ANTIMICROBIAL AGENTS
AND
CHEMOTHERAPY, Aug. 1976, P. 288-292
Copyright © 1976 American Society for Microbiology
Vol. 10, No. 2
Printed in U.S.A.
In Vitro Evaluation of the New Oral Cephalosporin Cefatrizine: Comparison with Other Cephalosporins C. CAROLINE BLACKWELL,* EARL H. FREIMER, AND GREGORY C. TUKE
Department of Medicine and Department of Microbiology,* Medical College of Ohio at Toledo, Toledo, Ohio 43614
Received for publication 23 February 1976
Cefatrizine (BL-S640), a semisynthetic, orally administered cephalosporin, found to have an in vitro spectrum of activity comparable to those of four other cephalosporins tested. It is as effective as cephalexin, the other orally administered cephalosporin evaluated, against most species, and it appears to be more effective than cephalexin against many Enterobacter, Haemophilus, and Proteus strains isolated in our hospital. It is not inactivated by the plasmiddetermined f-lactamases of 14 strains of ampicillin-resistant Salmonella typhimurium or the ampicillin resistance determinant of an H. influenza strain from the Center for Disease Control. No synergy was observed between cefatrizine and gentamicin, kanamycin, carbenicillin, or polymyxin when tested against selected strains. was
lates) and gram-positive (82 isolates) organisms were obtained from the clinical laboratory of the Hospital of the Medical College of Ohio at Toledo. All were stored in sterilized skimmed milk at -60°C. The strains to be tested were thawed and subcultured on solid medium to insure adequate growth. In addition, seven strains ofNeisseria meningitidis and an ampicillin-resistant Haemophilus influenzae strain sent to us from the Center for Disease Control, Atlanta, Ga., were included. Susceptibility studies. Minimal inhibitory concentrations (MICs) were determined by standard methods (6). Todd-Hewitt broth (BBL) was used to test Streptococcus species. Tryptic soy broth (Difco) was tried, but more reproducible results were obtained with the Todd-Hewitt medium. Clear GC medium used for Neisseria gonorrhoeae was composed of: soluble starch, 1 g; KH2PO4, 1 g; KCI, 5 g; K2HPO4, 4 g-, proteose peptone, 15 g-, water, 1 liter; glucose (20%, wt/vol), 25 ml; NaHCO3 (4.2%, wt/ MATERIALS AND METHODS vol), 10 ml; and supplement B (Difco), 10 ml. MuelAntibiotic agents. CF propylene glycolate (Bris- ler-Hinton medium (Difco) with 1% supplement B tol Laboratories, Syracuse, N.Y.) was supplied as was used for H. influenzae and N. meningitidis. the crystalline dipolar ion for use in antibiotic sus- Mueller-Hinton medium without any supplement ceptibility testing (2). KF, CL, CX, CZ, all from was used for all other strains tested. Twofold serial Lilly Research Laboratories, Eli Lilly & Co., Indian- dilutions of the antibiotics were prepared (128 to apolis, Ind., were used for comparative studies. 0.125 jig/ml). Tubes were inoculated with 105 orgaGentamicin sulfate (Garamycin, Schering Corp., nisms per ml and were incubated at 37°C for 18 to 24 Bloomfield, Mass.), disodium carbenicillin (Pyopen, h. Neisseria, Streptococcus, and Haemophilus Beecham-Massengil, Bristol, Tenn.), polymyxin sul- strains were incubated in 10% CO2. The MIC was fate (Aerosporin, Burroughs-Wellcome), and kana- recorded as the lowest concentration of antibiotic at mycin sulfate (Kantrex, Bristol Laboratories) were which there was no visible growth. Broth from tubes in which there was no visible used in synergy studies. All stock solutions of antibiotics were prepared daily. growth was inoculated to an agar plate by using a Bacterial isolates. Standard strains of Esche- calibrated platinum loop (3-mm diameter), and the richia coli ATCC 25922 and Staphylococcus aureus lowest concentration at which there was 99.9% killATCC 25923 were used in each trial as controls. ing of the inoculum was recorded as the minimal Representative strains of gram-negative (190 iso- bactericidal concentration (MBC). Duplicate plates 288
The need for an effective orally administered cephalosporin has been apparent for sometime. Cefatrizine (CF), BL-S640, is a new semisynthetic cephalosporim that is absorbed well from the gastrointestinal tract (2). The other orally administered cephalosporin in present use, cephalexin (CX), has less antibacterial activity than other members of this group of compounds: cephalothin (KF), cephaloridine (CL), cefazolin (CZ), and cephaloglycin (3, 7). In conjunction with clinical trials of this new cephalosporin, sensitivities of 272 local bacterial isolates were determined and compared for CF, KF, CL, CX, and CZ. In addition, Enterobacter, Pseudomonas, and Serratia strains were tested for synergistic effects of CF with other antibiotics.
VOL. 10, 1976
IN VITRO SUSCEPTIBILITIES TO CEFATRIZINE
289
were inoculated with 0.1 ml of an appropriate dilu- The strains that were susceptible to 8 ,ug/ml or tion to determine the number of colony-forming less usually had zones ranging from 18 to 30 units in the inoculum. Disk susceptibilities to each mm. The majority of strains that were resistant of the cephalosporins were determined by the Kirby- to 8 ug/ml or greater had zones ranging from 0 Bauer method (1). Zone sizes, indicating susceptibility or resistance, were recorded for KF, CL, CX and to 16 mm. Activity of cephalosporins against gramCZ. The size of the zone surrounding the CF disk positive isolates. Emphasis, reflected in the was recorded in millimeters. Synergy studies. Synergy studies were performed numbers of each species tested, was on the on Serratia, Pseudomonas, and Enterobacter species organisms that are frequently resistant to antiby the agar dilution method. Plates containing anti- biotics in common use. Each strain of Streptobiotic (0.125 to 128 ,ug/ml) or antibiotic plus one of coccus pyogenes and Streptococcus pneumoniae three concentrations of CF (0.25, 2, or 16 ,ug/ml) was sensitive to concentrations of CF readily were inoculated with a Steers replicator (9). Approximately 100 to 300 colony-forming units per spot obtainable in serum (Table 2). For both S. pywere inoculated by each prong of the replicator. ogenes and S. pneumoniae, the diameter of the Plates were prepared each day from fresh stock solu- zone of inhibition surrounding the disk containtions of antibiotics and dried for 2 h in an incubator ing 30 ,ug of CF per ml ranged from 20 to 30 before inoculation. The inoculated plates were incu- mm. The MBC was usually the same as the bated for 18 to 24 h at 37°C. Growth of fewer than MIC or within one- or two-tube dilutions of the five colonies per inoculum indicated susceptibility. MIC.
RESULTS Activity of cephalosporins against gramnegative isolates. Table 1 records the MICs for some of the more common gram-negative organisms. Each strain was tested at least twice under the conditions described above. Most strains of E. coli and Klebsiella were inhibited by low concentrations (128 ,g of each of the cephalosporins per ml. Each of 15 strains of S. typhimurium, 14 of which contained plasmids mediating resistance to ampicillin, was inhibited by 8 ,ug of BL-S640 per ml. Forty-two percent ofthe Proteus species tested were inhibited at this concentration of CF; similar values were found for KF (42%) and CZ (47%). CL and CX appeared less effective in their abilities to inhibit these strains. CF was not as effective against Enterobacter at 8 ,.g/ml (31% of strains inhibited) as CZ (50%) but definitely more so han the other three cephalosporins. Both N. meningitidis and gonorrhoeae were inhibited by low concentrations of CF. All isolates of H. influenzae were inhibited by 8 ,g of CF and KF per ml. CL and CZ inhibited 89% of the strains, but only 22% of the strains had MICs of 8 jig/ml or less with CX. An ampicillin-resistant strain (25 to 50 ug of ampicillin per ml) from the Center for Disease Control was sensitive at 8 ,ug of BL-S640 per ml, 2 ,ug of KF per ml, 8 ,g of CL per ml, 16 ,ug of CX per ml, and 8 ,tg of CZ per ml. MBCs were usually within one or two dilutions of the MIC. With disks containing 30 ,ug of CF per ml, the zone of inhibition ranged from 0 to 30 mm.
One strain of methicillin-resistant S. aureus, DU 4916, was resistant to >128 ,ug of each of the cephalosporins tested per ml, except for CL (MIC = 16 ,tg/ml). Zone diameters for those staphylococcal strains susceptible to c8 ug of CF per ml ranged from 18 to 40 mm. Those that required >8 jug of BL-S640 per ml had a diameter of 16 mm or less. MBC values varied; not all MBCs were within the one- or two-tube dilution range of the MIC. The only consistent observation for S. aureus was that size of the original inoculum influenced the MBC value; larger inocula usually resulted in higher values for both the MIC and MBC. The enterococci were resistant to these compounds, 16 ,g/ml being the lowest MIC. The MBC was not reached at the concentrations used. Zone diameters determined by the KirbyBauer method ranged from 10 to 16 mm, the majority being 14 or 15 mm. Synergy studies. Enterobacter, Pseudomonas, and Serratia strains were tested for synergistic effects of CF with other antibiotics. Pseudomonas and Serratia were examined for susceptibilities to gentamicin and carbenicillin and for the effect of the addition of CF at three concentrations (0.25, 2, and 16 ,mg/ml). As in the tube dilution experiments, each of the strains was resistant to CF. All but two strains, both Pseudomonas, were inhibited by 1 ug of gentamicin per ml; these two strains required 2 ,.g/ml. No synergistic effect was noted for the majority of the strains; in fact, in several instances, a slightly antagonistic effect was seen in the presence of CF. There was a slight amount of growth, a thin film or a few tiny colonies, on plates containing both gentamicin and CF, but no growth on gentamicin alone. The results obtained with carbenicillin were
TABLE 1. MICs of cephalosporins of gram-negative organisms Organismn and no. [ Antibiotic of isolates
Enterobacter sp.
T
No. of isolates susceptible at a cephalosporin concn (tg/ml) of: 64 | 128 1 2 4 8 1 16 | 32
0.125 0.25 ! 0.5
CF
5
3
1
2
3
1
1 3 4
1
4
3 1 1
8 8 5 5
(16)
KF CL CX CZ
Escherichia coli (47)
CF
3
KF CL CX CZ
9
Kkbsiella pneumoniae (18)
Neisseria gonorrhoeae (10)
N. meningitidis
(7)
Pseudomonas aeruginosa (31)
3
4
9
17
9
5
2
1 1
4 22 2 7
16 17 27 5
17 5 15 2
5 1 1
1 6 1
1
24
CF
Haemophilus influenzae (9)
1
4 1 4 1
3
KF CL CX CZ
6
CF KF CL CX CZ 4
KF CL CX CZ
1 9 7 10
CF KF CL cx CZ
Salmonella typhimurium
1
4
1
3 2 3
4
5
2
3
1
2
3
5 7 2 3
3 5 10
1 2 3 4
8 1 3
1
4
1
2
1
1
4 1 1 3
.
1
5
1
2
1 1
1
2
1
1 2 2 2
1
3
3 3
CF
31
KF CL cx CZ
31 31 31 31 8 7
1
4
10
9
13 6
1 3 1
8 7
1
1
1
6
3
5
3
2 5
2 4
1
11
KF CL CX
CZ .-A
=1
I
I 290
4
3 4 3
2 3 3 2 2
1 1 1
1
2.
4
3
CF
-1
2
1
KF CL CX CZ
Serratia marcescens (7)
2
2 1
6
CF
(15)
2
3 4
CF KF CL CX CZ
Proteus sp. (19)
1
6
8
CF
2
>128
i
I
7
i
I
I
7 7 7 7 I
VOL. 10, 1976
IN VITRO SUSCEPTIBILITIES TO CEFATRIZINE
291
TABLE 2. MICs of cephalosporins of gram-positive organisms Organism and no. of Antibiotisolates ics 0.125
Staphylococcus aureus (52)
CF KF CL cX CZ
Enterococci (13)
4 22 1 1
No. of isolates susceptible at a cephalosporin concn (,&g/ml) of: 0.25 0.5 1 2 4 8 16 32 64 128
1
2
13
12
17
6 12 1 5
19 11 11 19
19 6 12 14
2
1
10 8
14 4
6 1 1 2
CF KF CL CX CZ
Streptococcus pneumoniae (6)
S. pyogenes (10)
2
KF
2
CL CX CZ
6
CF KF CL
8 7 10
2 3
CZ
1
9
CX
1
5
7 3
12
10 1 10
3
11
CF
3
>128
2
1
4 3
1
3
9
similar to those obtained with gentamicin. No synergy between the two antibiotics was observed, but there was very slight, if any, antagonism. Enterobacter strains were tested for synergistic effects of gentamicin, kanamycin, and polymyxin B with CF. Again, no significant synergistic effect was observed with any of the antibiotics tested.
by Leitner et al. (5). This may be due to differences in the methods used; Stilwell et al. (10) observed that CF appeared less active in broth than in agar. The MICs of CF against our strains are similar to those obtained by Overturf using the broth dilution method. Overturf reported lower MICs for H. influenzae, ranging from 1.60 to 3.13 Ag/ml (8). The present study found MICs that ranged from 2 to 8 ,ugl ml. Since the medium used by both groups was DISCUSSION the same, Mueller-Hinton broth containing 1% Our findings with isolates from the clinical supplement B, differences may reflect varialaboratory of the Medical College of Ohio Hos- tions in the local strains. The MICs in these studies were somewhat pital are in agreement with reports of other groups (5, 8, 10, 11) that CF is a broad-spectrum higher for the streptococci, but this may reflect antimicrobial agent effective against pyogenic, the differences in the media. Our data agree gram-positive cocci and many members of the with the findings of Kayser (4), who reported Enterobacteriaceae. It is not inactivated by the that CL was the most effective cephalosporin ,8-lactamases from 14 strains of S. typhimu- tested against S. aureus. CZ is certainly as effective as CX, the orally rium or the ampicillin resistance determinants of H. influenzae. Its in vitro activity compared administered cephalosporin, in most species well with the activities of the four other cepha- evaluated, and it appears to be more effective losporins tested (5, 8). Against the gram-nega- than CX against many strains of Enterobacter, tive organisms, it was as active as CZ and Haemophilus, and Proteus isolated in our hosusually more active than CX or KF (Table pital. 1). These findings generally parallel those ACKNOWLEDGMENTS obtained by Watanakunakorn et al. (11), who This study was supported by a grant-in-aid from Bristol used the agar dilution method. Our MIC values Laboratories, Syracuse, N.Y. N. for N. meningitidis and gonorrhoeae apWe thank Cheryl Zimmerman for her assistance in preppear to be somewhat lower than those reported aration of this manuscript.
292
ANTIMICROB. AGENTS CHEMOTHER.
BLACKWELL, FREIMER, AND TUKE LITERATURE CITED 7.
1. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45:493-496. 2. Bristol Laboratories. 1974. Basic data brochure, oral BL-S640 PG. Bristol Laboratories, Syracuse, N.Y. 3. Finland, M. 1972. Oral and parenteral cephalosporins: the place of cephalexin in antibacterial therapy. Drugs 3:1-8. 4. Kayser, F. H. 1971. In vitro activity of cephalosporin antibiotics against gram positive bacteria. Postgrad. Med. J. 47(Suppl.):14-18. 5. Leitner, F., R. E. Buck, M. Misiek, T. A. Pursiano, and K. B. Price. 1975. BL-S640, a cephalosporin with a broad spectrum of antibacterial activity: properties in vitro. Antimicrob. Agents Chemother. 7:298-305. 6. Lennette, E. H., E. H. Spaulding, and J. P. Truant (ed.). 1974. Manual of clinical microbiology, p. 407-
8.
9.
10.
11.
442. American Society for-Microbiology, Washington, D.C. Levison, M. E., W. D. Johnson, T. S. Thornhill, and D. Kaye. 1969. Clinical and in vitro evaluation of cephalexin. J. Am. Med. Assoc. 209:1331-1336. Overturf, G. D., R. L. Ressler, P. B. Marengo, and J. Wilkins. 1975. In vitro evaluation of BIS640, a new oral cephalosporin antibiotic. Antimicrob. Agents Chemother. 8:305-310. Steers, E. E., E. L. Foltz, and B. S. Graves. 1959. Inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. Stilwell, G. A., H. G. Adams, and M. Turck. 1975. In vitro evaluation of a new oral cephalosporin, cefatrizine (BI.S640). Antimicrob. Agents Chemother. 8:751-753. Watanakunakorn, C., T. Bannister, and C. Glozbecker. 1975. Susceptibility of clinical isolates ofEnterobacteriaceae to BLS640, a new oral cephalosporin. Antimicrob. Agents Chemother. 7:381-385.
AND
CHEMOTHERAPY, Aug. 1976, P. 288-292
Copyright © 1976 American Society for Microbiology
Vol. 10, No. 2
Printed in U.S.A.
In Vitro Evaluation of the New Oral Cephalosporin Cefatrizine: Comparison with Other Cephalosporins C. CAROLINE BLACKWELL,* EARL H. FREIMER, AND GREGORY C. TUKE
Department of Medicine and Department of Microbiology,* Medical College of Ohio at Toledo, Toledo, Ohio 43614
Received for publication 23 February 1976
Cefatrizine (BL-S640), a semisynthetic, orally administered cephalosporin, found to have an in vitro spectrum of activity comparable to those of four other cephalosporins tested. It is as effective as cephalexin, the other orally administered cephalosporin evaluated, against most species, and it appears to be more effective than cephalexin against many Enterobacter, Haemophilus, and Proteus strains isolated in our hospital. It is not inactivated by the plasmiddetermined f-lactamases of 14 strains of ampicillin-resistant Salmonella typhimurium or the ampicillin resistance determinant of an H. influenza strain from the Center for Disease Control. No synergy was observed between cefatrizine and gentamicin, kanamycin, carbenicillin, or polymyxin when tested against selected strains. was
lates) and gram-positive (82 isolates) organisms were obtained from the clinical laboratory of the Hospital of the Medical College of Ohio at Toledo. All were stored in sterilized skimmed milk at -60°C. The strains to be tested were thawed and subcultured on solid medium to insure adequate growth. In addition, seven strains ofNeisseria meningitidis and an ampicillin-resistant Haemophilus influenzae strain sent to us from the Center for Disease Control, Atlanta, Ga., were included. Susceptibility studies. Minimal inhibitory concentrations (MICs) were determined by standard methods (6). Todd-Hewitt broth (BBL) was used to test Streptococcus species. Tryptic soy broth (Difco) was tried, but more reproducible results were obtained with the Todd-Hewitt medium. Clear GC medium used for Neisseria gonorrhoeae was composed of: soluble starch, 1 g; KH2PO4, 1 g; KCI, 5 g; K2HPO4, 4 g-, proteose peptone, 15 g-, water, 1 liter; glucose (20%, wt/vol), 25 ml; NaHCO3 (4.2%, wt/ MATERIALS AND METHODS vol), 10 ml; and supplement B (Difco), 10 ml. MuelAntibiotic agents. CF propylene glycolate (Bris- ler-Hinton medium (Difco) with 1% supplement B tol Laboratories, Syracuse, N.Y.) was supplied as was used for H. influenzae and N. meningitidis. the crystalline dipolar ion for use in antibiotic sus- Mueller-Hinton medium without any supplement ceptibility testing (2). KF, CL, CX, CZ, all from was used for all other strains tested. Twofold serial Lilly Research Laboratories, Eli Lilly & Co., Indian- dilutions of the antibiotics were prepared (128 to apolis, Ind., were used for comparative studies. 0.125 jig/ml). Tubes were inoculated with 105 orgaGentamicin sulfate (Garamycin, Schering Corp., nisms per ml and were incubated at 37°C for 18 to 24 Bloomfield, Mass.), disodium carbenicillin (Pyopen, h. Neisseria, Streptococcus, and Haemophilus Beecham-Massengil, Bristol, Tenn.), polymyxin sul- strains were incubated in 10% CO2. The MIC was fate (Aerosporin, Burroughs-Wellcome), and kana- recorded as the lowest concentration of antibiotic at mycin sulfate (Kantrex, Bristol Laboratories) were which there was no visible growth. Broth from tubes in which there was no visible used in synergy studies. All stock solutions of antibiotics were prepared daily. growth was inoculated to an agar plate by using a Bacterial isolates. Standard strains of Esche- calibrated platinum loop (3-mm diameter), and the richia coli ATCC 25922 and Staphylococcus aureus lowest concentration at which there was 99.9% killATCC 25923 were used in each trial as controls. ing of the inoculum was recorded as the minimal Representative strains of gram-negative (190 iso- bactericidal concentration (MBC). Duplicate plates 288
The need for an effective orally administered cephalosporin has been apparent for sometime. Cefatrizine (CF), BL-S640, is a new semisynthetic cephalosporim that is absorbed well from the gastrointestinal tract (2). The other orally administered cephalosporin in present use, cephalexin (CX), has less antibacterial activity than other members of this group of compounds: cephalothin (KF), cephaloridine (CL), cefazolin (CZ), and cephaloglycin (3, 7). In conjunction with clinical trials of this new cephalosporin, sensitivities of 272 local bacterial isolates were determined and compared for CF, KF, CL, CX, and CZ. In addition, Enterobacter, Pseudomonas, and Serratia strains were tested for synergistic effects of CF with other antibiotics.
VOL. 10, 1976
IN VITRO SUSCEPTIBILITIES TO CEFATRIZINE
289
were inoculated with 0.1 ml of an appropriate dilu- The strains that were susceptible to 8 ,ug/ml or tion to determine the number of colony-forming less usually had zones ranging from 18 to 30 units in the inoculum. Disk susceptibilities to each mm. The majority of strains that were resistant of the cephalosporins were determined by the Kirby- to 8 ug/ml or greater had zones ranging from 0 Bauer method (1). Zone sizes, indicating susceptibility or resistance, were recorded for KF, CL, CX and to 16 mm. Activity of cephalosporins against gramCZ. The size of the zone surrounding the CF disk positive isolates. Emphasis, reflected in the was recorded in millimeters. Synergy studies. Synergy studies were performed numbers of each species tested, was on the on Serratia, Pseudomonas, and Enterobacter species organisms that are frequently resistant to antiby the agar dilution method. Plates containing anti- biotics in common use. Each strain of Streptobiotic (0.125 to 128 ,ug/ml) or antibiotic plus one of coccus pyogenes and Streptococcus pneumoniae three concentrations of CF (0.25, 2, or 16 ,ug/ml) was sensitive to concentrations of CF readily were inoculated with a Steers replicator (9). Approximately 100 to 300 colony-forming units per spot obtainable in serum (Table 2). For both S. pywere inoculated by each prong of the replicator. ogenes and S. pneumoniae, the diameter of the Plates were prepared each day from fresh stock solu- zone of inhibition surrounding the disk containtions of antibiotics and dried for 2 h in an incubator ing 30 ,ug of CF per ml ranged from 20 to 30 before inoculation. The inoculated plates were incu- mm. The MBC was usually the same as the bated for 18 to 24 h at 37°C. Growth of fewer than MIC or within one- or two-tube dilutions of the five colonies per inoculum indicated susceptibility. MIC.
RESULTS Activity of cephalosporins against gramnegative isolates. Table 1 records the MICs for some of the more common gram-negative organisms. Each strain was tested at least twice under the conditions described above. Most strains of E. coli and Klebsiella were inhibited by low concentrations (128 ,g of each of the cephalosporins per ml. Each of 15 strains of S. typhimurium, 14 of which contained plasmids mediating resistance to ampicillin, was inhibited by 8 ,ug of BL-S640 per ml. Forty-two percent ofthe Proteus species tested were inhibited at this concentration of CF; similar values were found for KF (42%) and CZ (47%). CL and CX appeared less effective in their abilities to inhibit these strains. CF was not as effective against Enterobacter at 8 ,.g/ml (31% of strains inhibited) as CZ (50%) but definitely more so han the other three cephalosporins. Both N. meningitidis and gonorrhoeae were inhibited by low concentrations of CF. All isolates of H. influenzae were inhibited by 8 ,g of CF and KF per ml. CL and CZ inhibited 89% of the strains, but only 22% of the strains had MICs of 8 jig/ml or less with CX. An ampicillin-resistant strain (25 to 50 ug of ampicillin per ml) from the Center for Disease Control was sensitive at 8 ,ug of BL-S640 per ml, 2 ,ug of KF per ml, 8 ,g of CL per ml, 16 ,ug of CX per ml, and 8 ,tg of CZ per ml. MBCs were usually within one or two dilutions of the MIC. With disks containing 30 ,ug of CF per ml, the zone of inhibition ranged from 0 to 30 mm.
One strain of methicillin-resistant S. aureus, DU 4916, was resistant to >128 ,ug of each of the cephalosporins tested per ml, except for CL (MIC = 16 ,tg/ml). Zone diameters for those staphylococcal strains susceptible to c8 ug of CF per ml ranged from 18 to 40 mm. Those that required >8 jug of BL-S640 per ml had a diameter of 16 mm or less. MBC values varied; not all MBCs were within the one- or two-tube dilution range of the MIC. The only consistent observation for S. aureus was that size of the original inoculum influenced the MBC value; larger inocula usually resulted in higher values for both the MIC and MBC. The enterococci were resistant to these compounds, 16 ,g/ml being the lowest MIC. The MBC was not reached at the concentrations used. Zone diameters determined by the KirbyBauer method ranged from 10 to 16 mm, the majority being 14 or 15 mm. Synergy studies. Enterobacter, Pseudomonas, and Serratia strains were tested for synergistic effects of CF with other antibiotics. Pseudomonas and Serratia were examined for susceptibilities to gentamicin and carbenicillin and for the effect of the addition of CF at three concentrations (0.25, 2, and 16 ,mg/ml). As in the tube dilution experiments, each of the strains was resistant to CF. All but two strains, both Pseudomonas, were inhibited by 1 ug of gentamicin per ml; these two strains required 2 ,.g/ml. No synergistic effect was noted for the majority of the strains; in fact, in several instances, a slightly antagonistic effect was seen in the presence of CF. There was a slight amount of growth, a thin film or a few tiny colonies, on plates containing both gentamicin and CF, but no growth on gentamicin alone. The results obtained with carbenicillin were
TABLE 1. MICs of cephalosporins of gram-negative organisms Organismn and no. [ Antibiotic of isolates
Enterobacter sp.
T
No. of isolates susceptible at a cephalosporin concn (tg/ml) of: 64 | 128 1 2 4 8 1 16 | 32
0.125 0.25 ! 0.5
CF
5
3
1
2
3
1
1 3 4
1
4
3 1 1
8 8 5 5
(16)
KF CL CX CZ
Escherichia coli (47)
CF
3
KF CL CX CZ
9
Kkbsiella pneumoniae (18)
Neisseria gonorrhoeae (10)
N. meningitidis
(7)
Pseudomonas aeruginosa (31)
3
4
9
17
9
5
2
1 1
4 22 2 7
16 17 27 5
17 5 15 2
5 1 1
1 6 1
1
24
CF
Haemophilus influenzae (9)
1
4 1 4 1
3
KF CL CX CZ
6
CF KF CL CX CZ 4
KF CL CX CZ
1 9 7 10
CF KF CL cx CZ
Salmonella typhimurium
1
4
1
3 2 3
4
5
2
3
1
2
3
5 7 2 3
3 5 10
1 2 3 4
8 1 3
1
4
1
2
1
1
4 1 1 3
.
1
5
1
2
1 1
1
2
1
1 2 2 2
1
3
3 3
CF
31
KF CL cx CZ
31 31 31 31 8 7
1
4
10
9
13 6
1 3 1
8 7
1
1
1
6
3
5
3
2 5
2 4
1
11
KF CL CX
CZ .-A
=1
I
I 290
4
3 4 3
2 3 3 2 2
1 1 1
1
2.
4
3
CF
-1
2
1
KF CL CX CZ
Serratia marcescens (7)
2
2 1
6
CF
(15)
2
3 4
CF KF CL CX CZ
Proteus sp. (19)
1
6
8
CF
2
>128
i
I
7
i
I
I
7 7 7 7 I
VOL. 10, 1976
IN VITRO SUSCEPTIBILITIES TO CEFATRIZINE
291
TABLE 2. MICs of cephalosporins of gram-positive organisms Organism and no. of Antibiotisolates ics 0.125
Staphylococcus aureus (52)
CF KF CL cX CZ
Enterococci (13)
4 22 1 1
No. of isolates susceptible at a cephalosporin concn (,&g/ml) of: 0.25 0.5 1 2 4 8 16 32 64 128
1
2
13
12
17
6 12 1 5
19 11 11 19
19 6 12 14
2
1
10 8
14 4
6 1 1 2
CF KF CL CX CZ
Streptococcus pneumoniae (6)
S. pyogenes (10)
2
KF
2
CL CX CZ
6
CF KF CL
8 7 10
2 3
CZ
1
9
CX
1
5
7 3
12
10 1 10
3
11
CF
3
>128
2
1
4 3
1
3
9
similar to those obtained with gentamicin. No synergy between the two antibiotics was observed, but there was very slight, if any, antagonism. Enterobacter strains were tested for synergistic effects of gentamicin, kanamycin, and polymyxin B with CF. Again, no significant synergistic effect was observed with any of the antibiotics tested.
by Leitner et al. (5). This may be due to differences in the methods used; Stilwell et al. (10) observed that CF appeared less active in broth than in agar. The MICs of CF against our strains are similar to those obtained by Overturf using the broth dilution method. Overturf reported lower MICs for H. influenzae, ranging from 1.60 to 3.13 Ag/ml (8). The present study found MICs that ranged from 2 to 8 ,ugl ml. Since the medium used by both groups was DISCUSSION the same, Mueller-Hinton broth containing 1% Our findings with isolates from the clinical supplement B, differences may reflect varialaboratory of the Medical College of Ohio Hos- tions in the local strains. The MICs in these studies were somewhat pital are in agreement with reports of other groups (5, 8, 10, 11) that CF is a broad-spectrum higher for the streptococci, but this may reflect antimicrobial agent effective against pyogenic, the differences in the media. Our data agree gram-positive cocci and many members of the with the findings of Kayser (4), who reported Enterobacteriaceae. It is not inactivated by the that CL was the most effective cephalosporin ,8-lactamases from 14 strains of S. typhimu- tested against S. aureus. CZ is certainly as effective as CX, the orally rium or the ampicillin resistance determinants of H. influenzae. Its in vitro activity compared administered cephalosporin, in most species well with the activities of the four other cepha- evaluated, and it appears to be more effective losporins tested (5, 8). Against the gram-nega- than CX against many strains of Enterobacter, tive organisms, it was as active as CZ and Haemophilus, and Proteus isolated in our hosusually more active than CX or KF (Table pital. 1). These findings generally parallel those ACKNOWLEDGMENTS obtained by Watanakunakorn et al. (11), who This study was supported by a grant-in-aid from Bristol used the agar dilution method. Our MIC values Laboratories, Syracuse, N.Y. N. for N. meningitidis and gonorrhoeae apWe thank Cheryl Zimmerman for her assistance in preppear to be somewhat lower than those reported aration of this manuscript.
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1. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45:493-496. 2. Bristol Laboratories. 1974. Basic data brochure, oral BL-S640 PG. Bristol Laboratories, Syracuse, N.Y. 3. Finland, M. 1972. Oral and parenteral cephalosporins: the place of cephalexin in antibacterial therapy. Drugs 3:1-8. 4. Kayser, F. H. 1971. In vitro activity of cephalosporin antibiotics against gram positive bacteria. Postgrad. Med. J. 47(Suppl.):14-18. 5. Leitner, F., R. E. Buck, M. Misiek, T. A. Pursiano, and K. B. Price. 1975. BL-S640, a cephalosporin with a broad spectrum of antibacterial activity: properties in vitro. Antimicrob. Agents Chemother. 7:298-305. 6. Lennette, E. H., E. H. Spaulding, and J. P. Truant (ed.). 1974. Manual of clinical microbiology, p. 407-
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