was half as active as cefamandole against Enterobacter species, 2 to 4 times more active than cefamandole against all other species, and 4 to 25 times more active than cefoxitin against all species. In vivo experiments employed acute protection tests in infected mice, using five isolates each of the five genera. Drugs were administered intramuscularly in two doses 3 h apart at dosages of 2.5, 5, 10, 20, and 40 mg per mouse. In most instances, BL-S786 was the most efficacious drug, being some 1.3 to 9.1 times more active than cefoxitin in all experiments and 1.5 to 8.7 times more active than cefamandole in most experiments. BL-S786 and cefamandole were -comparable in activity in experiments with E. aerogenes, whereas BL-S786 was supenor in experiments with E. cloacae.
Cefamandole and BL-S786 are newly described, semisynthetic cephalosporin derivatives (4, 6), whereas cefoxitin is a semisynthetic cephamycin (5). All three have broad spectra of antibacterial activity against both gram-negative and gram-positive pathogens (4-7) and are partialy to markedly resistant to fl-lactamases (8, 10, 13). Furthermore, BL-S786 is as active in vivo, if not more so, as cephalothin, cephaloridine, or cefazolin in experimentally infected mice (8). Intramuscularly administered BL-S786 results in both higher serum concentrations and a longer biological half-life than comparable dosages of other cephalosporins (8). The studies reported here were performed to further define the in vitro and in vivo properties of BL-S786 as compared with cefoxitin and cefamandole.
MATERIALS AND METHODS Bacteria. In vitro studies were performed with 90 clinical isolates of gram-negative bacteria. These included Escherichia coli (21 isolates), Klebsiellapneumoniae (19 isolates), Proteus mirabilis (18 isolates), Enterobacter cloacae (17 isolates), and Enterobacter aerogenes (15 isolates). Identifications were made by using the API-20E system (Analytab Products, Inc.). Staphylococcus aureus ATCC 25923 was included for quality control purposes. t Reprint requests: MCV Box 85, Richmond, VA 23298.
Antibiotics and in vitro susceptibility testing. In vitro susceptibility tests were performed by using the WHO-ICS agar dilution procedure (3) and Mueller-Hinton agar (Baltimore Biological Laboratory, BioQuest). Test inocula were prepared from overnight broth cultures grown in Mueller-Hinton broth; densities of the inocula were standardized against a turbi-
dimetric standard as previously described (12). Dilutions of drug, prepared in saline from sterile stock solutions and containing 10 times the final desired concentrations, were added to 9 volumes of sterile, molten Mueller-Hinton agar. Final test concentrations ranged from 0.063 to 128 pg of active drug per ml. The drugs included BL-S786 (as the sodium salt, Bristol Laboratories, 75-L 1302), cefoxitin (as the sodium salt, Merck, Sharp & Dohme Research Laboratories, L-620338-01B), and cefamandole (as cefamandole naftate, Lilly Research Laboratories, CT-28836S). Prepared plates, both with drug and drug-free controls, were inoculated with a replicating device (Melrose Machine Shop) and were incubated at 35°C for 24 h. Susceptibility tests with isolates of P. mirabilis employed plates prepared with 4% agar. In vivo studies. Initially, 10 representative isolates of each genus were selected for in vivo studies to determine their infectivity for mice. Inocula were prepared from overnight broth cultures, washed three times in sterile saline, and adjusted to contain 107 cells per ml; these then were diluted to contain 107, lOi, 1i0, and 104 cells per ml. Six 18- to 22-g female ICR mice (Flow Laboratories, Inc.) were injected intraperitoneally with 1 ml of a diluted suspension. Infected mice were observed through 48 h. Five isolates of each
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that produced 100% lethality after 48 h in mice when injected at a concentration of 106 cells per ml were selected for further study. Acute mouse protection tests were performed with the five selected isolates of E. coli, K. pneumoniae, E. aerogenes, E. cloacae, and P. mirabilis. All 25 isolates were susceptible to cefamandole and BL-S786, whereas all 5 isolates of E. aerogenes and 1 isolate of E. cloacae were resistant to cefoxitin. Single experiments were performed with mice injected with one isolate and treated with the three drugs under study. Infective inocula were prepared as above and adjusted to contain 10' cells per ml. For each experiment, 180 mice were injected intraperitoneally with 1 ml of infective inoculum. The mice were then randomized into 15 treatment groups of 10 mice each; 30 mice were retained as controls. At 1 and 4 h after infection, mice in the treatment groups were injected intramuscularly with 0.2-ml volumes of drug prepared in saline. Dosages per injection were the same for all three drugs: 2.5, 5, 10, 20, and 40 mg/kg per 20-g mouse. Treated and untreated mice were maintained through 72 h with observation of survivors at 24, 48, and 72 h. Data analyses. In vitro data for each species were evaluated in terms of cumulative percent inhibition by each of the three drugs; geometric mean minimal inhibitory concentrations were determined. The in vitro activity, in terms of total numbers of organisms susceptible, of BL-S786 at 32 ug/ml was compared statistically (chi-square analysis) with those of cefoxigenus
413
tin and cefamandole at the same concentration (9). In vivo data were evaluated in terms of 50% protective dose (PD50) values based upon 48-h survival rates (11). The in vivo activity of BL-8786 was compared with those of cefoxitin and cefamandole in terms of relative potencies or ratios of PD50 values.
RESULTS
Generally, data from the in vitro studies (Table 1) with cefoxitin and cefamandole were in agreement with recently published values (1, 2), as were those for BL-S786 (6, 7, 8). BL-S786 was the most active of the three compounds against E. coli, K. pneumoniae, and P. mirabilis. BLS786 was, by far, the most active compound when tested against E. coli and K. pneumoniae, particularly at lower concentrations; cefoxitin was the least active at lower concentrations. One isolate of E. coli was resistant to cefamandole but not to the other two drugs, whereas one isolate of K. pneumoniae was resistant to both BL-S786 and cefamandole but not to cefoxitin. BL-S786 was the most active compound against P. mirabilis, whereas cefoxitin was the least active; 4 ,Ag of either BL-S786 or cefamandole per ml was inhibitory for 90% or better of isolates tested, whereas 4 jig of cefoxitin per ml was
TABLE 1. In vitro susceptibility of 90 clinical isolates of Enterobacteriaceae to BL-S786, cefoxitin, and cefamandole as determined by the WHO-ICS agar dilution procedure Organism (no. tested) and compound
Cumulative % inhibited isolates, at concentration Q(g/ml):
128
Ga
95
100
0.74 3.87 1.74
95
95
100
89
89
100
32
64
E. coli (21)
BL-S786 Cefoxitin Cefamandole
29
K. pneumoniae (19)
BL-S786 Cefoxitin Cefamandole P. mirabilis (18) BL-S786 Cefoxitin Cefamandole
79
26
0.62 2.7 1.8 0.46 10.9 1.9
100
E. aerogenes (15)
BL-S786 Cefoxitin Cefamandole
7b 7
67
80
80
80
87
87 7
100 100
3.8 80.6 2.61
E. cloacae (17) 82 88 100 3.83 41 71 82 18 29 53 BL-S786 12b 18 100 95.9 Cefoxitin 94 94 94 100 2.35 24 71 88 88 18 Cefamandole a Geometric mean minimal inhibitory concentration in micrograms per milliliter. b Differences between numbers of isolates inhibited by BL-S786 and cefoxitin were highly significant (chisquare, P < 0.001).
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ANTimICROB. AGENTS CHEMOTHER.
SHADOMY, WAGNER, AND CARVER
inhibitory for only 17%. No isolates of P. mirabilis were totally resistant to any of the three drugs. Data with Enterobacter species were varied. Cefoxitin was devoid of any activity against both E. cloacae and E. aerogenes at a concentration of 16 ,ug/ml or less. Both BL-S786 and cefamandole were active against E. aerogenes at lower concentrations with inhibition of 80% of isolates tested at 8 ,ug of either drug per ml. Cefamandole was someiwhat more active than BL-S786 against E. cloacae, but differences were marginal and nonsignificant. Differences between BL-S786 and cefoxitin when tested against both Enterobacter species were highly significant (chi-square analysis, P < 0.001). One isolate of E. aerogenes and two of E. cloacae were resistant to all three drugs. BL-S786 was found to be the most efficacious drug in vivo (Table 2). PD50 values for BL-S786 in mice infected with E. coli ranged from less than 2.5 mg per mouse in two experiments to 27 mg per mouse in one; BL-S786 failed in one experiment with E. coli. In terms of relative potencies, BL-S786 was 1.5 to 3.5 times more active than cefamandole and 1.3 to 3 times more active than cefoxitin. Cefamandole failed in three of five experiments with E. coli, whereas cefoxitin failed in none. BL-S786 also was the most active drug in experiments with K. pneumoniae, being 2 to 6 times more active than cefoxitin, the second most active drug and 4 to 16 times more active than cefamandole, which failed in four experiments. BL-S786 also was the most active drug in experiments with P. mirabilis, whereas cefamandole was the second most active. BL-S786 was 3.4 to 6.3 times more active than either cefamandole or cefoxitin in these latter experiments in which cefoxitin failed twice, whereas cefamandole failed once. In vivo data with Enterobacter appeared to be species specific, as differences in responses to the three drugs were of borderline significance (chi-square analysis, P < 0.10). PD5o values for BL-S786 were higher in experiments with E. aerogenes than in any of the above experiments, ranging from 9.1 to 31.2 mg per hmouse. In contrast, cefamandole, which had failed in 8 of 15 experiments with E. coli, K. pneumoniae and P. mirabilis, was protective in 4 of 5 experiments with E. aerogenes, with PD50 values ranging from 27.8 to 37.5 mg per mouse. Cefoxitin, which had been protective in all 10 experiments with E. coli and K. pneumoniae and in 3 of the 5 experiments with P. mirabilis, failed in 3 experiments with E. aerogenes. Overall, BL-S786 and cefamandole were comparable in activity against E. aerogenes in terms of PD50 values and were vastly superior to cefoxitin.
TABLE 2. In vivo efficacy of BL-S786, cefamandole, and cefoxitin in mice systemically infected with selected gram-negative pathogensa PD5o (doubled [mg/mouseb]) of: Isolate Organism S786
CMDc
CFX
3 4 6 7 9
11.6 27.1 >40 40 >40 15.4 3.5
25.2 33.9 24.3 7.9 7.0
1
2 17 31 32
11.2 6.5 40 27.2 >40 >40 >40
33.8 17.7 7.2 22.1 31.5
22 24 33 30 15
40 11.9
E. aerogenes
17 24 28 29 41
21.8 9.2 27.3 27.7 31.5
24.8 17.8 >40 37.5 34.7
20.0 26.2 >40 >40 >40
E. cloacae
3 12 13 14 25
10.0
24.2 >40 >40 >40 21.7
E. coli
K. pneumoniae
P. mirabilis
31.8 >40 >40 >40 22.8 a Mice infected with intraperitoneal challenges containing approximately 106 cells. All animals received two intramuscular injections of drug 1 and 4 h after >40 >40 >40
