ANTIMICROBiAi, AGENTS AND CHEMOTHERAY, Mar. 1977, P. 470-474 Copyright © 1977 American Society for Microbiology
Vol. 11, No. 3 Printed in U.S.A.
Comparison of In Vitro Activity of Cephalexin, Cephradine, and Cefaclor NANCY J. BILL
AND
JOHN A. WASHINGTON II*
Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901 Received for publication 27 October 1976
Inhibitory activity of cephalexin, cephradine, and cefaclor was compared by the WHO-ICS agar dilution technique. Cefaclor was substantially more active against staphylococci, streptococci, gonococci, meningococci, Haemophilus, Escherichia coli, Klebsiella pneumoniae, Citrobacter diversus, Proteus mirabilis, salmonellae, and shigellae than was cephalexin, which in turn was more active than cephradine. Cefaclor appeared to be less resistant to staphylococcal penicillinase than did the other two agents. None of these cephalosporins was active against Enterobacter, Serratia, indole-positive Proteeae, Pseudomonas, or Bacteroides fragilis. Cefaclor, 3-chloro-7-D-(2-phenylglycinamido)3-cephem4-carboxylic acid, is a new orally effective cephalosporin. The purpose of this study was to compare its activity in vitro with that of cephalexin and cephradine.
The plates were examined after 18 h of incubation at 35°C. The MIC was interpreted as the lowest concentration of antibiotic yielding no growth, a barely visible haze, or no more than one discrete colony. MHA supplemented with 1% IsoVitaleX (BBL) and 2% hemoglobin solution (Difco) was used to test Neisseria and Haemophilus (14). The inocula were MATERIALS AND METHODS prepared by scraping growth from chocolate blood Organisms. Most of the organisms used in this agar plates into Mueller-Hinton broth. The turbidstudy were recent isolates from the Clinical Microbi- ity of each suspension was adjusted to match that of ology Laboratory of the Mayo Clinic, Rochester, a one-half McFarland no. 1 standard. A 1:10 dilution Minn. Stock cultures of clinical isolates of salmonel- of the adjusted suspension was then delivered onto lae, shigellae, and providenciae that had been the surfaces of the agar plates with an inocula replistored on nutrient agar slants and overlayed with oil cator apparatus (9). To test the effect of increasing were used. Clinical isolates of anaerobic bacteria the inoculum size of Haemophilus, the turbidity of used for testing had been stored at -72°C in 10% the suspension was adjusted to match a no. 4 Mcskim milk (BBL). Ampicillin-resistant strains of Farland standard, and the suspension was directly Haemophilus influenzae were obtained from the inoculated onto the agar plates with the replicator Center for Disease Control, Atlanta, Ga. device. After inoculation, the plates were incubated Two reference strains of bacteria, Staphylococcus at 35°C in 5% CO2 for 18 h. The MICs were interaureus (ATCC 25923) and Escherichia coli (ATCC preted by the same criteria as above. 25922), and a strain of Pseudomonas aeruginosa The MICs of anaerobic bacteria were determined were used in each trial as controls when determin- according to the agar dilution procedure described in ing minimum inhibitory concentrations (MICs). the Wadsworth Anaerobic Bacteriology Manual (10). All organisms were subcultured to solid media to In this method, three to four colonies ofthe anaerobe check for purity before being inoculated into appro- were inoculated into supplemented thioglycolate priate broth media for testing. medium without indicator (BBL-135C) which was Antibiotics. Standard laboratory powders of ceph- enriched with hemin (5 ,ug/ml) before sterilization, alexin monohydrate and cefaclor (compound 99638) plus NaHCO3 (1 mg/ml) and vitamin K, (0.1 ,ug/ml) were kindly provided by Eli Lilly Laboratories for added just before use. After anaerobic overnight Clinical Research, Indianapolis, Ind. Cephradine incubation in a GasPak jar (BBL) at 35°C, the culwas kindly provided by Smith, Kline, and French ture was diluted with brucella broth containing 5% Co., Philadelphia, Pa. Fildes enrichment (Difco) and vitamin K, (0.1 ,ugl Susceptibility studies. MICs were determined by ml) so that its turbidity matched that of one-half the the agar dilution method (13). Generally, Mueller- turbidity of the no. 1 McFarland standard. Using Hinton agar (MHA) containing each antibiotic di- the inocula replicator device, the adjusted broth culluted according to the WHO-ICS scheme (2) was ture was applied to plates of brucella agar base inoculated with, unless indicated otherwise, a stan- containing serial twofold dilutions of antibiotic at dardized inoculum of 104 colony-forming units fimal concentrations ranging from 0.125 to 128 ,ug/ml (CFU) by using the replicator device of Steers et al. and supplemented with vitamin K, (10 jug/ml) and (9). For testing streptococci other than group D, 5% 5% laked blood. The plates were incubated at 35°C in defibrinated sheep blood was added to the MHA. GasPak jars (BBL) for approximately 48 h. The 470
471
COMPARISON OF THREE CEPHALOSPORINS
VOL. 11, 1977
MICs of each strain were read as the lowest concentration of drug yielding no growth, a barely visible haze, or no more than one discrete colony. Minimum bactericidal concentrations (MBCs) were performed according to the method described by Washington (12). Inoculum was prepared by subculturing portions of four or five isolated colonies of the organism into 5 ml of Mueller-Hinton broth and incubating the suspension for approximately 6 h. After the turbidity of the suspension was adjusted to match that of a no. 1 McFarland standard, it was diluted 1:10 to yield an inoculum of about 107 CFU/ ml and 1:1,000 to yield an inoculum of about 105 CFU/ml. Each of the tubes was inoculated with 0.5 ml of the adjusted broth culture. MICs were read after 18 h of incubation at 350C as the lowest concentration of antibiotic completely inhibiting visible growth. For the bactericidal phase, 0.5-ml aliquots from those tubes exhibiting no visible growth were used to prepare pour plates with brain heart infusion agar, which were read after 72 h of incubation at 350C. RESULTS
The susceptibilities of gram-positive cocci and gram-negative bacilli to the three cephalosporins are shown in Table 1. Cefaclor was generally more active than cephalexin or cephradine against both penicillin-susceptible and penicillin-resistant strains of S. aureus and S. epidermidis. At 2 ,ug/ml, cephalexin,
cephradine, and cefaclor inhibited 17, 9, and 32%, respectively, ofpenicillin-resistant strains of S. aureus and 50, 25, and 80%, respectively, of penicillin-susceptible strains. At 4 ,tg/ml, 95% of penicillin-susceptible strains of S. aureus were inhibited by cefaclor, in contrast to 80% of strains inhibited by the other two antibiotics. At least 93% of penicillin-resistant strains of S. aureus were inhibited by cephalexin and cefaclor at 8 ,ug/ml, in contrast to 84% inhibited by cephradine at this level. The effects of inoculum size ofS. aureus on the activities of the three cephalosporins are noted in Fig. 1. A factor for each antibiotic was calculated by dividing the MIC obtained with an inoculum of 107 CFU/ml by that obtained with an inoculum of 105 CFU/ml, as described elsewhere by Sabath et al. (7). The factors for cephalexin, cephradine, and cefaclor were c8, 16, and c64, respectively. Cefaclor was highly effective against group A and group B streptococci and inhibited all strains tested at 0.25 and 2.0 ug/ml, respectively. All strains of Streptococcus pneumoniae were inhibited by 1.0 ,ug/ ml. All of the 21 strains of pathogenic Neisseria were inhibited by 0.25 ,ug of cefaclor per ml, whereas 4 ,ug of cephalexin and 4 to 8 ,ug of cephradine per ml were required to achieve the same results.
TABLE 1. Susceptibility of gram-positive and gram-negative bacilli to cephalosporins MIC,0 (jAg/Mj)a No. of strains Organism Cephradine Cephalexin 8 16 82 resistaureus (penicillin Staphylococcus ant) 8 8 20 S. aureus (penicillin susceptible) 32 34 32 S. epidermidis (penicillin resistant) 4 4 18 S. epidermidis (penicillin susceptible) 0.5 0.5 17 Streptococcus, group A 4 2 20 Streptococcus, group B 128 128 67 Streptococcus, group D 4 2 15 S. pneumoniae 2 2 16 Neisseria gonorrhoeae 4 8 7 N. meningitidis 16 16 195 Escherichia coli 51 8 16 Klebsiella pneumoniae >128 >128 16 Enterobacter aerogenes >128 >128 25 E. cloacae >128 14 >128 Serratia marcescens >128 >128 32 Proteus mirabilis >128 >128 32 Proteus (indole positive) 8 16 21 Salmonella 8 16 20 Shigella 64 64 6 Citrobacter diversus >128 >128 6 C. freundii >128 >128 18 Providencia stuartii >128 >128 70 Pseudomonas aeruginosa >128 >128 6 P. maltophilia a Lowest concentration inhibiting at least 90% of strains tested.
-
Cefaclor 8
4 32 2 0.25
2 64 0.5 0.25 0.25
8 8 >128 >128 >128 >128 >128 2 8 8 >128 >128 >128 >128
472
ANTimicROB. AGIENTS CHEMOTHER.
BILL AND WASHINGTON
clor, with 83, 95, and 75%, respectively, being inhibited with 2 jig/ml. To obtain similar results, a four- to eightfold increase in the concentrations of cephalexin and cephradine was required for this purpose. Proteus mirabilis was more susceptible to cefaclor than cephalexin and cephradine, although 32 ,ug of cefaclor per ml was required to inhibit 75% of the 32 strains tested. The effects of inoculum size and the effect of the presence of,-lactamase on the susceptibility of H. influenzae to cephalexin, cephradine, and cefaclor are shown in Table 2. A significant inoculum effect was demonstrated for all three antibiotics with both /3-lactamase-positive and 3-lactamase-negative H. influenzae. The presence of (8-lactamase did not affect the activity of cefaclor but did affect the activity of cephalexin and cephradine. Inhibition ofH. influenzae was achieved with only 2 ,ug of cefaclor per ml, in 8 contrast to 8 Ag of cephalexin and 16 ,ug of cephradine per ml needed for ,B-lactamase-negU) 6 ative organisms, and 32 ,ug of cephalexin and 64 *-A Cephalexin co ,ug of cephradine per ml needed for /8-lacta0--o Cephradine organisms. mase-positive .) - Cefaclor The activities of the three antibiotics against 4 Bacteroides fragilis, B. melaninogenicus, and anaerobic gram-positive cocci are listed in Table 3. Cephalexin, cephradine, and especially 2 cefaclor had little effect onB. fragilis. At 64 ,ug/ ml, both cephalexin and cephradine inhibited 83% of the 18 strains of B. fragilis, whereas \ cefaclor inhibited only 11%. B. melaninogeniI I _I i 1 2 4 8 6 32 64128 cus was more susceptible to the cephalosporins than was B. fragilis, particularly if the strains / IC 10' col/ml of B. melaninogenicus were inhibited by peniFactor MIC 105 col/ml cillin at concentrations of '0.5 pAg/ml. CephaFIG. 1. Effect of inoculum size on MIC of three lexin, cephradine, and cefaclor required 1, 2, and 0.5 ,g/ml, respectively, for inhibition of B. cephalosporins for 10 strains of S. aureus.
The lowest concentrations ofthe three cephalosporins inhibiting at least 90% of facultatively anaerobic gram-negative bacilli are also shown in Table 1. At 4 ,ug/ml, cefaclor was more effective than cephalexin or cephradine against E. coli, with 83, 35, and 5%, respectively, of the 195 strains inhibited at this concentration. Cephalexin and cefaclor were more effective than cephradine against E. coli at 8 ,ug/ml, with 84, 93, and 55%, respectively, of the strains being inhibited. Similar results were obtained for Klebsiella pneumoniae. The three cephalosporins had little or no effect on Enterobacter aerogenes, E. cloacae, Serratia marcescens, indole-positive Proteus, Citrobacter freundii, Providencia stuartii, and Pseudomonas. Citrobacter diversus, Salmonella, and Shigella were highly susceptible to cefa-
hk.
.
..
.*
TABLE 2. Susceptibility ofHaemophilus influenzae to cephalosporins Cumulative * inhibited at increasing concn (^g/ml) Inoculum No of
j9-lactamase size (CFU/ +
+
-
-
ml) 104
106
104
106
st°in
Antibiotic
15
Cephalexin Cephradine Cefaclor
10
10
10
0.12
0.25
0.5
7
Cephalexin Cephradine Cefaclor Cephalexin Cephradine Cefaclor
Cephalexin Cephradine Cefaclor
1
2
4
8
67
87 93 100 67 93 93 100
16
32
64
128
73 100
10 10 10
10 20 30 20 20
60 60 30
50 40 30
60 40 30
80 40 50
80 80 70 80 50 50
50 100 50 100 30
90 100
20 10 40
30 40 10 40 40 40
473
COMPARISON OF THREE CEPHALOSPORINS
VOL. 11, 1977
TABLz 3. Susceptibility of anaerobic bacteria to cephalosporins Cumulative % inhibited at increasing concn (gg/ml)
No. o No of
Stral5
Antibiotic
SO.12
0.26
0.5
1
2
4
8
Peptococcus
17
Cephalexin Cephradine Cefaclor
12 12 29
18 18 35
29 29 53
29 35 71
53 47 76
76 76 82
82 82 76 82 82 88
82 94 100 88 94 100 94 100
Peptostreptococcus
10
Cephalexin Cephradine Cefaclor
10 10 10
10 20 20
20 20 30
30 30 40
40 40 60
70 70 70
70 80 70 80 80 80
90 100 90 100 80 80 100
Bacteroides fragilw
18
Cephalexin Cephradine Cefaclor
B. melaninogenicus (penicillin MIC, s0.5)
4
Cephalexin Cephradine Cefaclor
B. melaninogenicus (penicillin MIC, >0.5)
5
Cephalexin Cephradine Cefaclor
Organism
16
6 11 6 11
25 25 25
melaninogenicus with MIC values to penicillin of 0.5 ug/ml. Of the three antibiotics, cefaclor appeared to be slightly more effective against the anaerobic gram-positive cocci that were tested. At 2 ,ug/ ml, cephalexin, cephradine, and cefaclor inhibited 53, 47, and 76%, respectively, of the 17 strains of Peptococcus and 40, 40, and 60%, respectively, of the 10 strains of Peptostreptococcus.
Neither cefaclor nor cephradine produced 100% killing of 105 and 107 CFU of penicillinsusceptible and penicillin-resistant strains ofS. aureus per ml at concentrations of s 128 ug/ml. With one exception, neither cefaclor nor cephradine produced 99.9% killing of staphylococci in an inoculum of 107 CFU/ml. With one strain, cephradine produced 99.9% killing at 32 ,ug/ml. The MBC values of cefaclor against three strains of E. coli were the same as, two times (in the first two instances) and eight times the MIC values, respectively. With three strains ofK. pneumoniae, the MBC values were four times the MIC values in two instances and twice the MIC value in one instance. DISCUSSION Against both penicillinase- and non-penicillinase-producing strains of S. aureus, cefaclor was at least three times as active as cephradine at 2 ug/ml and at least one and a half times as active as cephalexin at this concentration. The effects of inoculum size of S. aureus on the
32
44 61
64
83 83 11
128
94 89 72
25 25 100 25 25 75 100 75 100
60 80 100 40 100 40 40 80 100 /~~~~~~~~~~~~~~~~~~~~~~~
activities of the three cephalosporins varied substantially (Fig. 1), with cephalexin being the least affected by penicillinase-producing strains and cefaclor being the most affected by such strains; cephradine was intermediate between the two. Similar studies by Sabath et al. (7) have shown that cephalexin was only slightly less resistant to staphylococcal penicillinase than was cephalothin. These data disagree with those reported by Fong et al. (3), who assayed for residual cephalosporin activity in broth cultures of S. aureus and found that cephalexin was completely inactivated by staphylococcal penicillinase within 12 h, in contrast to cephalothin, which underwent little inactivation. They also found that cephradine, which was not tested by Sabath et al. (7), was degraded more slowly than cephalexin, with 18% of the original concentration being measurable at 12 h. Cephradine was found to be more resistant than cephalexin to crude ,8-lactamase derived from S. aureus by Hubsher et al. (4). At 2 ug/ml, cefaclor inhibited all /8-lactamase-positive and -negative strains of H. influenzae at the generally recommended inoculum of 104 CFU/ml (11), in contrast to cephalexin and cephradine, which required substantially greater concentrations to achieve the same order of activity and which were also considerably affected by Haemophilus /8-lactamase. None of the cephalosporins inhibited a significant number of the strains of H. influenzae at the higher inoculum size. Our results with cephalexin and cephradine are similar to those reported by Emerson et al. (1). Whether or not cefaclor's
474
BILL AND WASHNGTON
greater activity against both 3-lactamase-positive and -negative strains at the lower inoculum is clinically significant remains to be seen. Cefaclor was also significantly more active against gonococci and meningococci than the other two agents and inhibited all strains at 0.25 jug/ml. The MIC values of cefaclor were at least 1/16 of those of the other two cephalosporins; however, no 84-lactamase-positive strains of Neisseria gonorrhoeae were studied. In general, cephalosporins have demonstrated broad ranges of MIC values against these two species of Neisseria. Cefaclor was substantially more active than cephalexin and cephradine against E. coli, K. pneumoniae, P. mirabilis, salmonellae, shigellae, and C. diversus. All three antibiotics were essentially inactive against Enterobacter, Serratia, indole-positive species of the Proteeae, Citrobacter freundii, and species of Pseudomonas. Klastersky et al. (5) and Scholand et al. (8) have previously reported that the antibacterial activity of cephradine was nearly identical to that of cephalexin. McGowan et al. (6), however, reported that cephradine was less active than cephalexin against blood culture isolates of E. coli, Klebsiella, and P. mirabilis. The methodologies used in each of these studies differed considerably from one another and in varying degrees from the reference WHO-ICS method (2, 11). It was, therefore, of interest to us to try to resolve these different results by testing each of the cephalosporins with the reference method. In general, we agree with McGowan et al. (6) that cephradine is less active than cephalexin. Moreover, our results demonstrate that neither antibiotic is as active as cefaclor against gram-positive and gram-negative aerobic and facultatively anaerobic bacteria. All three cephalosporins were active at clinically achievable serum levels (8 to 16 Ag/ml) against most anaerobic cocci, but they were essentially inactive at these levels againstBacteroides fragilis. With B. melaninogenicus their activity correlated closely with the penicillin susceptibility of the strains tested. The four strains that were inhibited by sO.5 Mug of penicillin per ml were also inhibited by 1, 2, and 0.5 MAg of cephalexin, cephradine, and cefaclor per ml, respectively. The five strains requiring >0.5 Mug of penicillin per ml for inhibition required 8, 4, and 32 Mug of cephalexin, cephradine, and cefaclor per ml, respectively, for complete inhibition. In conclusion, we have found cefaclor to be substantially more active than cephalexin, which in turn was more active than cephra-
ANTimicRoB. AGENTS CHEMOTHER.
dine, against staphylococci, streptococci, gonococci, meningococci, and certain species of the Enterobacteriaceae. Cefaclor was also highly active against both ,B-lactamase-positive and -negative strains of H. influenzae at the recommended inoculum size. Clinical studies are required to substantiate cefaclor's value in the therapy of infections due to these susceptible species. ACKNOWLEDGMENT This work was supported by a grant from the Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Ind. LITERATURE CITED 1. Emerson, B. B., A. L. Smith, A. L. Hading, and D. H. Smith. 1975. Haemophilus influensae type B susceptibility to 17 antibiotics. J. Pediatr. 86:617620. 2. Ericsson, H. M., and J. C. Sherris. 1971. Antibiotic sensitivity testing report of an international collaborative study. Acta Pathol. Microbiol. Scand. Sect. B Suppl. 217:1. 3. Fong, I. W., E. R. Engelking, and W. M. Kirby. 1976. Relative inactivation by Staphylococcus aureus of eight cephalosporn antibiotics. Antimicrob. Ag. Chemother. 9:939-944. 4. Hubeher, J. A., H. H. Gadebusch, and A. G. Itkin. 1976. Double-blind comparison of oephradine and cephalexin in the treatment of skin and soft-tissue infections due to Staphylococcus aureus. Curr. Ther. Res. Clin. Exp. 19:579-588. 5. Klasterak, J., D. Danea, and D. Weerts. 1973. Cepbradine: antibacterial activity and clinical effectiveness. Chemotherapy 18:191-204. 6. McGowan, J. E., Jr., C. Garner, C. Wilcox, and M. Finland. 1974. Antibiotic susceptibility of gramnegative bacilli isolated from blood cultures reults of tests with 35 agents and strains from 169 patients at Boston City Hospital during 1972. Am. J. Med. 57:225-238. 7. Sabath, L. D., C. Garner, C. Wilcox, and M. Finland. 1975. Effect of inoculum and ofbeta-lactamase on the anti-staphylococcal activity of thirteen penicill and cephaloporins, Antimicrob. Ag. Chemother. 8:344-349. 8. Scholand, J. F., G. R. Hodges, R. J. Fm, and B. Snlaw. 1974. Clinical evaluation of cepbradine, a new oral cephalo n. Am. J. Med. Sci. 267:111-116. 9. Steers, E., E. L. Foltz, and B. S. Graves. 1959. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 10. Sutter, V. L., V. L. Vargo, and S. M. Finegold. 1975. Wadsworth anaeobic bacteriolo manual, 2nd ed., p. 60-62. Wadsworth Hospital Center, Los Angeles. 11. Thornsberry, C., and L. A. Kirven. 1974. Antimicrobial susceptibility of Haemophilus influenzae. Antimicrob. Ag. Chemother. 6:620-624. 12. Washington, J. A., I. 1974. Laboratory procedures in clinical microbiology. Little, Brown and Co., Boston. 13. Washington, J. A., II, and A. L. Barry. 1974. Dilution test procedures, p. 410-417. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 14. Washington, J. A., II, R. J. Snyder, and P. C. Kohner. 1976. Spurious ampicillin resistance by testing Hae-
mophilus influenzae with agar containing supple-
ment C. Antimicrob. Ag. Chemother. 9:199-200.
Vol. 11, No. 3 Printed in U.S.A.
Comparison of In Vitro Activity of Cephalexin, Cephradine, and Cefaclor NANCY J. BILL
AND
JOHN A. WASHINGTON II*
Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901 Received for publication 27 October 1976
Inhibitory activity of cephalexin, cephradine, and cefaclor was compared by the WHO-ICS agar dilution technique. Cefaclor was substantially more active against staphylococci, streptococci, gonococci, meningococci, Haemophilus, Escherichia coli, Klebsiella pneumoniae, Citrobacter diversus, Proteus mirabilis, salmonellae, and shigellae than was cephalexin, which in turn was more active than cephradine. Cefaclor appeared to be less resistant to staphylococcal penicillinase than did the other two agents. None of these cephalosporins was active against Enterobacter, Serratia, indole-positive Proteeae, Pseudomonas, or Bacteroides fragilis. Cefaclor, 3-chloro-7-D-(2-phenylglycinamido)3-cephem4-carboxylic acid, is a new orally effective cephalosporin. The purpose of this study was to compare its activity in vitro with that of cephalexin and cephradine.
The plates were examined after 18 h of incubation at 35°C. The MIC was interpreted as the lowest concentration of antibiotic yielding no growth, a barely visible haze, or no more than one discrete colony. MHA supplemented with 1% IsoVitaleX (BBL) and 2% hemoglobin solution (Difco) was used to test Neisseria and Haemophilus (14). The inocula were MATERIALS AND METHODS prepared by scraping growth from chocolate blood Organisms. Most of the organisms used in this agar plates into Mueller-Hinton broth. The turbidstudy were recent isolates from the Clinical Microbi- ity of each suspension was adjusted to match that of ology Laboratory of the Mayo Clinic, Rochester, a one-half McFarland no. 1 standard. A 1:10 dilution Minn. Stock cultures of clinical isolates of salmonel- of the adjusted suspension was then delivered onto lae, shigellae, and providenciae that had been the surfaces of the agar plates with an inocula replistored on nutrient agar slants and overlayed with oil cator apparatus (9). To test the effect of increasing were used. Clinical isolates of anaerobic bacteria the inoculum size of Haemophilus, the turbidity of used for testing had been stored at -72°C in 10% the suspension was adjusted to match a no. 4 Mcskim milk (BBL). Ampicillin-resistant strains of Farland standard, and the suspension was directly Haemophilus influenzae were obtained from the inoculated onto the agar plates with the replicator Center for Disease Control, Atlanta, Ga. device. After inoculation, the plates were incubated Two reference strains of bacteria, Staphylococcus at 35°C in 5% CO2 for 18 h. The MICs were interaureus (ATCC 25923) and Escherichia coli (ATCC preted by the same criteria as above. 25922), and a strain of Pseudomonas aeruginosa The MICs of anaerobic bacteria were determined were used in each trial as controls when determin- according to the agar dilution procedure described in ing minimum inhibitory concentrations (MICs). the Wadsworth Anaerobic Bacteriology Manual (10). All organisms were subcultured to solid media to In this method, three to four colonies ofthe anaerobe check for purity before being inoculated into appro- were inoculated into supplemented thioglycolate priate broth media for testing. medium without indicator (BBL-135C) which was Antibiotics. Standard laboratory powders of ceph- enriched with hemin (5 ,ug/ml) before sterilization, alexin monohydrate and cefaclor (compound 99638) plus NaHCO3 (1 mg/ml) and vitamin K, (0.1 ,ug/ml) were kindly provided by Eli Lilly Laboratories for added just before use. After anaerobic overnight Clinical Research, Indianapolis, Ind. Cephradine incubation in a GasPak jar (BBL) at 35°C, the culwas kindly provided by Smith, Kline, and French ture was diluted with brucella broth containing 5% Co., Philadelphia, Pa. Fildes enrichment (Difco) and vitamin K, (0.1 ,ugl Susceptibility studies. MICs were determined by ml) so that its turbidity matched that of one-half the the agar dilution method (13). Generally, Mueller- turbidity of the no. 1 McFarland standard. Using Hinton agar (MHA) containing each antibiotic di- the inocula replicator device, the adjusted broth culluted according to the WHO-ICS scheme (2) was ture was applied to plates of brucella agar base inoculated with, unless indicated otherwise, a stan- containing serial twofold dilutions of antibiotic at dardized inoculum of 104 colony-forming units fimal concentrations ranging from 0.125 to 128 ,ug/ml (CFU) by using the replicator device of Steers et al. and supplemented with vitamin K, (10 jug/ml) and (9). For testing streptococci other than group D, 5% 5% laked blood. The plates were incubated at 35°C in defibrinated sheep blood was added to the MHA. GasPak jars (BBL) for approximately 48 h. The 470
471
COMPARISON OF THREE CEPHALOSPORINS
VOL. 11, 1977
MICs of each strain were read as the lowest concentration of drug yielding no growth, a barely visible haze, or no more than one discrete colony. Minimum bactericidal concentrations (MBCs) were performed according to the method described by Washington (12). Inoculum was prepared by subculturing portions of four or five isolated colonies of the organism into 5 ml of Mueller-Hinton broth and incubating the suspension for approximately 6 h. After the turbidity of the suspension was adjusted to match that of a no. 1 McFarland standard, it was diluted 1:10 to yield an inoculum of about 107 CFU/ ml and 1:1,000 to yield an inoculum of about 105 CFU/ml. Each of the tubes was inoculated with 0.5 ml of the adjusted broth culture. MICs were read after 18 h of incubation at 350C as the lowest concentration of antibiotic completely inhibiting visible growth. For the bactericidal phase, 0.5-ml aliquots from those tubes exhibiting no visible growth were used to prepare pour plates with brain heart infusion agar, which were read after 72 h of incubation at 350C. RESULTS
The susceptibilities of gram-positive cocci and gram-negative bacilli to the three cephalosporins are shown in Table 1. Cefaclor was generally more active than cephalexin or cephradine against both penicillin-susceptible and penicillin-resistant strains of S. aureus and S. epidermidis. At 2 ,ug/ml, cephalexin,
cephradine, and cefaclor inhibited 17, 9, and 32%, respectively, ofpenicillin-resistant strains of S. aureus and 50, 25, and 80%, respectively, of penicillin-susceptible strains. At 4 ,tg/ml, 95% of penicillin-susceptible strains of S. aureus were inhibited by cefaclor, in contrast to 80% of strains inhibited by the other two antibiotics. At least 93% of penicillin-resistant strains of S. aureus were inhibited by cephalexin and cefaclor at 8 ,ug/ml, in contrast to 84% inhibited by cephradine at this level. The effects of inoculum size ofS. aureus on the activities of the three cephalosporins are noted in Fig. 1. A factor for each antibiotic was calculated by dividing the MIC obtained with an inoculum of 107 CFU/ml by that obtained with an inoculum of 105 CFU/ml, as described elsewhere by Sabath et al. (7). The factors for cephalexin, cephradine, and cefaclor were c8, 16, and c64, respectively. Cefaclor was highly effective against group A and group B streptococci and inhibited all strains tested at 0.25 and 2.0 ug/ml, respectively. All strains of Streptococcus pneumoniae were inhibited by 1.0 ,ug/ ml. All of the 21 strains of pathogenic Neisseria were inhibited by 0.25 ,ug of cefaclor per ml, whereas 4 ,ug of cephalexin and 4 to 8 ,ug of cephradine per ml were required to achieve the same results.
TABLE 1. Susceptibility of gram-positive and gram-negative bacilli to cephalosporins MIC,0 (jAg/Mj)a No. of strains Organism Cephradine Cephalexin 8 16 82 resistaureus (penicillin Staphylococcus ant) 8 8 20 S. aureus (penicillin susceptible) 32 34 32 S. epidermidis (penicillin resistant) 4 4 18 S. epidermidis (penicillin susceptible) 0.5 0.5 17 Streptococcus, group A 4 2 20 Streptococcus, group B 128 128 67 Streptococcus, group D 4 2 15 S. pneumoniae 2 2 16 Neisseria gonorrhoeae 4 8 7 N. meningitidis 16 16 195 Escherichia coli 51 8 16 Klebsiella pneumoniae >128 >128 16 Enterobacter aerogenes >128 >128 25 E. cloacae >128 14 >128 Serratia marcescens >128 >128 32 Proteus mirabilis >128 >128 32 Proteus (indole positive) 8 16 21 Salmonella 8 16 20 Shigella 64 64 6 Citrobacter diversus >128 >128 6 C. freundii >128 >128 18 Providencia stuartii >128 >128 70 Pseudomonas aeruginosa >128 >128 6 P. maltophilia a Lowest concentration inhibiting at least 90% of strains tested.
-
Cefaclor 8
4 32 2 0.25
2 64 0.5 0.25 0.25
8 8 >128 >128 >128 >128 >128 2 8 8 >128 >128 >128 >128
472
ANTimicROB. AGIENTS CHEMOTHER.
BILL AND WASHINGTON
clor, with 83, 95, and 75%, respectively, being inhibited with 2 jig/ml. To obtain similar results, a four- to eightfold increase in the concentrations of cephalexin and cephradine was required for this purpose. Proteus mirabilis was more susceptible to cefaclor than cephalexin and cephradine, although 32 ,ug of cefaclor per ml was required to inhibit 75% of the 32 strains tested. The effects of inoculum size and the effect of the presence of,-lactamase on the susceptibility of H. influenzae to cephalexin, cephradine, and cefaclor are shown in Table 2. A significant inoculum effect was demonstrated for all three antibiotics with both /3-lactamase-positive and 3-lactamase-negative H. influenzae. The presence of (8-lactamase did not affect the activity of cefaclor but did affect the activity of cephalexin and cephradine. Inhibition ofH. influenzae was achieved with only 2 ,ug of cefaclor per ml, in 8 contrast to 8 Ag of cephalexin and 16 ,ug of cephradine per ml needed for ,B-lactamase-negU) 6 ative organisms, and 32 ,ug of cephalexin and 64 *-A Cephalexin co ,ug of cephradine per ml needed for /8-lacta0--o Cephradine organisms. mase-positive .) - Cefaclor The activities of the three antibiotics against 4 Bacteroides fragilis, B. melaninogenicus, and anaerobic gram-positive cocci are listed in Table 3. Cephalexin, cephradine, and especially 2 cefaclor had little effect onB. fragilis. At 64 ,ug/ ml, both cephalexin and cephradine inhibited 83% of the 18 strains of B. fragilis, whereas \ cefaclor inhibited only 11%. B. melaninogeniI I _I i 1 2 4 8 6 32 64128 cus was more susceptible to the cephalosporins than was B. fragilis, particularly if the strains / IC 10' col/ml of B. melaninogenicus were inhibited by peniFactor MIC 105 col/ml cillin at concentrations of '0.5 pAg/ml. CephaFIG. 1. Effect of inoculum size on MIC of three lexin, cephradine, and cefaclor required 1, 2, and 0.5 ,g/ml, respectively, for inhibition of B. cephalosporins for 10 strains of S. aureus.
The lowest concentrations ofthe three cephalosporins inhibiting at least 90% of facultatively anaerobic gram-negative bacilli are also shown in Table 1. At 4 ,ug/ml, cefaclor was more effective than cephalexin or cephradine against E. coli, with 83, 35, and 5%, respectively, of the 195 strains inhibited at this concentration. Cephalexin and cefaclor were more effective than cephradine against E. coli at 8 ,ug/ml, with 84, 93, and 55%, respectively, of the strains being inhibited. Similar results were obtained for Klebsiella pneumoniae. The three cephalosporins had little or no effect on Enterobacter aerogenes, E. cloacae, Serratia marcescens, indole-positive Proteus, Citrobacter freundii, Providencia stuartii, and Pseudomonas. Citrobacter diversus, Salmonella, and Shigella were highly susceptible to cefa-
hk.
.
..
.*
TABLE 2. Susceptibility ofHaemophilus influenzae to cephalosporins Cumulative * inhibited at increasing concn (^g/ml) Inoculum No of
j9-lactamase size (CFU/ +
+
-
-
ml) 104
106
104
106
st°in
Antibiotic
15
Cephalexin Cephradine Cefaclor
10
10
10
0.12
0.25
0.5
7
Cephalexin Cephradine Cefaclor Cephalexin Cephradine Cefaclor
Cephalexin Cephradine Cefaclor
1
2
4
8
67
87 93 100 67 93 93 100
16
32
64
128
73 100
10 10 10
10 20 30 20 20
60 60 30
50 40 30
60 40 30
80 40 50
80 80 70 80 50 50
50 100 50 100 30
90 100
20 10 40
30 40 10 40 40 40
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COMPARISON OF THREE CEPHALOSPORINS
VOL. 11, 1977
TABLz 3. Susceptibility of anaerobic bacteria to cephalosporins Cumulative % inhibited at increasing concn (gg/ml)
No. o No of
Stral5
Antibiotic
SO.12
0.26
0.5
1
2
4
8
Peptococcus
17
Cephalexin Cephradine Cefaclor
12 12 29
18 18 35
29 29 53
29 35 71
53 47 76
76 76 82
82 82 76 82 82 88
82 94 100 88 94 100 94 100
Peptostreptococcus
10
Cephalexin Cephradine Cefaclor
10 10 10
10 20 20
20 20 30
30 30 40
40 40 60
70 70 70
70 80 70 80 80 80
90 100 90 100 80 80 100
Bacteroides fragilw
18
Cephalexin Cephradine Cefaclor
B. melaninogenicus (penicillin MIC, s0.5)
4
Cephalexin Cephradine Cefaclor
B. melaninogenicus (penicillin MIC, >0.5)
5
Cephalexin Cephradine Cefaclor
Organism
16
6 11 6 11
25 25 25
melaninogenicus with MIC values to penicillin of 0.5 ug/ml. Of the three antibiotics, cefaclor appeared to be slightly more effective against the anaerobic gram-positive cocci that were tested. At 2 ,ug/ ml, cephalexin, cephradine, and cefaclor inhibited 53, 47, and 76%, respectively, of the 17 strains of Peptococcus and 40, 40, and 60%, respectively, of the 10 strains of Peptostreptococcus.
Neither cefaclor nor cephradine produced 100% killing of 105 and 107 CFU of penicillinsusceptible and penicillin-resistant strains ofS. aureus per ml at concentrations of s 128 ug/ml. With one exception, neither cefaclor nor cephradine produced 99.9% killing of staphylococci in an inoculum of 107 CFU/ml. With one strain, cephradine produced 99.9% killing at 32 ,ug/ml. The MBC values of cefaclor against three strains of E. coli were the same as, two times (in the first two instances) and eight times the MIC values, respectively. With three strains ofK. pneumoniae, the MBC values were four times the MIC values in two instances and twice the MIC value in one instance. DISCUSSION Against both penicillinase- and non-penicillinase-producing strains of S. aureus, cefaclor was at least three times as active as cephradine at 2 ug/ml and at least one and a half times as active as cephalexin at this concentration. The effects of inoculum size of S. aureus on the
32
44 61
64
83 83 11
128
94 89 72
25 25 100 25 25 75 100 75 100
60 80 100 40 100 40 40 80 100 /~~~~~~~~~~~~~~~~~~~~~~~
activities of the three cephalosporins varied substantially (Fig. 1), with cephalexin being the least affected by penicillinase-producing strains and cefaclor being the most affected by such strains; cephradine was intermediate between the two. Similar studies by Sabath et al. (7) have shown that cephalexin was only slightly less resistant to staphylococcal penicillinase than was cephalothin. These data disagree with those reported by Fong et al. (3), who assayed for residual cephalosporin activity in broth cultures of S. aureus and found that cephalexin was completely inactivated by staphylococcal penicillinase within 12 h, in contrast to cephalothin, which underwent little inactivation. They also found that cephradine, which was not tested by Sabath et al. (7), was degraded more slowly than cephalexin, with 18% of the original concentration being measurable at 12 h. Cephradine was found to be more resistant than cephalexin to crude ,8-lactamase derived from S. aureus by Hubsher et al. (4). At 2 ug/ml, cefaclor inhibited all /8-lactamase-positive and -negative strains of H. influenzae at the generally recommended inoculum of 104 CFU/ml (11), in contrast to cephalexin and cephradine, which required substantially greater concentrations to achieve the same order of activity and which were also considerably affected by Haemophilus /8-lactamase. None of the cephalosporins inhibited a significant number of the strains of H. influenzae at the higher inoculum size. Our results with cephalexin and cephradine are similar to those reported by Emerson et al. (1). Whether or not cefaclor's
474
BILL AND WASHNGTON
greater activity against both 3-lactamase-positive and -negative strains at the lower inoculum is clinically significant remains to be seen. Cefaclor was also significantly more active against gonococci and meningococci than the other two agents and inhibited all strains at 0.25 jug/ml. The MIC values of cefaclor were at least 1/16 of those of the other two cephalosporins; however, no 84-lactamase-positive strains of Neisseria gonorrhoeae were studied. In general, cephalosporins have demonstrated broad ranges of MIC values against these two species of Neisseria. Cefaclor was substantially more active than cephalexin and cephradine against E. coli, K. pneumoniae, P. mirabilis, salmonellae, shigellae, and C. diversus. All three antibiotics were essentially inactive against Enterobacter, Serratia, indole-positive species of the Proteeae, Citrobacter freundii, and species of Pseudomonas. Klastersky et al. (5) and Scholand et al. (8) have previously reported that the antibacterial activity of cephradine was nearly identical to that of cephalexin. McGowan et al. (6), however, reported that cephradine was less active than cephalexin against blood culture isolates of E. coli, Klebsiella, and P. mirabilis. The methodologies used in each of these studies differed considerably from one another and in varying degrees from the reference WHO-ICS method (2, 11). It was, therefore, of interest to us to try to resolve these different results by testing each of the cephalosporins with the reference method. In general, we agree with McGowan et al. (6) that cephradine is less active than cephalexin. Moreover, our results demonstrate that neither antibiotic is as active as cefaclor against gram-positive and gram-negative aerobic and facultatively anaerobic bacteria. All three cephalosporins were active at clinically achievable serum levels (8 to 16 Ag/ml) against most anaerobic cocci, but they were essentially inactive at these levels againstBacteroides fragilis. With B. melaninogenicus their activity correlated closely with the penicillin susceptibility of the strains tested. The four strains that were inhibited by sO.5 Mug of penicillin per ml were also inhibited by 1, 2, and 0.5 MAg of cephalexin, cephradine, and cefaclor per ml, respectively. The five strains requiring >0.5 Mug of penicillin per ml for inhibition required 8, 4, and 32 Mug of cephalexin, cephradine, and cefaclor per ml, respectively, for complete inhibition. In conclusion, we have found cefaclor to be substantially more active than cephalexin, which in turn was more active than cephra-
ANTimicRoB. AGENTS CHEMOTHER.
dine, against staphylococci, streptococci, gonococci, meningococci, and certain species of the Enterobacteriaceae. Cefaclor was also highly active against both ,B-lactamase-positive and -negative strains of H. influenzae at the recommended inoculum size. Clinical studies are required to substantiate cefaclor's value in the therapy of infections due to these susceptible species. ACKNOWLEDGMENT This work was supported by a grant from the Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Ind. LITERATURE CITED 1. Emerson, B. B., A. L. Smith, A. L. Hading, and D. H. Smith. 1975. Haemophilus influensae type B susceptibility to 17 antibiotics. J. Pediatr. 86:617620. 2. Ericsson, H. M., and J. C. Sherris. 1971. Antibiotic sensitivity testing report of an international collaborative study. Acta Pathol. Microbiol. Scand. Sect. B Suppl. 217:1. 3. Fong, I. W., E. R. Engelking, and W. M. Kirby. 1976. Relative inactivation by Staphylococcus aureus of eight cephalosporn antibiotics. Antimicrob. Ag. Chemother. 9:939-944. 4. Hubeher, J. A., H. H. Gadebusch, and A. G. Itkin. 1976. Double-blind comparison of oephradine and cephalexin in the treatment of skin and soft-tissue infections due to Staphylococcus aureus. Curr. Ther. Res. Clin. Exp. 19:579-588. 5. Klasterak, J., D. Danea, and D. Weerts. 1973. Cepbradine: antibacterial activity and clinical effectiveness. Chemotherapy 18:191-204. 6. McGowan, J. E., Jr., C. Garner, C. Wilcox, and M. Finland. 1974. Antibiotic susceptibility of gramnegative bacilli isolated from blood cultures reults of tests with 35 agents and strains from 169 patients at Boston City Hospital during 1972. Am. J. Med. 57:225-238. 7. Sabath, L. D., C. Garner, C. Wilcox, and M. Finland. 1975. Effect of inoculum and ofbeta-lactamase on the anti-staphylococcal activity of thirteen penicill and cephaloporins, Antimicrob. Ag. Chemother. 8:344-349. 8. Scholand, J. F., G. R. Hodges, R. J. Fm, and B. Snlaw. 1974. Clinical evaluation of cepbradine, a new oral cephalo n. Am. J. Med. Sci. 267:111-116. 9. Steers, E., E. L. Foltz, and B. S. Graves. 1959. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 10. Sutter, V. L., V. L. Vargo, and S. M. Finegold. 1975. Wadsworth anaeobic bacteriolo manual, 2nd ed., p. 60-62. Wadsworth Hospital Center, Los Angeles. 11. Thornsberry, C., and L. A. Kirven. 1974. Antimicrobial susceptibility of Haemophilus influenzae. Antimicrob. Ag. Chemother. 6:620-624. 12. Washington, J. A., I. 1974. Laboratory procedures in clinical microbiology. Little, Brown and Co., Boston. 13. Washington, J. A., II, and A. L. Barry. 1974. Dilution test procedures, p. 410-417. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 14. Washington, J. A., II, R. J. Snyder, and P. C. Kohner. 1976. Spurious ampicillin resistance by testing Hae-
mophilus influenzae with agar containing supple-
ment C. Antimicrob. Ag. Chemother. 9:199-200.
