Vol. 16, No. 1
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1979, p. 64-68 0066-4804/79/07-0064/05$02.00/0
Antibacterial Activity of a New Parenteral CephalosporinHR 756: Comparison with Cefamandole and Ceforanidet GEORGE W. COUNTS*
AND
MARVIN TURCK
Harborview Medical Center, Department of Medicine, University of Washington, Seattle, Washington 98104 Received for publication 7 May 1979
HR 756, a new parenteral cephalosporin that is beta-lactamase resistant, was tested against 271 bacterial isolates. Both agar and broth dilution testing were employed, using two media and two inoculum sizes of bacteria. Antibacterial activity of the drug was compared to that of cefamandole (CFM) and ceforanide (CFN). In agar, HR 756 was more active than CFM and CFN against all bacteria tested except isolates of Staphylococcus aureus, which were better inhibited by CFM. HR 756 exhibited some antipseudomonas activity in agar, although a marked inoculum effect was apparent. A comparison of median minimum inhibitory and bactericidal concentrations in broth showed again that HR 756 was the most active of these three drugs. HR 756 demonstrated enhanced antibacterial activity compared to CFM and CFN against bacteria sensitive to all three drugs as well as against more resistant isolates of Serratia marcescens, Enterobacter species, and indole-positive Proteus. As with other cephalosporins, results for most bacteria were affected by inoculum size, medium, and type of dilution test employed in in vitro studies. HR 756 is a new semisynthetic methoxyimino derivative of 7-amino-cephalosporanic acid (2). This parenteral drug is a representative of a newer group of cephalosporin antibiotics which shows relative resistance to beta-lactamases of gram-negative bacilli. This group includes cefoxitin, cefamandole (CFM), cefuroxime, and ceforanide (CFN)(7-11, 13). Due in part to this resistance, the newer cephalosporins may demonstrate activity against certain bacteria such as Enterobacter species, Seratia, and indole-positive Proteus strains, the majority of which are ordinarily resistant to other cephalosporin antibiotics. The purpose of this study was to compare the in vitro antibacterial activity of HR 756 to that of ceforanide and cefamandole. MATERIALS AND METHODS Bacterial isolates. A total of 271 clinical isolates of gram-negative and gram-positive bacteria was tested. Tested with all three antibiotics were 170 isolates of various genera of Enterobacteriaceae, 30 isolates of Staphylococcus aureus, and 27 isolates of enterococcus. The 28 strains of Enterobacter included representatives of the following species: E. cloacae, 9; E. aerogenes, 7; E. hafniae, 4; E. agglomerans, 3; and unidentified as to species, 5. The 25 indole-positive Proteus isolates included 8 strains of P. morganii, 7 strains of P. rettgeri, and 10 strains of P. vulgaris. An
additional 15 isolates of Serratia marcescens were selected for study. Finally, agar dilution testing was performed on 29 isolates of Pseudomonas aeruginosa using HR 756 and CFM. Antibiotics. Laboratory standard antibiotic powder was provided by the following: HR 756 by HoechstRoussel, Inc., Somerville, N.J.; CFN (BL-S786) by Bristol Laboratories, Syracuse, N. Y. and CFM by Eli Lilly & Co., Indianapolis, Ind. Susceptibility testing. Agar dilution and broth dilution minimum inhibitory concentrations (MICs) were determined using Mueller-Hinton agar and broth (MHA, MHB; Difco Laboratories, Detroit, Mich.) and nutrient agar (NA) and broth (Difco) according to previously described methods (3, 13). Inoculum sizes of 104 and 106 colony-forming units were used. Minimum bactericidal concentrations (MBCs) were obtained with these two broth media. Agar dilution testing employed a Steers replicating device (15). Criteria for endpoints for MIC and MBC values were as previously described (3). A laboratory reference strain of Escherichia coli inhibited by 0.125 ,ug of HR 756, 0.5 jg of CFN, and 0.5 jg of CFM, respectively, per ml was used as a control organism in all experiments.
RESULTS Agar dilution. Table 1 shows the range of agar dilution MICs found using MHA as well as the minimum drug concentration to inhibit 50 and 90% of tested isolates. There was no consistent difference in the results for various species of Enterobacter or indole-positive Proteus, and the results for each genera are presented in
t No reprints available. 64
VOL. 16, 1979
IN VITRO EVALUATION OF HR 756
composite. For most bacteria tested, HR 756 was the most active of the three cephalosporin antibiotics included in these studies. HR 756 inhibited 100% of isolates of E. coli and P. mirab-
65
ilis at a drug concentration of 0.125 tg of antibiotic per ml. At this concentration, neither of the other two drugs showed any inhibition of these two genera. For isolates of S. aureus, MICs
TABLE 1. MICs of HR 756, CFN, and CFM required to inhibit 50% (MICW) and 90%(MIC4r) of isolates of various bacteria tested in MHA using two inoculum sizes Organism MICro Inoculum MIC (jtg/ml) MIC90 Dru (no. of strains) g (CFU) range (jsg/ml) (pg/ml) E. coli (30) HR 756 104 0.03-0.06 0.03 0.06 106 0.03-0.125 0.06 0.125 CFN 104 0.25-1.0 0.25 0.5 106 0.25-5.0 1.0 *0.5 CFM 104 0.25-2.5 0.5 1.0 106 0.25-5.0 0.5 1.0 S. aureus (30)
HR 756
104
106 CFN
104 106
CFM
104
106
S. typhi (30)
HR 756
104 106
CFN
104
106 CFM P. mirabilis (27)
104
106
HR 756
104 106
CFN
i04 106 104 106
CFM
Proteus, indole-positive (25)
HR 756
104 106 104 106 104
CFN CFM
106
Enterobacter species (28)
HR 756 CFN CFM
Serratia (15)
Klebsiella (30)
HR 756
1
104
106 104 106 104 106
0.5-5.0 1.0-7.5 2.5-12.5 2.5-20 0.125-0.5 0.25-1.0
2.5 2.5 5.0 7.5 0.5 0.5
2.5 2.5 7.5 15 0.5 0.5
0.007-0.125 0.03-0.25 0.125-0.5 0.25-1.0 0.125-2.5 0.125-5.0
0.06 0.06 0.25 0.25 0.25 0.25
0.06 0.06 0.25 0.25 0.25 0.25
.015-0.125 .015-0.125 0.25-2.5 0.25-2.5 0.5-2.5 0.5-2.5
0.03 0.03 0.5 0.5 1.0 2.5
0.03 0.06 0.5 1.0 1.0 2.5
C0.007-2.5 0.015-5.0 0.125->100 0.125->100 0.125-20 0.125-100 0.015-50 0.03->100
0.5->100 1.0->100
0.5->100 1.0->100
0.03 0.06 5.0 20 1.0
2.5 0.125 0.25 2.5 12.5 2.5 2.5
CFN
l04 106 104
0.125-20 0.125-25 50->100
106
>100
CFM
104
10->100
1.0 5.0 >100 >100 >100
106
15->100
>100
HR 756 CFN CFM
104 106 104 106 104 106
0.03-0.25 0.06-0.25 0.125-5.0 0.25-5.0 0.25-5.0 1.0-15
0.06 0.06 0.25 0.5 1.0 1.0
0.125 0.125 100 >100 10 25 1.0
2.5 50 >100 50 >100 5.0 10 >100 >100 >100 >100
0.125 0.25 1.0
2.5 2.5 5.0
66
COUNTS AND TURCK
ANTIMICROB. AGENTS CHEMOTHER.
for CFM with both inoculum sizes were 5- to 30fold lower than those found with HR 756 and CFN. HR 756 inhibited approximately 80% of Enterobacter isolates at 1.0 ,ug of antibiotic per ml, whereas CFM and CFN inhibited approximately 30% of these same isolates at this concentration of drug. Only slight antibacterial activity was seen when the isolates of S. marcescens were tested against CFM and CFN. Median MICs were greater than 100 ,ug of antibiotic per ml for both drugs. However, a concentration of 5.0 ,ug of HR 756 per ml inhibited 90% of these isolates tested with the lower inoculum of bacterial cells and 50% with the higher inoculum. Moreover, a concentration of 10 ,ug of HR 756 per ml inhibited 14 of 15 Serratia isolates. For purposes of comparison with other studies, results found using Mueller-Hinton media are included in the tables. However, although not shown, there was considerable but inconsistent variation in results depending on whether NA or MHA was used as the test medium. Isolates of enterococci were resistant to all three antibiotics, and results are not presented. Considering P. aeruginosa, with the lower inoculum, 12.5 Ag of HR 756 per ml inhibited greater than 80% of the strains. Approximately twice that concentration of drug was required for similar inhibition when the higher inoculum was employed. At each concentration, the drug was more active in NA than in MHA. The widest discrepancy was at 15 ,ug/ml, where 70% of Pseudomonas isolates were inhibited in NA, but only
30% were inhibited in MHA. MICs for CFM with both inocula were greater than 250 ,ug of antibiotic per ml against P. aeruginosa and are not included in the table. Broth dilution. Table 2 shows median MICs for the three antibiotics in MHB with two inoculum sizes of bacterial cells. HR 756 was more active than CFN and CFM, with 10- to 100-fold difference in MICs. As was seen with studies in agar, CFM had greater activity against isolates of S. aureus. For most isolates of S. aureus, MICs of HR 756 were very similar to those of CFN. In general, MBCs in MHB were approximately the same as MICs. The only notable exception was with CFN activity against S. aureus isolates using the higher inoculum. The MBC was >100 jig of antibiotic per ml. With the lower inoculum, the order of activity in broth of these three drugs against isolates of Enterobacter species was HR 756 > CFM > CFN. With the higher inoculum of bacterial cells, CFN and CFM were approximately equal, with both having median MICs equal to 100 ,jg of drug per ml or greater. The activity of HR 756 against the isolates of Serratia, which was demonstrated in agar at both inoculum sizes of bacterial cells, was present in broth only with the lower inoculum of bacterial cells. With the higher inoculum, all three cephalosporins required at least 100 jig of antibiotic per ml for inhibition or killing. MICs and MBCs were determined in nutrient broth as well as MHB, and results revealed certain marked differences especially noted with
TABLE 2. Median MIC results for HR 756, CFN, and CFM using MHB and two inoculum sizes Organism (no. of strains)
E. coli (10)
S. aureus (10) Klebsiella (10) Enterobacter species (10) P. mirabilis (10)
Proteus, indole-positive (10) S. marcescens (10)
Inoculum
(CFU)a 10
0.06 1.0
1.0 7.5
CFM 1.0 5.0
104
10"
2.5 5.0
2.5 5.0
0.5 1.0
104 10
0.06
1.0
1.0
0.5
5.0
5.0
104
104
106 104 106
104 106
104 106
aCFU, colony-forming units.
HR 756
Median MIC (jig/ml) CFN
0.5 20
0.03 2.5
0.03
20
0.5 100
100
15
> 100
100
0.5
5.0
50
>100
1.0
2.5
7.5
100
>100
>100
>100
>100
VOL. 16, 1979
isolates of P. mirabilis. For example, with the higher inoculum, median MICs for HR 756 and CFN were 10-fold lower in nutrient broth compared to MHB. For other isolates, differences in MICs were not as great and drug activity in the two media varied according to organism and antibiotic.
DISCUSSION Among the gram-negative bacilli, some bacteria, such as species of Enterobacter and indolepositive Proteus as well as P. aeruginosa, have generally been resistant to the available cephalosporins. This resistance has been due in part to the production of beta-lactamases. More recently introduced cephalosporins such as CFM, cefoxitin, cefuroxime, and CFN have possessed some in vitro activity against isolates of Enterobacter and indole-positive Proteus, but not Pseudomonas (1, 10). HR 756 is one of this group of newer cephalosporin antibiotics resistant to some beta-lactamases produced by gram-negative bacilli (7, 8). Although beta-lactamases are not the only factors involved in resistance to cephalosporin antibiotics (12), one would ordinarily expect that beta-lactamase-resistant drugs would exhibit more antibacterial activity than antibiotics which were sensitive to these enzymes. HR 756 appears to demonstrate exceptional in vitro activity compared to other cephalosporin antibiotics (2, 9, 14, 16). In our studies, HR 756 exhibited enhanced antibacterial activity compared to CFM and CFN. The exception to this general finding was with isolates of S. aureus which were more susceptible to CFM. In Mueller-Hinton agar, the drug was approximately 4 times as active as CFM and CFN against isolates of E. coli, Salmonella, and Klebsiella, and approximately 10 times as active against isolates of P. mirabilis and indole-positive Proteus. Similarly, in broth, HR 756 was also more active than CFN and CFM, except for isolates of S. aureus. For all three cephalosporins, most MBCs were approximately the same as MICs. However, some isolates did show a substantial, i.e., threefold or greater, difference in median MBCs and MICs, especially in nutrient broth. This disparity occurred most often with CFM and least with HR 756. Isolates of Proteus most frequently exhibited this discrepancy. Similar to findings with other cephalosporins we have tested, results in these studies were influenced by inoculum size, medium, and type of dilutional test employed. Results with agar dilution testing varied according to whether NA or MHA was used. The variation was not consistent for the three antibiotics and eight genera
IN VITRO EVALUATION OF HR 756
67
of bacteria studied. For example, activity for all three drugs against isolates of S. aureus and P. mirabilis was greater in NA than in MHA. On the other hand, all three drugs were more active against isolates of Enterobacter species in MHA than in NA. For the remaining bacteria, results were more variable according to organism and antibiotic. CFN appeared less affected by difference in medium used than HR 756 and CFM. For some bacteria, e.g., E. coli, there was no difference between agar dilution and broth dilution MICs using the same medium. For other bacteria, such as isolates of Enterobacter, MICs were considerably lower in agar than in broth. Findell and Sherris provided evidence that the disparity between CFM MICs for Enterobacter isolates tested in agar and broth was due to a high frequency of of resistant mutants which were not detected with agar dilution methods (6). We also previously noted a similar disparity between Enterobacter agar and broth results with cefazaflur (3), CFM (1), and CFN (M. Silver, L. S. Tompkins, G. W. Counts, and M. Turck, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 17th, New York, Abstr. no. 73, 1977). In our current studies, the same type of discrepancy was seen, although differences in agar and broth results were not as marked with HR 756 as with the other two drugs. Other investigators have reported variable results with the testing of HR 756 against Enterobacter isolates. Chabbert and Lutz reported that median MIC for isolates of E. cloacae tested in agar was 30 pig of HR 756 per ml (2). Sosna et al., on the other hand, reported that 49 E. cloacae isolates tested by microtiter broth dilution had median MICs of less than 0.2 jig of HR 756 per ml (14). The nine isolates of E. cloacae included in our studies were inhibited by 12.5 ,ug of HR 756 per ml or less in agar and by 20 pug of HR 756 per ml or less in broth. Further studies are necessary to determine if the reported results simply reflect regional difference in susceptibility of Enterobacter isolates. Most isolates of S. marcescens are ordinarily resistant to cephalosporin antibiotics. A difference in the type of resistance has been described by Farrar and O'Dell (5), who found that those Serratia isolates that also were resistant to penicillins had a plasmid-mediated, noninducible cephalosporinase. The Serratia isolates that were more susceptible to penicillins possessed a chromosomally mediated, inducible cephalosporinase. The isolates tested by Wise et al. (16), which were susceptible to carbenicillin, and by Sosna et al. (14) were susceptible to HR 756. The S. marcescens isolates included in our studies were highly resistant to cephalothin, penicillins, and at least one aminoglycoside antibiotic as well. In our laboratory, the activity demon-
68
COUNTS AND TURCK
strated by HR 756 was confined to agar dilution testing and low-inoculum MHB studies. With higher inocula in broth, these isolates were not susceptible to HR 756, CFN, or CFM. A particularly interesting aspect of the spectrum of HR 756 is the in vitro activity against isolates of P. aeruginosa. Other investigators have reported that median MICs for isolates of P. aeruginosa were 8 (15) and 6.2 ,ug of HR 756 per ml (14). In our studies, a rather marked inoculum effect was noted which may modify any clinical application of these in vitro observations. Although the development of a cephalosporin antibiotic with antipseudomonas activity would represent an important therapeutic advance, additional laboratory studies and, if appropriate, clinical trials will be necessary to determine if this particular cephalosporin will be useful in this regard. The expected peak serum levels for these three drugs are roughly comparable for anticipated dosage recommendations. Intramuscular injection of 1 g of HR 756 and CFM is expected to yield peak serum levels of 20 to 25 ,ug/ml (Hoechst-Roussel, Inc., and Eli Lilly & Co.), a figure comparable to that achieved by a 500-mg injection of CFN (Bristol Laboratories). Studies of infections in experimental animals and preliminary results of human clinical trials are very encouraging as far as efficacy is concerned. In summary, HR 756 is a promising new cephalosporin which exhibited enhanced activity when compared in vitro to two other new parenteral cephalosporins. Although the drug does demonstrate some variability in activity according to test method, medium, and inoculum size, this variation is less pronounced than that seen with other cephalosporins we have studied. Whether the drug will be useful therapeutically awaits the results of further in vitro and clinical trials. ACKNOWLEDGMENTS This work was supported in part by grants from HoechstRoussel, Inc., Somerville, N.J., Bristol Laboratories, Syracuse, N.Y., and Eli Lilly & Co., Indianapolis, Ind. We gratefully acknowledge the excellent technical assistance of Dolores Zeleznik and Michael Silver. LITERATURE CITED 1. Adams, H. G., G. A. Stilwell, and M. Turek. 1976. In vitro evaluation of cefoxitin and cefamandole. Antimicrob. Agents Chemother. 9:1019-1024.
ANTIMICROB. AGENTS CHEMOTHER. 2. Chabbert, Y. A., and A. J. Lutz. 1978. HR 756, the syn isomer of a new methoxyimino cephalosporin with unusual antibacterial activity. Antimicrob. Agents Chemother. 14:749-754. 3. Counts, G. W., D. Gregory, D. Zeleznik, and M. Turck. 1977. Cefazaflur, a new parenteral cephalosporin: in vitro studies. Antimicrob. Agents Chemother. 11: 708-711. 4. Eykyn, S., C. Jenkins, A. King, and L. Phillips. 1976. Antibacterial activity of cefuroxime, a new cephalosporin antibiotic, compared with that of cephaloridine, cephalothin, and cefamandole. Antimicrob. Agents Chemother. 9:690-695. 5. Farrar, W. E., and N. M. O'Dell. 1976. Beta-lactamases and resistance to penicillins and cephalosporins in Serratia marcescens. J. Infect. Dis. 134:245-251. 6. Findell, C. M., and J. C. Sherris. 1976. Susceptibility of Enterobacter to cefamandole: evidence for a high mutation rate to resistance. Antimicrob. Agents Chemother. 9:970-974. 7. Fu, K. P., and H. C. Neu. 1978. A comparative study of the activity of cefamandole and other cephalosporins and analysis of the beta-lactamase stability and synergy of cefamandole with aminoglycosides. J. Infect. Dis.
137(suppl):S38-S48. 8. Fu, K. P., and H. C. Neu. 1978. Beta-lactamase stability of HR 756, a novel cephalosporin, compared to that of cefuroxime and cefoxitin. Antimicrob. Agents Chemother. 14:322-326. 9. Heymes, R., A. Lutz, and E. Schrinner. 1977. Experimental evaluation of HR 756, a new cephalosporin derivative: pre-clinical study. Infection 5:259-260. 10. Leitner, F., M. Misiek, T. A. Pursiano, R. E. Buck, D. R. Chisholm, R. G. DeRegis, Y. H. Tsai, and K. E. Price. 1976. Laboratory evaluation of BL-S786, a cephalosponn with broad-spectrum antibacterial activity. Antimicrob. Agents Chemother. 10:426-435. 11. Onishi, H. R., D. R. Daoust, S. B. Zimmerman, D. Hendlin, and E. 0. Stapley. 1974. Cefoxitin, a semisynthetic cephamycin antibiotic: resistance to beta-lactamase inactivation. Antimicrob. Agents Chemother. 5: 38-48. 12. Ott, J. L., J. R. Turner, and D. F. Mahoney. 1979. Lack of correlation between beta-lactamase production and susceptibility to cefamandole or cefoxitin among spontaneous mutants of Enterobacteriaceae. Antimicrob. Agents Chemother. 15:14-19. 13. Reller, L, B., W. W. Karney, H. N. Beaty, K. K. Holmes, and M. Turck, 1973. Evaluation of cefazolin, a new cephalosporin antibiotic. Antimicrob. Agents Chemother. 3:488-497. 14. Sosna, J. P., P. R. Murray, and G. Medoff. 1978. Comparison of the in vitro activities of HR 756 with cephalothin, cefoxitin, and cefamandole. Antimicrob. Agents Chemother. 14:876-879. 15. Steers, E., E. L. Foltz, B. S. Graves, and J. Riden. 1959. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 16. Wise, R., T. Roliason, M. Logan, J. M. Andrews, and K. A. Bedford. 1978. HR 756, a highly active cephalosporin: comparison with cefazolin and carbenicillin. Antimicrob. Agents Chemother. 14:807-811.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1979, p. 64-68 0066-4804/79/07-0064/05$02.00/0
Antibacterial Activity of a New Parenteral CephalosporinHR 756: Comparison with Cefamandole and Ceforanidet GEORGE W. COUNTS*
AND
MARVIN TURCK
Harborview Medical Center, Department of Medicine, University of Washington, Seattle, Washington 98104 Received for publication 7 May 1979
HR 756, a new parenteral cephalosporin that is beta-lactamase resistant, was tested against 271 bacterial isolates. Both agar and broth dilution testing were employed, using two media and two inoculum sizes of bacteria. Antibacterial activity of the drug was compared to that of cefamandole (CFM) and ceforanide (CFN). In agar, HR 756 was more active than CFM and CFN against all bacteria tested except isolates of Staphylococcus aureus, which were better inhibited by CFM. HR 756 exhibited some antipseudomonas activity in agar, although a marked inoculum effect was apparent. A comparison of median minimum inhibitory and bactericidal concentrations in broth showed again that HR 756 was the most active of these three drugs. HR 756 demonstrated enhanced antibacterial activity compared to CFM and CFN against bacteria sensitive to all three drugs as well as against more resistant isolates of Serratia marcescens, Enterobacter species, and indole-positive Proteus. As with other cephalosporins, results for most bacteria were affected by inoculum size, medium, and type of dilution test employed in in vitro studies. HR 756 is a new semisynthetic methoxyimino derivative of 7-amino-cephalosporanic acid (2). This parenteral drug is a representative of a newer group of cephalosporin antibiotics which shows relative resistance to beta-lactamases of gram-negative bacilli. This group includes cefoxitin, cefamandole (CFM), cefuroxime, and ceforanide (CFN)(7-11, 13). Due in part to this resistance, the newer cephalosporins may demonstrate activity against certain bacteria such as Enterobacter species, Seratia, and indole-positive Proteus strains, the majority of which are ordinarily resistant to other cephalosporin antibiotics. The purpose of this study was to compare the in vitro antibacterial activity of HR 756 to that of ceforanide and cefamandole. MATERIALS AND METHODS Bacterial isolates. A total of 271 clinical isolates of gram-negative and gram-positive bacteria was tested. Tested with all three antibiotics were 170 isolates of various genera of Enterobacteriaceae, 30 isolates of Staphylococcus aureus, and 27 isolates of enterococcus. The 28 strains of Enterobacter included representatives of the following species: E. cloacae, 9; E. aerogenes, 7; E. hafniae, 4; E. agglomerans, 3; and unidentified as to species, 5. The 25 indole-positive Proteus isolates included 8 strains of P. morganii, 7 strains of P. rettgeri, and 10 strains of P. vulgaris. An
additional 15 isolates of Serratia marcescens were selected for study. Finally, agar dilution testing was performed on 29 isolates of Pseudomonas aeruginosa using HR 756 and CFM. Antibiotics. Laboratory standard antibiotic powder was provided by the following: HR 756 by HoechstRoussel, Inc., Somerville, N.J.; CFN (BL-S786) by Bristol Laboratories, Syracuse, N. Y. and CFM by Eli Lilly & Co., Indianapolis, Ind. Susceptibility testing. Agar dilution and broth dilution minimum inhibitory concentrations (MICs) were determined using Mueller-Hinton agar and broth (MHA, MHB; Difco Laboratories, Detroit, Mich.) and nutrient agar (NA) and broth (Difco) according to previously described methods (3, 13). Inoculum sizes of 104 and 106 colony-forming units were used. Minimum bactericidal concentrations (MBCs) were obtained with these two broth media. Agar dilution testing employed a Steers replicating device (15). Criteria for endpoints for MIC and MBC values were as previously described (3). A laboratory reference strain of Escherichia coli inhibited by 0.125 ,ug of HR 756, 0.5 jg of CFN, and 0.5 jg of CFM, respectively, per ml was used as a control organism in all experiments.
RESULTS Agar dilution. Table 1 shows the range of agar dilution MICs found using MHA as well as the minimum drug concentration to inhibit 50 and 90% of tested isolates. There was no consistent difference in the results for various species of Enterobacter or indole-positive Proteus, and the results for each genera are presented in
t No reprints available. 64
VOL. 16, 1979
IN VITRO EVALUATION OF HR 756
composite. For most bacteria tested, HR 756 was the most active of the three cephalosporin antibiotics included in these studies. HR 756 inhibited 100% of isolates of E. coli and P. mirab-
65
ilis at a drug concentration of 0.125 tg of antibiotic per ml. At this concentration, neither of the other two drugs showed any inhibition of these two genera. For isolates of S. aureus, MICs
TABLE 1. MICs of HR 756, CFN, and CFM required to inhibit 50% (MICW) and 90%(MIC4r) of isolates of various bacteria tested in MHA using two inoculum sizes Organism MICro Inoculum MIC (jtg/ml) MIC90 Dru (no. of strains) g (CFU) range (jsg/ml) (pg/ml) E. coli (30) HR 756 104 0.03-0.06 0.03 0.06 106 0.03-0.125 0.06 0.125 CFN 104 0.25-1.0 0.25 0.5 106 0.25-5.0 1.0 *0.5 CFM 104 0.25-2.5 0.5 1.0 106 0.25-5.0 0.5 1.0 S. aureus (30)
HR 756
104
106 CFN
104 106
CFM
104
106
S. typhi (30)
HR 756
104 106
CFN
104
106 CFM P. mirabilis (27)
104
106
HR 756
104 106
CFN
i04 106 104 106
CFM
Proteus, indole-positive (25)
HR 756
104 106 104 106 104
CFN CFM
106
Enterobacter species (28)
HR 756 CFN CFM
Serratia (15)
Klebsiella (30)
HR 756
1
104
106 104 106 104 106
0.5-5.0 1.0-7.5 2.5-12.5 2.5-20 0.125-0.5 0.25-1.0
2.5 2.5 5.0 7.5 0.5 0.5
2.5 2.5 7.5 15 0.5 0.5
0.007-0.125 0.03-0.25 0.125-0.5 0.25-1.0 0.125-2.5 0.125-5.0
0.06 0.06 0.25 0.25 0.25 0.25
0.06 0.06 0.25 0.25 0.25 0.25
.015-0.125 .015-0.125 0.25-2.5 0.25-2.5 0.5-2.5 0.5-2.5
0.03 0.03 0.5 0.5 1.0 2.5
0.03 0.06 0.5 1.0 1.0 2.5
C0.007-2.5 0.015-5.0 0.125->100 0.125->100 0.125-20 0.125-100 0.015-50 0.03->100
0.5->100 1.0->100
0.5->100 1.0->100
0.03 0.06 5.0 20 1.0
2.5 0.125 0.25 2.5 12.5 2.5 2.5
CFN
l04 106 104
0.125-20 0.125-25 50->100
106
>100
CFM
104
10->100
1.0 5.0 >100 >100 >100
106
15->100
>100
HR 756 CFN CFM
104 106 104 106 104 106
0.03-0.25 0.06-0.25 0.125-5.0 0.25-5.0 0.25-5.0 1.0-15
0.06 0.06 0.25 0.5 1.0 1.0
0.125 0.125 100 >100 10 25 1.0
2.5 50 >100 50 >100 5.0 10 >100 >100 >100 >100
0.125 0.25 1.0
2.5 2.5 5.0
66
COUNTS AND TURCK
ANTIMICROB. AGENTS CHEMOTHER.
for CFM with both inoculum sizes were 5- to 30fold lower than those found with HR 756 and CFN. HR 756 inhibited approximately 80% of Enterobacter isolates at 1.0 ,ug of antibiotic per ml, whereas CFM and CFN inhibited approximately 30% of these same isolates at this concentration of drug. Only slight antibacterial activity was seen when the isolates of S. marcescens were tested against CFM and CFN. Median MICs were greater than 100 ,ug of antibiotic per ml for both drugs. However, a concentration of 5.0 ,ug of HR 756 per ml inhibited 90% of these isolates tested with the lower inoculum of bacterial cells and 50% with the higher inoculum. Moreover, a concentration of 10 ,ug of HR 756 per ml inhibited 14 of 15 Serratia isolates. For purposes of comparison with other studies, results found using Mueller-Hinton media are included in the tables. However, although not shown, there was considerable but inconsistent variation in results depending on whether NA or MHA was used as the test medium. Isolates of enterococci were resistant to all three antibiotics, and results are not presented. Considering P. aeruginosa, with the lower inoculum, 12.5 Ag of HR 756 per ml inhibited greater than 80% of the strains. Approximately twice that concentration of drug was required for similar inhibition when the higher inoculum was employed. At each concentration, the drug was more active in NA than in MHA. The widest discrepancy was at 15 ,ug/ml, where 70% of Pseudomonas isolates were inhibited in NA, but only
30% were inhibited in MHA. MICs for CFM with both inocula were greater than 250 ,ug of antibiotic per ml against P. aeruginosa and are not included in the table. Broth dilution. Table 2 shows median MICs for the three antibiotics in MHB with two inoculum sizes of bacterial cells. HR 756 was more active than CFN and CFM, with 10- to 100-fold difference in MICs. As was seen with studies in agar, CFM had greater activity against isolates of S. aureus. For most isolates of S. aureus, MICs of HR 756 were very similar to those of CFN. In general, MBCs in MHB were approximately the same as MICs. The only notable exception was with CFN activity against S. aureus isolates using the higher inoculum. The MBC was >100 jig of antibiotic per ml. With the lower inoculum, the order of activity in broth of these three drugs against isolates of Enterobacter species was HR 756 > CFM > CFN. With the higher inoculum of bacterial cells, CFN and CFM were approximately equal, with both having median MICs equal to 100 ,jg of drug per ml or greater. The activity of HR 756 against the isolates of Serratia, which was demonstrated in agar at both inoculum sizes of bacterial cells, was present in broth only with the lower inoculum of bacterial cells. With the higher inoculum, all three cephalosporins required at least 100 jig of antibiotic per ml for inhibition or killing. MICs and MBCs were determined in nutrient broth as well as MHB, and results revealed certain marked differences especially noted with
TABLE 2. Median MIC results for HR 756, CFN, and CFM using MHB and two inoculum sizes Organism (no. of strains)
E. coli (10)
S. aureus (10) Klebsiella (10) Enterobacter species (10) P. mirabilis (10)
Proteus, indole-positive (10) S. marcescens (10)
Inoculum
(CFU)a 10
0.06 1.0
1.0 7.5
CFM 1.0 5.0
104
10"
2.5 5.0
2.5 5.0
0.5 1.0
104 10
0.06
1.0
1.0
0.5
5.0
5.0
104
104
106 104 106
104 106
104 106
aCFU, colony-forming units.
HR 756
Median MIC (jig/ml) CFN
0.5 20
0.03 2.5
0.03
20
0.5 100
100
15
> 100
100
0.5
5.0
50
>100
1.0
2.5
7.5
100
>100
>100
>100
>100
VOL. 16, 1979
isolates of P. mirabilis. For example, with the higher inoculum, median MICs for HR 756 and CFN were 10-fold lower in nutrient broth compared to MHB. For other isolates, differences in MICs were not as great and drug activity in the two media varied according to organism and antibiotic.
DISCUSSION Among the gram-negative bacilli, some bacteria, such as species of Enterobacter and indolepositive Proteus as well as P. aeruginosa, have generally been resistant to the available cephalosporins. This resistance has been due in part to the production of beta-lactamases. More recently introduced cephalosporins such as CFM, cefoxitin, cefuroxime, and CFN have possessed some in vitro activity against isolates of Enterobacter and indole-positive Proteus, but not Pseudomonas (1, 10). HR 756 is one of this group of newer cephalosporin antibiotics resistant to some beta-lactamases produced by gram-negative bacilli (7, 8). Although beta-lactamases are not the only factors involved in resistance to cephalosporin antibiotics (12), one would ordinarily expect that beta-lactamase-resistant drugs would exhibit more antibacterial activity than antibiotics which were sensitive to these enzymes. HR 756 appears to demonstrate exceptional in vitro activity compared to other cephalosporin antibiotics (2, 9, 14, 16). In our studies, HR 756 exhibited enhanced antibacterial activity compared to CFM and CFN. The exception to this general finding was with isolates of S. aureus which were more susceptible to CFM. In Mueller-Hinton agar, the drug was approximately 4 times as active as CFM and CFN against isolates of E. coli, Salmonella, and Klebsiella, and approximately 10 times as active against isolates of P. mirabilis and indole-positive Proteus. Similarly, in broth, HR 756 was also more active than CFN and CFM, except for isolates of S. aureus. For all three cephalosporins, most MBCs were approximately the same as MICs. However, some isolates did show a substantial, i.e., threefold or greater, difference in median MBCs and MICs, especially in nutrient broth. This disparity occurred most often with CFM and least with HR 756. Isolates of Proteus most frequently exhibited this discrepancy. Similar to findings with other cephalosporins we have tested, results in these studies were influenced by inoculum size, medium, and type of dilutional test employed. Results with agar dilution testing varied according to whether NA or MHA was used. The variation was not consistent for the three antibiotics and eight genera
IN VITRO EVALUATION OF HR 756
67
of bacteria studied. For example, activity for all three drugs against isolates of S. aureus and P. mirabilis was greater in NA than in MHA. On the other hand, all three drugs were more active against isolates of Enterobacter species in MHA than in NA. For the remaining bacteria, results were more variable according to organism and antibiotic. CFN appeared less affected by difference in medium used than HR 756 and CFM. For some bacteria, e.g., E. coli, there was no difference between agar dilution and broth dilution MICs using the same medium. For other bacteria, such as isolates of Enterobacter, MICs were considerably lower in agar than in broth. Findell and Sherris provided evidence that the disparity between CFM MICs for Enterobacter isolates tested in agar and broth was due to a high frequency of of resistant mutants which were not detected with agar dilution methods (6). We also previously noted a similar disparity between Enterobacter agar and broth results with cefazaflur (3), CFM (1), and CFN (M. Silver, L. S. Tompkins, G. W. Counts, and M. Turck, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 17th, New York, Abstr. no. 73, 1977). In our current studies, the same type of discrepancy was seen, although differences in agar and broth results were not as marked with HR 756 as with the other two drugs. Other investigators have reported variable results with the testing of HR 756 against Enterobacter isolates. Chabbert and Lutz reported that median MIC for isolates of E. cloacae tested in agar was 30 pig of HR 756 per ml (2). Sosna et al., on the other hand, reported that 49 E. cloacae isolates tested by microtiter broth dilution had median MICs of less than 0.2 jig of HR 756 per ml (14). The nine isolates of E. cloacae included in our studies were inhibited by 12.5 ,ug of HR 756 per ml or less in agar and by 20 pug of HR 756 per ml or less in broth. Further studies are necessary to determine if the reported results simply reflect regional difference in susceptibility of Enterobacter isolates. Most isolates of S. marcescens are ordinarily resistant to cephalosporin antibiotics. A difference in the type of resistance has been described by Farrar and O'Dell (5), who found that those Serratia isolates that also were resistant to penicillins had a plasmid-mediated, noninducible cephalosporinase. The Serratia isolates that were more susceptible to penicillins possessed a chromosomally mediated, inducible cephalosporinase. The isolates tested by Wise et al. (16), which were susceptible to carbenicillin, and by Sosna et al. (14) were susceptible to HR 756. The S. marcescens isolates included in our studies were highly resistant to cephalothin, penicillins, and at least one aminoglycoside antibiotic as well. In our laboratory, the activity demon-
68
COUNTS AND TURCK
strated by HR 756 was confined to agar dilution testing and low-inoculum MHB studies. With higher inocula in broth, these isolates were not susceptible to HR 756, CFN, or CFM. A particularly interesting aspect of the spectrum of HR 756 is the in vitro activity against isolates of P. aeruginosa. Other investigators have reported that median MICs for isolates of P. aeruginosa were 8 (15) and 6.2 ,ug of HR 756 per ml (14). In our studies, a rather marked inoculum effect was noted which may modify any clinical application of these in vitro observations. Although the development of a cephalosporin antibiotic with antipseudomonas activity would represent an important therapeutic advance, additional laboratory studies and, if appropriate, clinical trials will be necessary to determine if this particular cephalosporin will be useful in this regard. The expected peak serum levels for these three drugs are roughly comparable for anticipated dosage recommendations. Intramuscular injection of 1 g of HR 756 and CFM is expected to yield peak serum levels of 20 to 25 ,ug/ml (Hoechst-Roussel, Inc., and Eli Lilly & Co.), a figure comparable to that achieved by a 500-mg injection of CFN (Bristol Laboratories). Studies of infections in experimental animals and preliminary results of human clinical trials are very encouraging as far as efficacy is concerned. In summary, HR 756 is a promising new cephalosporin which exhibited enhanced activity when compared in vitro to two other new parenteral cephalosporins. Although the drug does demonstrate some variability in activity according to test method, medium, and inoculum size, this variation is less pronounced than that seen with other cephalosporins we have studied. Whether the drug will be useful therapeutically awaits the results of further in vitro and clinical trials. ACKNOWLEDGMENTS This work was supported in part by grants from HoechstRoussel, Inc., Somerville, N.J., Bristol Laboratories, Syracuse, N.Y., and Eli Lilly & Co., Indianapolis, Ind. We gratefully acknowledge the excellent technical assistance of Dolores Zeleznik and Michael Silver. LITERATURE CITED 1. Adams, H. G., G. A. Stilwell, and M. Turek. 1976. In vitro evaluation of cefoxitin and cefamandole. Antimicrob. Agents Chemother. 9:1019-1024.
ANTIMICROB. AGENTS CHEMOTHER. 2. Chabbert, Y. A., and A. J. Lutz. 1978. HR 756, the syn isomer of a new methoxyimino cephalosporin with unusual antibacterial activity. Antimicrob. Agents Chemother. 14:749-754. 3. Counts, G. W., D. Gregory, D. Zeleznik, and M. Turck. 1977. Cefazaflur, a new parenteral cephalosporin: in vitro studies. Antimicrob. Agents Chemother. 11: 708-711. 4. Eykyn, S., C. Jenkins, A. King, and L. Phillips. 1976. Antibacterial activity of cefuroxime, a new cephalosporin antibiotic, compared with that of cephaloridine, cephalothin, and cefamandole. Antimicrob. Agents Chemother. 9:690-695. 5. Farrar, W. E., and N. M. O'Dell. 1976. Beta-lactamases and resistance to penicillins and cephalosporins in Serratia marcescens. J. Infect. Dis. 134:245-251. 6. Findell, C. M., and J. C. Sherris. 1976. Susceptibility of Enterobacter to cefamandole: evidence for a high mutation rate to resistance. Antimicrob. Agents Chemother. 9:970-974. 7. Fu, K. P., and H. C. Neu. 1978. A comparative study of the activity of cefamandole and other cephalosporins and analysis of the beta-lactamase stability and synergy of cefamandole with aminoglycosides. J. Infect. Dis.
137(suppl):S38-S48. 8. Fu, K. P., and H. C. Neu. 1978. Beta-lactamase stability of HR 756, a novel cephalosporin, compared to that of cefuroxime and cefoxitin. Antimicrob. Agents Chemother. 14:322-326. 9. Heymes, R., A. Lutz, and E. Schrinner. 1977. Experimental evaluation of HR 756, a new cephalosporin derivative: pre-clinical study. Infection 5:259-260. 10. Leitner, F., M. Misiek, T. A. Pursiano, R. E. Buck, D. R. Chisholm, R. G. DeRegis, Y. H. Tsai, and K. E. Price. 1976. Laboratory evaluation of BL-S786, a cephalosponn with broad-spectrum antibacterial activity. Antimicrob. Agents Chemother. 10:426-435. 11. Onishi, H. R., D. R. Daoust, S. B. Zimmerman, D. Hendlin, and E. 0. Stapley. 1974. Cefoxitin, a semisynthetic cephamycin antibiotic: resistance to beta-lactamase inactivation. Antimicrob. Agents Chemother. 5: 38-48. 12. Ott, J. L., J. R. Turner, and D. F. Mahoney. 1979. Lack of correlation between beta-lactamase production and susceptibility to cefamandole or cefoxitin among spontaneous mutants of Enterobacteriaceae. Antimicrob. Agents Chemother. 15:14-19. 13. Reller, L, B., W. W. Karney, H. N. Beaty, K. K. Holmes, and M. Turck, 1973. Evaluation of cefazolin, a new cephalosporin antibiotic. Antimicrob. Agents Chemother. 3:488-497. 14. Sosna, J. P., P. R. Murray, and G. Medoff. 1978. Comparison of the in vitro activities of HR 756 with cephalothin, cefoxitin, and cefamandole. Antimicrob. Agents Chemother. 14:876-879. 15. Steers, E., E. L. Foltz, B. S. Graves, and J. Riden. 1959. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 16. Wise, R., T. Roliason, M. Logan, J. M. Andrews, and K. A. Bedford. 1978. HR 756, a highly active cephalosporin: comparison with cefazolin and carbenicillin. Antimicrob. Agents Chemother. 14:807-811.
