THE JOURNAL OF INFECTIOUS DISEASES • VOL. 133, NO.3· © 1976 by the University of Chicago. All rights reserved.
MARCH 1976
Activity of Combinations of Antimicrobial Agents against Bacteroides Jragilis From the Research and Medical Services, Wadsworth Hospital Center, Veterans Administration, and the Department of Medicine, University of California, Los Angeles, California
David F. Busch, Vera L. Sutter, and Sydney M. Finegold
these combinations of antimicrobial agents on the inhibition of B. fragilis has not yet been defined. B. fragilis has been recovered from patients with endocarditis as well [3, 4]. Administration of agents with bactericidal activity to patients with endocarditis is considered to be desirable, and antibiotic combinations are often employed to achieve or enhance the bactericidal effect against other organisms such as Streptococcus faecalis. Of the antimicrobial agents effective in vitro against B. fragilis, only metronidazole has been consistently inhibitory and bactericidal at achievable concentrations [5-7], but clinical evaluation is not yet sufficient to permit recommendation of metronidazole as a single agent for treatment of B. fragilis septicemia or endocarditis, conditions for which a bactericidal effect may be desirable [8]. Several reports have suggested in vitro activity of a combination of metronidazole and spiramycin against anaerobic organisms other than B. fragilis, and clinical efficacy of this combination in certain anaerobic infections has been observed [9-14]. Weare unaware of reports of tests of combinations of antimicrobial agents other than clindamycin plus gentamicin against B. fragilis [15, 16]. This report describes results of in vitro tests of 32 clinical isolates of B. fragilis against nine pairs of antimicrobials; most pairs (table 1) were se-
Bacteroides fragilis, a dominant member of the
lower intestinal flora, is frequently implicated in infections related to loss of integrity of the bowel mucosa. B. fragilis is often recovered in mixed culture from these infections. Members of the family Bacteroidaceae have also been recovered from 5.4 % -11. 6 % of all bacteremias in recent series, and B. fragilis accounts for up to 88% of those isolates [1]. As a result, antimicrobial programs designed for treatment of both intraabdominal infections and septic conditions of undetermined etiology often include agents active against B. fragilis in addition to antibiotics active against other components of the intestinal flora and other potential pathogens. Commonly, an aminoglycoside is paired with a penicillin, a cephalosporin, clindamycin, or chloramphenicol for antibiotic coverage of these infections. One group has long advocated the use of penicillin and tetracycline for this purpose [2]. The possible interaction of
Received for publication August 11, 1975, and in revised form November 3, 1975. The authors acknowledge the excellent technical assistance of Ms. Phyllis Stewart. Please address requests for reprints to Dr. Sydney M. Finegold, Infectious Disease Section, Wadsworth Hospital Center, Los Angeles, California 90073.
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Thirty-two clinical isolates of Bacteroides fragilis were tested against nine pairs of antimicrobial agents by means of an agar dilution technique. A synergistic effect was observed with the combination of clindamycin and metronidazole. End points were achieved with 17 strains. Of these strains, 13 (76%) were inhibited by the combination of clindamycin and metronidazole; each drug was present at a concentration of ::::;;25 % of its minimal inhibitory concentration when tested alone. This combination also showed synergistic bactericidal activity against three of six strains examined by a tube dilution technique. No antagonism was noted with any strain. The other eight combinations tested failed to show a consistent synergistic effect, although no antagonism was observed. These in vitro data indicate that antagonism is not likely to be encountered when combination therapy is used for B. fragilis infections. For selected B. fragilis infections, the combination of clindamycin and metronidazole may be useful.
322
Busch, Sutter, and Finegold
Table 1. Effect of nine combinations of antimicrobial agents against Bacteroides /ragilis. Agents in combination
Concentrations tested *
Synergism ratiot
0.125-8.0 0.125-16.0
12/17 (76)
Erythromycin Metronidazole
0.25-64.0 0.125-8.0
10/30 (33)
Clindamycin Chloramphenicol
0.125-8.0 0.25-16.0
6/19 (32)
Carbenicillin Metronidazole Cefoxitin Metronidazole
1.0-128.0 0.125-8.0 2.0-64.0 0.125-8.0
Penicillin Tetracycline
0.5-128.0 0.25-32.0
2/27 (8)
Amikacin Carbenicillin
2.0-32.0 1.0-256.0
0/32
Amikacin Cefoxitin
2.0-32.0 1.0-64.0
0/32
Amikacin Clindamycin
2.0-32.0 0.125-8.0
0/32
4/28 (14) 3/30 (10)
* Concentrations are given as J,tg/ml except for that of penicillin, which is given as units/ml. t Synergism ratio number of strains showing synergism/number of strains with interpretable results (percentage showing synergism). Results could not be interpreted when the MIC for either drug was at the lowest or next to the lowest concentration tested for that drug.
=
lected to represent either combinations commonly used in clinical practice or combinations poten.. tially useful for the treatment of serious infections due to B. fragilis. Materials and Methods
Organisms. All organisms tested except one were clinical isolates of B. fragilis either recovered at the Wadsworth Veterans Administration Hospital or referred to the hospital for identification between 1968 and 1974. The anatomical sites of isolation included blood (eight isolates), intraab.. dominal abscess or infection (1 7 isolates), extraperitoneal and soft tissue infections (five isolates), and osteomyelitis (one isolate). One of the clinical isolates was a strain (no. WAL 1887) used in this laboratory as a control for susceptibility testing. The final isoiate was an organism used as a reference strain for antibiotic susceptibility testing at the Virginia Polytechnic Institute (obtained from
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Clindamycin Metronidazole
Tracy Wilkins, Virginia Polytechnic Institute, Blacksburg, Va.). Organisms were identified and subspeciated according to methods previously described [17]. Twenty-one isolates were Bacteroides fragilis subspecies fragilis, five were subspecies thetaiotaomicron, five were subspecies distasonis, and one was subspecies vulgatus. Antimicrobial agents. Stock solutions of the following antimicrobials were prepared with use of susceptibility testing powder supplied by the respective manufacturers and sterile distilled water: amikacin (Bristol Laboratories, Syracuse, N.Y.), carbenicillin (Roerig, New York, N.Y.), cefoxitin (Merck Sharp & Dohme, West Point, Pa.), chloramphenicol (Parke-Davis, Detroit, Mich.) , clindamycin (Upjohn, Kalamazoo, Mich.), metronidazole (Amersham/Searle, Arlington Heights, Ill.), penicillin G (Eli Lilly and Co., Indianapolis, Ind.), and tetracycline (Pfizer, New York, N.Y.). Erythromycin (Eli Lilly) was prepared in 95 % ethanol. Stock solutions were stored at - 20 C for a maximum of four weeks prior to use, except for carbenicillin, which was stored at - 70 C, and metronidazole, which was freshly prepared on the day of testing. AntimicroMal plates for bacteriostatic tests. After thawing, antimicrobial stock solutions were diluted in serial twofold steps with sterile distilled water; appropriate dilutions were added to tubes containing brucella agar (Pfizer), laked sheep blood (5%), and vitamin K 1 (10 llg/ml). The final volume of 20 ml was poured into 100- X 15-mm petri plates; plates were dried at 35 C for 30 min before inoculation. For each combination tested, plates were prepared to complete a checkerboard series of twofold dilutions of antimicrobials within the concentration ranges indicated in table 1. Plates were also prepared with the antimicrobial agents incorporated singly for determination of individual MICs. All plates were prepared on the day of inoculation. Control plates were also prepared with use of sterile distilled water in place of antimicrobial solutions. Inoculation, incubation, and interpretation. Organisms were prepared according to procedures outlined in the Wadsworth Anaerobic Bacteriology Manual [17], and plates were inoculated with use of a Steers replicator [18]. Control plates were inoculated before and after the inoculation of
323
Drug Combinations against B. fragilis
Results
Bacteriostatic tests. MICs obtained with the individual antimicrobial agents correlated well
with results previously reported from this laboratory [5, 20-22]. Specifically, all strains of B. fragilis tested were inhibited by concentrations of clindamycin (~4 ~g/ml), cefoxitin (~64 ~g/ ml), chloramphenicol (~16 ~g/ml), and metronidazole (~8 I-lg/ml) achievable in serum with standard dosage regimens. Two of the 32 strains tested were resistant to 256 I-lg of carbenicillin/ ml and also to 256 units of penicillin/ml. The remaining 30 strains were all inhibited by ~64 ~g of carbenicillin/ml and by ~64 units of penicillin/m!. A bimodal distribution of susceptibility to tetracycline was seen, with only 15 of the 32 strains inhibited at concentrations of ~8 ~g/ml. Certain isolates required as much as 16 ~g of erythromycin/ml for inhibition. Amikacin was inactive against all strains at concentrations of ~512 ~g/ml.
Of the nine combinations tested, three (amikacin plus clindamycin, amikacin plus carbenicillin, and amikacin plus cefoxitin) showed complete indifference in their activity against B. fragilis. Amikacin neither enhanced nor antagonized the activity of the other three antibiotics. Three combinations demonstrated synergism with only a few strains by the criterion of a fourfold reduction in the MIC for each drug; individual isobolograms constructed for these organisms also consistently indicated synergism. Two of 27 strains yielding interpretable data showed synergism of penicillin and tetracycline, three of 30 showed synergism of cefoxitin and metronidazole, and four of 28 showed synergism of carbenicillin and metronidazole. Only one isolate showed synergism of two of these three combinations. Table 2 describes the distribution of synergistic strains among the four subspecies of B. tragilis tested. No antagonism was seen. Composite data indicating indifference of these three combinations is represented in isobolograms (figure 1). With all but two of 19 strains that had an end point with clindamycin plus chloramphenicol, these drugs showed at least an additive effect; results with six strains met the criteria for synergism (figure 2, top left). Erythromycin and metronidazole were synergistic against 10 of 30 strains with interpretable results (figure 2, bottom left).
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each antimicrobial series. One set of control plates was incubated aerobically. Parallel sets of control plates and all antimicrobial plates were placed in GasPak® jars (Baltimore Biological Laboratories, Baltimore, Md.); anaerobiosis was achieved with GasPak envelopes (BBL). Plates were read after incubation for 42-48 hr at 35 C. The MIC for an organism was considered to be the lowest concentration of drug yielding no growth, a barely visible haze, or one discrete colony [19]. For evaluation of antimicrobial combinations, MICs were determined for each drug at all tested concentrations of the second drug. Evaluation of drug interaction. For individual strains, an effect was considered to be synergistic when an organism was inhibited by a combination of two agents, each at a concentration of ~25 % of its MIC when tested alone. Results could not be interpreted when the MIC for either drug was at the lowest or next to lowest concentration tested for that drug. The effect of each combination on the panel of organisms as a whole was evaluated further by construction of an isobologram using the geometric mean of the MICs of each interpretable test for one agent at various concentrations of the other agent. These isobolograms were compared to theoretical "additive" or "indifferent" straight-line isobolograms developed by connecting points on the axes representing the geometric mean MICs for the individual antimicrobial agents. An isobologram approximating this straight line indicates a composite additive or indifferent effect of the antimicrobial combination on the panel of organisms. Bowing of the isobologram toward the origin is indicative of synergism, while bowing away from the origin would suggest antagonism. Bactericidal tests. The test procedure described by Nastro and Finegold [5] was used for the study of six isolates of B. fragilis for bactericidal end points with clindamycin and metronidazole alone and in combination. (Clindamycin was tested in a concentration of 2 ~g/ml, and the concentration of metronidazole was varied from 4 ~g/ml to 0.125 ~g/ml in twofold steps.)
324
Busch, Sutter, and Finegold
Table 2.
Inhibition of subspecies of Bacteroides fragilis by six combinations of antimicrobial agents. Antimicrobial combinations tested *
Subspecies of B. fragilis
Penicillin + tetracycline
21 5 5 1 32
1/17 0/5 1/5 0/0 2/27
fragilis thetaiotaomicron distasonis vulgatus Total
Cefoxitin + metronidazole
1/20 0/5 2/5 0/0 3/30
Carbenicillin + metronidazole
Chloramphenicol + clindamycin
Erythromycin+ metronidazole
3/18 1/5 0/5 0/0 4/28
3/9 3/5 0/5 0/0 6/19
7/20 0/5 3/5 0/0 10/30
Clindamycin+ metronidazole
7/8 2/4 3/5 0/0 12/17
'" Data are given as number of strains demonstrating synergism/number of strains with interpretable results. Results could not be interpreted when the MIC for either drug was at the lowest or next to lowest concentration tested for that drug.
Bfra9.i!!.§.
6
28 STRAINS
8. tragi/is 27 STRAINS
e.....
~
~ 30 ~
U
4-
z
~
a:
20
~
10
+---r---,.----,.------~___,
.25.5
1
2
METRONIDAZOLE (Pv/ml) 20 30 PENICILLIN (units/mil
40
Figure 1. Isobolograms showing inhibitory activity of three nonsynergistic combinations of antimicrobial agents against strains of Bacteroides fragilis that achieved end points. Points indicate the geometric mean MIC for one antimicrobial agent (ordinate) at the concentration of the second agent, shown on the abscissa.
B. fragpis 30 STRAINS loJ
5
~
1
z fE I-
loJ
:E
.5 .25
,'125
2
4
6
8
16
CEFOXITIN }Ig/ml
The combination of c1indamycin and metronidazole demonstrated synergism against 76% of the strains that had an end point with this pair of drugs. Synergism was identified in strains of
B. fragilis subspecies fragilis, thetaiotaomicron, and distasonis (table 2). These two drugs in combination had at least an additive effect against all strains with interpretable results and in no case were antagonistic. All 32 strains tested were inhibited in the presence of the combination of 0.5 I-lg of clindamycin/ml plus 0.5 I-lg of metronidazole/ml (figure 2, top right). Bactericidal tests. Table 3 shows the results of the bactericidal tests with clindamycin, metronidazole, and the combination of the two drugs. All six strains had minimal bactericidal concentrations (MBCs) of ~2-4 I-lg/ml with each drug singly. For three of the six strains, the combination was synergistic with respect to
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No. of strains tested
Drug Combinations against B. fragilis
325
1.25
1 8. fragi/is
E .....
8. frogjlis 19 STRAINS
17 STRAINS
co
5 z 0 >~
« 0 z
.5
0
.25 .125
•
~
•
+--r--,----,r------,-----==~..,
.25.5
I
2
4
8
CHLORAMPHENICOL (JlQ/mf)
30 STRAINS
2
01 ~
+---r------,r---......,.--~--..,..;~I.__,
2 3 4 ERYTHROMYCIN (JoIo/m!)
I
1.75
METRONIDAZOLE (J'Q/ml)
Figure 2. Isobolograms showing inhibitory activity against Bacteroides fragilis of three antimicrobial combinations that demonstrated synergism against individual strains. Points indicate the geometric mean MIC for one antimicrobial agent (ordinate) at the concentration of the second agent, shown on the abscissa.
Bfrogpis
E .......
.125.25.5
5
6
MBC; the combination was neither synergistic nor antagonistic for one strain; and in two cases the results were not interpretable. All six strains were killed in the presence of 2 !lg of c1indamycin/ml plus 1 !lg of metronidazole/ml. Discussion
The complex nature of the flora of the lower intestine often leads to the use of multiple antibiotic agents in the treatment of infections due to disruption of intestinal mucosal barriers. Often, treatment must be initiated empirically before results of cultures are known. The aminoglycosides, which are commonly used in this setting and are represented in this report by amikacin, have broad-spectrum activity against gram-negative
aerobic and facultatively anaerobic bacteria but are recognized to be essentially inactive by themselves against most anaerobes -and particularly against B. fragilis [22]. Aminoglycosides often enhance the activity of other antibiotics, such as the penicillins and cephalosporins, against organisms such as streptococci [23, 24], staphylococci [25], and enteric bacilli [26]. Whereas Fass et al. [15] have reported in vitro synergism of the combination of c1indamycin and the aminoglycoside gentamicin against a single strain of B. fragilis, in the present study synergism was not observed against B. fragilis with the combination of c1indamycin and amikacin. Leigh [16] has likewise indicated an indifferent interaction (neither synergism nor antagonism) of clindamycin and gentamicin against B. fragilis. These findings are comparable also to those of Sabath and Toftegaard [27], who demonstrated in vitro indifference of clindamycin plus gentamicin against Clostridium perfringens, a c1indamycin-resistant isolate of Staphylococcus aureus, and certain gram-negative bacilli. A similar lack of interaction was seen when amikacin was combined with carbenicillin and when amikacin was combined with cefoxitin, a new cephamycin that has shown consistent in vitro activity against B. fragilis [20, 28]. The absence of antagonism demonstrated with these
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::::i
\
Busch, Sutter, and Finegold
326
Table 3.
Bactericidal activity of clindamycin and metronidazole against six strains of Bacteroides fragilis. Minimal bactericidal concentration (r-tg/ml) of
WAL * strain no. (subspecies of B. fragilis)
(thetaiotaomicron) (fragilis) (distasonis) (thetaiotaomicron) (thetaiotaomicron) (thetaiotaomicron)
* WAL =
>4 >4 4 >4 4 >4
Metronidazole 2 1
4 2
4 4
0.5 1 :::;;0.125 0.25 0.5 1
Wadsworth Anaerobic Bacteriology Laboratory.
combinations in this in vitro study is reassuring in view of their extensive use in the treatment of abdominal infections in which B. fragilis is often only one of a number of potentially pathogenic bacteria that may be recovered from a single specimen. Combinations of antimicrobial agents are employed in these infections since there is insufficient information concerning the relative importance of individual organisms in mixed infections to allow treatment that does not cover both the anaerobic and aerobic organisms encountered; in addition, there are no currently available antibiotics that individually encompass this spectrum of organisms adequately. These in vitro data indicate that the commonly used aminoglycosides are not likely to antagonize the action of clindamycin, carbenicillin, or cefoxitin against B. fragiZis. The general lack of synergism seen with combinations including the cell wall-active agents (represented here by penicillin, carbenicillin, and cefoxitin) was somewhat surprising. In particular, the combination of penicillin and tetracycline showed indifferent activity against B. fragilis. In some medical centers this combination has long been advocated in therapy of intraabdominal infections [2]. In recent years up to 70% of isolates of B. fragilis have been found to be resistant to tetracycline [5, 21, 29]. Since penicillin does not (at least in in vitro tests) improve the activity of tetracycline against B. fragilis, this combination would seem to be inappropriate for treatment of serious intraabdominal infections in the absence of information about the antimicrobial susceptibility of individual isolates and in the absence of clear-cut clinical proof of effectiveness of this combination.
In contrast to the penicillins and cefoxitin, erythromycin and clindamycin act synergistically in vitro in combination with certain other agents. The combination of clindamycin and chloramphenicol showed synergism against 32% of strains of B. fragilis that demonstrated an end point in this study; however, these drugs have characteristics that will probably limit any potential clinical usefulness of the combination. The combinations of clindamycin plus metronidazole and erythromycin plus metronidazole were synergistic. With the presence of metronidazole in these pairs, a bactericidal effect could be anticipated with either combination [5]. In this study, the combination of clindamycin and metronidazole did in fact demonstrate bactericidal synergism against three of six strains examined. The mechanism of this interaction is speculative at present. Clindamycin acts by binding to the 50S ribosomal subunit, thus inhibiting binding of amino acids to the ribosomes with resultant suppression of bacterial protein synthesis [30]. Clindamycin may also interact with peptide chain initiation associated with the formation of the 30S ribosomal complex [31]. The mechanism of action of metronidazole against anaerobic bacteria is not fully understood, although it is thought to act as an electron acceptor in phosphoroclastic reactions specific to anaerobic organisms [32, 33]. Ings has shown labeled metronidazole to be bound to the DNA and protein of Trichomonas vaginaZis but not to the RNA of the organism [34]. These studies suggest inhibition of nucleic acid synthesis by metronidazole. Some investigators suggest that an intermediate compound such as hydroxyalanine [34] or a nitrofurazone [35] may actually be responsible for the inhibition. Thus
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2380 2461 2661 2750 2923 2926
Clindamycin
Metronidazole in the presence of 2 r-tg of clindamycin/ml
Drug Combinations against B. fragilis
References 1. Sonnenwirth, A. C. Incidence of intestinal anaerobes in blood cultures. In A. Balows, R. M. DeHaan, V. R. Dowell, Jr., and L. B. Guze [ed.]. Anaerobic bacteria, role in disease. Charles C Thomas, Springfield, Ill., 1968, p. 157-172. 2. Fullen, W. D., Hunt, J., Altemeier, W. A. Prophylactic antibiotics in penetrating wounds of the abdomen. J. Trauma 12:282-289, 1972. 3. FeIner, J. M., Dowell, V. R., Jr. Anaerobic bacterial endocarditis. N. Engl. J. Med. 283: 1188-1192, 1970. 4. Nastro, L. J., Finegold, S. M. Endocarditis due to anaerobic gram negative bacilli. Am. J. Med. 54: 482-496, 1973. 5. Nastro, L. J., Finegold, S. M. Bactericidal activity of five antimicrobial agents against Bacteroides fragilis. J. Infect. Dis. 126: 104-107, 1972. 6. Whelan, J. P. F., Hale, J. H. Bactericidal activity of metronidazole against Bacteroides fragilis. J. Clin. Pathol. 26:393-395, 1973. 7. Ueno, K., Ninomiya, K., Suzuki, S. Antibacterial activity of metronidazole against anaerobic bacteria. Chemotherapy (Japan) 19: 111-114, 1971. 8. Tally, F. P., Sutter, V. L., Finegold, S. M. Treatment of anaerobic infections with metronidazole. Antimicrob. Agents Chemother. 7:672-675, 1975.
9. Videau, D. Association metronidazole-spiramycine sur les germes anaerobies. Pathol. BioI. (Paris) 19:661-666, 1971. 10. Cros, P., Freidel, M., Jacquemard, R. Interet de l'association synergique metronidazole-spiramycine en pratique courante odonto-stomatologique. Rev. Fr. Odontostomatol. 18: 1039-1048, 1971. 11. Chikhani, P. Etude d'une association antibacterienne hautement synergique. Rev. Stomatol. Chir. Maxillofac. 72: 513-522, 1971. 12. LaUfer, J., Mignon, H., Videau, D. L'association metronidazole-spiramYcine: concentrations et synergie in situ comparees aux CMI de la flore buccale. Rev. Stomatol. Chir. Maxillofac. 74:387392, 1974. 13. Videau, D., Blanchard, J.-c., Sebald, M. Flore buccodentaire: sensibilite aux antibiotiques et interet de l'association spiramycine-metronidazole. Ann. Microbiol. (Paris) 124:505-516, 1973. 14. Garcia Perla, A., Gomez de la Mata, J. Experiencias clinicas con la asociacion spiramicina-metronidazol. An. Esp. Odontoestomatol. 32:239-242, 1973. 15. Fass, R. J., Rotilie, C. A., Prior, R. B. Interaction of clindamycin and gentamicin in vitro. Antimicrob. Agents Chemother. 6:582-587, 1974. 16. Leigh, D. A. Bacteroides infections. Lancet 2: 10811082, 1973. 17. Sutter, V. L., Vargo, V. L., Finegold, S. M. Wadsworth anaerobic bacteriology manual. 2nd ed. University of California, Los Angeles, Extension Division, 1975. 106 p. 18. Steers, E., Foltz, E. L., Graves, B. S. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311, 1959. 19. Ericsson, H. M., Sherris, J. C. Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol. Microbiol. Scand. [Suppl.] 217:1-90,1971. 20. Sutter, V. L., Finegold, S. M. Susceptibility of anaerobic bacteria to carbenicillin, cefoxitin, and related drugs. J. Infect. Dis. 131:417-422, 1975. 21. Sutter, V. L., Kwok, Y.-Y., Finegold, S. M. Susceptibility of Bacteroides fragilis to six antibiotics determined by standardized antimicrobial disc susceptibility testing. Antimicrob. Agents Chemother. 3: 188-193, 1973. 22. Finegold, S. M., Sutter, V. L. Susceptibility of gramnegative anaerobic bacilli to gentamicin and other aminoglycosides. J. Infect. Dis. 124(Suppl.) :S56S58, 1971. 23. Durack, D. T., Pelletier, L. L., Petersdorf, R. G. Chemotherapy of experimental streptococcal endoc;arditis. II. Synergism between penicillin and streptomycin against penicillin-sensitive streptococci. J. Clin. Invest. 53:829-833, 1974. 24. Sapico, F: L., Keys, T. F., Hewitt, W. L. Experimental enterococcal endocarditis. II. Study of in vivo synergism of penicillin and streptomycin. Am. J. Med. Sci. 263: 128-135, 1972.
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the combination of clindamycin and metronida~ zole may be synergistic by virtue of action at sites of both nucleic acid synthesis and protein synthesis. The data from in vitro tests reported in this communication extend the observation of antimicrobial interactions against anaerobic bacteria to B. fragilis, a dominant member of the bowel flora and a pathogen frequently encountered in intraabdominal infections, gynecological infections, and abdominal sepsis and occasionally encountered in soft-tissue infections and endocarditis. It is of interest that no antagonism was observed. Although essentially all strains of B. fragilis can currently be inhibited by easily obtainable concentrations of several antimicrobial agents, combinations such as clindamycin plus metronidazole may prove useful in the treatment of selected infections such as endocarditis, septic thrombophlebitis, and osteomyelitis, in which B. fragilis is implicated as a single or primary pathogen and for which maximal antibacterial activity is desired. The combination might also become useful in the event of increased resistance of B. fragilis to individual antimicrobial agents.
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328
30. Weisblum, B., Davies, J. Antibiotic inhibitors of the bacterial ribosome. Bacteriol. Rev. 32:493-528, 1968. 31. Reusser, F. Effect of lincomycin and clindamycin on peptide chain initiation. Antimicrob. Agents Chemother. 7:32-37, 1975. 32. O'Brien, R. W., Morris, J. G. Effect of metronidazole on hydrogen production by Clostridium acetobutylicum. Arch. Mikrobiol. 84:225-233, 1972. 33. Edwards, D. 1., Dye, M., Crane, H. The selective toxicity of antimicrobial nitroheterocyclic drugs. J. Gen. Microbiol. 76: 135-145, 1973. 34. Ings, R. M. J., McFadzean, J. A., Ormerod, W. E. The mode of action of metronidazole in Trichomonas vaginalis and other micro-organisms. Biochem. Pharmacol. 23: 1421-1429, 1974. 35. Tanowitz, H. B., Wittner, M., Rosenbaum, R. M., Kress, Y. In vitro studies on the differential toxicity of metronidazole in protozoa and mammalian cells. Ann. Trop. Med. Parasitol. 69: 19-28, 1975.
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25. Sande, M. A., Johnson, M. L. Antimicrobial therapy of experimental endocarditis caused by Staphylococcus aureus. J. Infect. Dis. 131:367-375, 1975. 26. Klastersky, J., Cappel, R., Daneau, D. Clinical significance of in vitro synergism between antibiotics in gram negative infections. Antimicrob. Agents Chemother. 2:470-475, 1972. 27. Sabath, L. D., Toftegaard, 1. Rapid microassays for clindamycin and gentamicin when present together and the effect of pH and of each on the antibacterial activity of the other. Antimicrob. Agents Chemother. 6:54-59, 1974. 28. Tally, F. P., Jacobus, N. V., Bartlett, J. G., Gorbach, S. L. Susceptibility of anaerobes to cefoxitin and other cephalosporins. Antimicrob. Agents Chemother. 7: 128-132, 1975. 29. Martin, W. J., Gardner, M., Washington, J. A., II. In vitro antimicrobial susceptibility of anaerobic bacteria isolated from clinical specimens. Antimicrob. Agents Chemother. 1: 148-158, 1972.
Busch, Sutter, and Finegold
MARCH 1976
Activity of Combinations of Antimicrobial Agents against Bacteroides Jragilis From the Research and Medical Services, Wadsworth Hospital Center, Veterans Administration, and the Department of Medicine, University of California, Los Angeles, California
David F. Busch, Vera L. Sutter, and Sydney M. Finegold
these combinations of antimicrobial agents on the inhibition of B. fragilis has not yet been defined. B. fragilis has been recovered from patients with endocarditis as well [3, 4]. Administration of agents with bactericidal activity to patients with endocarditis is considered to be desirable, and antibiotic combinations are often employed to achieve or enhance the bactericidal effect against other organisms such as Streptococcus faecalis. Of the antimicrobial agents effective in vitro against B. fragilis, only metronidazole has been consistently inhibitory and bactericidal at achievable concentrations [5-7], but clinical evaluation is not yet sufficient to permit recommendation of metronidazole as a single agent for treatment of B. fragilis septicemia or endocarditis, conditions for which a bactericidal effect may be desirable [8]. Several reports have suggested in vitro activity of a combination of metronidazole and spiramycin against anaerobic organisms other than B. fragilis, and clinical efficacy of this combination in certain anaerobic infections has been observed [9-14]. Weare unaware of reports of tests of combinations of antimicrobial agents other than clindamycin plus gentamicin against B. fragilis [15, 16]. This report describes results of in vitro tests of 32 clinical isolates of B. fragilis against nine pairs of antimicrobials; most pairs (table 1) were se-
Bacteroides fragilis, a dominant member of the
lower intestinal flora, is frequently implicated in infections related to loss of integrity of the bowel mucosa. B. fragilis is often recovered in mixed culture from these infections. Members of the family Bacteroidaceae have also been recovered from 5.4 % -11. 6 % of all bacteremias in recent series, and B. fragilis accounts for up to 88% of those isolates [1]. As a result, antimicrobial programs designed for treatment of both intraabdominal infections and septic conditions of undetermined etiology often include agents active against B. fragilis in addition to antibiotics active against other components of the intestinal flora and other potential pathogens. Commonly, an aminoglycoside is paired with a penicillin, a cephalosporin, clindamycin, or chloramphenicol for antibiotic coverage of these infections. One group has long advocated the use of penicillin and tetracycline for this purpose [2]. The possible interaction of
Received for publication August 11, 1975, and in revised form November 3, 1975. The authors acknowledge the excellent technical assistance of Ms. Phyllis Stewart. Please address requests for reprints to Dr. Sydney M. Finegold, Infectious Disease Section, Wadsworth Hospital Center, Los Angeles, California 90073.
321
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Thirty-two clinical isolates of Bacteroides fragilis were tested against nine pairs of antimicrobial agents by means of an agar dilution technique. A synergistic effect was observed with the combination of clindamycin and metronidazole. End points were achieved with 17 strains. Of these strains, 13 (76%) were inhibited by the combination of clindamycin and metronidazole; each drug was present at a concentration of ::::;;25 % of its minimal inhibitory concentration when tested alone. This combination also showed synergistic bactericidal activity against three of six strains examined by a tube dilution technique. No antagonism was noted with any strain. The other eight combinations tested failed to show a consistent synergistic effect, although no antagonism was observed. These in vitro data indicate that antagonism is not likely to be encountered when combination therapy is used for B. fragilis infections. For selected B. fragilis infections, the combination of clindamycin and metronidazole may be useful.
322
Busch, Sutter, and Finegold
Table 1. Effect of nine combinations of antimicrobial agents against Bacteroides /ragilis. Agents in combination
Concentrations tested *
Synergism ratiot
0.125-8.0 0.125-16.0
12/17 (76)
Erythromycin Metronidazole
0.25-64.0 0.125-8.0
10/30 (33)
Clindamycin Chloramphenicol
0.125-8.0 0.25-16.0
6/19 (32)
Carbenicillin Metronidazole Cefoxitin Metronidazole
1.0-128.0 0.125-8.0 2.0-64.0 0.125-8.0
Penicillin Tetracycline
0.5-128.0 0.25-32.0
2/27 (8)
Amikacin Carbenicillin
2.0-32.0 1.0-256.0
0/32
Amikacin Cefoxitin
2.0-32.0 1.0-64.0
0/32
Amikacin Clindamycin
2.0-32.0 0.125-8.0
0/32
4/28 (14) 3/30 (10)
* Concentrations are given as J,tg/ml except for that of penicillin, which is given as units/ml. t Synergism ratio number of strains showing synergism/number of strains with interpretable results (percentage showing synergism). Results could not be interpreted when the MIC for either drug was at the lowest or next to the lowest concentration tested for that drug.
=
lected to represent either combinations commonly used in clinical practice or combinations poten.. tially useful for the treatment of serious infections due to B. fragilis. Materials and Methods
Organisms. All organisms tested except one were clinical isolates of B. fragilis either recovered at the Wadsworth Veterans Administration Hospital or referred to the hospital for identification between 1968 and 1974. The anatomical sites of isolation included blood (eight isolates), intraab.. dominal abscess or infection (1 7 isolates), extraperitoneal and soft tissue infections (five isolates), and osteomyelitis (one isolate). One of the clinical isolates was a strain (no. WAL 1887) used in this laboratory as a control for susceptibility testing. The final isoiate was an organism used as a reference strain for antibiotic susceptibility testing at the Virginia Polytechnic Institute (obtained from
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Clindamycin Metronidazole
Tracy Wilkins, Virginia Polytechnic Institute, Blacksburg, Va.). Organisms were identified and subspeciated according to methods previously described [17]. Twenty-one isolates were Bacteroides fragilis subspecies fragilis, five were subspecies thetaiotaomicron, five were subspecies distasonis, and one was subspecies vulgatus. Antimicrobial agents. Stock solutions of the following antimicrobials were prepared with use of susceptibility testing powder supplied by the respective manufacturers and sterile distilled water: amikacin (Bristol Laboratories, Syracuse, N.Y.), carbenicillin (Roerig, New York, N.Y.), cefoxitin (Merck Sharp & Dohme, West Point, Pa.), chloramphenicol (Parke-Davis, Detroit, Mich.) , clindamycin (Upjohn, Kalamazoo, Mich.), metronidazole (Amersham/Searle, Arlington Heights, Ill.), penicillin G (Eli Lilly and Co., Indianapolis, Ind.), and tetracycline (Pfizer, New York, N.Y.). Erythromycin (Eli Lilly) was prepared in 95 % ethanol. Stock solutions were stored at - 20 C for a maximum of four weeks prior to use, except for carbenicillin, which was stored at - 70 C, and metronidazole, which was freshly prepared on the day of testing. AntimicroMal plates for bacteriostatic tests. After thawing, antimicrobial stock solutions were diluted in serial twofold steps with sterile distilled water; appropriate dilutions were added to tubes containing brucella agar (Pfizer), laked sheep blood (5%), and vitamin K 1 (10 llg/ml). The final volume of 20 ml was poured into 100- X 15-mm petri plates; plates were dried at 35 C for 30 min before inoculation. For each combination tested, plates were prepared to complete a checkerboard series of twofold dilutions of antimicrobials within the concentration ranges indicated in table 1. Plates were also prepared with the antimicrobial agents incorporated singly for determination of individual MICs. All plates were prepared on the day of inoculation. Control plates were also prepared with use of sterile distilled water in place of antimicrobial solutions. Inoculation, incubation, and interpretation. Organisms were prepared according to procedures outlined in the Wadsworth Anaerobic Bacteriology Manual [17], and plates were inoculated with use of a Steers replicator [18]. Control plates were inoculated before and after the inoculation of
323
Drug Combinations against B. fragilis
Results
Bacteriostatic tests. MICs obtained with the individual antimicrobial agents correlated well
with results previously reported from this laboratory [5, 20-22]. Specifically, all strains of B. fragilis tested were inhibited by concentrations of clindamycin (~4 ~g/ml), cefoxitin (~64 ~g/ ml), chloramphenicol (~16 ~g/ml), and metronidazole (~8 I-lg/ml) achievable in serum with standard dosage regimens. Two of the 32 strains tested were resistant to 256 I-lg of carbenicillin/ ml and also to 256 units of penicillin/ml. The remaining 30 strains were all inhibited by ~64 ~g of carbenicillin/ml and by ~64 units of penicillin/m!. A bimodal distribution of susceptibility to tetracycline was seen, with only 15 of the 32 strains inhibited at concentrations of ~8 ~g/ml. Certain isolates required as much as 16 ~g of erythromycin/ml for inhibition. Amikacin was inactive against all strains at concentrations of ~512 ~g/ml.
Of the nine combinations tested, three (amikacin plus clindamycin, amikacin plus carbenicillin, and amikacin plus cefoxitin) showed complete indifference in their activity against B. fragilis. Amikacin neither enhanced nor antagonized the activity of the other three antibiotics. Three combinations demonstrated synergism with only a few strains by the criterion of a fourfold reduction in the MIC for each drug; individual isobolograms constructed for these organisms also consistently indicated synergism. Two of 27 strains yielding interpretable data showed synergism of penicillin and tetracycline, three of 30 showed synergism of cefoxitin and metronidazole, and four of 28 showed synergism of carbenicillin and metronidazole. Only one isolate showed synergism of two of these three combinations. Table 2 describes the distribution of synergistic strains among the four subspecies of B. tragilis tested. No antagonism was seen. Composite data indicating indifference of these three combinations is represented in isobolograms (figure 1). With all but two of 19 strains that had an end point with clindamycin plus chloramphenicol, these drugs showed at least an additive effect; results with six strains met the criteria for synergism (figure 2, top left). Erythromycin and metronidazole were synergistic against 10 of 30 strains with interpretable results (figure 2, bottom left).
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each antimicrobial series. One set of control plates was incubated aerobically. Parallel sets of control plates and all antimicrobial plates were placed in GasPak® jars (Baltimore Biological Laboratories, Baltimore, Md.); anaerobiosis was achieved with GasPak envelopes (BBL). Plates were read after incubation for 42-48 hr at 35 C. The MIC for an organism was considered to be the lowest concentration of drug yielding no growth, a barely visible haze, or one discrete colony [19]. For evaluation of antimicrobial combinations, MICs were determined for each drug at all tested concentrations of the second drug. Evaluation of drug interaction. For individual strains, an effect was considered to be synergistic when an organism was inhibited by a combination of two agents, each at a concentration of ~25 % of its MIC when tested alone. Results could not be interpreted when the MIC for either drug was at the lowest or next to lowest concentration tested for that drug. The effect of each combination on the panel of organisms as a whole was evaluated further by construction of an isobologram using the geometric mean of the MICs of each interpretable test for one agent at various concentrations of the other agent. These isobolograms were compared to theoretical "additive" or "indifferent" straight-line isobolograms developed by connecting points on the axes representing the geometric mean MICs for the individual antimicrobial agents. An isobologram approximating this straight line indicates a composite additive or indifferent effect of the antimicrobial combination on the panel of organisms. Bowing of the isobologram toward the origin is indicative of synergism, while bowing away from the origin would suggest antagonism. Bactericidal tests. The test procedure described by Nastro and Finegold [5] was used for the study of six isolates of B. fragilis for bactericidal end points with clindamycin and metronidazole alone and in combination. (Clindamycin was tested in a concentration of 2 ~g/ml, and the concentration of metronidazole was varied from 4 ~g/ml to 0.125 ~g/ml in twofold steps.)
324
Busch, Sutter, and Finegold
Table 2.
Inhibition of subspecies of Bacteroides fragilis by six combinations of antimicrobial agents. Antimicrobial combinations tested *
Subspecies of B. fragilis
Penicillin + tetracycline
21 5 5 1 32
1/17 0/5 1/5 0/0 2/27
fragilis thetaiotaomicron distasonis vulgatus Total
Cefoxitin + metronidazole
1/20 0/5 2/5 0/0 3/30
Carbenicillin + metronidazole
Chloramphenicol + clindamycin
Erythromycin+ metronidazole
3/18 1/5 0/5 0/0 4/28
3/9 3/5 0/5 0/0 6/19
7/20 0/5 3/5 0/0 10/30
Clindamycin+ metronidazole
7/8 2/4 3/5 0/0 12/17
'" Data are given as number of strains demonstrating synergism/number of strains with interpretable results. Results could not be interpreted when the MIC for either drug was at the lowest or next to lowest concentration tested for that drug.
Bfra9.i!!.§.
6
28 STRAINS
8. tragi/is 27 STRAINS
e.....
~
~ 30 ~
U
4-
z
~
a:
20
~
10
+---r---,.----,.------~___,
.25.5
1
2
METRONIDAZOLE (Pv/ml) 20 30 PENICILLIN (units/mil
40
Figure 1. Isobolograms showing inhibitory activity of three nonsynergistic combinations of antimicrobial agents against strains of Bacteroides fragilis that achieved end points. Points indicate the geometric mean MIC for one antimicrobial agent (ordinate) at the concentration of the second agent, shown on the abscissa.
B. fragpis 30 STRAINS loJ
5
~
1
z fE I-
loJ
:E
.5 .25
,'125
2
4
6
8
16
CEFOXITIN }Ig/ml
The combination of c1indamycin and metronidazole demonstrated synergism against 76% of the strains that had an end point with this pair of drugs. Synergism was identified in strains of
B. fragilis subspecies fragilis, thetaiotaomicron, and distasonis (table 2). These two drugs in combination had at least an additive effect against all strains with interpretable results and in no case were antagonistic. All 32 strains tested were inhibited in the presence of the combination of 0.5 I-lg of clindamycin/ml plus 0.5 I-lg of metronidazole/ml (figure 2, top right). Bactericidal tests. Table 3 shows the results of the bactericidal tests with clindamycin, metronidazole, and the combination of the two drugs. All six strains had minimal bactericidal concentrations (MBCs) of ~2-4 I-lg/ml with each drug singly. For three of the six strains, the combination was synergistic with respect to
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No. of strains tested
Drug Combinations against B. fragilis
325
1.25
1 8. fragi/is
E .....
8. frogjlis 19 STRAINS
17 STRAINS
co
5 z 0 >~
« 0 z
.5
0
.25 .125
•
~
•
+--r--,----,r------,-----==~..,
.25.5
I
2
4
8
CHLORAMPHENICOL (JlQ/mf)
30 STRAINS
2
01 ~
+---r------,r---......,.--~--..,..;~I.__,
2 3 4 ERYTHROMYCIN (JoIo/m!)
I
1.75
METRONIDAZOLE (J'Q/ml)
Figure 2. Isobolograms showing inhibitory activity against Bacteroides fragilis of three antimicrobial combinations that demonstrated synergism against individual strains. Points indicate the geometric mean MIC for one antimicrobial agent (ordinate) at the concentration of the second agent, shown on the abscissa.
Bfrogpis
E .......
.125.25.5
5
6
MBC; the combination was neither synergistic nor antagonistic for one strain; and in two cases the results were not interpretable. All six strains were killed in the presence of 2 !lg of c1indamycin/ml plus 1 !lg of metronidazole/ml. Discussion
The complex nature of the flora of the lower intestine often leads to the use of multiple antibiotic agents in the treatment of infections due to disruption of intestinal mucosal barriers. Often, treatment must be initiated empirically before results of cultures are known. The aminoglycosides, which are commonly used in this setting and are represented in this report by amikacin, have broad-spectrum activity against gram-negative
aerobic and facultatively anaerobic bacteria but are recognized to be essentially inactive by themselves against most anaerobes -and particularly against B. fragilis [22]. Aminoglycosides often enhance the activity of other antibiotics, such as the penicillins and cephalosporins, against organisms such as streptococci [23, 24], staphylococci [25], and enteric bacilli [26]. Whereas Fass et al. [15] have reported in vitro synergism of the combination of c1indamycin and the aminoglycoside gentamicin against a single strain of B. fragilis, in the present study synergism was not observed against B. fragilis with the combination of c1indamycin and amikacin. Leigh [16] has likewise indicated an indifferent interaction (neither synergism nor antagonism) of clindamycin and gentamicin against B. fragilis. These findings are comparable also to those of Sabath and Toftegaard [27], who demonstrated in vitro indifference of clindamycin plus gentamicin against Clostridium perfringens, a c1indamycin-resistant isolate of Staphylococcus aureus, and certain gram-negative bacilli. A similar lack of interaction was seen when amikacin was combined with carbenicillin and when amikacin was combined with cefoxitin, a new cephamycin that has shown consistent in vitro activity against B. fragilis [20, 28]. The absence of antagonism demonstrated with these
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::::i
\
Busch, Sutter, and Finegold
326
Table 3.
Bactericidal activity of clindamycin and metronidazole against six strains of Bacteroides fragilis. Minimal bactericidal concentration (r-tg/ml) of
WAL * strain no. (subspecies of B. fragilis)
(thetaiotaomicron) (fragilis) (distasonis) (thetaiotaomicron) (thetaiotaomicron) (thetaiotaomicron)
* WAL =
>4 >4 4 >4 4 >4
Metronidazole 2 1
4 2
4 4
0.5 1 :::;;0.125 0.25 0.5 1
Wadsworth Anaerobic Bacteriology Laboratory.
combinations in this in vitro study is reassuring in view of their extensive use in the treatment of abdominal infections in which B. fragilis is often only one of a number of potentially pathogenic bacteria that may be recovered from a single specimen. Combinations of antimicrobial agents are employed in these infections since there is insufficient information concerning the relative importance of individual organisms in mixed infections to allow treatment that does not cover both the anaerobic and aerobic organisms encountered; in addition, there are no currently available antibiotics that individually encompass this spectrum of organisms adequately. These in vitro data indicate that the commonly used aminoglycosides are not likely to antagonize the action of clindamycin, carbenicillin, or cefoxitin against B. fragiZis. The general lack of synergism seen with combinations including the cell wall-active agents (represented here by penicillin, carbenicillin, and cefoxitin) was somewhat surprising. In particular, the combination of penicillin and tetracycline showed indifferent activity against B. fragilis. In some medical centers this combination has long been advocated in therapy of intraabdominal infections [2]. In recent years up to 70% of isolates of B. fragilis have been found to be resistant to tetracycline [5, 21, 29]. Since penicillin does not (at least in in vitro tests) improve the activity of tetracycline against B. fragilis, this combination would seem to be inappropriate for treatment of serious intraabdominal infections in the absence of information about the antimicrobial susceptibility of individual isolates and in the absence of clear-cut clinical proof of effectiveness of this combination.
In contrast to the penicillins and cefoxitin, erythromycin and clindamycin act synergistically in vitro in combination with certain other agents. The combination of clindamycin and chloramphenicol showed synergism against 32% of strains of B. fragilis that demonstrated an end point in this study; however, these drugs have characteristics that will probably limit any potential clinical usefulness of the combination. The combinations of clindamycin plus metronidazole and erythromycin plus metronidazole were synergistic. With the presence of metronidazole in these pairs, a bactericidal effect could be anticipated with either combination [5]. In this study, the combination of clindamycin and metronidazole did in fact demonstrate bactericidal synergism against three of six strains examined. The mechanism of this interaction is speculative at present. Clindamycin acts by binding to the 50S ribosomal subunit, thus inhibiting binding of amino acids to the ribosomes with resultant suppression of bacterial protein synthesis [30]. Clindamycin may also interact with peptide chain initiation associated with the formation of the 30S ribosomal complex [31]. The mechanism of action of metronidazole against anaerobic bacteria is not fully understood, although it is thought to act as an electron acceptor in phosphoroclastic reactions specific to anaerobic organisms [32, 33]. Ings has shown labeled metronidazole to be bound to the DNA and protein of Trichomonas vaginaZis but not to the RNA of the organism [34]. These studies suggest inhibition of nucleic acid synthesis by metronidazole. Some investigators suggest that an intermediate compound such as hydroxyalanine [34] or a nitrofurazone [35] may actually be responsible for the inhibition. Thus
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2380 2461 2661 2750 2923 2926
Clindamycin
Metronidazole in the presence of 2 r-tg of clindamycin/ml
Drug Combinations against B. fragilis
References 1. Sonnenwirth, A. C. Incidence of intestinal anaerobes in blood cultures. In A. Balows, R. M. DeHaan, V. R. Dowell, Jr., and L. B. Guze [ed.]. Anaerobic bacteria, role in disease. Charles C Thomas, Springfield, Ill., 1968, p. 157-172. 2. Fullen, W. D., Hunt, J., Altemeier, W. A. Prophylactic antibiotics in penetrating wounds of the abdomen. J. Trauma 12:282-289, 1972. 3. FeIner, J. M., Dowell, V. R., Jr. Anaerobic bacterial endocarditis. N. Engl. J. Med. 283: 1188-1192, 1970. 4. Nastro, L. J., Finegold, S. M. Endocarditis due to anaerobic gram negative bacilli. Am. J. Med. 54: 482-496, 1973. 5. Nastro, L. J., Finegold, S. M. Bactericidal activity of five antimicrobial agents against Bacteroides fragilis. J. Infect. Dis. 126: 104-107, 1972. 6. Whelan, J. P. F., Hale, J. H. Bactericidal activity of metronidazole against Bacteroides fragilis. J. Clin. Pathol. 26:393-395, 1973. 7. Ueno, K., Ninomiya, K., Suzuki, S. Antibacterial activity of metronidazole against anaerobic bacteria. Chemotherapy (Japan) 19: 111-114, 1971. 8. Tally, F. P., Sutter, V. L., Finegold, S. M. Treatment of anaerobic infections with metronidazole. Antimicrob. Agents Chemother. 7:672-675, 1975.
9. Videau, D. Association metronidazole-spiramycine sur les germes anaerobies. Pathol. BioI. (Paris) 19:661-666, 1971. 10. Cros, P., Freidel, M., Jacquemard, R. Interet de l'association synergique metronidazole-spiramycine en pratique courante odonto-stomatologique. Rev. Fr. Odontostomatol. 18: 1039-1048, 1971. 11. Chikhani, P. Etude d'une association antibacterienne hautement synergique. Rev. Stomatol. Chir. Maxillofac. 72: 513-522, 1971. 12. LaUfer, J., Mignon, H., Videau, D. L'association metronidazole-spiramYcine: concentrations et synergie in situ comparees aux CMI de la flore buccale. Rev. Stomatol. Chir. Maxillofac. 74:387392, 1974. 13. Videau, D., Blanchard, J.-c., Sebald, M. Flore buccodentaire: sensibilite aux antibiotiques et interet de l'association spiramycine-metronidazole. Ann. Microbiol. (Paris) 124:505-516, 1973. 14. Garcia Perla, A., Gomez de la Mata, J. Experiencias clinicas con la asociacion spiramicina-metronidazol. An. Esp. Odontoestomatol. 32:239-242, 1973. 15. Fass, R. J., Rotilie, C. A., Prior, R. B. Interaction of clindamycin and gentamicin in vitro. Antimicrob. Agents Chemother. 6:582-587, 1974. 16. Leigh, D. A. Bacteroides infections. Lancet 2: 10811082, 1973. 17. Sutter, V. L., Vargo, V. L., Finegold, S. M. Wadsworth anaerobic bacteriology manual. 2nd ed. University of California, Los Angeles, Extension Division, 1975. 106 p. 18. Steers, E., Foltz, E. L., Graves, B. S. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311, 1959. 19. Ericsson, H. M., Sherris, J. C. Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol. Microbiol. Scand. [Suppl.] 217:1-90,1971. 20. Sutter, V. L., Finegold, S. M. Susceptibility of anaerobic bacteria to carbenicillin, cefoxitin, and related drugs. J. Infect. Dis. 131:417-422, 1975. 21. Sutter, V. L., Kwok, Y.-Y., Finegold, S. M. Susceptibility of Bacteroides fragilis to six antibiotics determined by standardized antimicrobial disc susceptibility testing. Antimicrob. Agents Chemother. 3: 188-193, 1973. 22. Finegold, S. M., Sutter, V. L. Susceptibility of gramnegative anaerobic bacilli to gentamicin and other aminoglycosides. J. Infect. Dis. 124(Suppl.) :S56S58, 1971. 23. Durack, D. T., Pelletier, L. L., Petersdorf, R. G. Chemotherapy of experimental streptococcal endoc;arditis. II. Synergism between penicillin and streptomycin against penicillin-sensitive streptococci. J. Clin. Invest. 53:829-833, 1974. 24. Sapico, F: L., Keys, T. F., Hewitt, W. L. Experimental enterococcal endocarditis. II. Study of in vivo synergism of penicillin and streptomycin. Am. J. Med. Sci. 263: 128-135, 1972.
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the combination of clindamycin and metronida~ zole may be synergistic by virtue of action at sites of both nucleic acid synthesis and protein synthesis. The data from in vitro tests reported in this communication extend the observation of antimicrobial interactions against anaerobic bacteria to B. fragilis, a dominant member of the bowel flora and a pathogen frequently encountered in intraabdominal infections, gynecological infections, and abdominal sepsis and occasionally encountered in soft-tissue infections and endocarditis. It is of interest that no antagonism was observed. Although essentially all strains of B. fragilis can currently be inhibited by easily obtainable concentrations of several antimicrobial agents, combinations such as clindamycin plus metronidazole may prove useful in the treatment of selected infections such as endocarditis, septic thrombophlebitis, and osteomyelitis, in which B. fragilis is implicated as a single or primary pathogen and for which maximal antibacterial activity is desired. The combination might also become useful in the event of increased resistance of B. fragilis to individual antimicrobial agents.
327
328
30. Weisblum, B., Davies, J. Antibiotic inhibitors of the bacterial ribosome. Bacteriol. Rev. 32:493-528, 1968. 31. Reusser, F. Effect of lincomycin and clindamycin on peptide chain initiation. Antimicrob. Agents Chemother. 7:32-37, 1975. 32. O'Brien, R. W., Morris, J. G. Effect of metronidazole on hydrogen production by Clostridium acetobutylicum. Arch. Mikrobiol. 84:225-233, 1972. 33. Edwards, D. 1., Dye, M., Crane, H. The selective toxicity of antimicrobial nitroheterocyclic drugs. J. Gen. Microbiol. 76: 135-145, 1973. 34. Ings, R. M. J., McFadzean, J. A., Ormerod, W. E. The mode of action of metronidazole in Trichomonas vaginalis and other micro-organisms. Biochem. Pharmacol. 23: 1421-1429, 1974. 35. Tanowitz, H. B., Wittner, M., Rosenbaum, R. M., Kress, Y. In vitro studies on the differential toxicity of metronidazole in protozoa and mammalian cells. Ann. Trop. Med. Parasitol. 69: 19-28, 1975.
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25. Sande, M. A., Johnson, M. L. Antimicrobial therapy of experimental endocarditis caused by Staphylococcus aureus. J. Infect. Dis. 131:367-375, 1975. 26. Klastersky, J., Cappel, R., Daneau, D. Clinical significance of in vitro synergism between antibiotics in gram negative infections. Antimicrob. Agents Chemother. 2:470-475, 1972. 27. Sabath, L. D., Toftegaard, 1. Rapid microassays for clindamycin and gentamicin when present together and the effect of pH and of each on the antibacterial activity of the other. Antimicrob. Agents Chemother. 6:54-59, 1974. 28. Tally, F. P., Jacobus, N. V., Bartlett, J. G., Gorbach, S. L. Susceptibility of anaerobes to cefoxitin and other cephalosporins. Antimicrob. Agents Chemother. 7: 128-132, 1975. 29. Martin, W. J., Gardner, M., Washington, J. A., II. In vitro antimicrobial susceptibility of anaerobic bacteria isolated from clinical specimens. Antimicrob. Agents Chemother. 1: 148-158, 1972.
Busch, Sutter, and Finegold
