Journal of Antimicrobial Chemotherapy (1978) 4 {Suppl. B), 15-32
Cefoxitin, a semi-synthetic cephamycin: a microbiological overview
J. Birnbaum, E. O. Stapley, A. K. Miller, H. Wallick, D. Hendlin and H. B. Woodruff
Cefoxitin is a chemically modified form of a member of a new family of P-lactam antibiotics, the cephamycins. Cefoxitin resembles the cephalosporins, but due to its 7amethoxyl group (a unique structural feature of the cephamycin family), it possesses important biochemical characteristics not found in other P-lactam antibiotics. The methoxyl group provides the molecule with a remarkable degree of resistance to all P-lactamases. This resistance provides the antibiotic with a broad antibacterial spectrum, including activity against indole-positive Proteus strains, Serratia spp. and Bacteroides spp. (including Bacteroides fragilis and all its subspecies) as well as against all the bacterial pathogens normally sensitive to the cephalosporins. In addition, cefoxitin is active against many strains of Gram-negative bacteria that have become refractory to other P-lactam antibiotics. Cefoxitin is a cell-wall-active agent and is bactericidal in its action. Marked resistance to enzymatic degradation, high bactericidal activity over a large range of inoculum levels, and favorable metabolic disposition makes cefoxitin a highly effective agent in vivo. Thus, cefoxitin is a major advance over other P-lactam antibiotics in current use. Introduction In 1972, workers at the Merck Sharp & Dohme Research Laboratories described the discovery of a new family of P-lactam antibiotics, the cephamycins (Stapley, Jackson, Hernandez, Zimmerman, Currie, Mochales, Mata, Woodruff & Hendlin, 1972; Miller, Goegelman, Weston, PutteT & Wolf, 1972). Although this new family is related to cephalosporin C, its members have two important structural differences from it (Figure 1): all are characterized by the presence of a methoxyl group, in addition to r>a aminoadipic acid, on the 7-carbon of the cephem nucleus; secondly, the cephamycins have various substitutions on the 3-carbon, in contrast to cephalosporin C, which has an acetoxy group at that locus. The cephamycins are produced by Streptomyces species (filamentous bacteria), whereas cephalosporin C is produced by Cephalosporium acremonium (fungus). In keeping with the precedents in the naming of antibiotic substances,* the term 'cephamycin' was coined for this new family of P-lactam antibiotics. This name has been well accepted in the scientific literature, as evidenced by the many articles that refer to •According to the piocedures in the U.S. Adopted Names Dictionary of Drug Names, Rockville, U.S. Pharmacopoeial Convention, Inc., 1976, pp. 346-349; and in International Non-Proprietary Names (INN) for Pharmaceutical Substances, World Health Organization, 1976, pp. 309-310. 15
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Merck Sharp A. Dohme Research Laboratories, Rahway, New Jersey, U.S.A.
16
J. Bimbaum et al. Cephamycm
Slreptomyces (filamentous boctenum) products
A
i
D - a — ominoodipote-HN
OCH 3
:
-OH CO,H
C*
-0-C-NH2
O - o —ominoodipote - H N 0 11
-CH2-O-C-CH3 CO2H Cepholospofin C
Figure 1. Structure of cephamycins A, B and C* in comparison to cephalosporin C. •Cephamycin C was described independently by Nagarajan et al. (1971).
this group of compounds (Hamilton-Miller, Kerry & Brumfitt, 1974; Jackson, Thomas & Alford, 1977; Neu, 1974; Norrby, Brorsson & Seeberg, 1976). Soon afteT the discovery of the new family, it became evident that cephamycin C had the best overall activity of the cephamycins (Miller, Celozzi, Pelak, Stapley & Hendlin, 1972a; Miller et al., 1972A). Its spectrum of antimicrobial activity is considerably broader than that of cephalosporin C, the agent is nontoxic even at very high doses, and it provides good efficacy in laboratory animal models. A particularly notable part of the expanded spectrum of cephamycin C is the activity against strains of Gram-negative bacteria that produce a p"-lactamase. When Daoust and associates (Daoust, Onishi, Wallick, Hendlin & Stapley, 1973) evaluated cephamycin C against many clinical isolates that contained a (J-lactamase, they found that the new antibiotic was distinctly more resistant to degradation than either cephaloridine or cephalothin. Daoust et al. (1973) hypothesized that it was its resistance to P-lactamase that expanded the Gram-negative spectrum of cephamycin C. Cefoxitin, a semi-synthetic cephamycin In spite of good Gram-negative activity, cephamycin C has one major drawback—it lacks activity against Gram-positive organisms (Miller et al., 1972a, 19726). Accordingly, a major programme in chemistry was undertaken to prepare semi-synthetic derivatives of cephamycin. It was soon found that several new chemical reactions had to be developed (Firestone, Schelechow, Johnston & Christensen, 1972; KaTady et al., 1972) to permit the preparation of chemical derivatives of the cephamycins. Cefoxitin sodium was one of the most promising semi-synthetic analogues of cephamycin.
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Cephalosponum (fungus Iproducf
Microbiological overview
17
The roles of the 7-(J-thienylaceryl, 7a-methoxyl, and 3-carbamoyl groups
In comparison with cephamycin C, cefoxitin retains the 7a-methoxyl group and the 3-carbamoyl function and incorporates a thienylacetyl moiety in place of the D-a aminoadipic acid of the natural product (Figure 2). The introduction of the thienylacetyl group overcomes the lack of Gram-positive activity that limits the utility of cephamycin C (Wallick & Hendlin, 1974).
II
-CH,-C
thienylacetyl
cephem nucltut
3-corbamoyl
Figure 2. Structure of cefoxitin. Cefoxitin sodium: Sodium 3-{hydroxymethyl)-7a-methoxy-8-oxo-7[2-(2-thienyl)acetamidol)-5-thia-l-azabicyclo-[4.2.0]oct-2-ene-2-carboxylate carbamate (ester).
The contributions of the thienylacetyl group to biological activity are shown clearly in Table I, in which the minimum inhibitory concentrations (MTCs) of cephamycin C and cefoxitin for several organisms are compared. The thienylacetyl group dramatically enhances activity against Gram-positive organisms and improves activity against many Gram-negative organisms, though less dramatically. Table I. Antibacterial spectra of cephamycin C and cefoxitin Strain
MICO*g/ml) Cephamycin C Cefoxitin
Gram-positive Staphylococcus aureus 2949 3147 Streptococcus pyogenes 3009 3176 Strep, pneumoniae 3377
>100 >100 >100 >100 >100
6-25 6-25 0-78 6-25 312
25 25
6-25 6-25 312 1-56 6-25 6-25 6-25 1-56
Gram-negative Escherichia coli 2017 3349 Klebsiella pneumoniae 3068 Proteus mirabilis 3343 3255 P. morganii 3376 3202 P. vulgaris 1810
12-5 312 312 25 25
1-56
Determined by agar dilution, using a multiple-inoculum replicating device to deposit 104 to 10* cells of each test organism on the surface of the antibiotic, which is contained in Mueller-Hinton agar petri plates.
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
o
18
J. Birnbaum tt al
The importance of the 7a-methoxyl group is evident from a comparison of cefoxitin with its 7-H counterpart, desmethoxycefoxitin (Table II). Removal of the 7-methoxyl group from cefoxitin improves the activity in vitro against Staphylococcus aureus, but it markedly decreases the activity against some GTam-negative bacteria, particularly two strains of Proteus morganii, an organism known to produce fMactamase. These findings, as well as those of Daoust et al. (1973), suggested that the 7ct-methoxyl group had the unique ability to protect the cephem nucleus from hydrolytic attack by P-lactamases. Table II. Comparison of the antibacterial activities of cefoxitin and its 7-H counterpart, desmethoxycefoxitin LJII alii
Cefoxitin
MIC Og/ml) Desmethoxycefoxitin
Gram-positive Staphylococcus aureus 2949 3147 Streptococcus pyogenes 3009 Strep, agalactiae 1934
6-25 6-25 0-78 0-78
019 0-39 019 019
6-25 6-25 12-5
12-5 >100 50 50 1-56 6-25 >100 >100 12-5
Gram-negative Escherichia coli 2017 3349 Klebsiella pneumoniae C17 3083 Proteus mirabilis 3343 3255 P. morganii 3376 3202 P. vulgar is 1810
312 1-56 6-25 6-25 6-25 1-56
Determined by agar dilution, using a multiple-inoculum replicating device to deposit 104 to 10* cells of each test organism on the surface of the antibiotic, which is contained in Mueller-Hinton agar petri plates.
Table III shows the contribution made by the 7a-methoxyl group to resistance to fHactamase action. With a methoxyl gTOup on the 7-carbon of the cephem nucleus, cefoxitin is totally resistant to the enzyme from Enterobacter cloacae. Substitution of a 7a-methoxyl group for the 7a-hydrogen on the cephalothin molecule improves its stability in the presence of enzyme too, but not as much as for cefoxitin, indicating that the 3carbamoyl group makes some minor contribution to resistance to P-lactamase action. Table in. Relationship of the 7-a-methoxyl group to p-lactamase resistance 7-a group
Antibiotic Cephalothin Cephalothin analog Cefoxitin Cefoxitin analog
-H -OCH, -OCH, -H
Per cent destruction by P-Iactamase from E. coli 2885
E. cloacae 2646
>99 16 0 >99
74 16 0 >99
Methods described by Onishi el al. (1974).
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Strain
Microbiological overview
19
In addition to enhancing Gram-positive activity, the thienylacetyl group contributes to the stability of the antibiotic in the presence of P-lactamase (Table IV). As shown by their rates of hydrolysis, cefoxitin is more resistant than its natural product precursor, cephamycin C, to a p-lactamase from an Enterobacter species, just as cephalothin is more resistant than its precursor, cephalosporin C. From the results of a large chemical effort mounted to exploit the finding that cephamycins are resistant to P-lactamase (Cama, Table IV. Enzyme kinetics of Enterobacter cloacae P-lactamase Rate of hydrolysis V B1 ,(xl0-«)» * m (xl0-«) (/tmol)
Cephamycin C Cefoxitin Cephalosporin C Cephalothin
3-3 0019 200 91
2-4 0-6 14-3 0-6
•|imol/min/mg protein. Methods are described in Onishi et al. (1974).
Leanza, Beattie & Christensen, 1972), it became apparent that any group larger than methyl at the 7-position was able to confer some level of stability in the presence of P-lactamase. Of all the chemical groups introduced, however, the naturally occurring methoxyl group was unique because it was the only group that served to prevent hydrolysis by P-lactamase while allowing the cephem nucleus to retain its full antimicrobial potency (Stapley el al., 1978). Though other groups provided stability of the antibiotic in the presence of the enzyme, they reduced the biological potency significantly. From this structure-activity correlation, it became apparent that stability to P-lactamase action is conferred by steric hindrance by the methoxyl group of the cephamycins at the active site of the enzyme. The cephalosporins, with only a hydrogen atom in the 7-position, provide no structural hindrance and are, therefore, susceptible to attack by the enzyme. The contribution of the 3-carbamoyl group to the activity of cefoxitin is best seen in the results of experimental infections in mice (Table V). TheTe is little difference among all 4 organisms employed between susceptibility in vitro to the acetoxy and carbamoyl analogues of cefoxitin. There are marked differences, however, in susceptibility in vivo; Table V. The importance of the 3-carbamoyl group to activity in vivo
Infecting organism Staphylococcus aureus 2949 Streptococcus pyogenes 3009 Proteus morganii 3376 Escherichia coli 2017
Activity in vivo and in vitro Cefoxitin analog Cefoxitin (3-acetoxy) (3-carbamoyl) EDM MIC EDM MIC
>20 10 >20 >20
6-25 1 56 12-5
0-25
25
0-53
006 015
6-25
312 6-25 6-25
E D " = Dose (SC x 2) in mg per animal that protects 50% of the mice. Procedures as described by Miller et al. (1974). MIC given in M8/rn.
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Antibiotic
20
J. Birnbaum et at.
Table VI. Effect of 3-carbamoyl group on urinary recovery of antibiotic in mice and monkeys , ... . AntiDiouc
Substituent in position 3
Cephalothin Cephalothin analog Cefoxitin Cefoxitin analog
Acetoxy Carbamoyl Carbamoyl Acetoxy
Urinary recovery (%) Mice* Monkeyf 32 64 72 11
52 ND 93 36
*Dosc was 20 mg/kg, recovery assayed 4 h later. tDosc was 10 mg/kg, recovery assayed 7 h later. Methods according to Miller et al. (1974). ND — not done.
Antibacterial spectrum of cefoxitin Wallick & Hendlin (1974) presented the first detailed report on the activity of cefoxitin in vitro. Table VII shows that, for Gram-negative bacteria generally susceptible to cephalothin (e.g., Escherichia ioli, Proteus mirabilis, Klebsiella), cefoxitin was significantly more active than cephalothin. Of greater interest is the susceptibility to cefoxitin of some isolates of indole-positive Proteus species and Providencia, 2 groups of organisms normally resistant to cephalothin (Griffith & Block, 1964; Wick & Preston, 1972). At least 90% of the 59 isolates comprising these groups had MIC values for cefoxitin of 12-5 ug/ml or less. Gram-positive micro-organisms, on the other hand, were less susceptible to cefoxitin than to cephalothin (Figure 3). More than 90% of the strains of Staphylococcus aureus weTe inhibited by cefoxitin at concentrations of 312 ug/ml or less, whereas cephalothin was effective against 100 % of the cultures at lower concentrations. The streptococci were more susceptible to cefoxitin than were the staphylococci (MICs < 0 - 6 3 ug/ml). The patterns of susceptibility of S. aureus and streptococci to cefoxitin are similar to those for cephalexin, with cephalexin being more active in the MIC determination by only a single dilution. Wallick & Hendlin (1974) showed also that the MICs for 5 pneumoccccal isolates were 312 ug/ml for cefoxitin and 9 0 % of their B. fragilis strains contain a P-lactamase.
Table XII. P-Lactam substrates for a cephalosporinase from Bacteroides fragilis
Substrates Penicillin Benzylpenicillin Cephalosporins Cephaloridine Cephalothin Cefazolin Cefuroxime Cefamandole Cephamycin Cefoxitin
Relative rates of hydrolysis Enzyme of Enzyme of strain 3272 strain 3389 1
7
100 66 75 28 24
100 43 279 182 32
0
0
Procedures as described by Darland & Bimbaum (1977).
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Cefoxitin
28
J. Bimbaum et al.
Interaction of cefoxitin with the bacterial cell
The behavior of cefoxitin is typical of a cell-wall-active p-lactam antibiotic. At subinhibitory levels of cefoxitin (less than the MIC), cell wall effects are noted, i.e., cells elongate during growth and eventually produce longfilaments(Zimmerman & Stapley, 1976). Filament formation also has been noted in the presence of most of the cephalosporins and penicillins. Zimmerman & Stapley (1976) observed a possible structure: activity relationship among the P-lactam antibiotics that inducefilamentformation when used at concentrations lower than the MIC. Antibiotics that contained a non-aromatic substituent in the 6- or 7-position did not induce filamentous growth, whereas most of the antibiotics tested that contained an aromatic substituent in either of those positions did. Because all the antibiotics tested are known to be effective therapeutically, it is not likely that filament formation has clinical significance. Under conditions of high osmolarity, spheroplasts are induced when bacteria are grown in the presence of cefoxitin at levels equal to or higher than the MIC (Onishi, Daoust, Zimmerman, Hendlin & Stapley, 1974; Chapman & Russell, 1976). The spheroplasts induced by cefoxitin are osmotically sensitive and can be readily lysed by dilution of the medium. This pattern of cefoxitin activity is typical of penicillin G, cephalothin, and other cell-wall-active bactericidal agents.
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Other anaerobes After the good antibacterial activity of cefoxitin had become apparent, several surveys were undertaken to measure the activity of the antibiotic against other anaerobic pathogens. Tally, Jacobus, Bartlett & Gorbach (1975) found that when an MIC of 16 ug/ml is used as a limit for sensitivity, a large majority of anaerobes is considered susceptible to cefoxitin. Among 43 strains of Gram-negative cocci, 42 were inhibited by cefoxitin at 16 ug/ml. All 10 strains of Eubacterium were sensitive. Strains of Clostridium perfringens were inhibited by cefoxitin at 4 ug/ml or less, and 81 % of other Clostridium species weTe inhibited at 16 ug/ml. Sutjer & Finegold (1975), in a similar study, found that, in addition to the excellent activity against Bdcteroides, Fusobacterium, Peptococcus, Peptostreptococcus, Clostridium, and Eubacterium, cefoxitin also had good activity against Veillonella, Bifidobacterium, and Propiortibacterium acnes. In all these studies, cefoxitin was equal in potency to the cephalosporins cephalothin, cefazolin, and cephaloridine. A marked difference between cefoxitin and the cephalosporins was seen only for B.fragilis and several other strains of Bacteroides. These organisms contain a potent cephalosporinase with a broad substrate range, and only cefoxitin appears able to resist hydrolysis by this enzyme (Darland & Birnbaum, 1977); only cefoxitin has good antibacterial activity against these organisms. The importance of the P-lactamase of B. fragilis in a mixed anaerobic infection was illustrated by Hackman & Wilkins (1975). B.fragilis protects the anaerobe Fusobacterium necrophorum from penicillin and cephalothin in vivo. F. necrophorum is susceptible to cephalothin in vitro, but is not susceptible in vivo when B. fragilis is present. Cefoxitin, which is active against both organisms and is resistant to the P-lactamase of B.fragilis, was able to protect mice against such a mixed infection. Thus, in addition to having excellent activity against aerobic bacteria, cefoxitin is the only antibiotic that has activity against all the important Gram-positive and Gram-negative anaerobic pathogens, both in vitro and in vivo.
Microbiological overview
29
Conclusions The extensive data obtained by many medical microbiology laboratories around the world show that cefoxitin provides a major advance over presently used (Mactam antibiotics. Cefoxitin has a broader spectrum of activity, one that includes the entire spectrum of the cephalosporins and, in addition, is active against Proteus morganii, P. vulgaris, P. rettgeri, Serratia spp., and Bacteroides spp., including B. fragilis. It is active against many strains of E. coli and Klebsiella that have become resistant to cephalosporins. The broader spectrum of cefoxitin, as compared with the spectra of the cephalosporins and most penicillins, can be accounted for by its almost complete resistance to P-lactamase. The presence of a P-lactamase with a broad range of substrates is responsible for the development of resistance in many organisms, particularly the Gram-negative aerobic and anaerobic bacilli. Cefoxitin is bactericidal, is unaffected by variations in inoculum size, and is metabolically stable. It is, therefore, a highly reliable antibiotic for use in vivo. The correlation between sensitivity of organisms to cefoxitin in vitro and activity of cefoxitin against these organisms in vivo has been established by Miller et al. (1977). Studies in man have also shown the reliability and the high frequency of clinical improvement that can be obtained with cefoxitin therapy (McCloskey, 1977).
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Onishi, Zimmerman & Stapley (1974) reported on the binding of cefoxitin to Gramnegative and Gram-positive bacteria. They found that between 4 and 9 pmol/mg of cell was bound by bacteria. Cephaloridine, cephalothin, and penicillin competed very poorly with cefoxitin for binding sites on Gram-negative cells, but were highly effective in competing with cefoxitin for binding sites on the Gram-positive bacterium, S. aureus. Spratt (1975, 1977) also studied the interaction of cefoxitin with the cell. Cefoxitin showed a narrow range between the concentration at which cell division was inhibited and that at which lysis took place. For cephaloridine, inhibition and lysis occurred at the same concentration. The implication of this finding is that cefoxitin and cephaloridine may be more bactericidal than cephalexin, ampicillin, or benzyl penicillin—the other antibiotics used in this study. Spratt (1975) showed that penicillin binds to 6 proteins in the cell membrane of E. coli. Inhibition of proteins no. 1 and no. 3 by penicillin is the event that is responsible for cell death and lysis. Cefoxitin binds to 5 of these 6 sites, including proteins no. 1 and no. 3. Protein no. 2 does not bind cefoxitin and is not involved in bactericidal action. Spratt (1977) showed that penicillin binds in a reversible manneT to the binding proteins, whereas the binding of cefoxitin to the major proteins is largely irreversible. The failure of penicillin to compete with cefoxitin in Gram-negative cells, as shown by Onishi, Zimmerman & Stapley (1974), is probably due to the reversible nature of penicillin protein binding and the non-reversibility of the binding of cefoxitin to the major proteins. The irreversible nature of cefoxitin protein binding may have important implications for the effectiveness of the antibiotic, and may account for its strong bactericidal action. For example, Goodwin & Hill (1977) showed that cefoxitin produced lysis of heavy suspensions of all strains of E. coli and Klebsiella that they tested, and suppressed regrowth at a low multiple of its MIC. Cephalothin and cefuroxime also caused some lysis, but several of the organisms tested showed regrowth, even at high multiples of the MICs. In addition to its strong lytic effects, the good binding of cefoxitin to cell wall enzymes and its resistance to P-lactamase may account for the favorable MIC/MBC ratios and the minimal effects of variations in inoculum size.
30
J. Bimbaum et al.
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
References Adams, H. G., Stilwcll, G. A. & Turck, M. In vitro evaluation of cefoxitin and cefamandole. Antimicrobial Agents and Chemotherapy 9: 1019-24 (1976). Anderson, J. D. & Sykes, R. B. Characterization of a P-lactamase obtained from a strain of Bacteroides fragilis resistant to |J-lactam antibiotics. Journal of Medical Microbiology 6: 201-6 (1973). Bach, V. T., Roy, I. & Thadepalli, H. Susceptibility of anaerobic bacteria to cefoxitin and related compounds. Antimicrobial Agents and Chemotherapy 11: 912-3 (1977). Bauer, W. W., Kirby, W. M. M., Sherris, J. C. & Turck, M. Antibiotic susceptibility testing by a standardized single-disc method. American Journal of Clinical Pathology 45: 493-6 (1966). Brumfitt, W., Kosmidis, J., Hamilton-Milltr, J. M. T. & Gilchrist, J. N. Cefoxitin and cephalothin: antimicrobial activity, human pharmacokinetics and toxicology. Antimicrobial Agents and Chemotherapy 6: 290-9 (1974). Buhs, R. P., Maxim, T. E., Allen, N., Jacob, T. A. & Wolf, F. J. Analysis of cefoxitin, cephalothin and their deacylated metabolites in human'urine by high performance liquid chromatography. Journal of Chromatography 99: 609-18 (1974). Cama, L. D., Leanza, W. J., Beattie, T. R. & Christensen, B. G. Substituted penicillin and cephalosporin derivatives. I. Stereo-specific introduction of the C-6 (7) methoxy group. Journal of the American Chemical Society 94: 1408-10 (1972). Chapman, D. G. & Russell, A. D. Effect of cefoxitin on Proteus species. Microbios Letters 2: 51-3 (1976). Daoust, D. R., Onishi, H. R., Wallick, H., Hendlin, D. & Stapley, E. O. Cephamycins, a new family of P-lactam antibiotics: antibacterial activity and resistance to P-lactamasc degradation. Antimicrobial Agents and Chemotherapy 3: 254—61 (1973). Darland, G. & Birnbaum, J. Cefoxitin resistance to P-lactamase: a major factor for susceptibility of Bacteroides to the antibiotic. Antimicrobial Agents and Chemotherapy 11: 725-34 (1977). Del Bene, V. E. & Farrar, W. E. Cephalosporinase activity in Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 3: 369-72 (1973). Eickhoff, T. C. & Ehret, J. M. In vitro comparison of cefoxitin, cefamandole, cephalexin and cephalothin. Antimicrobial Agents and Chemotherapy 9: 994-9 (1976). Ernst, E. C , Berger, S., Barza, M., Jacobus, N. V. & Tally, F. P. Activity of cefamandole and other cephalosporins against aerobic and anaerobic bacteria. Antimicrobial Agents and Chemotherapy 9: 852-5 (1976). Farrar, W. E. & Gramling, P. K. Antistaphylococcal activity and P-lactamase resistance of newer cephalosporins. Journal of Infectious Diseases 133: 691-5 (1976). Firestone, R. A., Schelechow, N., Johnston, D. B. R. & Christensen, B. G. Substituted penicillins and cephalosporins. II. C-6(7>alkyl derivatives. Tetrahedron Letters: 375-8 (1972). Fong, I. W., Engelking, E. R. & Kirby, W. M. M. Relative inactivation by Staphylococcus aureus of eight cephalosporin antibiotics. Antimicrobial Agents and Chemotherapy 9: 93SM4(1976). Goldner, M., Glass, D. G. & Fleming, P. C. Spontaneous mutant with loss of P-lactamase in Aerobacter cloacae. Journal of Bacteriology: 961-3 (1969). Goodwin, C. S. & Hill, J. P. Lysis of Enterobacteria by cefoxitin, cefuroxime, and cephalothin. Antimicrobial Agents and Chemotherapy 11: 26-30 (1977). Griffith, R. S. & Block, H. R. Cephalothin, a new antibiotic. Journal of the American Medical Association 189: 823-8 (1964). Hackman, A. S. & Wilkins, T. D. Comparison of cefoxitin and cephalothin therapy of a mixed Bacteroides fragilis and Fusobacterium necrophorum infection in mice. Antimicrobial Agents and Chemotherapy 8: 224-5 (1975). Hamilton-Miller, J. M. T., Kerry, D. W. & Brumfitt, W. An in vitro comparison of cefoxitin, a semisynthetic cephamycin, with cephalothin. Journal of Antibiotics, 27: 42-8 (1974). Jackson, R. T., Thomas, F. E. & Alford, R. H. Cefoxitin activity against multiply antibioticresistant Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 11: 84-7 (1977). Jones, R. N., Thornsberry, C, Barry, A. L., Fuchs, P. C , Gavan, T. L. & Gerlach, E. H. BL-S786, a new parenteral cephalosporin. II. In vitro antimicrobial activity comparison with six related cephalosporins. Journal of Antibiotics 30: 583-92 (1977).
Microbiological overview
31
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Kammer, R. B., Preston, D. A., Turner, J. R. & Hawley, L. C. Rapid detection of ampicillinresistant Haemophilus influenzae and their susceptibility to sixteen antibiotics. Antimicrobial Agents and Chemotherapy 8: 91-4 (1975). Karady, S., Pines, S. H., Weinstock, L. M., Roberts, F. E., Brenner, G. S., Hoinowski, A. M., Cheng, T. Y. & Sletzinger, M. Semisynthetic cephalosporins via a novel acyl exchange reaction. Journal of the American Chemical Society 94: 1410-11 (1972). Kirby, W. M. M., de Maine, J. B. & Serrill, W. S. Pharmacokinetics of the cephalosporins in healthy volunteers and uremic patients. Postgraduate Medical Journal 47 (Feb. Suppl.): 41-6(1971). Kosmidis, J., Hamilton-Miller, J. M. T., Gilchrist, J. N. G., Kerry, D. W. & Brumfitt, W. Cefoxitin, a new semi-synthetic cephamycin: an in vitro and in vivo comparison with cephalothin. British Medical Journal iv: 653-5 (1973). Lewis, R. P., Meyer, R. D. & Kraus, L. L. Antibacterial activity of selected p-lactam and aminoglycoside antibiotics against cephalothin-resistant Enterobacteriaceae. Antimicrobial Agents and Chemotherapy 9: 780-6 (1976). McCloskey, R. V. Results of a clinical trial of cefoxitin, a new cephamycin antibiotic. Antimicrobial Agents and Chemotherapy 12: 636-41 (1977). Miller, A. K., Celozzi, E., Pelak, B. A., Stapley, E. O. & Hendlin, D. Cephamycins, a new family of P-lactam antibiotics. III. In vitro studies. Antimicrobial Agents and Chemotherapy 2: 281-6 (1972a). Miller, A. K., Celozzi, E., Kong, Y., Pelak, B. A., Kropp, H., Stapley, E. O. & Hendlin, D. Cephamycins, a new family of P-lactam antibiotics. IV. In vivo studies. Antimicrobial Agents and Chemotherapy 2: 287-90 (1972A). Miller, A. K., Celozzi, E., Kong, Y., Pelak, B. A., Hendlin, D. & Stapley, E. O. Cefoxitin, a semisynthetic cephamycin antibiotic: In vivo evaluation. Antimicrobial Agents and Chemotherapy 5: 33-7 (1974). Miller, A. K., Celozzi, E., Pelak, B. A., Birnbaum, J. & Stapley, E. O. Comparison of in vitro and in vivo activities of cefoxitin, cefamandole, cephalothin and cefazolin. Proceedings, 10th International Congress of Chemotherapy, Zurich, Sept. 18-23, 1977 (in press). Miller, T. W., Goegelman, R. T., Weston, R. G., Putter, I. & Wolf, F. J. Cephamycins, a new family of P-lactam antibiotics. II. Isolation and chemical characterization. Antimicrobial Agents and Chemotherapy 2: 132-5 (1972). Moellering, R. C , Dray, M. & Kunz, L. J. Susceptibility of clinical isolates of bacteria to cefoxitin and cephalothin. Antimicrobial Agents and Chemotherapy 6: 320-4 (1974). Nagarajan, R., Boeck, L. D., Gorman, M., Hamill, R. L., Higgins, C. E., Hoehn, M. M., Stark, W. M. & Whitney, J. G. P-Lactam antibiotics from Streptomyces. Journal of the American Chemical Society 93: 2308-10 (1971). Neu, H. C. Cefoxitin, a semisynthetic cephamycin antibiotic: Antibacterial spectrum and resistance to hydrolysis by Gram-negative fJ-lactamases. Antimicrobial Agents and Chemotherapy 6: 170-6 (1974). Norrby, R., Brorsson, J. E. & Seeberg, S. Comparative study of the in vitro antibacterial activity of cefoxitin, cefuroxime and cephaloridine. Antimicrobial Agents and Chemotherapy 9: 506-10 (1976). O'Callaghan, C. H., Sykes, R. B., Griffiths, A. & Thornton, J. E. Cefuroxime, a new cephalosporin antibiotic: Activity in vitro. Antimicrobial Agents and Chemotherapy 9: 511-19 (1976). Olsson, B., Dornbusch, K. & Nord, C. E. Susceptibility to beta-lactam antibiotics and production of fi-lactamases in Bacteroides fragilis. Medical Microbiology and Immunology 163: 183-94 (1977). Olsson, B., Nord, C. E. & Wadstrom, T. Formation of beta-lactamase in Bacteroides fragilis: cell-bound and extracellular activity. Antimicrobial Agents and Chemotherapy 9: 727-35 (1976). Onishi, H. R., Daoust, D. R., Zimmerman, S. B., Hendlin, D. & Stapley, E. O. Cefoxitin, a semisynthetic cephamycin antibiotic: resistance to 3-lactamase inactivation. Antimicrobial Agents and Chemotherapy 5: 38-48 (1974). Onishi, H. R., Zimmerman, S. B. & Stapley, E. O. Observations on the mode of action of cefoxitin. Annals of the New York Academy of Science 235: 406-25 (1974).
32
J. Birnbaum tt al.
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Phillips, I., King, A., Warren, C , Watts, B. & Stoatc, M. W. The activity of penicillin and eight cephalosporins on Neisseria gonorrhoeae. Journal of Antimicrobial Chemotherapy 2: 31-9 (1976). Schrogie, J. J., Davies, R. O., Kwan, K. C , Rogers, D., Holmes, G. I., Skeggs, H. & Martin, C. M. Bioavailability and pharmacokinetics of cefoxitin. Journal of Antimicrobial Chemotherapy 4 (Suppl. B): 69-78 (1978). Shah, P. M., Zwischenbrugger, H. & Stille, W. In vitro activity of cefoxitin, a new cephalosporin. Munchener Medizinische Wochenschrift 118: 1469-72 (1976). Sonneville, P. F.F Kartodirdjo, R. R., Skeggs, H., Till, A. E. & Martin, C. M. Comparative clinical pharmacology of intravenous cefoxitin and ccphalothin. European Journal of Clinical Pharmacology 9: 397-403 (1976). Spratt, B. G. Distinct penicillin-binding proteins involved in the division, elongation, and shape of Escherichia coli K12. Proceedings, National Academy of Science, U.S.A. 92: 2999-3003 (1975). Spratt, B. G. Properties of the penicillin-binding proteins of Escherichia coli K12. European Journal of Biochemistry 72: 341-52 (1977). Stapley, E. O., Jackson, M., Hernandez, S., Zimmerman, S. B., Carrie, S. A., Mochales, S. Mata, J. M., Woodruff, H. B. & Hendlin, D. Cephamycins, a new family of P-lactam antibiotics. I. Production by actinomycetes, including Streptomyces lactamdurans sp. n. Antimicrobial Agents and Chemotherapy 2: 122-31 (1972). Stapley, E. O., Daoust, D. R., Hendlin, D., Miller, A. K., Zimmerman, S. B., Birnbaum, J., Cama, L. D. & Christensen, B. G. Cefoxitin: mechanism of activity against cephalosporinresistant bacteria. Proceedings, Symposium on Microbial Resistance, Tokyo, Oct. 1977. In the press (1978). Stewart, D. & Bodey, G. P. Comparative in vitro activity of cephalosporins. Journal of Antibiotics 29: 181-6(1976). Sutter, V. L. & Finegold, S. M. Susceptibility of anaerobic bacteria to carbenicillin, cefoxitin and related drugs. Journal of Infectious Diseases 131: 147-22 (1975). Tally, F. P., Jacobus, N. V., Bartlett, J. G. & Gorbach, S. L. Susceptibility of anaerobes to cefoxitin and other cephalosporins. Antimicrobial Agents and Chemotherapy 7: 128-32 (1975). Verbist, L. Comparison of the antibacterial activity of nine cephalosporins against Enterobacteriaceae and nonfermentative Gram-negative bacilli. Antimicrobial Agents and Chemotherapy 10: 657-63 (1976). Wallick, H. & Hendlin, D. Cefoxitin, a semisynthetic cephamycin antibiotic: susceptibility studies. Antimicrobial Agents and Chemotherapy 5: 25-32 (1974). Washington, J. A. The in vitro spectrum of cephalosporins. Mayo Clinic Proceedings 51: 237-50 (1976). Wick, W. E. In vitro and in vivo laboratory comparison of cephalothin and desacetylcephalothin. Antimicrobial Agents and Chemotherapy—1964: 870-5 (1965). Wick, W. E. & Preston, D. A. Biological properties of three 3-heterocyclicthiomethyl cephalosporin antibiotics. Antimicrobial Agents and Chemotherapy 1: 221-34 (1972). Yourassowsky, E. & Schoutens, E. In vitro bacteriostatic and bactericidal activities of seven cephalosporin antibiotics on Haemophilus influenzae. International Journal of Clinical Pharmacology 12: 433-6 (1975). Yourassowsky, E., Schoutens, E. & Vanderlinen, M. P. Antibacterial activity of eight cephalosporins against Haemophilus influenzae and Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 2: 55-9 (1976). Zimmerman, S. B. & Stapley, E. O. Relative morphological effects induced by cefoxitin and other P-lactam antibiotics in vitro. Antimicrobial Agents and Chemotherapy 9: 318-26(1976).
Cefoxitin, a semi-synthetic cephamycin: a microbiological overview
J. Birnbaum, E. O. Stapley, A. K. Miller, H. Wallick, D. Hendlin and H. B. Woodruff
Cefoxitin is a chemically modified form of a member of a new family of P-lactam antibiotics, the cephamycins. Cefoxitin resembles the cephalosporins, but due to its 7amethoxyl group (a unique structural feature of the cephamycin family), it possesses important biochemical characteristics not found in other P-lactam antibiotics. The methoxyl group provides the molecule with a remarkable degree of resistance to all P-lactamases. This resistance provides the antibiotic with a broad antibacterial spectrum, including activity against indole-positive Proteus strains, Serratia spp. and Bacteroides spp. (including Bacteroides fragilis and all its subspecies) as well as against all the bacterial pathogens normally sensitive to the cephalosporins. In addition, cefoxitin is active against many strains of Gram-negative bacteria that have become refractory to other P-lactam antibiotics. Cefoxitin is a cell-wall-active agent and is bactericidal in its action. Marked resistance to enzymatic degradation, high bactericidal activity over a large range of inoculum levels, and favorable metabolic disposition makes cefoxitin a highly effective agent in vivo. Thus, cefoxitin is a major advance over other P-lactam antibiotics in current use. Introduction In 1972, workers at the Merck Sharp & Dohme Research Laboratories described the discovery of a new family of P-lactam antibiotics, the cephamycins (Stapley, Jackson, Hernandez, Zimmerman, Currie, Mochales, Mata, Woodruff & Hendlin, 1972; Miller, Goegelman, Weston, PutteT & Wolf, 1972). Although this new family is related to cephalosporin C, its members have two important structural differences from it (Figure 1): all are characterized by the presence of a methoxyl group, in addition to r>a aminoadipic acid, on the 7-carbon of the cephem nucleus; secondly, the cephamycins have various substitutions on the 3-carbon, in contrast to cephalosporin C, which has an acetoxy group at that locus. The cephamycins are produced by Streptomyces species (filamentous bacteria), whereas cephalosporin C is produced by Cephalosporium acremonium (fungus). In keeping with the precedents in the naming of antibiotic substances,* the term 'cephamycin' was coined for this new family of P-lactam antibiotics. This name has been well accepted in the scientific literature, as evidenced by the many articles that refer to •According to the piocedures in the U.S. Adopted Names Dictionary of Drug Names, Rockville, U.S. Pharmacopoeial Convention, Inc., 1976, pp. 346-349; and in International Non-Proprietary Names (INN) for Pharmaceutical Substances, World Health Organization, 1976, pp. 309-310. 15
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Merck Sharp A. Dohme Research Laboratories, Rahway, New Jersey, U.S.A.
16
J. Bimbaum et al. Cephamycm
Slreptomyces (filamentous boctenum) products
A
i
D - a — ominoodipote-HN
OCH 3
:
-OH CO,H
C*
-0-C-NH2
O - o —ominoodipote - H N 0 11
-CH2-O-C-CH3 CO2H Cepholospofin C
Figure 1. Structure of cephamycins A, B and C* in comparison to cephalosporin C. •Cephamycin C was described independently by Nagarajan et al. (1971).
this group of compounds (Hamilton-Miller, Kerry & Brumfitt, 1974; Jackson, Thomas & Alford, 1977; Neu, 1974; Norrby, Brorsson & Seeberg, 1976). Soon afteT the discovery of the new family, it became evident that cephamycin C had the best overall activity of the cephamycins (Miller, Celozzi, Pelak, Stapley & Hendlin, 1972a; Miller et al., 1972A). Its spectrum of antimicrobial activity is considerably broader than that of cephalosporin C, the agent is nontoxic even at very high doses, and it provides good efficacy in laboratory animal models. A particularly notable part of the expanded spectrum of cephamycin C is the activity against strains of Gram-negative bacteria that produce a p"-lactamase. When Daoust and associates (Daoust, Onishi, Wallick, Hendlin & Stapley, 1973) evaluated cephamycin C against many clinical isolates that contained a (J-lactamase, they found that the new antibiotic was distinctly more resistant to degradation than either cephaloridine or cephalothin. Daoust et al. (1973) hypothesized that it was its resistance to P-lactamase that expanded the Gram-negative spectrum of cephamycin C. Cefoxitin, a semi-synthetic cephamycin In spite of good Gram-negative activity, cephamycin C has one major drawback—it lacks activity against Gram-positive organisms (Miller et al., 1972a, 19726). Accordingly, a major programme in chemistry was undertaken to prepare semi-synthetic derivatives of cephamycin. It was soon found that several new chemical reactions had to be developed (Firestone, Schelechow, Johnston & Christensen, 1972; KaTady et al., 1972) to permit the preparation of chemical derivatives of the cephamycins. Cefoxitin sodium was one of the most promising semi-synthetic analogues of cephamycin.
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Cephalosponum (fungus Iproducf
Microbiological overview
17
The roles of the 7-(J-thienylaceryl, 7a-methoxyl, and 3-carbamoyl groups
In comparison with cephamycin C, cefoxitin retains the 7a-methoxyl group and the 3-carbamoyl function and incorporates a thienylacetyl moiety in place of the D-a aminoadipic acid of the natural product (Figure 2). The introduction of the thienylacetyl group overcomes the lack of Gram-positive activity that limits the utility of cephamycin C (Wallick & Hendlin, 1974).
II
-CH,-C
thienylacetyl
cephem nucltut
3-corbamoyl
Figure 2. Structure of cefoxitin. Cefoxitin sodium: Sodium 3-{hydroxymethyl)-7a-methoxy-8-oxo-7[2-(2-thienyl)acetamidol)-5-thia-l-azabicyclo-[4.2.0]oct-2-ene-2-carboxylate carbamate (ester).
The contributions of the thienylacetyl group to biological activity are shown clearly in Table I, in which the minimum inhibitory concentrations (MTCs) of cephamycin C and cefoxitin for several organisms are compared. The thienylacetyl group dramatically enhances activity against Gram-positive organisms and improves activity against many Gram-negative organisms, though less dramatically. Table I. Antibacterial spectra of cephamycin C and cefoxitin Strain
MICO*g/ml) Cephamycin C Cefoxitin
Gram-positive Staphylococcus aureus 2949 3147 Streptococcus pyogenes 3009 3176 Strep, pneumoniae 3377
>100 >100 >100 >100 >100
6-25 6-25 0-78 6-25 312
25 25
6-25 6-25 312 1-56 6-25 6-25 6-25 1-56
Gram-negative Escherichia coli 2017 3349 Klebsiella pneumoniae 3068 Proteus mirabilis 3343 3255 P. morganii 3376 3202 P. vulgaris 1810
12-5 312 312 25 25
1-56
Determined by agar dilution, using a multiple-inoculum replicating device to deposit 104 to 10* cells of each test organism on the surface of the antibiotic, which is contained in Mueller-Hinton agar petri plates.
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
o
18
J. Birnbaum tt al
The importance of the 7a-methoxyl group is evident from a comparison of cefoxitin with its 7-H counterpart, desmethoxycefoxitin (Table II). Removal of the 7-methoxyl group from cefoxitin improves the activity in vitro against Staphylococcus aureus, but it markedly decreases the activity against some GTam-negative bacteria, particularly two strains of Proteus morganii, an organism known to produce fMactamase. These findings, as well as those of Daoust et al. (1973), suggested that the 7ct-methoxyl group had the unique ability to protect the cephem nucleus from hydrolytic attack by P-lactamases. Table II. Comparison of the antibacterial activities of cefoxitin and its 7-H counterpart, desmethoxycefoxitin LJII alii
Cefoxitin
MIC Og/ml) Desmethoxycefoxitin
Gram-positive Staphylococcus aureus 2949 3147 Streptococcus pyogenes 3009 Strep, agalactiae 1934
6-25 6-25 0-78 0-78
019 0-39 019 019
6-25 6-25 12-5
12-5 >100 50 50 1-56 6-25 >100 >100 12-5
Gram-negative Escherichia coli 2017 3349 Klebsiella pneumoniae C17 3083 Proteus mirabilis 3343 3255 P. morganii 3376 3202 P. vulgar is 1810
312 1-56 6-25 6-25 6-25 1-56
Determined by agar dilution, using a multiple-inoculum replicating device to deposit 104 to 10* cells of each test organism on the surface of the antibiotic, which is contained in Mueller-Hinton agar petri plates.
Table III shows the contribution made by the 7a-methoxyl group to resistance to fHactamase action. With a methoxyl gTOup on the 7-carbon of the cephem nucleus, cefoxitin is totally resistant to the enzyme from Enterobacter cloacae. Substitution of a 7a-methoxyl group for the 7a-hydrogen on the cephalothin molecule improves its stability in the presence of enzyme too, but not as much as for cefoxitin, indicating that the 3carbamoyl group makes some minor contribution to resistance to P-lactamase action. Table in. Relationship of the 7-a-methoxyl group to p-lactamase resistance 7-a group
Antibiotic Cephalothin Cephalothin analog Cefoxitin Cefoxitin analog
-H -OCH, -OCH, -H
Per cent destruction by P-Iactamase from E. coli 2885
E. cloacae 2646
>99 16 0 >99
74 16 0 >99
Methods described by Onishi el al. (1974).
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Strain
Microbiological overview
19
In addition to enhancing Gram-positive activity, the thienylacetyl group contributes to the stability of the antibiotic in the presence of P-lactamase (Table IV). As shown by their rates of hydrolysis, cefoxitin is more resistant than its natural product precursor, cephamycin C, to a p-lactamase from an Enterobacter species, just as cephalothin is more resistant than its precursor, cephalosporin C. From the results of a large chemical effort mounted to exploit the finding that cephamycins are resistant to P-lactamase (Cama, Table IV. Enzyme kinetics of Enterobacter cloacae P-lactamase Rate of hydrolysis V B1 ,(xl0-«)» * m (xl0-«) (/tmol)
Cephamycin C Cefoxitin Cephalosporin C Cephalothin
3-3 0019 200 91
2-4 0-6 14-3 0-6
•|imol/min/mg protein. Methods are described in Onishi et al. (1974).
Leanza, Beattie & Christensen, 1972), it became apparent that any group larger than methyl at the 7-position was able to confer some level of stability in the presence of P-lactamase. Of all the chemical groups introduced, however, the naturally occurring methoxyl group was unique because it was the only group that served to prevent hydrolysis by P-lactamase while allowing the cephem nucleus to retain its full antimicrobial potency (Stapley el al., 1978). Though other groups provided stability of the antibiotic in the presence of the enzyme, they reduced the biological potency significantly. From this structure-activity correlation, it became apparent that stability to P-lactamase action is conferred by steric hindrance by the methoxyl group of the cephamycins at the active site of the enzyme. The cephalosporins, with only a hydrogen atom in the 7-position, provide no structural hindrance and are, therefore, susceptible to attack by the enzyme. The contribution of the 3-carbamoyl group to the activity of cefoxitin is best seen in the results of experimental infections in mice (Table V). TheTe is little difference among all 4 organisms employed between susceptibility in vitro to the acetoxy and carbamoyl analogues of cefoxitin. There are marked differences, however, in susceptibility in vivo; Table V. The importance of the 3-carbamoyl group to activity in vivo
Infecting organism Staphylococcus aureus 2949 Streptococcus pyogenes 3009 Proteus morganii 3376 Escherichia coli 2017
Activity in vivo and in vitro Cefoxitin analog Cefoxitin (3-acetoxy) (3-carbamoyl) EDM MIC EDM MIC
>20 10 >20 >20
6-25 1 56 12-5
0-25
25
0-53
006 015
6-25
312 6-25 6-25
E D " = Dose (SC x 2) in mg per animal that protects 50% of the mice. Procedures as described by Miller et al. (1974). MIC given in M8/rn.
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Antibiotic
20
J. Birnbaum et at.
Table VI. Effect of 3-carbamoyl group on urinary recovery of antibiotic in mice and monkeys , ... . AntiDiouc
Substituent in position 3
Cephalothin Cephalothin analog Cefoxitin Cefoxitin analog
Acetoxy Carbamoyl Carbamoyl Acetoxy
Urinary recovery (%) Mice* Monkeyf 32 64 72 11
52 ND 93 36
*Dosc was 20 mg/kg, recovery assayed 4 h later. tDosc was 10 mg/kg, recovery assayed 7 h later. Methods according to Miller et al. (1974). ND — not done.
Antibacterial spectrum of cefoxitin Wallick & Hendlin (1974) presented the first detailed report on the activity of cefoxitin in vitro. Table VII shows that, for Gram-negative bacteria generally susceptible to cephalothin (e.g., Escherichia ioli, Proteus mirabilis, Klebsiella), cefoxitin was significantly more active than cephalothin. Of greater interest is the susceptibility to cefoxitin of some isolates of indole-positive Proteus species and Providencia, 2 groups of organisms normally resistant to cephalothin (Griffith & Block, 1964; Wick & Preston, 1972). At least 90% of the 59 isolates comprising these groups had MIC values for cefoxitin of 12-5 ug/ml or less. Gram-positive micro-organisms, on the other hand, were less susceptible to cefoxitin than to cephalothin (Figure 3). More than 90% of the strains of Staphylococcus aureus weTe inhibited by cefoxitin at concentrations of 312 ug/ml or less, whereas cephalothin was effective against 100 % of the cultures at lower concentrations. The streptococci were more susceptible to cefoxitin than were the staphylococci (MICs < 0 - 6 3 ug/ml). The patterns of susceptibility of S. aureus and streptococci to cefoxitin are similar to those for cephalexin, with cephalexin being more active in the MIC determination by only a single dilution. Wallick & Hendlin (1974) showed also that the MICs for 5 pneumoccccal isolates were 312 ug/ml for cefoxitin and 9 0 % of their B. fragilis strains contain a P-lactamase.
Table XII. P-Lactam substrates for a cephalosporinase from Bacteroides fragilis
Substrates Penicillin Benzylpenicillin Cephalosporins Cephaloridine Cephalothin Cefazolin Cefuroxime Cefamandole Cephamycin Cefoxitin
Relative rates of hydrolysis Enzyme of Enzyme of strain 3272 strain 3389 1
7
100 66 75 28 24
100 43 279 182 32
0
0
Procedures as described by Darland & Bimbaum (1977).
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Cefoxitin
28
J. Bimbaum et al.
Interaction of cefoxitin with the bacterial cell
The behavior of cefoxitin is typical of a cell-wall-active p-lactam antibiotic. At subinhibitory levels of cefoxitin (less than the MIC), cell wall effects are noted, i.e., cells elongate during growth and eventually produce longfilaments(Zimmerman & Stapley, 1976). Filament formation also has been noted in the presence of most of the cephalosporins and penicillins. Zimmerman & Stapley (1976) observed a possible structure: activity relationship among the P-lactam antibiotics that inducefilamentformation when used at concentrations lower than the MIC. Antibiotics that contained a non-aromatic substituent in the 6- or 7-position did not induce filamentous growth, whereas most of the antibiotics tested that contained an aromatic substituent in either of those positions did. Because all the antibiotics tested are known to be effective therapeutically, it is not likely that filament formation has clinical significance. Under conditions of high osmolarity, spheroplasts are induced when bacteria are grown in the presence of cefoxitin at levels equal to or higher than the MIC (Onishi, Daoust, Zimmerman, Hendlin & Stapley, 1974; Chapman & Russell, 1976). The spheroplasts induced by cefoxitin are osmotically sensitive and can be readily lysed by dilution of the medium. This pattern of cefoxitin activity is typical of penicillin G, cephalothin, and other cell-wall-active bactericidal agents.
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Other anaerobes After the good antibacterial activity of cefoxitin had become apparent, several surveys were undertaken to measure the activity of the antibiotic against other anaerobic pathogens. Tally, Jacobus, Bartlett & Gorbach (1975) found that when an MIC of 16 ug/ml is used as a limit for sensitivity, a large majority of anaerobes is considered susceptible to cefoxitin. Among 43 strains of Gram-negative cocci, 42 were inhibited by cefoxitin at 16 ug/ml. All 10 strains of Eubacterium were sensitive. Strains of Clostridium perfringens were inhibited by cefoxitin at 4 ug/ml or less, and 81 % of other Clostridium species weTe inhibited at 16 ug/ml. Sutjer & Finegold (1975), in a similar study, found that, in addition to the excellent activity against Bdcteroides, Fusobacterium, Peptococcus, Peptostreptococcus, Clostridium, and Eubacterium, cefoxitin also had good activity against Veillonella, Bifidobacterium, and Propiortibacterium acnes. In all these studies, cefoxitin was equal in potency to the cephalosporins cephalothin, cefazolin, and cephaloridine. A marked difference between cefoxitin and the cephalosporins was seen only for B.fragilis and several other strains of Bacteroides. These organisms contain a potent cephalosporinase with a broad substrate range, and only cefoxitin appears able to resist hydrolysis by this enzyme (Darland & Birnbaum, 1977); only cefoxitin has good antibacterial activity against these organisms. The importance of the P-lactamase of B. fragilis in a mixed anaerobic infection was illustrated by Hackman & Wilkins (1975). B.fragilis protects the anaerobe Fusobacterium necrophorum from penicillin and cephalothin in vivo. F. necrophorum is susceptible to cephalothin in vitro, but is not susceptible in vivo when B. fragilis is present. Cefoxitin, which is active against both organisms and is resistant to the P-lactamase of B.fragilis, was able to protect mice against such a mixed infection. Thus, in addition to having excellent activity against aerobic bacteria, cefoxitin is the only antibiotic that has activity against all the important Gram-positive and Gram-negative anaerobic pathogens, both in vitro and in vivo.
Microbiological overview
29
Conclusions The extensive data obtained by many medical microbiology laboratories around the world show that cefoxitin provides a major advance over presently used (Mactam antibiotics. Cefoxitin has a broader spectrum of activity, one that includes the entire spectrum of the cephalosporins and, in addition, is active against Proteus morganii, P. vulgaris, P. rettgeri, Serratia spp., and Bacteroides spp., including B. fragilis. It is active against many strains of E. coli and Klebsiella that have become resistant to cephalosporins. The broader spectrum of cefoxitin, as compared with the spectra of the cephalosporins and most penicillins, can be accounted for by its almost complete resistance to P-lactamase. The presence of a P-lactamase with a broad range of substrates is responsible for the development of resistance in many organisms, particularly the Gram-negative aerobic and anaerobic bacilli. Cefoxitin is bactericidal, is unaffected by variations in inoculum size, and is metabolically stable. It is, therefore, a highly reliable antibiotic for use in vivo. The correlation between sensitivity of organisms to cefoxitin in vitro and activity of cefoxitin against these organisms in vivo has been established by Miller et al. (1977). Studies in man have also shown the reliability and the high frequency of clinical improvement that can be obtained with cefoxitin therapy (McCloskey, 1977).
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Onishi, Zimmerman & Stapley (1974) reported on the binding of cefoxitin to Gramnegative and Gram-positive bacteria. They found that between 4 and 9 pmol/mg of cell was bound by bacteria. Cephaloridine, cephalothin, and penicillin competed very poorly with cefoxitin for binding sites on Gram-negative cells, but were highly effective in competing with cefoxitin for binding sites on the Gram-positive bacterium, S. aureus. Spratt (1975, 1977) also studied the interaction of cefoxitin with the cell. Cefoxitin showed a narrow range between the concentration at which cell division was inhibited and that at which lysis took place. For cephaloridine, inhibition and lysis occurred at the same concentration. The implication of this finding is that cefoxitin and cephaloridine may be more bactericidal than cephalexin, ampicillin, or benzyl penicillin—the other antibiotics used in this study. Spratt (1975) showed that penicillin binds to 6 proteins in the cell membrane of E. coli. Inhibition of proteins no. 1 and no. 3 by penicillin is the event that is responsible for cell death and lysis. Cefoxitin binds to 5 of these 6 sites, including proteins no. 1 and no. 3. Protein no. 2 does not bind cefoxitin and is not involved in bactericidal action. Spratt (1977) showed that penicillin binds in a reversible manneT to the binding proteins, whereas the binding of cefoxitin to the major proteins is largely irreversible. The failure of penicillin to compete with cefoxitin in Gram-negative cells, as shown by Onishi, Zimmerman & Stapley (1974), is probably due to the reversible nature of penicillin protein binding and the non-reversibility of the binding of cefoxitin to the major proteins. The irreversible nature of cefoxitin protein binding may have important implications for the effectiveness of the antibiotic, and may account for its strong bactericidal action. For example, Goodwin & Hill (1977) showed that cefoxitin produced lysis of heavy suspensions of all strains of E. coli and Klebsiella that they tested, and suppressed regrowth at a low multiple of its MIC. Cephalothin and cefuroxime also caused some lysis, but several of the organisms tested showed regrowth, even at high multiples of the MICs. In addition to its strong lytic effects, the good binding of cefoxitin to cell wall enzymes and its resistance to P-lactamase may account for the favorable MIC/MBC ratios and the minimal effects of variations in inoculum size.
30
J. Bimbaum et al.
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
References Adams, H. G., Stilwcll, G. A. & Turck, M. In vitro evaluation of cefoxitin and cefamandole. Antimicrobial Agents and Chemotherapy 9: 1019-24 (1976). Anderson, J. D. & Sykes, R. B. Characterization of a P-lactamase obtained from a strain of Bacteroides fragilis resistant to |J-lactam antibiotics. Journal of Medical Microbiology 6: 201-6 (1973). Bach, V. T., Roy, I. & Thadepalli, H. Susceptibility of anaerobic bacteria to cefoxitin and related compounds. Antimicrobial Agents and Chemotherapy 11: 912-3 (1977). Bauer, W. W., Kirby, W. M. M., Sherris, J. C. & Turck, M. Antibiotic susceptibility testing by a standardized single-disc method. American Journal of Clinical Pathology 45: 493-6 (1966). Brumfitt, W., Kosmidis, J., Hamilton-Milltr, J. M. T. & Gilchrist, J. N. Cefoxitin and cephalothin: antimicrobial activity, human pharmacokinetics and toxicology. Antimicrobial Agents and Chemotherapy 6: 290-9 (1974). Buhs, R. P., Maxim, T. E., Allen, N., Jacob, T. A. & Wolf, F. J. Analysis of cefoxitin, cephalothin and their deacylated metabolites in human'urine by high performance liquid chromatography. Journal of Chromatography 99: 609-18 (1974). Cama, L. D., Leanza, W. J., Beattie, T. R. & Christensen, B. G. Substituted penicillin and cephalosporin derivatives. I. Stereo-specific introduction of the C-6 (7) methoxy group. Journal of the American Chemical Society 94: 1408-10 (1972). Chapman, D. G. & Russell, A. D. Effect of cefoxitin on Proteus species. Microbios Letters 2: 51-3 (1976). Daoust, D. R., Onishi, H. R., Wallick, H., Hendlin, D. & Stapley, E. O. Cephamycins, a new family of P-lactam antibiotics: antibacterial activity and resistance to P-lactamasc degradation. Antimicrobial Agents and Chemotherapy 3: 254—61 (1973). Darland, G. & Birnbaum, J. Cefoxitin resistance to P-lactamase: a major factor for susceptibility of Bacteroides to the antibiotic. Antimicrobial Agents and Chemotherapy 11: 725-34 (1977). Del Bene, V. E. & Farrar, W. E. Cephalosporinase activity in Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 3: 369-72 (1973). Eickhoff, T. C. & Ehret, J. M. In vitro comparison of cefoxitin, cefamandole, cephalexin and cephalothin. Antimicrobial Agents and Chemotherapy 9: 994-9 (1976). Ernst, E. C , Berger, S., Barza, M., Jacobus, N. V. & Tally, F. P. Activity of cefamandole and other cephalosporins against aerobic and anaerobic bacteria. Antimicrobial Agents and Chemotherapy 9: 852-5 (1976). Farrar, W. E. & Gramling, P. K. Antistaphylococcal activity and P-lactamase resistance of newer cephalosporins. Journal of Infectious Diseases 133: 691-5 (1976). Firestone, R. A., Schelechow, N., Johnston, D. B. R. & Christensen, B. G. Substituted penicillins and cephalosporins. II. C-6(7>alkyl derivatives. Tetrahedron Letters: 375-8 (1972). Fong, I. W., Engelking, E. R. & Kirby, W. M. M. Relative inactivation by Staphylococcus aureus of eight cephalosporin antibiotics. Antimicrobial Agents and Chemotherapy 9: 93SM4(1976). Goldner, M., Glass, D. G. & Fleming, P. C. Spontaneous mutant with loss of P-lactamase in Aerobacter cloacae. Journal of Bacteriology: 961-3 (1969). Goodwin, C. S. & Hill, J. P. Lysis of Enterobacteria by cefoxitin, cefuroxime, and cephalothin. Antimicrobial Agents and Chemotherapy 11: 26-30 (1977). Griffith, R. S. & Block, H. R. Cephalothin, a new antibiotic. Journal of the American Medical Association 189: 823-8 (1964). Hackman, A. S. & Wilkins, T. D. Comparison of cefoxitin and cephalothin therapy of a mixed Bacteroides fragilis and Fusobacterium necrophorum infection in mice. Antimicrobial Agents and Chemotherapy 8: 224-5 (1975). Hamilton-Miller, J. M. T., Kerry, D. W. & Brumfitt, W. An in vitro comparison of cefoxitin, a semisynthetic cephamycin, with cephalothin. Journal of Antibiotics, 27: 42-8 (1974). Jackson, R. T., Thomas, F. E. & Alford, R. H. Cefoxitin activity against multiply antibioticresistant Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 11: 84-7 (1977). Jones, R. N., Thornsberry, C, Barry, A. L., Fuchs, P. C , Gavan, T. L. & Gerlach, E. H. BL-S786, a new parenteral cephalosporin. II. In vitro antimicrobial activity comparison with six related cephalosporins. Journal of Antibiotics 30: 583-92 (1977).
Microbiological overview
31
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Kammer, R. B., Preston, D. A., Turner, J. R. & Hawley, L. C. Rapid detection of ampicillinresistant Haemophilus influenzae and their susceptibility to sixteen antibiotics. Antimicrobial Agents and Chemotherapy 8: 91-4 (1975). Karady, S., Pines, S. H., Weinstock, L. M., Roberts, F. E., Brenner, G. S., Hoinowski, A. M., Cheng, T. Y. & Sletzinger, M. Semisynthetic cephalosporins via a novel acyl exchange reaction. Journal of the American Chemical Society 94: 1410-11 (1972). Kirby, W. M. M., de Maine, J. B. & Serrill, W. S. Pharmacokinetics of the cephalosporins in healthy volunteers and uremic patients. Postgraduate Medical Journal 47 (Feb. Suppl.): 41-6(1971). Kosmidis, J., Hamilton-Miller, J. M. T., Gilchrist, J. N. G., Kerry, D. W. & Brumfitt, W. Cefoxitin, a new semi-synthetic cephamycin: an in vitro and in vivo comparison with cephalothin. British Medical Journal iv: 653-5 (1973). Lewis, R. P., Meyer, R. D. & Kraus, L. L. Antibacterial activity of selected p-lactam and aminoglycoside antibiotics against cephalothin-resistant Enterobacteriaceae. Antimicrobial Agents and Chemotherapy 9: 780-6 (1976). McCloskey, R. V. Results of a clinical trial of cefoxitin, a new cephamycin antibiotic. Antimicrobial Agents and Chemotherapy 12: 636-41 (1977). Miller, A. K., Celozzi, E., Pelak, B. A., Stapley, E. O. & Hendlin, D. Cephamycins, a new family of P-lactam antibiotics. III. In vitro studies. Antimicrobial Agents and Chemotherapy 2: 281-6 (1972a). Miller, A. K., Celozzi, E., Kong, Y., Pelak, B. A., Kropp, H., Stapley, E. O. & Hendlin, D. Cephamycins, a new family of P-lactam antibiotics. IV. In vivo studies. Antimicrobial Agents and Chemotherapy 2: 287-90 (1972A). Miller, A. K., Celozzi, E., Kong, Y., Pelak, B. A., Hendlin, D. & Stapley, E. O. Cefoxitin, a semisynthetic cephamycin antibiotic: In vivo evaluation. Antimicrobial Agents and Chemotherapy 5: 33-7 (1974). Miller, A. K., Celozzi, E., Pelak, B. A., Birnbaum, J. & Stapley, E. O. Comparison of in vitro and in vivo activities of cefoxitin, cefamandole, cephalothin and cefazolin. Proceedings, 10th International Congress of Chemotherapy, Zurich, Sept. 18-23, 1977 (in press). Miller, T. W., Goegelman, R. T., Weston, R. G., Putter, I. & Wolf, F. J. Cephamycins, a new family of P-lactam antibiotics. II. Isolation and chemical characterization. Antimicrobial Agents and Chemotherapy 2: 132-5 (1972). Moellering, R. C , Dray, M. & Kunz, L. J. Susceptibility of clinical isolates of bacteria to cefoxitin and cephalothin. Antimicrobial Agents and Chemotherapy 6: 320-4 (1974). Nagarajan, R., Boeck, L. D., Gorman, M., Hamill, R. L., Higgins, C. E., Hoehn, M. M., Stark, W. M. & Whitney, J. G. P-Lactam antibiotics from Streptomyces. Journal of the American Chemical Society 93: 2308-10 (1971). Neu, H. C. Cefoxitin, a semisynthetic cephamycin antibiotic: Antibacterial spectrum and resistance to hydrolysis by Gram-negative fJ-lactamases. Antimicrobial Agents and Chemotherapy 6: 170-6 (1974). Norrby, R., Brorsson, J. E. & Seeberg, S. Comparative study of the in vitro antibacterial activity of cefoxitin, cefuroxime and cephaloridine. Antimicrobial Agents and Chemotherapy 9: 506-10 (1976). O'Callaghan, C. H., Sykes, R. B., Griffiths, A. & Thornton, J. E. Cefuroxime, a new cephalosporin antibiotic: Activity in vitro. Antimicrobial Agents and Chemotherapy 9: 511-19 (1976). Olsson, B., Dornbusch, K. & Nord, C. E. Susceptibility to beta-lactam antibiotics and production of fi-lactamases in Bacteroides fragilis. Medical Microbiology and Immunology 163: 183-94 (1977). Olsson, B., Nord, C. E. & Wadstrom, T. Formation of beta-lactamase in Bacteroides fragilis: cell-bound and extracellular activity. Antimicrobial Agents and Chemotherapy 9: 727-35 (1976). Onishi, H. R., Daoust, D. R., Zimmerman, S. B., Hendlin, D. & Stapley, E. O. Cefoxitin, a semisynthetic cephamycin antibiotic: resistance to 3-lactamase inactivation. Antimicrobial Agents and Chemotherapy 5: 38-48 (1974). Onishi, H. R., Zimmerman, S. B. & Stapley, E. O. Observations on the mode of action of cefoxitin. Annals of the New York Academy of Science 235: 406-25 (1974).
32
J. Birnbaum tt al.
Downloaded from http://jac.oxfordjournals.org/ at Monash University on April 12, 2015
Phillips, I., King, A., Warren, C , Watts, B. & Stoatc, M. W. The activity of penicillin and eight cephalosporins on Neisseria gonorrhoeae. Journal of Antimicrobial Chemotherapy 2: 31-9 (1976). Schrogie, J. J., Davies, R. O., Kwan, K. C , Rogers, D., Holmes, G. I., Skeggs, H. & Martin, C. M. Bioavailability and pharmacokinetics of cefoxitin. Journal of Antimicrobial Chemotherapy 4 (Suppl. B): 69-78 (1978). Shah, P. M., Zwischenbrugger, H. & Stille, W. In vitro activity of cefoxitin, a new cephalosporin. Munchener Medizinische Wochenschrift 118: 1469-72 (1976). Sonneville, P. F.F Kartodirdjo, R. R., Skeggs, H., Till, A. E. & Martin, C. M. Comparative clinical pharmacology of intravenous cefoxitin and ccphalothin. European Journal of Clinical Pharmacology 9: 397-403 (1976). Spratt, B. G. Distinct penicillin-binding proteins involved in the division, elongation, and shape of Escherichia coli K12. Proceedings, National Academy of Science, U.S.A. 92: 2999-3003 (1975). Spratt, B. G. Properties of the penicillin-binding proteins of Escherichia coli K12. European Journal of Biochemistry 72: 341-52 (1977). Stapley, E. O., Jackson, M., Hernandez, S., Zimmerman, S. B., Carrie, S. A., Mochales, S. Mata, J. M., Woodruff, H. B. & Hendlin, D. Cephamycins, a new family of P-lactam antibiotics. I. Production by actinomycetes, including Streptomyces lactamdurans sp. n. Antimicrobial Agents and Chemotherapy 2: 122-31 (1972). Stapley, E. O., Daoust, D. R., Hendlin, D., Miller, A. K., Zimmerman, S. B., Birnbaum, J., Cama, L. D. & Christensen, B. G. Cefoxitin: mechanism of activity against cephalosporinresistant bacteria. Proceedings, Symposium on Microbial Resistance, Tokyo, Oct. 1977. In the press (1978). Stewart, D. & Bodey, G. P. Comparative in vitro activity of cephalosporins. Journal of Antibiotics 29: 181-6(1976). Sutter, V. L. & Finegold, S. M. Susceptibility of anaerobic bacteria to carbenicillin, cefoxitin and related drugs. Journal of Infectious Diseases 131: 147-22 (1975). Tally, F. P., Jacobus, N. V., Bartlett, J. G. & Gorbach, S. L. Susceptibility of anaerobes to cefoxitin and other cephalosporins. Antimicrobial Agents and Chemotherapy 7: 128-32 (1975). Verbist, L. Comparison of the antibacterial activity of nine cephalosporins against Enterobacteriaceae and nonfermentative Gram-negative bacilli. Antimicrobial Agents and Chemotherapy 10: 657-63 (1976). Wallick, H. & Hendlin, D. Cefoxitin, a semisynthetic cephamycin antibiotic: susceptibility studies. Antimicrobial Agents and Chemotherapy 5: 25-32 (1974). Washington, J. A. The in vitro spectrum of cephalosporins. Mayo Clinic Proceedings 51: 237-50 (1976). Wick, W. E. In vitro and in vivo laboratory comparison of cephalothin and desacetylcephalothin. Antimicrobial Agents and Chemotherapy—1964: 870-5 (1965). Wick, W. E. & Preston, D. A. Biological properties of three 3-heterocyclicthiomethyl cephalosporin antibiotics. Antimicrobial Agents and Chemotherapy 1: 221-34 (1972). Yourassowsky, E. & Schoutens, E. In vitro bacteriostatic and bactericidal activities of seven cephalosporin antibiotics on Haemophilus influenzae. International Journal of Clinical Pharmacology 12: 433-6 (1975). Yourassowsky, E., Schoutens, E. & Vanderlinen, M. P. Antibacterial activity of eight cephalosporins against Haemophilus influenzae and Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 2: 55-9 (1976). Zimmerman, S. B. & Stapley, E. O. Relative morphological effects induced by cefoxitin and other P-lactam antibiotics in vitro. Antimicrobial Agents and Chemotherapy 9: 318-26(1976).
