Comparative in vitro activity of cefoxitin, cefotaxime alone, and in combination with desacetylcefotaxime against the Bacteroides species

33

DIAGN MICROBIOLINFECTDIS 1989;12:33-37

Comparative In Vitro Activity of Cefoxitin, Cefotaxime Alone, and in Combination with Desacetylcefotaxime Against the Bacteroides Species Hanna N. Canawati

The agar dilution method was used to determine the inhibitory activity of cefotaxime (CTX) alone, desacetylcefotaxime (dCTX) alone, CTX plus dCTX, and cefoxitin against 74 clinical isolates of the Bacteroides species recovered from diabetic patients with foot ulcers. The study concluded that the addition of dCTX to CTX increased the inhibitory activity from 45% to 73% for all strains tested and from 50% to 81% among the 32 strains of Bacteroides fragilis. This synergistic interaction against B. fragilis resulted in a four- to nine-fold reduction in the MIC of seven strains (64-128 tzg/ml, resist-

ant category MICs). While the lowest CTX MIC for B. fragilis was 2 tzg/ml (four strains), the addition of dCTX also produced a remarkable reduction in susceptible range CTX MICs to 0.05-2 ~g/ml in 16 strains (50%). The overall susceptibility to cefoxitin and CTX plus dCTX was as follows: 100% and 100% for Bacteroides vulgatus, 50% and 66% for Bacteroides thetaiotaomicron, 100% and 33% for Bacteroides ovatus, and 83% and 82% for Bacteriodes species other than the B. fragilis group.

INTRODUCTION

of bacterial strains including anaerobes (Neu, 1982; Jones et al., 1984; Aldridge et al., 1984; Chin and Neu, 1984). The purpose of this study was 1) to compare the in vitro activity of CTX alone and in combination with dCTX and determine if synergistic interactions of this drug combination do occur against the clinical strains of B. fragilis; and 2) to compare the inhibitory activity of cefoxitin, CTX alone, and CTX plus dCTX against these isolates of the Bacteroides species.

Cefotaxime (CTX) is a "third-generation" cephalosporin with an unusually broad spectrum of activity against Gram-positive bacteria (Jones and Thornsberry, 1982) and Gram-negative bacteria, especially members of Enterobacteriaceae (Jorgensen et al., 1980; Jones and Packer, 1982). Cefotaxime is also relatively active against anaerobic bacteria including Bacteroides fragilis (Jenkins et al., 1982; Tally et al., 1983; Cuchural et al., 1984; O'Keefe et al., 1987). Cefotaxime is partially deacetylated at the position 3 (Fu et al., 1979), and desacetylcefotaxime (dCTX) is formed as the principal metabolite. Numerous reports have shown that dCTX interacts synergistically in vitro with its parent compound (CTX) against several types From the U.S.C. Schoolof Medicine, Los Angeles, and MicrobiologyService, Rancho Los AmigosMedicalCenter, Downey, California. Address reprint requests to: Hanna Canawati, Ph.D., SM(AAM), RanchoLos AmigosMedical Center, 7601 E. Imperial Highway,Downey, CA 90242. Received July 29, 1988; accepted August 18, 1988. © 1989 ElsevierScience Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/89/$3.50

MATERIALS A N D METHODS Organisms All Bacteroides species tested were clinical isolates recovered from foot wounds or decubitus ulcers of patients with diabetes mellitus at the rehabilitation institute of Rancho Los Amigos Medical Center (Downey, CA). The organisms tested were: Bacteroides fragilis (32 strains), B. vulgatus (8 strains), B. ovatus (6 strains), and other Bacteroides species (22 strains). Bacterial strains were isolated from prereduced blood media supplemented with vitamin K

34

H. Canawati

and heroin in the anaerobic glove chamber. Identification of bacteria was performed by conventional methods using API anaerobic strips and other recognized differential criteria. B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741 (American Type Culture Collection, Rockville, MD) were used as control organisms with each test run. Stock organisms were kept in freezer at - 70°C until required for testing.

Antibiotics CTX and dCTX powders were provided by Dr. William Novick, Jr., of Hoechst-Roussel Pharmaceuticals, Inc. (Somerville, NJ). Cefoxitin was supplied by Merck and Co., Inc. (Rahway, NJ). Antibiotics were reconstituted and diluted according to the manufacturer's instructions.

Antimicrobial Susceptibility Tests Minimal inhibitory concentration values of cefoxitin, CTX alone, dCTX alone, and CTX plus dCTX were determined by the agar dilution procedure described by the National Committee of Clinical Laboratory Standards (NCCLS, 1985). The antimicrobics were diluted in unsupplemented Wilkins-Chalgren agar (Oxoid, Columbia, MD) in serial twofold dilutions with the following concentration ranges: cefoxitin, CTX, and dCTX, 0.05-128 p,g/ml. The dCTX metabolite was added in a fixed concentration (8 p,g/ml) to all CTX agar dilutions as recommended previously (Smith et al., 1986). All MICs were determined after 48 hr of anaerobic incubation. Breakpoints for susceptibilities considered in this study were those values suggested by the manufacturer (~<32 ~g/ml for CTX and ~<16 ~g/ml for cefoxitin).

Interpretation of Synergy Tests of synergy for CTX alone, dCTX alone, and CTX plus dCTX were performed against all strains tested. Synergy was determined by calculating the fractional inhibitory concentration (FIC) as applied by others (Aldridge et al., 1984; Smith et al., 1986). MICc+D FIC - - - + MICc

MICD+c MICD

where MICc+D = the MIC of CTX plus dCTX, MICc = the MIC of CTX alone, MICo+c = the MIC of dCTX in combination with CTX, and MICD = the MIC of dCTX alone. Interpretive criteria for FIC results were used in a scale suggested by others (Neu et al., 1977; Hall

et al., 1983) as follows: <0.5, full synergy; 0.5-0.75, partial synergy; >10.76, additive.

RESULTS Comparison of susceptibility of the Bacteroides species to cefoxitin, CTX alone, and in combination with dCTX is shown in Table 1. A marked reduction in CTX MIC was demonstrated in most strains tested when dCTX metabolite was added. This MIC reduction was most notable in B. fragilis where 16 strains (50%) demonstrated an MIC of <~32 btg/ml (susceptible category) for CTX alone, whereas the number of susceptible strains was increased to 26 (81%) with the addition of dCTX to CTX. With CTX alone, the lowest MIC value for B. fragilis was 2 lag/ml, but with the combination of CTX and dCTX 16 strains (50%) exhibited much lower MIC values that ranged from 0.05 to 0.5 ~,g/ml. Similarly, combination of the metabolite and the parent drug had an increased inhibitory effect against B. vulgatus (63-100% susceptible) and the Bacteroides species (64-82% susceptible). The results of the antimicrobial interactions of CTX alone, dCTX alone, and CTX in combination against 32 strains of B. fragilis are shown in Tables 2 and 3. A total of 16 B. fragilis strains (50%) exhibited MIC of 64-~>128 ~,g/ml (resistant category) to CTX alone. Of these, seven strains (22%) had a remarkably low MIC of 0.2-8 btg/ml when dCTX was added. This MIC reduction in the presence of the metabolite was a fourfold to ninefold decrease. Again, 16 strains of B. fragilis were susceptible to CTX alone (MIC of 0.5-32 ~g/ml). Seven of these showed a reduction in MIC (0.05-0.2 ~,g/ml) with the addition of dCTX to CTX. Estimation of synergistic interaction of CTX and dCTX against B. fragilis is shown in Table 4. Full synergy, partial synergy, and additive values for the 32 B. fragilis strains tested were 17/32 (53%), 12/32 (37%), and 3/32 (9%), respectively. The inhibitory effects of CTX alone, dCTX alone, and CTX in combination with dCTX was also tested against the ATCC control organisms (Table 5). A synergistic interaction with CTX plus dCTX was observed for B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741 strains where the CTX MIC was reduced to 0.5 },g/ml and 8 ~g/ml, respectively. Against cefoxitin, our strains of Bacteroides demonstrated a strain to strain variation in antimicrobial activity (Table 6). The activity of cefoxitin was 78% and 50% susceptibility for B. fragilis and B. thetaiotaomicron strains, respectively. It is of interest to mention that when the dCTX metabolite was added to the parent drug compound, a striking increase in activity was observed in nearly all strains tested,

35

CTX/dCTX vs. Bacteroides

TABLE 1.

C o m p a r i s o n of Activity of Cefoxitin, Cefotaxime (CTX) Alone, a n d in C o m b i n a t i o n w i t h Desacetylcefotaxime (dCTX) Against the Bacteroides Species MICs (Ixg/ml)

Organism (n)

~<0.5

B. fragilis (32) Cefoxitin CTX CTX + dCTX B. vulgatus (8) Cefoxitin CTX CTX + dCTX B. thetaiotaomicron (6) Cefoxitin CTX CTX + dCTX B. ovatus (6) Cefoxitin CTX CTX + dCTX Bacteroides species other (22)a Cefoxitin CTX CTX + dCTX

0.1

0.2

0.5

7

1 3

1

2 2

4

2

4

8

16

32

64

5 5 4

9 2 2

7

2 6

5 2 3

2 4

2

2 2

1 1

1

2

2 2

2 1 2

1 3 1

2 1

1

1

2

2

4 3 3

2 3 5

1 4 3

1 1

4

1

2 2

1

3

1 1

3

2

128

~>128

4 2

8 4

1

2 4

2 1

2 4

1

1

2 3

7 2

4

3

qncluding strains of B. bivius, B. capillosus, B. disiens and B. ureolyticus

except B. ovatus. This i n h a n c e d activity w a s m o s t similar or e v e n slightly s u p e r i o r to that activity of cefoxitin.

DISCUSSION The inhibitory effects of CTX against the Bacteroides species in general, a n d against B. fragilis in particular, h a v e b e e n r e p o r t e d b y n u m e r o u s a u t h o r s (Jenkins et al., 1982; Tally et al., 1983; C u c h u r a l et al., 1984). O u r s t u d y of CTX a n d its metabolite dCTX w a s p e r f o r m e d on a n u m b e r of Bacteroides strains r e c o v e r e d f r o m diabetic patients h a v i n g foot ulcers and/or p r e s s u r e sores. At the time, this investigation w a s in its p r e l i m i n a r y stage, a w o r k w i t h CTX a n d

TABLE 2.

dCTX interaction on anaerobic bacteria w a s reported, a n d the addition of 8 Ixg/ml of dCTX in a fixed concentration to CTX that m a y possibly yield the best satisfactory inhibitory effect w a s r e c o m m e n d e d (Smith et al., 1986). Therefore, w e selected 8 ~tg/ml of dCTX in a fixed concentration t h r o u g h o u t this s t u d y since it is the a m o u n t achievable in tissues (Novick, 1982). The results of o u r s t u d y indicated that the activity of CTX alone a n d in c o m b i n a t i o n with dCTX against the Bacteroides species confirm the w o r k of others that CTX a n d dCTX do act in a synergistic m a n n e r (Aldridge et al., 1984; Jones et al., 1984; Smith et al., 1986). Against B. fragilis isolates, it is of interest to note f r o m this s t u d y that the lowest CTX MIC o b t a i n e d

C o m p a r a t i v e Inhibitory Activity of 32 Strains of B. fragilis in Presence of Cefoxitin, Cefotaxime (CTX) Alone, Desacetylcefotaxime (dCTX) Alone, a n d in CTX Plus dCTX Tested in A g a r Dilution MICs (~g/ml)

Antibiotic

0.5

Cefoxitin CTX dCTX CTX + dCTX

2

2

0.1

4

0.2

7

0.5

1

2

4

8

16

32

64

128

5 5

9 2

7

1

2 6 8

1 4 11

3

4

2

1

5 2 1 3

1 4 7 2

>128

8 5 4

36

H. Canawati

TABLE 3.

Variations of MIC Values (~,g/ml) for Certain B. fragilis Strains in the Presence of Cefotaxime (CTX) Alone and CTX in Combination with Desacetylcefotaxime (dCTX)

TABLE 5.

Inhibitory Effect of Cefotaxime (CTX) Alone, Desacetylcefotaxime (dCTX) Alone, and CTX in Combination with dCTX Against ATCC Control Organisms

No. of Organisms

MICcTx

MICcTx+dCTX

Organism

MICc°

MICD

MICc +D

1 3 2 1 2 2 2 1

>128 128 64 64 32 16 4 2

8 0.2 4 2 0.2 0.2 0.2 0.05

B. fragilis ATCC 25285 B. thetaiotaomicron ATCC 29741

8 32

16 32

0.5 8

was 0.5 ~g/ml, and this value was observed only in one strain, but the addition of dCTX to CTX had lowered the MIC value to 0.05 ~g/ml and 16 strains (50%) demonstrated a reduction in MIC that ranged from 0.05-0.5 txg/ml (Table 2). Again, 18 strains (56%) with CTX MIC of 64-~>128p,g/ml (resistant category) displayed resistance to only six strains (19%) when dCTX was added. Like other reports (Aldridge et al., 1984), there were significant differences in drug interaction of CTX and dCTX among the various other species in the B. fragilis group. Synergistic action of CTX and its metabolite against our B. fragilis strains has resulted in a marked increase in antimicrobial activity. Forty percent of strains exhibited full synergy between CTX and dCTX against B. fragilis group in the report by Aldridge et al. (1984). Our study showed 53% full synergy interactions with B. fragilis strains. Against other strains of B. fragilis group, there was also a considerable inhanced inhibitory interaction between CTX and dCTX especially against B. vulgatus, but not B. thetaiotaomicron or B. ovatus. Although cefoxitin demonstrated a greater inhibitory activity than CTX against all Bacteroides species that we tested, the addition of dCTX remarkably improved the activity against most strains. Comparative susceptibility of cefoxitin and CTX against the Bacteroides species were previously reported from numerous medical centers in the United States (Jenkins et al., 1982; Tally et al., 1983;

aMICc = MICof CTX;M I C D of CTX + dCTX.

=

MICof dCTX;M I C c + D

=

M1C

Cuchural et al., 1984). Studies from VA Wadsworth (Los Angeles, CA) showed that 93% and 59% of B. fragilis isolates were inhibited by cefoxitin and CTX, respectively (Chuchural et al., 1984). The resuits of our studies on cefoxitin, however, indicated a lower activity (78%) against this group of bacteria. The comparative results of CTX and dCTX combination versus cefoxitin against B. fragilis strains confirm the report of other workers where the addition of dCTX to CTX demonstrated a marked reduction in CTX MIC90 (12.5 ~g/ml) and the susceptibility results became similar to cefoxitin activity when tested against B. fragilis (Chin et al., 1984). A moderate susceptibility of CTX was also shown against all isolates we tested (51%), but the addition of dCTX has raised the CTX activity to 81% for B. fragilis and Bacteroides species. Since CTX and dCTX interaction is now well established, this may have some clinical implications where pharmcokinetic properties of CTX indicated high serum levels that exceed the MICs of most bacteria 8-12 hr after an i.v. injection (Luthy et al., 1979), and the presence of dCTX has already been recognized in human serum (Coombs, 1982; Doluisio, 1982) and in neonatal CSF (Trang et al., 1985). This situation of CTX deacetylation may ex-

TABLE 6.

Comparison of Activity of Cefoxitin and Cefotaxime CTX Alone and in Combination with dCTX Against the Bacteroides Species % Susceptible to

TABLE 4.

Organism Number %

Antibiotic Interaction of Cefotaxime (CTX) and Desacetylcefotaxime (dCTX) Against 32 Strains of B. fragilis Synergy

Partial Synergy

Additive

17/32 53

12/32 37

3/32 9

Organism (n)

Cefoxitin

CTX

CTX + dCTX

B. fragilis (32) B. vulgatus (8) B.thetaiotaomicron (6) B. ovatus (6) Bacteroides other (22)

78 100 50 100 83

50 63 17 33 64

81 100 66 33 82

CTX/dCTX vs. Bacteroides

plain the clinical cure of an interesting case of s p o n t a n e o u s bacterial peritonitis in a patient with alcoholic liver disease at our institution w h o was treated with a course of CTX against Enterobacter cloacae isolated from the patient's ascites that had an elevated white cell c o u n t and despite the organism resistance to CTX (MIC of >64 ~g/ml). This

37

clinical case initiated us to run a pilot project of CTX alone and CTX plus dCTX metabolite against a n u m b e r of CTX-resistant isolates of Enterobacteriaceae m e m b e r s that were recently r e c o v e r e d from patients with diabetes or liver disease. The results of the inhibitory interaction trial will be r e p o r t e d shortly.

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Jones RN, Thornsberry C (1982) Cefotaxime: A review of in vitro antimicrobial properties and spectrum of activity. Rev Infect Dis 4:$300. Jorgensen JH, Crawford SHA, Alexander GA (1980) In vitro activities of CTX and moxalactam (LY127935)against aerobic gram-negative bacilli. Antimicrob Agents Chemother 17:937. Luthy R, Much R, Blaser J, Bhend H, Siegenthaler W (1979) Human pharmacology of cefotaxime (HR 756), a new cephalosporin. Antimicrob Agents Chemother 16:127. National Committee for Clinical Laboratory Standards (1985) Reference Agar Dilution Procedure for Antimicrobial Susceptibility Testing of Anaerobic Bacteria, vol. 5, no. 18. National Committee for Clinical Laboratory Standards, Villanova, PA. Neu HC (1977) Mecillinam--an amidino penicillin which acts synergistically with other ~-lactam compounds. J Antimicrob Chemother 3(suppl):43. Neu HC (1982) Antimicrobial activity of desacetylcefotaxime alone and in combination with cefotaxime. Rev Infect Dis 4(suppl):S374. Novick WJ (1982) Levels of cefotaxime in body fluids and tissues: A review. Rev Infect Dis 4(suppl):S346. O'Keefe JP, Venezio FR, Di Vincenzo CA, Shatzer KL (1987) Activity of newer f~-lactum agents against clinical isolates of Bacteroides species. Antimicrob Agents Chemother 31:2002. Smith JA, Henry D, Ngui-Yen J, Castell A, Coderre S (1986) Comparison of agar dilution, microdilution, and disk elution methods for measuring the synergy of cefotoxime and its metabolite against anaerobes. J Clin Microbiol 23:1104. Tally FP, Cuchural GJ, Jacobus NV, Gorbach SL, Aldridge K, Cleary T, Finegold SM, Hill G, Iannini P, O'Keefe JP, Pierson C (1983) Nationwide study of the susceptibility of the Bacteroides fragilis group in the United States. Antimicrob Agents Chemother 28:675. Trang JM, Jacobs RF, Kearns GL, Brown AL, et al (1985) Cefotaxime and desacelylcefotaxime pharmacokinetics in infants and children with meningitis. Antimicrob Agents Chemother 28:791.