ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1978, p. 609-613

0066-4804/78/0014-0609$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 14, No. 4

Printed in U.S.A.

Comparative Susceptibilities of Anaerobic Bacteria to Metronidazole, Ornidazole, and SC-28538 ELLIE J. C. GOLDSTEIN,t VERA L. SUTTER, AND SYDNEY M. FINEGOLD* Medical and Research Services, Wadsworth Hospital Center, Veterans Administration, and the University of California School of Medicine, Los Angeles, California 90073 Received for publication 11 May 1978

The susceptibilities of 284 anaerobic bacteria, including 55 strains of the Bacteroides fragilis group, were determined by an agar dilution technique to metronidazole and two newer nitroimidazoles, ornidazole and SC-28538. All three agents showed marked in vitro activity against virtually all anaerobic bacteria tested. At concentrations c1 ,ig/ml, SC-28538 was significantly more active than either rnetronidazole or ornidazole. At concentrations of >1 ,ig/ml, the activities of all three agents were comparable. Propionibacterium and Actinomyces showed significant resistance to all three agents. Anaerobic and microaerophilic members of the genus Streptococcus were also often resistant, in contrast to Peptococcus and Peptostreptococcus strains. In addition, the bactericidal activities of ornidazole and SC-28538 were determined against 27 strains of the B. fragilis group by a broth dilution technique. The minimal inhibitory and minimal bactericidal concentrations of each agent were very close. At concentrations of c0.5 jg/ml, SC-28538 showed greater bactericidal activity; at concentrations of -2 jLg/ml, the activies of both agents were similar.

Although penicillin is the drug of choice in most non-Bacteroides fragilis anaerobic infections, a number of patients are allergic to penicillin. Both chloramphenicol and clindamycin. alternative agents active against anaerobic bacteria (including B. fragilis), produce infrequent but significant toxicity. Consequently, there is a need for the development of safe and effective antimicrobial agents for therapy of anaerobic infections. Metronidazole was the first 5-nitroimidazole compound shown to be active in vitro against most anaerobic bacteria (10, 15, 19). It is presently available for therapy of anaerobic infections in Europe (6, 7) and is undergoing clinical trials in the United States (8, 15). Several other nitroimidazole compounds have been developed more recently. We report the in vitro susceptibilities of 284 strains of anaerobic bacteria, including 55 strains of the B. fragilis group, to metronidazole and two newer nitroimidazole compounds, ornidazole and SC-28538 (Searle). We also determined the bactericidal activities of ornidazole and SC-28538 against 27 strains of the B. fragilis group. MATERIALS AND METHODS Bacterial strains. A total of 284 strains of anaerobic bacteria was used in this study (Table 1). The B. t Present address: Infectious Disease Division, SUNYDownstate Medical Center, Brooklyn, NY 11203.

fragilis group consisted of B. distasonis, B. fragilis, B. thetaiotaomicron, and B. vulgatus species. All isolates were recovered from clinical specimens received in 1975 and 1976. These bacteria were identified according to the criteria of the Wadsworth Anaerobic Bacteriology Manual (14) and the Virginia Polytechnic Institute's Anaerobe Laboratory Manual (4). Antimicrobial agents. (i) Metronidazole. Metronidazole (Fig. 1) was introduced as an antitrichomonad in 1959 (2). Subsequently, it was shown to be active in vitro (3, 13, 15, 19, 27) and in vivo (6-8, 15) against a wide variety of anaerobic bacteria. Its pharmacokinetics have been well studied previously (9, 11, 16). (ii) Ornidazole. Ornidazole (Fig. 1) is a recently synthesized nitroimidazole derivative with antitrichomonad activity (12). It has been previously tested and found to be active against several strains of anaerobic bacteria (20). The pharmacokinetics (pKa, protein binding, volume of distribution, and peak serum levels) and metabolism of ornidazole are similar to those of metronidazole (11). It is a light-yellow, crystalline substance with a melting range of 74 to 79°C. A 1% aqueous solution has a pH of approximately 6.6. In water its solubility is 2.4% at 25°C, whereas in 95% ethanol its solubility is >50%. Ornidazole is slightly more soluble in water than metronidazole. The mean half-life of elimination from human plasma is 1.7 times greater than that of metronidazole (14.4 h for ornidazole; 8.4 h for metronidazole); this difference is statistically significant (11). (iii) SC-28538. SC-28538 was prepared as outlined previously (17, 18). Its solubility in water at pH 7 is approximately 0.1%, whereas its solubility in organic 609

610

A

GOLDSTEIN, SUTTER, AND FINEGOLD

S

iii

I

_-

_

_-

ANTIMICROB. AGENTS CHEMOTHER.

18888111888888888888 4 v-4 v_ _4 _4

-4

v-

"-

v_

"4

_-

_-

_-

II

I I

I I

r-

eq

I

I I I

I

I

I

I

I

I

I

I

I

I

I

I

I

I I

II I

I

I I

I I

I

OI

O

II

I

I II

k0 0

0II

.2 Cs .0

..

laCC

0 Co

0 0

9 SL 8

,,,

II

L

7

I

OC

18

C,,

IaI

8 8 "MO99'RO_II g l

8

t 0C0

- -C

I

I

I

0

elC' l CIRc

IC o

Ct Co

I I I

I

8 88 ,,,, II, 1111 ,,,

-4

0

oo C.

II

I

co

0

0

'

I

.I~0

I

'''18

I

C.) co

co C

9

g

craq ; e 0I 0I

0 C

0

C

E0N

0.

- -

0Gb eq10

0 0. 0

C)

O~ clc CV 0)O u:7 0)

, 0

ula

esI

I

10t"ilp to t

I

0CoCoa: 0 a:0

troi W

2

O0

Ob ob

0 0 CJ4Z-

co

o

01q

I?

1 rt- 0

I. ql

0

0

eq

_ in t0

L

C',0)t-fl10eto "

o

o

eq r- eq

CooI,

I II

00)55550

H

N

cims eys

0)

C.1

I

0

I

I1

0 I 1G bC oeoeq 1

10

t t. t-

0

0

I

10 eq

t-

D05qCOCOCD4104'1

|

'.

I

_ CD

0 m

eD

la )C

C

.0

Ca

O0 i

iIjto

Cl to

1-0

co

co

co

cr

C9

co

Ceq

"-4

I"

CD

4)

z

~

~

~

.2n

OS0

Ca

D to

0

} i i 9.03.l

0

0

t

ANAEROBIC BACTERIAL SUSCEPTIBILITIES

VOL. 14, 1978 H-C -N4 n C-CH N02-C - N

CH2CH20H Metronidazole ( F1agyl1), 1-( 2-hydroxyethyl) 2-methyl5-nitroimidazole.

H-C - Nw II

N02-C - N'

C-CH3

CH2CHOH - CH2Cl

Ornidazole ( Tiberal (a), 1-(3-chloro-2-hydroxypropyl) 2-methyl-5 nitroimidazole COO Na+

H-C - N4 N

N02 -C-_N

C

-

S(H)2 CH)

0

CH3 Searle SC 28538, 4-[ 2- (1-methyl-5 nitro-2-imidazolylthio) ethoxy] benzoate

FIG. 1. Structures of the three nitroimidazole derivatives.

611

spectively, whereas 94.5% were susceptible to SC-28538. Most of the other bacteria were susceptible to metronidazole concentrations of 16 Lg/ml, including B. melaninogenicus, fusobacteria, and clostridia. Only 90.9% of Lactobacillus species and 50% of Actinomyces species tested were susceptible to metronidazole at 16 ,ug/ml. None of the five strains of anaerobic or microaerophilic streptococci and none of the four strains of Propionibacterium tested were susceptible to metronidazole at this level. The activities of ornidazole and SC-28538 at levels of 2 ,ig/ml or greater were essentially comparable to that of metronidazole for the bacteria tested. At levels of 16,ug/ml, 90.9% of Lactobacillus species were susceptible to both ormidazole and SC-28538, whereas 75 and 25% of Actinomyces species were susceptible to ornidazole and SC-28538, respectively. At the same level, all five strains of anaerobic or microaerophilic cocci were resistant to ornidazole, whereas three (60%) were susceptible to SC-28538. Broth dilutions. The susceptibilities of 27 strains of the B. fragilis group to ornidazole and SC-28538 are illustrated in Fig. 2. MIC and MBC curves for each agent correlated closely. The MBC was frequently one dilution greater than the MIC. At concentrations of 2 ytg/ml or greater, the activities of both agents were similar; however, at concentrations of 0.5 yg/ml or less, there was much greater activity of SC28538. At 0.5,/g/ml, 88% of strains of B. fragilis group were inhibited (MIC) by SC-28538, whereas only 48% of strains were inhibited (MIC) by ornidazole; about 30% were killed by both drugs at this concentration.

solvents is limited (personal communication, R. D. Muir, G. D. Searle and Co., Chicago, Ill.). The melting point is not sharp and is over 280°C. The metabolism and pharmacokinetics of SC-28538 have not been studied previously but are thought to be similar to those of other nitroimidazole compounds (R. D. Muir, personal communication). Ornidazole was kindly supplied by Hoffman-LaRoche and Co., Nutley, N. J., and metronidazole and SC-28538 were supplied by G. D. Searle and Co., Chicago, Ill. Antimicrobial susceptibility tests. Minimal inhibitory concentrations (MICs) of all three agents were determined by the agar dilution method as described previously (14). Antibiotic solutions were freshly prepared for each test, using sterilized distilled DISCUSSION water as the solvent. In addition, 27 strains of B. Metronidazole has significant in vitro activity fragilis were tested by a previously described twofold broth dilution method (14) for MIC and minimal bac- against virtually all obligate anaerobes at readily tericidal concentrations (MBCs) of ornidazole and SC- achievable blood levels (13, 20). Previously the 28538. B. fragilis (ATCC 29327) was used as a con- microaerophilic and anaerobic streptococci, Actrol organism for fast growers, and Peptococcus mag- tinomyces and Propionibacterium have been renus (ATCC 29328) was used for slow growers. ported to be resistant (13); our data are in agreement with those observations. Although a recent RESULTS report (5) notes relative reistance of one strain Agar dilution MICs. The activities of the of B. fragilis to metronidazole, none of our isothree drugs against the anaerobes tested are lates demonstrated resistance. shown in Table 1. At higher concentrations, Metronidazole is the standard against which there was little difference in the percent suscep- other nitromidazole compounds are compared. tibility, whereas at lower concentrations, there On a weight basis at concentrations >1 ,ug/ml, was a marked difference in percent susceptibil- the activity of both ornidazole and SC-28538 was ity. At a 16-,ug/ml concentration, a level achiev- comparable to that of metronidazole. At concenable in the blood with metronidazole, all 55 trations s1 ,tg/ml, SC-28538 was significantly strains of the B. fragilis group were susceptible more active than either ornidazole or metronito all three agents. However, at 1-,ug/ml concen- dazole; however, since the pharmacokinetics of tration, only 43.6 and 61.8% of the strains were SC-28538 have not been delineated, it is uncersusceptible to metronidazole and ornidazole, re- tain whether this will present any clinical advan-

612

GOLDSTEIN, SUTTER, AND FINEGOLD

ANTIMICROB. AGENTS CHEMOTHER.

27-

0

Lii

20-

I z (I-)

z

I5

A

ORNIDAZOLE

X

MBC

°

H

SC 28538

(I) 0

IL

0

z

MIC A MBC A

0

A

X

a

/

31. 0.062

.125

2.0 4.0 5 1.0 .25 ANTIMICROBIAL CONC (,jgm/ml)

8.

160

32.0

FIG. 2. Cumulative susceptibility of 27 strains of B. fragilis (MIC, MBC) to ornidazole and SC-28538 by broth dilution technique.

tage. The longer serum half-life of ornidazole (11) would permit a more convenient (i.e., longer) dosage interval than would metronidazole. Ornidazole has had limited clinical trials (1). One report of ornidazole therapy of a case of B. fragilis meningitis showed good cerebrospinal fluid penetration and a successful clinical response (1). The MIC and MBC curves for the susceptibilities of 27 strains of the B. fragilis group to ornidazole and SC-28538 were closely correlated. This correlation was comparable to that of metronidazole as reported previously (10), where the MIC and MBC for 57 strains of B. fragilis were identical or one tube dilution apart. Wurst (20) reported the susceptibilities of 114 strains of anaerobes to ornidazole and metronidazole. All of his strains, except seven isolates of P. acnes, were inhibited by c3.1 jig of both agents. Our data showed that a number of strains of the B. fragilis group, B. melaninogenicus, Peptococcus, Clostridium species, and Eubacterium species were not inhibited at c4 ,ig/ml. The Propionibacterium, Actinomyces, and anaerobic and microaerophilic streptococcal strains tested also showed significant resistance to ornidazole and SC-28538. However, SC-28538 showed greater activity against the microaerophilic and anaerobic streptococci. Both ornidazole and SC-28538 showed marked in vitro activity against virtually all anaerobes tested and may prove valuable in treatment of anaerobic infections.

a

ACKNOWLEDGMENTS This study was supported by the Veterans Administration Medical Research Service and grants from G. D. Searle and Co., Chicago, Ill., and Hoffman-LaRoche and Co., Nutley, N.J. LITERATURE CITED 1. Cadoz, M., B. Deborne, C. LaFaix, et al. 1977. Traitement par l'ornidazole injectable d'une meningite A Bacteroides fragilis. Nouv. Presse Med. 6:2438-2439. 2. Corsar, C., and L. Julou. 1959. Activite de 1'(hydroxy-2 ethyl)-1-methyl-2-nitro-5-imidazole (8.823 R.P.) vis-a-

3.

4.

5. 6.

7.

8.

9.

vis des infections experimentales a Trichomonas vaginalis. Ann. Inst. Pasteur 96:238-241. Dornbusch, K., C-E. Nord, and T. Wadstrom. 1974. Biochemical characterization and in vitro determination of antibiotic suscetibility of clinical isolates of Bacteroides fragilis. Scand. J. Infect. Dis. 6:253-258. Holdeman, L. V., and W. E. C. Moore (ed.). 1975. Anaerobe laboratory manual, 3rd ed. Virginia Polytechnic Institute and State University, Blacksburg. Ingham, H. R., S. Eaton, C. W. Venables, and P. Adams. 1978. Bacteroides fragilis resistant to metronidazole after long-term therapy. Lancet i:214. Ingham, R., G. E. Rich, J. B. Selkon, et al. 1975. Treatment with metronidazole of three patients with serious infections due to Bacteroides fragilis. J. Antimicrob. Chemother. 1:235-242. Ingham, H. R., J. B. Selkon, and C. M. Roxby. 1977. Bacteriological study of otogenic cerebral abscesses: chemotherapeutic role of metronidazole. Br. Med. J. 2:991-993. Ledger, W. J., C. Gee, P. A. Pollin, et al. 1976. A new approach to patients with suspected anaerobic postpartum pelvic infections. Transabdominal uterine aspiration for culture and metronidazole for treatment. Am. J. Obstet. Gynecol. 126:1-6. Muller, M., D. G. Lindmock, and J. McLaughlin. 1977. Mode of action of metronidazole on anaerobic organisms, p. 12-19. In S. M. Finegold, J. McFadzean, and F. J. C. Roe (ed.), Metronidazole. Proceedings of the International Metronidazole Conference. Excerpta

VOL. 14, 1978

ANAEROBIC BACTERIAL SUSCEPTIBILITIES

Medica, Princton, N. J. 10. Nastro, L. J., and S. M. Finegold. 1972. Bactericidal activity of five antimicrobial agents against Bacteroides fragilis. J. Infect. Dis. 126:104-107. 11. Schwartz, D. E., and F. Jenney. 1976. Comparative pharmacokinetic studies of ornidazole and metronidazole in man. Chemotherapy 22:19-29. 12. Skold, M., H. Gnarpe, and L. Hillstrom. 1977. Ornidazole: a new antiprotozoal compound for treatment of Trichomonas vaginalis infection. Br. J. Vener. Dis. 53:44-48. 13. Sutter, V. L., and S. M. Finegold. 1976. Susceptibility of anaerobic bacteria to 23 antimicrobial agents. Antimicrob. Agents Chemother. 10:736-752. 14. Sutter, V. L., V. L. Vargo, and S. M. Finegold. 1975. Wadsworth anaerobic bacteriology manual, 2nd ed. Department of Continuing Education, University Extension and School of Medicine, University of California, Los Angeles. 15. Tally, F. P., V. L. Sutter, and S. M. Finegold. 1972.

16.

17.

613

Metronidazole versus anaerobes. In vitro data and initial clinical observations. Calif. Med. 117:22-26. Templeton, R. 1977. Metabolism and pharmacokinetics of metronidazole: a review, p. 28-49. In S. M. Finegold, J. A. McFadzean, and F. J. C. Roe (ed.), Metronidazole. Proceedings of the International Metronidazole Conference. Excerpta Medica, Princeton, N. J. Tweit, R. C., E. M. Kreider, and R. D. Muir. 1973. Synthesis of antimicrobial nitroimidazolyl 2-sulfides, sulfoxides, and -sulfones. J. Med. Chem. 16:1161-1169. Tweit, R. C., R. D. Muir, and S. Ziecina. 1977. Nitroimidazoles with antibacterial activity against Neisseria gonorrhoeae. J. Med. Chem. 20:1697-1700. Ueno, K., K. Ninomiya, and S. Suzuki. 1971. Antibacterial activity of metronidazole against anaerobic bacteria. Chemotherapy (Tokyo) 19:111-114. Wurst, J. 1977. Susceptibility of anaerobic bacteria to metronidazole, ornidazole, and tinidazole and routine susceptibility testing by standardized methods. Antimicrob. Agents Chemother. 11:631-637. -

18. 19.

20.