Vol. 14, No. 1
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUly 1978, P. 1-5 0066-4804/78/0014-0001$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
In Vitro Activity and Beta-Lactamase Stability of BL-S786 Compared with Those of Other Cephalosporins NALINEE ASWAPOKEE, PRASIT ASWAPOKEE, KWUNG P. FU,
AND
HAROLD C. NEU*
Division of Infectious Diseases, Departments of Medicine and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032
Received for publication 14 February 1978
In vitro activity of BL-S786, a new parenterally semisynthetic cephalosporin, investigated against 570 bacterial isolates. BL-S786 inhibited most Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Salmonella. It inhibited some Enterobacter and indole-positive Proteus, but it was less active against these later species than was cefamandole, cefuroxime, or cefoxitin. It was not active against Serratia marcescens, Pseudomonas aeruginosa, or Bacteroides fragilis. BL-S786 was the least active new cephalosporin tested against staphylococci and was less active than cephalothin against streptococcal species. The activity of BL-S786 was not altered by the type of assay medium nor by 50% serum. The size of the test inoculum altered the miniimal inhibitory and bactericidal concentrations for inhibition of some organisms, particularly those with Richmond type I /-lactamases. BL-S786 was not hydrolyzed by the R-factormediated, Richmond type III f8-lactamase, but it was hydrolyzed by type I 1,lactamases. was
Bacteria. Bacterial strains tested were recent clinical isolates from patients hospitalized at the Columbia-Presbyterian Medical Center, New York City, or selected ,B-lactamase-producing isolates which had been retained frozen from previous studies with other compounds (5, 6). Susceptibility testing. Antimicrobial activity was measured by agar or broth dilution methods as noted in the text, using Mueller-Hinton broth or agar (BBL). An inoculum of 105 colony-forming units (CFU) was used unless otherwise specified. Serial twofold dilutions of antibiotic were prepared, and an overnight culture of bacteria was applied with an inoculating device. Organisms were incubated at 35°C for 18 h. The minimal inhibitory concentration (MIC) of an antibiotic was defined as the lowest concentration that inhibited development of visible growth on agar or turbidity in a tube. Minimal bactericidal concentration (MBC) was determined by plating 0.01 ml from clear tubes of broth onto blood agar. The MBC was the concentration at which less than five colonies grew. The effect of growth medium on activity of BL-S786 was determined using brain heart infusion, nutrient broth, and Mueller-Hinton broth (BBL), and the effect of inoculum size was determined using 103, 105, and 107 CFU. Activity against anaerobic bacteria was determined on Mueller-Hinton agar supplemented with sheep blood and vitamin K. Plates were incubated at 350C for 48 h in GasPak jars (BBL). Haemophilus were tested using chocolate agar, as were the Neisseria, which were incubated in the presence of 5%
Cephalosporin antibiotics are used clinically in many infections because of their broad antibacterial spectrum, which includes both the common gram-positive and gram-negative bacteria. Recently new agents, cefamandole, cefoxitin, and cefuroxime, have been studied in vitro and in the treatment of infection (5, 6, 8). These compounds extend the spectrum of the firstgeneration cephalosporins, but have no major pharmacological advantage over cephalothin and cefazolin. BL-S786, 7-[(a-2-aminomethylphenyl)acetamido]-3-[ (1- carboxymethyltetrazol - 5 - ylthio )methyl]-3-cepham-4-carboxylic acid, a new parenterally administered semisynthetic cephalosporin, has been reported to be more active against certain members of the Enterobacteriaceae than its older congeners (1-3). This in vitro study was undertaken to compare the overall antibacterial activities of BL-S786 with those of the other cephalosporins cephalothin, cefazolin, cefoxitin, cefamandole, and cefuroxime in regard to in vitro activity and resistance to gram-negative ,8-lactamases. MATERIALS AND METHODS Antibiotics. BL-S786 was supplied by K. Price of Bristol Laboratories. Cephalothin, cefazolin, and cefamandole were gifts from Eli Lilly and Co. Cefoxitin was obtained from Merck Sharp & Dohme Research Laboratories, and cefuroxime came from Glaxo Pharmaceuticals. Fresh antibiotic dilutions were made daily in sterile distilled water or broth.
CO2Assay of antibiotics. Standard and sample solutions to be assayed for antibiotic were placed in agar wells on glass plates (14 by 14 inches, ca. 35.6 by 35.6 cm) prepared with 300 ml of antibiotic medium 2 1
2
ANTIMICROB. AGENTS CHEMOTHER.
ASWAPOKEE ET AL.
Staphylococcus epidernidis, BL-S786 was the least active agent, requiring a concentration of 6.2 ,tg/ml to inhibit 50% of staphylococcal isolates, compared to 0.1 ,ug/ml for several of the other agents. None of the cephalosporins inhibited Streptococcus faecalis at concentrations below 12.5 ,ug/ml. The Escherichia coli strains shown were selected for resistance to cephalothin. Against these E. coli isolates, BL-S786 was the most active agent tested. Against Klebsiella, BL-S786 was equivalent in activity to cephalothin, cefazolin, cefamandole, and cefuroxime, but less active than cefoxitin. Against Enterobacter, BL-S786 was more active than cephalothin, cefazolin, and cefoxitin, but it was much less active than cefamandole or cefuroxime. BL-S786 was the most active agent tested against Proteus mirabilis, but the differences in activity among the agents were minor. BL-S786 was more active than cephalothin or cefazolin, and similar to cefoxitin, against Citrobacter. Cefoxitin was the most active agent against indole-positive Proteus, followed by cefamandole and cefuroxime. BL-S786 inhibited 50% of the Proteus rettgeri and Proteus vulgaris at 25 ,ug/ml, but the MICs against Proteus morganii were usually greater than 100 ,ug/ml. BL-S786 inhibited most of the Providencia, which were also inhibited by cefoxitin, cefamandole, and cefuroxime. BL-S786 was less active than the newer agents against Acinetobacter, and it had no activity against Serratia marcescens or Pseudomonas aeruginosa. Against Bacteroides fragilis, BL-S786 was no more active than cephalothin and less active than cefamandole, cefuroxime, and cefazolin. Only cefoxitin had activity against the B. fragilis used in these experiments. Since it is known that type of medium and inoculum size affect the MICs obtained with cephalosporin antibiotics, these factors were investigated. There was no consistent effect (5, 6) of the type of medium used (Mueller-Hinton broth, brain heart infusion broth, or nutrient broth) on the MIC of BL-S786 against either S. aureus or the Enterobacteriaceae. Most of the
(Difco). The assay organism was Staphylococcus auATCC 25293. Zones of inhibition of growth were measured, and the concentration of antibiotic was determined from curves constructed from plots of a standard run in an identical method. Hydrolysis of antibiotics. Bacterial strains were grown in Mueller-Hinton broth to a concentration of 109 CFU. These were removed by centrifugation and then washed with 0.05 M potassium phosphate buffer (pH 7.0). Bacterial cells were resuspended in 0.05 M potassium phosphate buffer (pH 7.0) to which BLS786 at a concentration of 30 pg/ml was added, and the mixtures were incubated at 370C for 1 h. Bacteria were removed by high-speed centrifugation, and the amount of unhydrolyzed antibiotic in the medium was determined with the agar-well technique described. ,t-Lactamase assays. The enzymes were classified according to the schema of Richmond and Sykes (9). The f8-lactamase assay procedures used were either the microiodometric or spectrophotometric, as previously described (4). /3-Lactamase was detected in clinical isolates by the chromogenic technique (7). ,B-Lactamase assays were performed using purified (4) or partially purified enzymes that had been prepared by sonic disruption of bacteria, followed by centrifugation to remove debris and chromatography on Sephadex G50.
reus
RESULTS
BL-S786 inhibited the majority (88%) of betahemolytic streptococci, Streptococcus pyogenes, and Streptococcus agalactiae and the majority (71%) of Streptococcus pneumoniae at a concentration of 0.4 ,tg/ml (Table 1). In contrast, only 45% of Streptococcus viridans isolates were inhibited at this concentration. All of the Haemophilus influenzae and Neisseria gonorrhoeae were inhibited at a level of 3.1 ,ug/ml. This included two ,B-lactamase-producing H. influenzae and two ,B-lactamase-producing N. gonorrhoeae. Two Haemophilus parainfluenzae strains and two Neisseria meningitidis strains were inhibited at 1.6 Ag/ml. The activity of BL-S786 is compared with other cephalosporin antibiotics in Table 2. The concentration of antibiotic that inhibited 50 and 90% of isolates is listed for simplicity. Against S. aureus and
TABLE 1. Activity of BL-S786 against selected gram-positive and gram-negative organisms % Inhibited at MIC Organism Organism
Streptococcus pneumoniae Streptococcus, beta-hemolytic (group A, B) Streptococcus viridans Haemophilus influenzae Haemophilus parainfluenzae Neisseria meningitidis Neisseria gonorrhoeae
(JLg/ml):
~No. of
isolates 0.2
0.4
7 25
57
71 88
11 9 2 2 12
9
45
0.8
64
1.6
3.1
86 96
100
73 22 100 100 50
82 100 100
3
ACTIVITY OF BL-S786
VOL. 14, 1978
TABLE 2. Comparative activity of cephalosporins against gram-positive and gram-negative bacteria MIC (ug/ml) for 50 and 90% of test strains Organiism (no. of strains) BL-S786 Cephalothin Cefazolin Cefoxitin Cefamandole Cefuroxime -a 0.2 0.1 1.6 0.1 6.2 Staphylococcus aureus (29) 1.6 0.8 3.1 0.2 12.5 0.1 1.6 0.1 0.2 0.1 6.2 Staphylococcus epidermidis (30) 6.2 12.5 0.2 0.8 0.2 400 25 25 12.5 50 Streptococcus faecalis (32)
Escherichia coli (32) Klebsiella pneumoniae (32) Enterobacter (31) Proteus mirabilis (32)
1.6 12.5 1.6 25 25 >400 0.8 50
Proteus, indole-positive (32)
100
Citrobacter (32)
200 3.1 >400
Providencia (28)
12.5
50 >400
3.1 50 200 >400 6.2 100 >400
Acinetobacter (13)
200
25 >400 >400
>400
>400
200
>400
Shigella (32)
0.2 6.2 3.1 100
Bacteroides (29) a -,
200 >400
6.3 50 25 >400 50 200
12.5 50 1.6 6.2 50 200 3.1 12.5 6.2 12.5 6.2
>400
100
200 >400 >400
100
100 >400
>400
Salmonella (32)
6.2 200 200 >400
>400
Serratia (30)
12.5 >400 3.1 200 >400
50 200
6.2 50 200 100 >400
1.6 3.1 6.3 50 6.2 100
6.2 100
0.4 25 3.1 100
1.6 12.5
3.1
>400 0.2 0.8 12.5 50 >400 50 >400 3.1 6.3 1.6 6.3 50 200
6.2 100
1.6 12.5 6.2 12.5 1.6 12.5 25 200 3.1 12.5 25 200 50 200 25 >400
100 >400
Not determined.
differences in the MICs were only twofold, although occasional four- or eightfold differences in MICs among the media were observed. Agar dilution results were similar to MICs obtained in broth. The size of the inoculum did not affect the MICs of S. aureus, but it did affect the MICs achieved against a number of the members of the Enterobacteriaceae. For example, at 103
CFU Enterobacter strain 3671 had a BL-S786 MIC of 6.3 ,tg/ml, but with 107 CFU the MIC was greater than 400 ,ug/ml. Similar results were found for some of the E. coli, Citrobacter, and indole-positive Proteus isolates tested at inocula of 103 and 107 CFU. Determination of the MICs in 50% serum did not result in an increase of the MICs of S. aureus, E. coli, Klebsiella, Enterobacter, P. mirabilis, or P. vulgaris. In general, the MICs and MBCs obtained with an inoculum of 105 CFU differed only twofold for most organisms. However, the MICs and MBCs of Enterobacter cloacae, some indole-positive Proteus, and some Citrobacter differed by 8- to 16-fold. The activity of BL-S786, cefamandole, cefoxitin, and cefuroxime was compared against iso-
lates that contained Richmond classification type I ,B-lactamases resistant to cephalothin (Table 3). There was no consistent sensitivity pattern by which it was possible to state that if an organism were sensitive or resistant to one cephalosporin it would have a similar sensitivity to the others. For example, the MICs of BLS786 and cefoxitin were similar against one Citrobacter isolate and not against the other Citrobacter isolate. The same was true in examining the relationship of BL-S786 and cefamandole or cefuroxime. Thus, knowledge of the activity of one of these newer cephalosporins against type I,-lactamases fails to allow for generalizations concerning in vitro activity. Organisms that showed a significant effect of the inoculum size on MICs or had shown large differences between MICs and MBCs were selected for study to determine the degree of hydrolysis of BL-S786 by intact bacteria. Organisms that did not possess those properties also were selected for comparison. There was significant hydrolysis of BL-S786 by the organisms which either had a discrepancy in MIC-MBC value or had much greater MICs at a larger
4
ASWAPOKEE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
inoculum size (Table 4). Hydrolysis occurred with strains that did not have ,8-lactamase activity detectable when assayed with a chromogenic substrate, but which were known to have inducible f3-lactamase activity. The stability of BL-S786 to fB-lactamases isolated from different bacteria is given in Table 5. BL-S786 was relatively stable against type I (Richmond) fl-lactamases of both induced and noninduced types as compared with cephalothin
cephaloridine. It was much less stable than cefoxitin or cefuroxime. Against a type III ,Blactamase, the most common R-factor type, which is primarily active against penicillins, it was as stable as cefazolin and twofold more stable than cephalothin. BL-S786 was about as stable as cefoxitin against a type I plasmid-mediated /8-lactamase that can hydrolyze oxacillin. These observations would indicate that resistance to /3-lactamase contributes in part to the in or
TABLE 3. Comparison of MICs of BL-S786 and other cephalosporins against f3-lactamase-producin,g organisms MIC
Isolate'
Enterobacter 1645 Citrobacter 2642 Citrobacter 2847 Acinetobacter 2284 Providencia 3392 Providencia 3389 Proteus rettgeri 2729 Serratia 2792 a All strains contain type I
BL-S786
Cefamandole
100 50 >400 1.6 12.5 50 100 >400
12.5 0.8 3.1 3.1 50 12.5 3.1 12.5
(tg/ml) of compound: Cefuroxime 100 3.1
1.6 3.1 50 25 6.2 12.5
Cefoxitin
200 50 100 3.1 12.5 25 3.1 6.2
Cephalothin >400 400 400 400 400 400 200 >400
i-lactamases.
TABLE 4. Susceptibility of organisms in comparison to the amount of antibiotic hydrolyzed in intact
microorganisms
Organism
,-Lactamase'
MIC
(,ug/ml)
MBC
(pg/ml)
+ 6.3 1.6 Staphylococcus aureus 3539 + 25 3.1 Staphylococcus aureus 3830 + 6.3 100 Escherichia coli 4018 + 3.1 200 Escherichia coli 4019 -C 400 >400 Enterobacter 3965 + 3.1 3.1 Klebsiella 2984 + 12.5 Citrobacter 3375 6.3 200 200 Proteus rettgeri 3755 _C >400 400 Proteus morganii 2869 _C 0.4 0.4 Proteus mirabilis 2401 a Determined by use of chromogenic method (8). b The MIC at 107 CFU was eightfold or greater than that found at 103 CFU. c/8-Lactamase activity is inducible.
Inoculum
effecth + + + + +
+
-
% PL-S786
hydrolyzed 0 0 31 35 24 33 59 38 59 0
TABLE 5. /3-Lactamase hydrolysis of BL-S786 compared with other cephalosporinsa Relative hydrolysis rate of antibiotic:
Organism
Type of
b
Cefazolin Cefoxitin BL-S786 Cephalothin ridine Citrobacter I 100 99 60 0 20 Proteus morganii I 100 209 48 0 26 III Pseudomonas 1822 100 23 11 0 13 Klebsiella IV 100 62 28 0 6 V Shigella 100 18 8 0 3 Pseudomonas Induced Id 100 260 127 0 26 a'/-Lactamase activity was determined by the spectrophotometric method by measuring optical density decrease at 255 nm. Reaction mixture contained 0.5 ml of 0.4 mM cephalosporin plus 0.5 ml of phosphate buffer (0.05 M, pH 7). Hydrolysis rate was relative to the rate of hydrolysis of cephaloridine, which is given a value of 100. b Richmond classification.
VOL. 14, 1978
vitro activity of BL-S786 against some of the members of the Enterobacteriaceae that contain f,-lactamases.
DISCUSSION BL-S786 has been shown to inhibit growth of E. coli, Klebsiella pneumoniae, P. mirabilis, and Salmonella. In contrast to previous studies, we found that BL-S786 was not active against many E. cloacae (1, 3). This may be due to the fact that we used primarily E. cloacae and had few Enterobacter aerogenes isolates among our test organisms. Like the older cephalosporins, BL-S786 was not active against S. marcescens, P. aeruginosa, and B. fragilis. When compared to newer cephalosporins, BL-S786 had activity comparable to cefamandole and cefuroxime against E. coli, Klebsiella, and P. mirabilis. It was less active than cefoxitin or cefamandole against indole-positive Proteus and Providencia. The compound was the least active parenteral cephalosporin tested against S. epidermidis and S. aureus, and some strains of these grampositive cocci were inhibited only at levels of 12.5 mg/ml. The activity of BL-S786 against streptococcal isolates was also less than that of both the older cephalosporins and the new agents. BL-S786 was active against E. coli, H. influenzae, and N. gonorrhoeae which conIII tained a Richmond type Wf-lactamase, and it was not destroyed by a purified preparation of this enzyme type. The activity of BL-S786 was not influenced by the type of medium used in the tests or by serum. Its MICs and MBCs, similar to those found with- other cephalosporins, were influenced by increase in the inoculum size. The difference between inhibitory and bactericidal levels appeared to be related to ,B-lactamase destruction of the agent. Hydrolysis of the compound by bacterial strains that showed significant difference in MIC and MBC or had an inoculum effect was an indirect evidence of this possibility. BL-S786 was considerably more stable to hydrolysis by type I f3-lactamases than was cephalothin or cefazolin, and this relative ,8-lactamase stability would seem to explain some of its increased in vitro activity, but the
ACTIVITY OF BL-S786
5
agent showed relatively poor activity against Pseudomonas, Citrobacter, and indole-positive Proteus, all of which contain this type of enzyme. BL-S786 does not extend the spectrum of the cephalosporins to the degree that cefamandole, cefuroxime, and cefoxitin have. Its lower activity against gram-positive cocci, as well as its lack of anaerobic activity, would indicate that the agent's merit may have to be related to pharmacological properties. Further studies may clarify whether these in vitro findings are of clinical importance. ACKNOWLEDGMENTS We thank Ket W. Kung for her technical assistance and Iris P. Rivera for her manuscript preparation.
LITERATURE CITED 1. Jones, R. N., P. C. Fuchs, T. L. Gavan, E. H. Gerlach, A. Barry, and C. Thornberry. 1977. BL-S786, a new parenteral cephalosporin. I. A collaborative in vitro susceptibility comparison to cephalothin against 5,762 clinical bacterial isolates. J. Antibiot. 30:576-582. 2. Jones, R. N., C. Thornberry, A. L Barry, P. C. Fuchs, T. L Gavan, and E. H. Gerlach. 1977. BL-S786, a new parenteral cephalosporin. II. In vitro antimicrobial activity comparison with six related cephalosporins. J. Antibiot. 30:583-592. 3. Leitner, F., M. Misiek, T. A. Pursiano, R. E. Buck, D. R. Chishohn, R. G. DeRegis, Y. H. Tsai, and K. E. Price. 1976. Laboratory evaluation of BL-S786, a cephalosporin with broad-spectrum antibacterial activity. Antimicrob. Agents Chemother. 10:426-435. 4. Neu, H. C. 1971. Beta-lactamase production by Pseudomonas aeruginosa, p. 534-538. Antimicrob. Agents Chemother. 1970. 5. Neu, H. C. 1974. Cefoxitin, a semisynthetic cephamycin antibiotic: antibacterial spectrum and resistance to hydrolysis by gram-negative beta-lactamases. Antimicrob. Agents Chemother. 6:170-176. 6. Neu, H. C. 1974. Cefamandole, a cephalosporin antibiotic with an unusually wide spectrum of activity. Antimicrob. Agents Chemother. 6:177-182. 7. O'Caliaghan, C. H., A. Morris, S. M. Kirby, and A. H. Shingler. 1972. Novel method for detection of ,-lactamases .by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1:283-288. 8. O'Callaghan, C. H., R. B. Sykes, A. Griffiths, and J. E. Thornton. 1976. Cefuroxime, a new cephalosporin antibiotic: activity in vitro. Antimicrob. Agents Chemother. 9:511-519. 9. Richmond, M. H., and R. B. Sykes. 1973. The f8-lactamases of gram-negative bacteria and their possible physiological role. Adv. Microb. Physiol. 9:31-88.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUly 1978, P. 1-5 0066-4804/78/0014-0001$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
In Vitro Activity and Beta-Lactamase Stability of BL-S786 Compared with Those of Other Cephalosporins NALINEE ASWAPOKEE, PRASIT ASWAPOKEE, KWUNG P. FU,
AND
HAROLD C. NEU*
Division of Infectious Diseases, Departments of Medicine and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032
Received for publication 14 February 1978
In vitro activity of BL-S786, a new parenterally semisynthetic cephalosporin, investigated against 570 bacterial isolates. BL-S786 inhibited most Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Salmonella. It inhibited some Enterobacter and indole-positive Proteus, but it was less active against these later species than was cefamandole, cefuroxime, or cefoxitin. It was not active against Serratia marcescens, Pseudomonas aeruginosa, or Bacteroides fragilis. BL-S786 was the least active new cephalosporin tested against staphylococci and was less active than cephalothin against streptococcal species. The activity of BL-S786 was not altered by the type of assay medium nor by 50% serum. The size of the test inoculum altered the miniimal inhibitory and bactericidal concentrations for inhibition of some organisms, particularly those with Richmond type I /-lactamases. BL-S786 was not hydrolyzed by the R-factormediated, Richmond type III f8-lactamase, but it was hydrolyzed by type I 1,lactamases. was
Bacteria. Bacterial strains tested were recent clinical isolates from patients hospitalized at the Columbia-Presbyterian Medical Center, New York City, or selected ,B-lactamase-producing isolates which had been retained frozen from previous studies with other compounds (5, 6). Susceptibility testing. Antimicrobial activity was measured by agar or broth dilution methods as noted in the text, using Mueller-Hinton broth or agar (BBL). An inoculum of 105 colony-forming units (CFU) was used unless otherwise specified. Serial twofold dilutions of antibiotic were prepared, and an overnight culture of bacteria was applied with an inoculating device. Organisms were incubated at 35°C for 18 h. The minimal inhibitory concentration (MIC) of an antibiotic was defined as the lowest concentration that inhibited development of visible growth on agar or turbidity in a tube. Minimal bactericidal concentration (MBC) was determined by plating 0.01 ml from clear tubes of broth onto blood agar. The MBC was the concentration at which less than five colonies grew. The effect of growth medium on activity of BL-S786 was determined using brain heart infusion, nutrient broth, and Mueller-Hinton broth (BBL), and the effect of inoculum size was determined using 103, 105, and 107 CFU. Activity against anaerobic bacteria was determined on Mueller-Hinton agar supplemented with sheep blood and vitamin K. Plates were incubated at 350C for 48 h in GasPak jars (BBL). Haemophilus were tested using chocolate agar, as were the Neisseria, which were incubated in the presence of 5%
Cephalosporin antibiotics are used clinically in many infections because of their broad antibacterial spectrum, which includes both the common gram-positive and gram-negative bacteria. Recently new agents, cefamandole, cefoxitin, and cefuroxime, have been studied in vitro and in the treatment of infection (5, 6, 8). These compounds extend the spectrum of the firstgeneration cephalosporins, but have no major pharmacological advantage over cephalothin and cefazolin. BL-S786, 7-[(a-2-aminomethylphenyl)acetamido]-3-[ (1- carboxymethyltetrazol - 5 - ylthio )methyl]-3-cepham-4-carboxylic acid, a new parenterally administered semisynthetic cephalosporin, has been reported to be more active against certain members of the Enterobacteriaceae than its older congeners (1-3). This in vitro study was undertaken to compare the overall antibacterial activities of BL-S786 with those of the other cephalosporins cephalothin, cefazolin, cefoxitin, cefamandole, and cefuroxime in regard to in vitro activity and resistance to gram-negative ,8-lactamases. MATERIALS AND METHODS Antibiotics. BL-S786 was supplied by K. Price of Bristol Laboratories. Cephalothin, cefazolin, and cefamandole were gifts from Eli Lilly and Co. Cefoxitin was obtained from Merck Sharp & Dohme Research Laboratories, and cefuroxime came from Glaxo Pharmaceuticals. Fresh antibiotic dilutions were made daily in sterile distilled water or broth.
CO2Assay of antibiotics. Standard and sample solutions to be assayed for antibiotic were placed in agar wells on glass plates (14 by 14 inches, ca. 35.6 by 35.6 cm) prepared with 300 ml of antibiotic medium 2 1
2
ANTIMICROB. AGENTS CHEMOTHER.
ASWAPOKEE ET AL.
Staphylococcus epidernidis, BL-S786 was the least active agent, requiring a concentration of 6.2 ,tg/ml to inhibit 50% of staphylococcal isolates, compared to 0.1 ,ug/ml for several of the other agents. None of the cephalosporins inhibited Streptococcus faecalis at concentrations below 12.5 ,ug/ml. The Escherichia coli strains shown were selected for resistance to cephalothin. Against these E. coli isolates, BL-S786 was the most active agent tested. Against Klebsiella, BL-S786 was equivalent in activity to cephalothin, cefazolin, cefamandole, and cefuroxime, but less active than cefoxitin. Against Enterobacter, BL-S786 was more active than cephalothin, cefazolin, and cefoxitin, but it was much less active than cefamandole or cefuroxime. BL-S786 was the most active agent tested against Proteus mirabilis, but the differences in activity among the agents were minor. BL-S786 was more active than cephalothin or cefazolin, and similar to cefoxitin, against Citrobacter. Cefoxitin was the most active agent against indole-positive Proteus, followed by cefamandole and cefuroxime. BL-S786 inhibited 50% of the Proteus rettgeri and Proteus vulgaris at 25 ,ug/ml, but the MICs against Proteus morganii were usually greater than 100 ,ug/ml. BL-S786 inhibited most of the Providencia, which were also inhibited by cefoxitin, cefamandole, and cefuroxime. BL-S786 was less active than the newer agents against Acinetobacter, and it had no activity against Serratia marcescens or Pseudomonas aeruginosa. Against Bacteroides fragilis, BL-S786 was no more active than cephalothin and less active than cefamandole, cefuroxime, and cefazolin. Only cefoxitin had activity against the B. fragilis used in these experiments. Since it is known that type of medium and inoculum size affect the MICs obtained with cephalosporin antibiotics, these factors were investigated. There was no consistent effect (5, 6) of the type of medium used (Mueller-Hinton broth, brain heart infusion broth, or nutrient broth) on the MIC of BL-S786 against either S. aureus or the Enterobacteriaceae. Most of the
(Difco). The assay organism was Staphylococcus auATCC 25293. Zones of inhibition of growth were measured, and the concentration of antibiotic was determined from curves constructed from plots of a standard run in an identical method. Hydrolysis of antibiotics. Bacterial strains were grown in Mueller-Hinton broth to a concentration of 109 CFU. These were removed by centrifugation and then washed with 0.05 M potassium phosphate buffer (pH 7.0). Bacterial cells were resuspended in 0.05 M potassium phosphate buffer (pH 7.0) to which BLS786 at a concentration of 30 pg/ml was added, and the mixtures were incubated at 370C for 1 h. Bacteria were removed by high-speed centrifugation, and the amount of unhydrolyzed antibiotic in the medium was determined with the agar-well technique described. ,t-Lactamase assays. The enzymes were classified according to the schema of Richmond and Sykes (9). The f8-lactamase assay procedures used were either the microiodometric or spectrophotometric, as previously described (4). /3-Lactamase was detected in clinical isolates by the chromogenic technique (7). ,B-Lactamase assays were performed using purified (4) or partially purified enzymes that had been prepared by sonic disruption of bacteria, followed by centrifugation to remove debris and chromatography on Sephadex G50.
reus
RESULTS
BL-S786 inhibited the majority (88%) of betahemolytic streptococci, Streptococcus pyogenes, and Streptococcus agalactiae and the majority (71%) of Streptococcus pneumoniae at a concentration of 0.4 ,tg/ml (Table 1). In contrast, only 45% of Streptococcus viridans isolates were inhibited at this concentration. All of the Haemophilus influenzae and Neisseria gonorrhoeae were inhibited at a level of 3.1 ,ug/ml. This included two ,B-lactamase-producing H. influenzae and two ,B-lactamase-producing N. gonorrhoeae. Two Haemophilus parainfluenzae strains and two Neisseria meningitidis strains were inhibited at 1.6 Ag/ml. The activity of BL-S786 is compared with other cephalosporin antibiotics in Table 2. The concentration of antibiotic that inhibited 50 and 90% of isolates is listed for simplicity. Against S. aureus and
TABLE 1. Activity of BL-S786 against selected gram-positive and gram-negative organisms % Inhibited at MIC Organism Organism
Streptococcus pneumoniae Streptococcus, beta-hemolytic (group A, B) Streptococcus viridans Haemophilus influenzae Haemophilus parainfluenzae Neisseria meningitidis Neisseria gonorrhoeae
(JLg/ml):
~No. of
isolates 0.2
0.4
7 25
57
71 88
11 9 2 2 12
9
45
0.8
64
1.6
3.1
86 96
100
73 22 100 100 50
82 100 100
3
ACTIVITY OF BL-S786
VOL. 14, 1978
TABLE 2. Comparative activity of cephalosporins against gram-positive and gram-negative bacteria MIC (ug/ml) for 50 and 90% of test strains Organiism (no. of strains) BL-S786 Cephalothin Cefazolin Cefoxitin Cefamandole Cefuroxime -a 0.2 0.1 1.6 0.1 6.2 Staphylococcus aureus (29) 1.6 0.8 3.1 0.2 12.5 0.1 1.6 0.1 0.2 0.1 6.2 Staphylococcus epidermidis (30) 6.2 12.5 0.2 0.8 0.2 400 25 25 12.5 50 Streptococcus faecalis (32)
Escherichia coli (32) Klebsiella pneumoniae (32) Enterobacter (31) Proteus mirabilis (32)
1.6 12.5 1.6 25 25 >400 0.8 50
Proteus, indole-positive (32)
100
Citrobacter (32)
200 3.1 >400
Providencia (28)
12.5
50 >400
3.1 50 200 >400 6.2 100 >400
Acinetobacter (13)
200
25 >400 >400
>400
>400
200
>400
Shigella (32)
0.2 6.2 3.1 100
Bacteroides (29) a -,
200 >400
6.3 50 25 >400 50 200
12.5 50 1.6 6.2 50 200 3.1 12.5 6.2 12.5 6.2
>400
100
200 >400 >400
100
100 >400
>400
Salmonella (32)
6.2 200 200 >400
>400
Serratia (30)
12.5 >400 3.1 200 >400
50 200
6.2 50 200 100 >400
1.6 3.1 6.3 50 6.2 100
6.2 100
0.4 25 3.1 100
1.6 12.5
3.1
>400 0.2 0.8 12.5 50 >400 50 >400 3.1 6.3 1.6 6.3 50 200
6.2 100
1.6 12.5 6.2 12.5 1.6 12.5 25 200 3.1 12.5 25 200 50 200 25 >400
100 >400
Not determined.
differences in the MICs were only twofold, although occasional four- or eightfold differences in MICs among the media were observed. Agar dilution results were similar to MICs obtained in broth. The size of the inoculum did not affect the MICs of S. aureus, but it did affect the MICs achieved against a number of the members of the Enterobacteriaceae. For example, at 103
CFU Enterobacter strain 3671 had a BL-S786 MIC of 6.3 ,tg/ml, but with 107 CFU the MIC was greater than 400 ,ug/ml. Similar results were found for some of the E. coli, Citrobacter, and indole-positive Proteus isolates tested at inocula of 103 and 107 CFU. Determination of the MICs in 50% serum did not result in an increase of the MICs of S. aureus, E. coli, Klebsiella, Enterobacter, P. mirabilis, or P. vulgaris. In general, the MICs and MBCs obtained with an inoculum of 105 CFU differed only twofold for most organisms. However, the MICs and MBCs of Enterobacter cloacae, some indole-positive Proteus, and some Citrobacter differed by 8- to 16-fold. The activity of BL-S786, cefamandole, cefoxitin, and cefuroxime was compared against iso-
lates that contained Richmond classification type I ,B-lactamases resistant to cephalothin (Table 3). There was no consistent sensitivity pattern by which it was possible to state that if an organism were sensitive or resistant to one cephalosporin it would have a similar sensitivity to the others. For example, the MICs of BLS786 and cefoxitin were similar against one Citrobacter isolate and not against the other Citrobacter isolate. The same was true in examining the relationship of BL-S786 and cefamandole or cefuroxime. Thus, knowledge of the activity of one of these newer cephalosporins against type I,-lactamases fails to allow for generalizations concerning in vitro activity. Organisms that showed a significant effect of the inoculum size on MICs or had shown large differences between MICs and MBCs were selected for study to determine the degree of hydrolysis of BL-S786 by intact bacteria. Organisms that did not possess those properties also were selected for comparison. There was significant hydrolysis of BL-S786 by the organisms which either had a discrepancy in MIC-MBC value or had much greater MICs at a larger
4
ASWAPOKEE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
inoculum size (Table 4). Hydrolysis occurred with strains that did not have ,8-lactamase activity detectable when assayed with a chromogenic substrate, but which were known to have inducible f3-lactamase activity. The stability of BL-S786 to fB-lactamases isolated from different bacteria is given in Table 5. BL-S786 was relatively stable against type I (Richmond) fl-lactamases of both induced and noninduced types as compared with cephalothin
cephaloridine. It was much less stable than cefoxitin or cefuroxime. Against a type III ,Blactamase, the most common R-factor type, which is primarily active against penicillins, it was as stable as cefazolin and twofold more stable than cephalothin. BL-S786 was about as stable as cefoxitin against a type I plasmid-mediated /8-lactamase that can hydrolyze oxacillin. These observations would indicate that resistance to /3-lactamase contributes in part to the in or
TABLE 3. Comparison of MICs of BL-S786 and other cephalosporins against f3-lactamase-producin,g organisms MIC
Isolate'
Enterobacter 1645 Citrobacter 2642 Citrobacter 2847 Acinetobacter 2284 Providencia 3392 Providencia 3389 Proteus rettgeri 2729 Serratia 2792 a All strains contain type I
BL-S786
Cefamandole
100 50 >400 1.6 12.5 50 100 >400
12.5 0.8 3.1 3.1 50 12.5 3.1 12.5
(tg/ml) of compound: Cefuroxime 100 3.1
1.6 3.1 50 25 6.2 12.5
Cefoxitin
200 50 100 3.1 12.5 25 3.1 6.2
Cephalothin >400 400 400 400 400 400 200 >400
i-lactamases.
TABLE 4. Susceptibility of organisms in comparison to the amount of antibiotic hydrolyzed in intact
microorganisms
Organism
,-Lactamase'
MIC
(,ug/ml)
MBC
(pg/ml)
+ 6.3 1.6 Staphylococcus aureus 3539 + 25 3.1 Staphylococcus aureus 3830 + 6.3 100 Escherichia coli 4018 + 3.1 200 Escherichia coli 4019 -C 400 >400 Enterobacter 3965 + 3.1 3.1 Klebsiella 2984 + 12.5 Citrobacter 3375 6.3 200 200 Proteus rettgeri 3755 _C >400 400 Proteus morganii 2869 _C 0.4 0.4 Proteus mirabilis 2401 a Determined by use of chromogenic method (8). b The MIC at 107 CFU was eightfold or greater than that found at 103 CFU. c/8-Lactamase activity is inducible.
Inoculum
effecth + + + + +
+
-
% PL-S786
hydrolyzed 0 0 31 35 24 33 59 38 59 0
TABLE 5. /3-Lactamase hydrolysis of BL-S786 compared with other cephalosporinsa Relative hydrolysis rate of antibiotic:
Organism
Type of
b
Cefazolin Cefoxitin BL-S786 Cephalothin ridine Citrobacter I 100 99 60 0 20 Proteus morganii I 100 209 48 0 26 III Pseudomonas 1822 100 23 11 0 13 Klebsiella IV 100 62 28 0 6 V Shigella 100 18 8 0 3 Pseudomonas Induced Id 100 260 127 0 26 a'/-Lactamase activity was determined by the spectrophotometric method by measuring optical density decrease at 255 nm. Reaction mixture contained 0.5 ml of 0.4 mM cephalosporin plus 0.5 ml of phosphate buffer (0.05 M, pH 7). Hydrolysis rate was relative to the rate of hydrolysis of cephaloridine, which is given a value of 100. b Richmond classification.
VOL. 14, 1978
vitro activity of BL-S786 against some of the members of the Enterobacteriaceae that contain f,-lactamases.
DISCUSSION BL-S786 has been shown to inhibit growth of E. coli, Klebsiella pneumoniae, P. mirabilis, and Salmonella. In contrast to previous studies, we found that BL-S786 was not active against many E. cloacae (1, 3). This may be due to the fact that we used primarily E. cloacae and had few Enterobacter aerogenes isolates among our test organisms. Like the older cephalosporins, BL-S786 was not active against S. marcescens, P. aeruginosa, and B. fragilis. When compared to newer cephalosporins, BL-S786 had activity comparable to cefamandole and cefuroxime against E. coli, Klebsiella, and P. mirabilis. It was less active than cefoxitin or cefamandole against indole-positive Proteus and Providencia. The compound was the least active parenteral cephalosporin tested against S. epidermidis and S. aureus, and some strains of these grampositive cocci were inhibited only at levels of 12.5 mg/ml. The activity of BL-S786 against streptococcal isolates was also less than that of both the older cephalosporins and the new agents. BL-S786 was active against E. coli, H. influenzae, and N. gonorrhoeae which conIII tained a Richmond type Wf-lactamase, and it was not destroyed by a purified preparation of this enzyme type. The activity of BL-S786 was not influenced by the type of medium used in the tests or by serum. Its MICs and MBCs, similar to those found with- other cephalosporins, were influenced by increase in the inoculum size. The difference between inhibitory and bactericidal levels appeared to be related to ,B-lactamase destruction of the agent. Hydrolysis of the compound by bacterial strains that showed significant difference in MIC and MBC or had an inoculum effect was an indirect evidence of this possibility. BL-S786 was considerably more stable to hydrolysis by type I f3-lactamases than was cephalothin or cefazolin, and this relative ,8-lactamase stability would seem to explain some of its increased in vitro activity, but the
ACTIVITY OF BL-S786
5
agent showed relatively poor activity against Pseudomonas, Citrobacter, and indole-positive Proteus, all of which contain this type of enzyme. BL-S786 does not extend the spectrum of the cephalosporins to the degree that cefamandole, cefuroxime, and cefoxitin have. Its lower activity against gram-positive cocci, as well as its lack of anaerobic activity, would indicate that the agent's merit may have to be related to pharmacological properties. Further studies may clarify whether these in vitro findings are of clinical importance. ACKNOWLEDGMENTS We thank Ket W. Kung for her technical assistance and Iris P. Rivera for her manuscript preparation.
LITERATURE CITED 1. Jones, R. N., P. C. Fuchs, T. L. Gavan, E. H. Gerlach, A. Barry, and C. Thornberry. 1977. BL-S786, a new parenteral cephalosporin. I. A collaborative in vitro susceptibility comparison to cephalothin against 5,762 clinical bacterial isolates. J. Antibiot. 30:576-582. 2. Jones, R. N., C. Thornberry, A. L Barry, P. C. Fuchs, T. L Gavan, and E. H. Gerlach. 1977. BL-S786, a new parenteral cephalosporin. II. In vitro antimicrobial activity comparison with six related cephalosporins. J. Antibiot. 30:583-592. 3. Leitner, F., M. Misiek, T. A. Pursiano, R. E. Buck, D. R. Chishohn, R. G. DeRegis, Y. H. Tsai, and K. E. Price. 1976. Laboratory evaluation of BL-S786, a cephalosporin with broad-spectrum antibacterial activity. Antimicrob. Agents Chemother. 10:426-435. 4. Neu, H. C. 1971. Beta-lactamase production by Pseudomonas aeruginosa, p. 534-538. Antimicrob. Agents Chemother. 1970. 5. Neu, H. C. 1974. Cefoxitin, a semisynthetic cephamycin antibiotic: antibacterial spectrum and resistance to hydrolysis by gram-negative beta-lactamases. Antimicrob. Agents Chemother. 6:170-176. 6. Neu, H. C. 1974. Cefamandole, a cephalosporin antibiotic with an unusually wide spectrum of activity. Antimicrob. Agents Chemother. 6:177-182. 7. O'Caliaghan, C. H., A. Morris, S. M. Kirby, and A. H. Shingler. 1972. Novel method for detection of ,-lactamases .by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1:283-288. 8. O'Callaghan, C. H., R. B. Sykes, A. Griffiths, and J. E. Thornton. 1976. Cefuroxime, a new cephalosporin antibiotic: activity in vitro. Antimicrob. Agents Chemother. 9:511-519. 9. Richmond, M. H., and R. B. Sykes. 1973. The f8-lactamases of gram-negative bacteria and their possible physiological role. Adv. Microb. Physiol. 9:31-88.
