ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1975, p. 305-310 Copyright i 1975 American Society for Microbiology
Vol. 8, No. 3
Printed in U.S.A.
In Vitro Evaluation of BL-S640, a New Oral Cephalosporin Antibiotic GARY D. OVERTURF,* RONALD L. RESSLER, PAUL B. MARENGO, AND JEANETTE WILKINS The Department of Pediatrics and the Hastings Foundation Infectious Disease Laboratory, Los Angeles County/University of Southern California Medical Center, and the University of Southern California School of Medicine, Los Angeles, California 90033 Received for publication 18 April 1975
BL-S640, a new oral cephalosporin analogue, was evaluated in vitro against 102 gram-negative and 80 gram-positive bacteria. The antimicrobial spectrum was similar to that of previous cephalosporin analogues. Good antimicrobial activity against strains of Escherichia coli, Klebsiella, staphylococci, and streptococci was demonstrated. Relatively poor activity and/or resistance was noted among most strains of Proteus, Providencia, Pseudomonas, and Serratia. In comparative studies BL-S640 -had better activity against strains of Hemophilus influenzae, Staphylococcus aureus, and Enterobacteriaceae than many cephalosporin analogues. Variation of susceptibility results was dependent upon the type of media and inoculum size. Cross-resistance between BL-S640 cephalexin, cephalothin, and cefazolin was demonstrated. Among strains of Klebsiella the more rapid selection of resistance to other cephalosporins was in contrast to BL-S640. Experience in vitro with BL-S640 has documented its antimicrobial activity,and further studies of pharmacokinetics and therapeutic efficacy are indicated.
Since the introduction of cephalothin in 1962 be administered orally, intramuscularly, or in(1, 11) structural modification of the 7- travenously. BL-S640 17-D-alpha-amino-alpha- [4-hydroxyaminocephalosporanic acid nucleus has provided analogues for clinical use with improved phenylJacetamido [3-(1H-1,2,3-triazol-5-thio)physical-chemical, pharmacologic, or antimi- methyl]-3-cephem-4-carboxylic acidi is a new crobial properties (3-5, 9, 10). In particular, the semisynthetic cephalosporin (2). The structure need for cephalosporins suitable for oral admin- is shown in Fig. 1. A water-soluble preparation istration has been recognized. Cephaloglycin is available as a propylene glycolate, since aqueand cephalexin were the first acid-stable deriva- ous solutions are unstable and have been shown tives suitable for oral administration; in addi- to lose up to 50% of antimicrobial activity at pH tion, cephradine was introduced in 1974. How- 7.4 in several hours. (They are stable, however, ever, cephaloglycin is unstable at alkaline pH at -16 C for up to 10 days.) In anticipation that and is rapidly metabolized in the liver to the less this cephalosporin analogue may be suitable as potent desacetylcephaloglycin; therefore, oral an oral preparation, its in vitro antimicrobial administration results in low serum antibacte- spectrum and activity are reported. rial activity (5, 12). This has prohibited its use MATERIALS AND METHODS in systemic infections and confined it to treatment of urinary tract infections. In contrast, Antibiotics. BL-S640 propylene glycolate (Bristol cephalexin is totally absorbed after oral admin- Laboratories, Syracuse, N.Y.) was supplied as the istration and is excreted unchanged by the crystalline zwitterion for use in antibiotic susceptibilkidney (4, 8). Although it possesses desirable ity testing (2). Reference standard was prepared in pharmacological properties, cephalexin has less water in a concentration of 1,000 jig of BL-S640 per ml antimicrobial activity than cephalothin, cepha- and used immediately after preparation. Standard of cephalexin (Lilly Laboratories), cephloridine, cefazolin, or cephaloglycin (4, 8). De- preparations alothin (Lilly Laboratories), and cefazolin (Lilly Labspite this limitation, cephalexin has been used oratories) were used for appropriate comparative extensively since more active oral cephalospo- studies. rins are unavailable. A recently licensed anaBacterial isolates. All bacteria were recovered logue, cephradine, differs only slightly from from the blood, urine cerebrospinal fluid, or wounds of cephalexin in its antimicrobial activity and patients hospitalized in the Los Angeles County/ pharmacological properties (12, 5), and it can University of Southern California Medical Center. 305
306
OVERTURF ET AL.
ANrimICROB. AGENTS CHEMnomER. 0
NO
4< \
/
C
-
S
H N
~~~NH2
04_x~
CH2-
N
C -OH
S
N
H
11 0
FIG. 1. Chemical structure of BL-S640. Microorganisms, stored in either Mueller-Hinton (Enterobacteriaceae, Pseudomonras sp., staphylococci), tryptic soy (streptococci, pneumococci), or MuellerHinton plus 1% supplement B (Hemophilus influenzae) broth at -20 C, were thawed and subcultured on appropriate solid media to insure adequate growth. Susceptibility studies. The determinations of minimal inhibitory concentrations (MICs) were performed by standard methods (7). Mueller-Hinton broth (Difco) was utilized in susceptibility studies with Enterobacteriaceae, Pseudomonas sp., and staphylococci. Supplement B (Difco) in a concentration of 1% was added to Mueller-Hinton broth for use with H. influenzae. Tryptic soy broth (Difco) was used for studies with Streptococcus pneumoniae and other streptococci. Each cephalosporin was diluted serially twofold, from 200 to 0.1 pg/ml, except for studies with streptococci where concentrations from 200 to 0.025 pggml were utilized. After overnight incubation at 37 C, cultures were diluted to prepare a standard inoculum of 10' organisms. Streptococci, S. pneumoniae, and Hemophilus microorganisms were always incubated in 10% CO. The MIC was defined as the lowest concentration of antibiotic which allowed no visible turbidity after incubation for 18 to 20 h at 37 C. Tubes without turbidity were inoculated on appropriate solid media with a 3-mm loop and the lowest concentration yielding 10 colonies or less was defined as the minimal bactericidal concentration (MBC). In susceptibility studies against selected staphylococci, Pseudomonas sp., and Enterobacteriaceae, results with Mueller-Hinton agar or broth (Difco) and nutrient agar or broth (Difco) were compared. BL-S640 in twofold dilutions (200 to 0.1 pg/ml) was added to agar at 47 C. Antibiotic plates were inoculated immediately after their preparation. The inocula were varied in parallel broth and agar studies to contain 10', 106, or 107 microorganisms. Antibiotic stability study. Antibiotic stability was evaluated by determination of the MIC against selected microorganisms in twofold dilutions of BL-S640 that had been maintained at room temperature for 0, 24, 48, and 72 h.
Cross-resistance study. Representative grampositive and/or gram-negative microorganisms were selected to study cross-resistance between BL-8640 and cephalothin, cephalexin, or cefazolin. First, selected strains with demonstrated susceptibility to each of the cephalosporins were inoculated into seri-
ally diluted concentrations of BL-S640 as previously described. Those microorganisms surviving the highest concentration of BL-S640 after overnight incubation were re-inoculated into appropriate broth. After another 18 to 20 h of incubation, these microorganisms were used to prepare the inoculum for similar tubes containing the serially diluted BL-S640. After seven such passages in BL-S640, determinations of MICs for each strain were repeated to BL-S640, cephalexin, and cephalothin. In addition, cefazolin was included in cross-resistance studies with strains
of Klebsiella. To test the stability of the resistance acquired after serial passage in BL-S640, all strains were subcultured for 10 days on Mueller-Hinton media without added antibiotic. Change in antibiotic resistance was screened for by daily disk susceptibility tests to cephalothin and documented by the determination of MICs to all of the selected cephalosporins.
RESULTS Antimicrobial spectrum. Most strains of Escherichia coli and Klebsiella were susceptible to low concentrations of BL-S640; 30 of 35 strains (85%) were inhibited at concentrations of .12.5 pg of BL-S640 per ml (Table 1). However, the remaining five strains were resistant at concentrations of >50 pAg of BL-S640 per ml. Strains of Citrobacter were inhibited at concentrations of .6.25 pAg of BL-S640 per ml. Strains of Serratia and Pseudomonas uniformly required concentrations of > 50 ug of BL-S640 per ml for inhibition of growth (Table 1). Enterobacter and Providencia frequently required > 12.5 pg of BL-S640 per ml to inhibit growth. The MICs of Proteus strains were separated sharply into those < 1.60 pg of BL-S640 per ml and those .12.5 pg of BL-S640 per ml. However, the susceptibility of the 14 strains varied and did not correlate with indole production. Strains of methicillin-susceptible staphylococci had a median MIC and MBC of 3.13 and 12.5 pg of BL-S640 per ml, respectively (Table 2). In contrast, methicillin-resistant staphylococci were uniformly resistant, requiring concentrations of . 25 ,g BL-S640 per ml to inhibit growth. Streptococci were uniformly susceptible
307
IN VITRO EVALUATION OF BL-S640
VOL. 8, 1975
TABLE 1. Cumulative MICs and MBCs of 79 strains of Enterobacteriaceae and Pseudomonas for BL-S640 BL-S 640 (,ug/ml)
No.
Deter-
Species Escherichia coli
tested 20
mination MIC
200
12.50 > 200
11 14
200 0.025-25 0.1-12.5 0.1-0.4
>200 0.2 6.25 0.2
Species
8 24
a Concentrations in micrograms of BL-S640 per milliliter. Methicillin-resistant staphylococci. c Beta-hemolytic streptococci, non-group A or D.
to low concentrations of BL-S640 with the exception of group D organisms (Table 2). Six of 11 group D streptococci had MICs and MBCs of 25 and > 200 Mg of BL-S640 per ml, respectively. Frequent persistence of streptococci on concentrations greater than the MIC of BL-S640 resulted in a wide disparity between MICs and MBCs. S. pneumoniae were susceptible at low concentrations; all 24 strains were inhibited by 12.5 Mg of BL-S640 per ml for a bactericidal effect. Eight strains of nontypable H. influenzae were tested; all strains were inhibited at low concentrations with a median MIC and MBC of 1.60 and 3.13 MAg of BL-S640 per ml, respectively. Stability studies. Aqueous solutions of
BL-S640 were unstable when maintained at 37 C for 24, 48, or 72 h. After 24 h, MICs against gram-negative and gram-positive organisms increased two- to threefold; an additional two- to threefold increase in MICs was obtained after holding at 37 C for an additional 48 to 72 h. No variation in MICs was noted when stand! ard antibiotic had been frozen at -20 C up to 4 weeks. Effect of media and inoculum size. The variation of inoculum size against susceptible microorganisms had little effect on the observed MTCs to BL-S640 when performed on MuellerHinton or nutrient agar (Table 3). No difference in MICs greater than twofold was noted with inocula of 101 or 105 bacteria. With one exception, MICs were comparable by either agar dilution method for each inoculum. However, lower inhibitory concentrations were noted against Staphylococcus aureus when nutrient agar was utilized. With inocula of 10' or 107 microorganisms,
308
OVERTURF ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
variation was noted in the MICs obtained in nutrient versus Mueller-Hinton broth (Table 4). The larger inoculum resulted in a three- to sixfold-higher MIC in Mueller-Hinton broth and a fourfold-higher MIC in nutrient broth. In addition, inhibitory concentrations were noted to be from two- to fourfold less in nutrient than in Mueller-Hinton broth with the same inocula against the same organism. The exceptions were in the study of two strains of Klebsiella, where inhibitory concentrations for BL-S640 were higher in nutrient than in Mueller-Hinton broth (Table 4). Cross-resistance and comparison of potency to other cephalosporins. The antimicrobial potencies of BL-S640, cephalothin, cefazolin, and cephalexin were compared (Table 5). BL-S640 demonstrated generally equivalent or greater activity than cefazolin, cephalothin, or cephalexin against Klebsiella, Proteus, and E. coli. All four cephalosporins had minimal or no activity against most strains of Pseudomonas, Enterobacter, Serratia, and Providencia (not shown). Although inhibitory concentrations of cefazolin and cephalothin were consistently two- to fourfold less than BL-S640 against TABLE 3. Comparison of MICs of BL-S640 on nutrient or Mueller-Hinton agar with various inocula Nutrient agar
Mueller-Hinton agar
Strain
Escherichia coli Klebsiella Citrobacter Staphylococ-
105
108
105
10'
Bacteria
Bacteria
Bacteria
Bacteria
1.60a
1.60
1.60
1.60
1.60 6.25 3.13
1.60 3.13 1.60
1.60 6.25 0.80
1.60 6.25 0.40
methicillin-susceptible staphylococci, cephalexin was notably less potent than BL-S640 against this microorganism. The selection of microorganisms resistant to BL-S640 after serial passage in increasing concentrations of BL-S640 occurred readily as depicted by results with representative strains (Table 6). With the exception of strains of S. aureus, the increased resistance to cephalexin and cephalothin paralleled selection of resistance to BL-S640. An unexpected development of resistance to cephalothin with many strains of Klebsiella was examined in more detail (Table 7). After seven passages in BL-S640, eight of 10 strains of Klebsiella remained sensitive to BL-S640. In contrast, significant resistance (>fourfold difference in MIC) was demonstrated to cephalothin in five of these 10 strains. Additional investigation revealed that resistance had developed to cefazolin and cephalexin in four of the 10 strains, respectively. However, the development of resistance to each cephalosporin occurred randomly. The acquired resistance of strains of E. coli and Klebsiella was stable and persisted at the same level after 10 passages in Mueller-Hinton broth without BL-S640. The stability of resistance among strains of staphylococci, Proteus, and Klebsiella was more variable and did not correlate with the degree of resistance attained. For example, Klebsiella strain no. 73-284 (Table 7) reverted to the susceptibility of the parent strain by the sixth passage in broth without added BL-S640. In contrast, Klebsiella strain no. 73-241 maintained its high level of resistance through 10 such passages. DISCUSSION
cus aureus
The in vitro antimicrobial spectrum of a Concentrations in micrograms of BL-S640 per BL-S640 was shown to be similar to other milliliter. TABLE 4. Comparison of MICs of BL-S640 in nutrient or Mueller-Hinton broth with various inocula Nutrient broth
Mueller-Hinton broth Strain
Staphylococcus aureus Escherichia coli
Citrobacter Klebsiella Proteus
Enterobacter a
10' Bacteria 3.13a 12.50 1.60 50.0 6.25 50.0 1.6 50.0 1.6 100.0
107 Bacteria
12.5 50.0 100.0 > 200.0 > 200.0 > 200.0 100.0 200.0 12.5 > 200.0
Concentrations expressed as micrograms of BL-S640 per milliliter.
105 Bacteria
107 Bacteria
0.4 1.6 3.13 12.50 6.25 100.0 12.5 25.0 0.4 25.0
12.50 6.25 100.0 100.0 > 200.0 100.0 > 200.0 0.8 100.0
1.6
IN VITRO EVALUATION OF BL-S640
VOL. 8, 1975
309
TABLE 5. Comparison of MICs of cephalothin and cephalexin or cefazolin to BL-S640 against representative strains MICa Strain no.
Bacteria
Escherichia coli Klebsiella Proteus
Staphylococcus aureus
a
73-149 73-212 73-391 73-127 73-213 73-123 73-181 73-382 74-192 74-319
BL-S640
Cephalexin
Cefazolin
Cephalothin
100 1.6
50 6.25
50 1.6
.200 12.5
6.25
6.25
6.25
50.0 0.80 50.0 12.50 3.13 6.25 0.80
100 3.13 200 12.50 6.25 25.0 3.13
200 1.6 200 25 1.60 3.13 0.20
50.0 200 3.13 > 200 50 0.80 0.80 .0.10
Concentrations expressed as micrograms of antibiotic per milliliter. TABLE 6. Cross-resistance of BL-S640 to cephalosporins after serial passage through BL-S640 MICa Strain
7th passage through BL-S640
Parent strains
BL-S640
Cephalexin
Cephalothin
BL-S640
Cephalexin
3.13 3.13
6.25 6.25
0.40 0.40
100.0 12.50
200.0 25.0
12.50
6.25
200.0
100.0
200.0
1.60
6.25
3.13
12.5
12.5
3.13
6.25
6.25
50 0
50.0
Cephalothin
Staphylococcus aureus 73-382 R 73-359 Proteus 73-181
12.5
3.13 0.80
Klebsiella 74-337
1.60
Escherichia coli 73-212 a
25.0
Concentrations expressed as micrograms of antibiotic per milliliter.
TABLE 7. Cross-resistance of BL-S640 and cephalosporins among Klebsiella strains after serial passage through BL-S640 Parent strains Strain no.
73-177 73-205 73-213 73-259 73-284 74-124 74-224 74-436 74-55 74-241 a
7th passage through BL-S640
BL-S 640
Cephalothin
Cephalexin
Cefazolin
BL-S 640
Cephalothin
Cephalexin
Cefazolin
1.6a 1.6 1.6 1.6 6.25 1.6 0.8 0.8 1.6 3.13
1.6 3.13 1.6 3.13 25.0 3.13 1.6 1.6 12.5 6.25
6.25 3.13 3.13 6.25 12.5 3.13 3.13 3.13 6.25 3.13
1.6 1.6 1.6 1.6 50.0 1.6 1.6 1.6 6.25 6.25
3.13 1.6 3.13 6.25 50.0 1.6 3.13 1.6 3.13 50.0
50.0 3.13 6.25 100.0 100.0 3.13 25.0 3.13 12.5 100.0
6.25 3.13 6.25 50.0 25.0 3.13 25.0 6.25 25.0 25.0
12.5 1.6 1.6 50.0 > 100.0 1.6 6.25 1.6 6.25 > 100.0
Concentrations expressed as micrograms of antibiotic per milliliter.
available cephalosporins (1,3-5, 9). Strains of Pseudomonas and Serratia were markedly resistant, as were many strains of Proteus, Providencia, and Enterobacter. Streptococci, exclud-
ing group D organisms, were susceptible at low concentrations. Despite the requirement of high concentrations for bactericidal activity against occasional strains, BL-S640 showed greater po-
310
OVERTURF ET AL.
tency against H. influenzae, type b, microorganisms than many other previously studied cephalosporins (3, 10). In addition, greater activity of BL-S640 against staphylococci, E. coli, and Klebsiella microorganisms as compared to cephalexin was demonstrated in this study. In preliminary in vitro studies, MICs four- to eightfold less were obtained with BL-S640 than with cephalexin with strains from these genera (2). This magnitude of difference in potency between BL-S640 and cephalexin was not consistently present in this study. In both studies, inocula sizes and media were comparable and the difference in results can not be explained by a variation in these factors. Development of cross-resistance between BL-S640 and other cephalosporins was demonstrated. Among strains of staphylococci, resistance acquired to BL-S640 paralleled resistance to cephalothin and cephalexin; the same was true for strains of Proteus and E. coli. The stability of acquired resistance for most strains was variable. In contrast, strains of Klebsiella retained their susceptibility after multiple passages in BL-S640 despite the frequent development of resistance to cephalothin, cephalexin, or cefazolin. The mechanism for this differential selection of resistance is unexplained, and requires further study. This phenomenon could occur more frequently than previously appreciated since only those bacterial strains demonstrating resistance against one cephalosporin are routinely chosen for cross-resistance studies. In pharmacologic studies with BL-S640 propylene glycolate, absorption after oral administration has been demonstrated (1). The preliminary comparative studies with BL-S640 and cephalexin in animals indicated that BL-S640 was absorbed slower, achieved lower peak levels, and had a longer half-life than cephalexin. Further studies in adult male volunteers demonstrated that peak concentrations of 5 to 7 and 11 to 12 ,g of BL-S640 per ml can be obtained at 1 to 2 h after doses of 0.50 and 1.0 g, respectively, of BL-S640 propylene glycolate. A pharmacological half-life of approximately 1.5 h for BL-S640 was calculated. In parallel oral absorption studies in humans, cephalexin yielded peak serum concentrations four times higher than BL-S640 and total absorption of cephalexin was superior as reflected by areas under absorption curves. Higher concentrations were attained in the urine in the first 24 h after oral administration of cephalexin, and approximately 90% of this antibiotic was recovered in the urine. This was contrasted to the recovery of only 20% of a given dose of BL-S640 in the urine in 24 h. However, 10 of 16 subjects had detecta-
ANTIMICROB. AGENTS CHEMOTHER.
ble levels of BL-S640 in the urine for >48 h. In summary, studies with BL-S640 have confirmed its broad antimicrobial spectrum, which is similar to other cephalosporins. Preliminary pharmacological studies demonstrating lesser peak serum levels and more prolonged urinary concentrations than cephalexin suggest that BL-S640 may be useful as an oral agent for the treatment of urinary tract infections. The potentially greater antimicrobial potency of BL-S640 compared to other presently available oral agents is promising and may warrant further evaluation of the use of BL-S640 in systemic infections. The significance of these in vitro data of antimicrobial activity must be correlated with subsequent studies of pharmacokinetics as well as studies of efficacy in experimental or clinical infections. ACKNOWLEDGMENTS This study was supported by a grant-in-aid from Bristol Laboratories, Syracuse, N.Y., and by the Hastings Foundation Fund.
LITERATURE CITED 1. Boniece, W. S., W. E. Wick, D. H. Holmes, and C. D. Redman. 1962. In vitro and in vivo laboratory evaluation of cephalothin, a new broad spectrum antibiotic. J.Bacteriol. 84:1292-1299. 2. Bristol Laboratories. 1974. Basic data brochure, Oral BL-
S640 PG. Bristol Laboratories, Syracuse, N.Y. 3. Eykyn, S., C. Jenkins, A. King, and I. Phillips. 1973. Antibacterial activity of cefamandole, a new cephalosporin antibiotic, compared with that of cephaloridine,
cephalothin, and cephalexin. Ant imicrob. Agents Chemother. 3:657-661. 4. Finland, M. 1972. Oral and parenteral cephalosporins. The place of cephalexin in antibacterial therapy. Drugs 3:1-8. 5. Johnson, W. D., J. M. Applestein, and D. Kaye. 1968. Cephaloglycin: clinical and laboratory experience with an orally administered cephalosporin. J. Am. Med.
Assoc. 206:2698-2702. 6. Klastersky, J., D. Daneau, and D. Weerts. 1973. Cephradine: antibacterial activity and clinical effectiveness. Chemotherapy 18:191-204. 7. Anderson, T. G., J. E. Blair, E. H. Lennette, and J. P. Truant (ed.). 1970. Manual of clinical microbiology, p. 299-312. American Society for Microbiology, Bethesda, Md. 8. 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. 9. Ries, K., M. E. Levison, and D. Kaye. 1973. Clinical and in vitro evaluation of cefazolin, a new cephalosporin antibiotic. Antimicrob. Agents Chemother. 3:168-174. 10. Sabath, L. D., C. Wilcox, C. Gamer, and M. Finland. 1973. In vitro activity of cefazolin against recent clinical bacterial isolates. J. Infect. Dis.
128(Suppl):S320-S326. 11. Turck, M., K. N. Anderson, R. H. Smith, J. F. Wallace,
and R. G. Petersdorf. 1965. Laboratory and clinical evaluation of a new antibiotic: cephalothin. Ann. Int. Med. 63:199-211. 12. Wick, W. E., and W. S. Boniece. 1965. In vitro and in vivo laboratory evaluation of cephaloglycin and cephaloridine. Appl. Microbiol. 13:248-253.
Vol. 8, No. 3
Printed in U.S.A.
In Vitro Evaluation of BL-S640, a New Oral Cephalosporin Antibiotic GARY D. OVERTURF,* RONALD L. RESSLER, PAUL B. MARENGO, AND JEANETTE WILKINS The Department of Pediatrics and the Hastings Foundation Infectious Disease Laboratory, Los Angeles County/University of Southern California Medical Center, and the University of Southern California School of Medicine, Los Angeles, California 90033 Received for publication 18 April 1975
BL-S640, a new oral cephalosporin analogue, was evaluated in vitro against 102 gram-negative and 80 gram-positive bacteria. The antimicrobial spectrum was similar to that of previous cephalosporin analogues. Good antimicrobial activity against strains of Escherichia coli, Klebsiella, staphylococci, and streptococci was demonstrated. Relatively poor activity and/or resistance was noted among most strains of Proteus, Providencia, Pseudomonas, and Serratia. In comparative studies BL-S640 -had better activity against strains of Hemophilus influenzae, Staphylococcus aureus, and Enterobacteriaceae than many cephalosporin analogues. Variation of susceptibility results was dependent upon the type of media and inoculum size. Cross-resistance between BL-S640 cephalexin, cephalothin, and cefazolin was demonstrated. Among strains of Klebsiella the more rapid selection of resistance to other cephalosporins was in contrast to BL-S640. Experience in vitro with BL-S640 has documented its antimicrobial activity,and further studies of pharmacokinetics and therapeutic efficacy are indicated.
Since the introduction of cephalothin in 1962 be administered orally, intramuscularly, or in(1, 11) structural modification of the 7- travenously. BL-S640 17-D-alpha-amino-alpha- [4-hydroxyaminocephalosporanic acid nucleus has provided analogues for clinical use with improved phenylJacetamido [3-(1H-1,2,3-triazol-5-thio)physical-chemical, pharmacologic, or antimi- methyl]-3-cephem-4-carboxylic acidi is a new crobial properties (3-5, 9, 10). In particular, the semisynthetic cephalosporin (2). The structure need for cephalosporins suitable for oral admin- is shown in Fig. 1. A water-soluble preparation istration has been recognized. Cephaloglycin is available as a propylene glycolate, since aqueand cephalexin were the first acid-stable deriva- ous solutions are unstable and have been shown tives suitable for oral administration; in addi- to lose up to 50% of antimicrobial activity at pH tion, cephradine was introduced in 1974. How- 7.4 in several hours. (They are stable, however, ever, cephaloglycin is unstable at alkaline pH at -16 C for up to 10 days.) In anticipation that and is rapidly metabolized in the liver to the less this cephalosporin analogue may be suitable as potent desacetylcephaloglycin; therefore, oral an oral preparation, its in vitro antimicrobial administration results in low serum antibacte- spectrum and activity are reported. rial activity (5, 12). This has prohibited its use MATERIALS AND METHODS in systemic infections and confined it to treatment of urinary tract infections. In contrast, Antibiotics. BL-S640 propylene glycolate (Bristol cephalexin is totally absorbed after oral admin- Laboratories, Syracuse, N.Y.) was supplied as the istration and is excreted unchanged by the crystalline zwitterion for use in antibiotic susceptibilkidney (4, 8). Although it possesses desirable ity testing (2). Reference standard was prepared in pharmacological properties, cephalexin has less water in a concentration of 1,000 jig of BL-S640 per ml antimicrobial activity than cephalothin, cepha- and used immediately after preparation. Standard of cephalexin (Lilly Laboratories), cephloridine, cefazolin, or cephaloglycin (4, 8). De- preparations alothin (Lilly Laboratories), and cefazolin (Lilly Labspite this limitation, cephalexin has been used oratories) were used for appropriate comparative extensively since more active oral cephalospo- studies. rins are unavailable. A recently licensed anaBacterial isolates. All bacteria were recovered logue, cephradine, differs only slightly from from the blood, urine cerebrospinal fluid, or wounds of cephalexin in its antimicrobial activity and patients hospitalized in the Los Angeles County/ pharmacological properties (12, 5), and it can University of Southern California Medical Center. 305
306
OVERTURF ET AL.
ANrimICROB. AGENTS CHEMnomER. 0
NO
4< \
/
C
-
S
H N
~~~NH2
04_x~
CH2-
N
C -OH
S
N
H
11 0
FIG. 1. Chemical structure of BL-S640. Microorganisms, stored in either Mueller-Hinton (Enterobacteriaceae, Pseudomonras sp., staphylococci), tryptic soy (streptococci, pneumococci), or MuellerHinton plus 1% supplement B (Hemophilus influenzae) broth at -20 C, were thawed and subcultured on appropriate solid media to insure adequate growth. Susceptibility studies. The determinations of minimal inhibitory concentrations (MICs) were performed by standard methods (7). Mueller-Hinton broth (Difco) was utilized in susceptibility studies with Enterobacteriaceae, Pseudomonas sp., and staphylococci. Supplement B (Difco) in a concentration of 1% was added to Mueller-Hinton broth for use with H. influenzae. Tryptic soy broth (Difco) was used for studies with Streptococcus pneumoniae and other streptococci. Each cephalosporin was diluted serially twofold, from 200 to 0.1 pg/ml, except for studies with streptococci where concentrations from 200 to 0.025 pggml were utilized. After overnight incubation at 37 C, cultures were diluted to prepare a standard inoculum of 10' organisms. Streptococci, S. pneumoniae, and Hemophilus microorganisms were always incubated in 10% CO. The MIC was defined as the lowest concentration of antibiotic which allowed no visible turbidity after incubation for 18 to 20 h at 37 C. Tubes without turbidity were inoculated on appropriate solid media with a 3-mm loop and the lowest concentration yielding 10 colonies or less was defined as the minimal bactericidal concentration (MBC). In susceptibility studies against selected staphylococci, Pseudomonas sp., and Enterobacteriaceae, results with Mueller-Hinton agar or broth (Difco) and nutrient agar or broth (Difco) were compared. BL-S640 in twofold dilutions (200 to 0.1 pg/ml) was added to agar at 47 C. Antibiotic plates were inoculated immediately after their preparation. The inocula were varied in parallel broth and agar studies to contain 10', 106, or 107 microorganisms. Antibiotic stability study. Antibiotic stability was evaluated by determination of the MIC against selected microorganisms in twofold dilutions of BL-S640 that had been maintained at room temperature for 0, 24, 48, and 72 h.
Cross-resistance study. Representative grampositive and/or gram-negative microorganisms were selected to study cross-resistance between BL-8640 and cephalothin, cephalexin, or cefazolin. First, selected strains with demonstrated susceptibility to each of the cephalosporins were inoculated into seri-
ally diluted concentrations of BL-S640 as previously described. Those microorganisms surviving the highest concentration of BL-S640 after overnight incubation were re-inoculated into appropriate broth. After another 18 to 20 h of incubation, these microorganisms were used to prepare the inoculum for similar tubes containing the serially diluted BL-S640. After seven such passages in BL-S640, determinations of MICs for each strain were repeated to BL-S640, cephalexin, and cephalothin. In addition, cefazolin was included in cross-resistance studies with strains
of Klebsiella. To test the stability of the resistance acquired after serial passage in BL-S640, all strains were subcultured for 10 days on Mueller-Hinton media without added antibiotic. Change in antibiotic resistance was screened for by daily disk susceptibility tests to cephalothin and documented by the determination of MICs to all of the selected cephalosporins.
RESULTS Antimicrobial spectrum. Most strains of Escherichia coli and Klebsiella were susceptible to low concentrations of BL-S640; 30 of 35 strains (85%) were inhibited at concentrations of .12.5 pg of BL-S640 per ml (Table 1). However, the remaining five strains were resistant at concentrations of >50 pAg of BL-S640 per ml. Strains of Citrobacter were inhibited at concentrations of .6.25 pAg of BL-S640 per ml. Strains of Serratia and Pseudomonas uniformly required concentrations of > 50 ug of BL-S640 per ml for inhibition of growth (Table 1). Enterobacter and Providencia frequently required > 12.5 pg of BL-S640 per ml to inhibit growth. The MICs of Proteus strains were separated sharply into those < 1.60 pg of BL-S640 per ml and those .12.5 pg of BL-S640 per ml. However, the susceptibility of the 14 strains varied and did not correlate with indole production. Strains of methicillin-susceptible staphylococci had a median MIC and MBC of 3.13 and 12.5 pg of BL-S640 per ml, respectively (Table 2). In contrast, methicillin-resistant staphylococci were uniformly resistant, requiring concentrations of . 25 ,g BL-S640 per ml to inhibit growth. Streptococci were uniformly susceptible
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IN VITRO EVALUATION OF BL-S640
VOL. 8, 1975
TABLE 1. Cumulative MICs and MBCs of 79 strains of Enterobacteriaceae and Pseudomonas for BL-S640 BL-S 640 (,ug/ml)
No.
Deter-
Species Escherichia coli
tested 20
mination MIC
200
12.50 > 200
11 14
200 0.025-25 0.1-12.5 0.1-0.4
>200 0.2 6.25 0.2
Species
8 24
a Concentrations in micrograms of BL-S640 per milliliter. Methicillin-resistant staphylococci. c Beta-hemolytic streptococci, non-group A or D.
to low concentrations of BL-S640 with the exception of group D organisms (Table 2). Six of 11 group D streptococci had MICs and MBCs of 25 and > 200 Mg of BL-S640 per ml, respectively. Frequent persistence of streptococci on concentrations greater than the MIC of BL-S640 resulted in a wide disparity between MICs and MBCs. S. pneumoniae were susceptible at low concentrations; all 24 strains were inhibited by 12.5 Mg of BL-S640 per ml for a bactericidal effect. Eight strains of nontypable H. influenzae were tested; all strains were inhibited at low concentrations with a median MIC and MBC of 1.60 and 3.13 MAg of BL-S640 per ml, respectively. Stability studies. Aqueous solutions of
BL-S640 were unstable when maintained at 37 C for 24, 48, or 72 h. After 24 h, MICs against gram-negative and gram-positive organisms increased two- to threefold; an additional two- to threefold increase in MICs was obtained after holding at 37 C for an additional 48 to 72 h. No variation in MICs was noted when stand! ard antibiotic had been frozen at -20 C up to 4 weeks. Effect of media and inoculum size. The variation of inoculum size against susceptible microorganisms had little effect on the observed MTCs to BL-S640 when performed on MuellerHinton or nutrient agar (Table 3). No difference in MICs greater than twofold was noted with inocula of 101 or 105 bacteria. With one exception, MICs were comparable by either agar dilution method for each inoculum. However, lower inhibitory concentrations were noted against Staphylococcus aureus when nutrient agar was utilized. With inocula of 10' or 107 microorganisms,
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OVERTURF ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
variation was noted in the MICs obtained in nutrient versus Mueller-Hinton broth (Table 4). The larger inoculum resulted in a three- to sixfold-higher MIC in Mueller-Hinton broth and a fourfold-higher MIC in nutrient broth. In addition, inhibitory concentrations were noted to be from two- to fourfold less in nutrient than in Mueller-Hinton broth with the same inocula against the same organism. The exceptions were in the study of two strains of Klebsiella, where inhibitory concentrations for BL-S640 were higher in nutrient than in Mueller-Hinton broth (Table 4). Cross-resistance and comparison of potency to other cephalosporins. The antimicrobial potencies of BL-S640, cephalothin, cefazolin, and cephalexin were compared (Table 5). BL-S640 demonstrated generally equivalent or greater activity than cefazolin, cephalothin, or cephalexin against Klebsiella, Proteus, and E. coli. All four cephalosporins had minimal or no activity against most strains of Pseudomonas, Enterobacter, Serratia, and Providencia (not shown). Although inhibitory concentrations of cefazolin and cephalothin were consistently two- to fourfold less than BL-S640 against TABLE 3. Comparison of MICs of BL-S640 on nutrient or Mueller-Hinton agar with various inocula Nutrient agar
Mueller-Hinton agar
Strain
Escherichia coli Klebsiella Citrobacter Staphylococ-
105
108
105
10'
Bacteria
Bacteria
Bacteria
Bacteria
1.60a
1.60
1.60
1.60
1.60 6.25 3.13
1.60 3.13 1.60
1.60 6.25 0.80
1.60 6.25 0.40
methicillin-susceptible staphylococci, cephalexin was notably less potent than BL-S640 against this microorganism. The selection of microorganisms resistant to BL-S640 after serial passage in increasing concentrations of BL-S640 occurred readily as depicted by results with representative strains (Table 6). With the exception of strains of S. aureus, the increased resistance to cephalexin and cephalothin paralleled selection of resistance to BL-S640. An unexpected development of resistance to cephalothin with many strains of Klebsiella was examined in more detail (Table 7). After seven passages in BL-S640, eight of 10 strains of Klebsiella remained sensitive to BL-S640. In contrast, significant resistance (>fourfold difference in MIC) was demonstrated to cephalothin in five of these 10 strains. Additional investigation revealed that resistance had developed to cefazolin and cephalexin in four of the 10 strains, respectively. However, the development of resistance to each cephalosporin occurred randomly. The acquired resistance of strains of E. coli and Klebsiella was stable and persisted at the same level after 10 passages in Mueller-Hinton broth without BL-S640. The stability of resistance among strains of staphylococci, Proteus, and Klebsiella was more variable and did not correlate with the degree of resistance attained. For example, Klebsiella strain no. 73-284 (Table 7) reverted to the susceptibility of the parent strain by the sixth passage in broth without added BL-S640. In contrast, Klebsiella strain no. 73-241 maintained its high level of resistance through 10 such passages. DISCUSSION
cus aureus
The in vitro antimicrobial spectrum of a Concentrations in micrograms of BL-S640 per BL-S640 was shown to be similar to other milliliter. TABLE 4. Comparison of MICs of BL-S640 in nutrient or Mueller-Hinton broth with various inocula Nutrient broth
Mueller-Hinton broth Strain
Staphylococcus aureus Escherichia coli
Citrobacter Klebsiella Proteus
Enterobacter a
10' Bacteria 3.13a 12.50 1.60 50.0 6.25 50.0 1.6 50.0 1.6 100.0
107 Bacteria
12.5 50.0 100.0 > 200.0 > 200.0 > 200.0 100.0 200.0 12.5 > 200.0
Concentrations expressed as micrograms of BL-S640 per milliliter.
105 Bacteria
107 Bacteria
0.4 1.6 3.13 12.50 6.25 100.0 12.5 25.0 0.4 25.0
12.50 6.25 100.0 100.0 > 200.0 100.0 > 200.0 0.8 100.0
1.6
IN VITRO EVALUATION OF BL-S640
VOL. 8, 1975
309
TABLE 5. Comparison of MICs of cephalothin and cephalexin or cefazolin to BL-S640 against representative strains MICa Strain no.
Bacteria
Escherichia coli Klebsiella Proteus
Staphylococcus aureus
a
73-149 73-212 73-391 73-127 73-213 73-123 73-181 73-382 74-192 74-319
BL-S640
Cephalexin
Cefazolin
Cephalothin
100 1.6
50 6.25
50 1.6
.200 12.5
6.25
6.25
6.25
50.0 0.80 50.0 12.50 3.13 6.25 0.80
100 3.13 200 12.50 6.25 25.0 3.13
200 1.6 200 25 1.60 3.13 0.20
50.0 200 3.13 > 200 50 0.80 0.80 .0.10
Concentrations expressed as micrograms of antibiotic per milliliter. TABLE 6. Cross-resistance of BL-S640 to cephalosporins after serial passage through BL-S640 MICa Strain
7th passage through BL-S640
Parent strains
BL-S640
Cephalexin
Cephalothin
BL-S640
Cephalexin
3.13 3.13
6.25 6.25
0.40 0.40
100.0 12.50
200.0 25.0
12.50
6.25
200.0
100.0
200.0
1.60
6.25
3.13
12.5
12.5
3.13
6.25
6.25
50 0
50.0
Cephalothin
Staphylococcus aureus 73-382 R 73-359 Proteus 73-181
12.5
3.13 0.80
Klebsiella 74-337
1.60
Escherichia coli 73-212 a
25.0
Concentrations expressed as micrograms of antibiotic per milliliter.
TABLE 7. Cross-resistance of BL-S640 and cephalosporins among Klebsiella strains after serial passage through BL-S640 Parent strains Strain no.
73-177 73-205 73-213 73-259 73-284 74-124 74-224 74-436 74-55 74-241 a
7th passage through BL-S640
BL-S 640
Cephalothin
Cephalexin
Cefazolin
BL-S 640
Cephalothin
Cephalexin
Cefazolin
1.6a 1.6 1.6 1.6 6.25 1.6 0.8 0.8 1.6 3.13
1.6 3.13 1.6 3.13 25.0 3.13 1.6 1.6 12.5 6.25
6.25 3.13 3.13 6.25 12.5 3.13 3.13 3.13 6.25 3.13
1.6 1.6 1.6 1.6 50.0 1.6 1.6 1.6 6.25 6.25
3.13 1.6 3.13 6.25 50.0 1.6 3.13 1.6 3.13 50.0
50.0 3.13 6.25 100.0 100.0 3.13 25.0 3.13 12.5 100.0
6.25 3.13 6.25 50.0 25.0 3.13 25.0 6.25 25.0 25.0
12.5 1.6 1.6 50.0 > 100.0 1.6 6.25 1.6 6.25 > 100.0
Concentrations expressed as micrograms of antibiotic per milliliter.
available cephalosporins (1,3-5, 9). Strains of Pseudomonas and Serratia were markedly resistant, as were many strains of Proteus, Providencia, and Enterobacter. Streptococci, exclud-
ing group D organisms, were susceptible at low concentrations. Despite the requirement of high concentrations for bactericidal activity against occasional strains, BL-S640 showed greater po-
310
OVERTURF ET AL.
tency against H. influenzae, type b, microorganisms than many other previously studied cephalosporins (3, 10). In addition, greater activity of BL-S640 against staphylococci, E. coli, and Klebsiella microorganisms as compared to cephalexin was demonstrated in this study. In preliminary in vitro studies, MICs four- to eightfold less were obtained with BL-S640 than with cephalexin with strains from these genera (2). This magnitude of difference in potency between BL-S640 and cephalexin was not consistently present in this study. In both studies, inocula sizes and media were comparable and the difference in results can not be explained by a variation in these factors. Development of cross-resistance between BL-S640 and other cephalosporins was demonstrated. Among strains of staphylococci, resistance acquired to BL-S640 paralleled resistance to cephalothin and cephalexin; the same was true for strains of Proteus and E. coli. The stability of acquired resistance for most strains was variable. In contrast, strains of Klebsiella retained their susceptibility after multiple passages in BL-S640 despite the frequent development of resistance to cephalothin, cephalexin, or cefazolin. The mechanism for this differential selection of resistance is unexplained, and requires further study. This phenomenon could occur more frequently than previously appreciated since only those bacterial strains demonstrating resistance against one cephalosporin are routinely chosen for cross-resistance studies. In pharmacologic studies with BL-S640 propylene glycolate, absorption after oral administration has been demonstrated (1). The preliminary comparative studies with BL-S640 and cephalexin in animals indicated that BL-S640 was absorbed slower, achieved lower peak levels, and had a longer half-life than cephalexin. Further studies in adult male volunteers demonstrated that peak concentrations of 5 to 7 and 11 to 12 ,g of BL-S640 per ml can be obtained at 1 to 2 h after doses of 0.50 and 1.0 g, respectively, of BL-S640 propylene glycolate. A pharmacological half-life of approximately 1.5 h for BL-S640 was calculated. In parallel oral absorption studies in humans, cephalexin yielded peak serum concentrations four times higher than BL-S640 and total absorption of cephalexin was superior as reflected by areas under absorption curves. Higher concentrations were attained in the urine in the first 24 h after oral administration of cephalexin, and approximately 90% of this antibiotic was recovered in the urine. This was contrasted to the recovery of only 20% of a given dose of BL-S640 in the urine in 24 h. However, 10 of 16 subjects had detecta-
ANTIMICROB. AGENTS CHEMOTHER.
ble levels of BL-S640 in the urine for >48 h. In summary, studies with BL-S640 have confirmed its broad antimicrobial spectrum, which is similar to other cephalosporins. Preliminary pharmacological studies demonstrating lesser peak serum levels and more prolonged urinary concentrations than cephalexin suggest that BL-S640 may be useful as an oral agent for the treatment of urinary tract infections. The potentially greater antimicrobial potency of BL-S640 compared to other presently available oral agents is promising and may warrant further evaluation of the use of BL-S640 in systemic infections. The significance of these in vitro data of antimicrobial activity must be correlated with subsequent studies of pharmacokinetics as well as studies of efficacy in experimental or clinical infections. ACKNOWLEDGMENTS This study was supported by a grant-in-aid from Bristol Laboratories, Syracuse, N.Y., and by the Hastings Foundation Fund.
LITERATURE CITED 1. Boniece, W. S., W. E. Wick, D. H. Holmes, and C. D. Redman. 1962. In vitro and in vivo laboratory evaluation of cephalothin, a new broad spectrum antibiotic. J.Bacteriol. 84:1292-1299. 2. Bristol Laboratories. 1974. Basic data brochure, Oral BL-
S640 PG. Bristol Laboratories, Syracuse, N.Y. 3. Eykyn, S., C. Jenkins, A. King, and I. Phillips. 1973. Antibacterial activity of cefamandole, a new cephalosporin antibiotic, compared with that of cephaloridine,
cephalothin, and cephalexin. Ant imicrob. Agents Chemother. 3:657-661. 4. Finland, M. 1972. Oral and parenteral cephalosporins. The place of cephalexin in antibacterial therapy. Drugs 3:1-8. 5. Johnson, W. D., J. M. Applestein, and D. Kaye. 1968. Cephaloglycin: clinical and laboratory experience with an orally administered cephalosporin. J. Am. Med.
Assoc. 206:2698-2702. 6. Klastersky, J., D. Daneau, and D. Weerts. 1973. Cephradine: antibacterial activity and clinical effectiveness. Chemotherapy 18:191-204. 7. Anderson, T. G., J. E. Blair, E. H. Lennette, and J. P. Truant (ed.). 1970. Manual of clinical microbiology, p. 299-312. American Society for Microbiology, Bethesda, Md. 8. 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. 9. Ries, K., M. E. Levison, and D. Kaye. 1973. Clinical and in vitro evaluation of cefazolin, a new cephalosporin antibiotic. Antimicrob. Agents Chemother. 3:168-174. 10. Sabath, L. D., C. Wilcox, C. Gamer, and M. Finland. 1973. In vitro activity of cefazolin against recent clinical bacterial isolates. J. Infect. Dis.
128(Suppl):S320-S326. 11. Turck, M., K. N. Anderson, R. H. Smith, J. F. Wallace,
and R. G. Petersdorf. 1965. Laboratory and clinical evaluation of a new antibiotic: cephalothin. Ann. Int. Med. 63:199-211. 12. Wick, W. E., and W. S. Boniece. 1965. In vitro and in vivo laboratory evaluation of cephaloglycin and cephaloridine. Appl. Microbiol. 13:248-253.
