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Effectiveness of cefotaxime alone and in combination with desacetylcefotaxime against Bacteroides fragilis
39
DIAGN MICROBIOLINFECTDIS 1989;12:39-43
Effectiveness of Cefotaxime Alone and in Combination with Desacetylcefotaxime Against
Bacteroides fragilis Benedict L. Wasilauskas
The synergistic activity of the combination cefotaxime-desacetylcefotaxime (CTX/dCTX) was compared to the effectiveness of seven other antimicrobial agents: cefoxitin (CFOX), cefotetan (CTAN), ceftizoxime (CTIZ), chloramphenicol (CLOR), clindamycin (CLIND), metronidazole (METR), and ampicillin-sulbactam (A/S) tested against 100 clinical isolates belonging to the Bacteroides fragilis group. All tests were performed using the NCCLS reference agar-dilution method. The overall susceptibility of these organisms to CTX/dCTX was 84% compared to CFOX at 78% or CTAN at 66%. The other
antimicrobials inhibited greater than 90% of these isolates. There was no difference between the susceptibility rates of CTX/dCTX and CTX with the B fragilis (85%) or B. distasonis (75%) strains. Bacteroides thetaiotaomicron showed a 11% greater susceptibility to CTX/dCTX than to CTX. Of the 100 isolates tested, 40% showed either synergy or partial synergistic interactions between CTX and dCTX. Most of the isolates showed indifference (52%), while 8% demonstrated antagonism; a relatively unique finding to date.
INTRODUCTION
eration cephalosporins and combinations of betalactamase inhibitors with penicillins, our laboratory was interested in evaluating the efficacy of CTX and dCTX alone and in combination (CTX/dCTX) in a comparative study with other antimicrobial agents against strains of Bacteroides.
The antimicrobial activity of cefotaxime (CTX) against various anaerobes and Bacteroides is well known (Jorgensen et al., 1980; Borobio et al., 1980; Jacobus et al., 1980). Since CTX is metabolized to a desacetyl form similar to other cephalosporins (cephalothin and cephapirim), the activity of this metabolite has also been studied (Schrinner et al., 1984; Limbert et al., 1982). In vitro aerobic activity similar to second-generation cephalosporins has been ascribed to desacetylcefotaxime (dCTX) (Schrinner et al., 1984; Neu, 1982). More recently, significant synergistic activity using both compounds against various microorganisms has also been observed (Jones et al., 1982; Chin and Neu 1984). With the introduction of newer third-gen-
From the Department of Pathology,BowmanGray School of Medicine, WakeForest University, Winston-Salem,North Carolina Address reprint requests to: Dr. BenedictL. Wasilauskas,Department of PathologyWake Forest University:BowmanGray School of Medicine, Winston-Salem,NC 27103. Received August 30, 1988; revised and accepted September 1, 1988. © 1989 Elsevier Science PublishingCo., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/88/$3.50
MATERIALS AND METHODS Organisms One hundred isolates of the Bacteroides fragilis group were tested. The following species were included: 62 B. fragilis, 28 B. thetaiotaomicron, 8 B. distasonis, and 2 B. bivius. Of this group, 90 were beta-lactamase positive by the chromogenic cephalosporin assay (Cefinase, BBL, Cockeysville, MD). The remaining ten beta-lactamase negative isolates consisted of six B. fragilis and four B. distasonis strains.
Susceptibility Testing The National Committee for Clinical Laboratory Standards (NCCLS, 1985) reference agar dilution method as outlined in their approved standard M11A was used for all testing. Briefly, this consisted of using Wilkins-Chalgren agar without additional
40
s u p p l e m e n t s . Serial twofold dilutions of the antimicrobial agents from 64 ~g/ml through 1 ~g/ml were used. The i n o c u l u m w a s p r e p a r e d by g r o w i n g each isolate in thioglycollate b r o t h s u p p l e m e n t e d with vitamin K a n d hemin. The turbidity of g r o w t h w a s adjusted to a 0.5 b a r i u m sulfate s t a n d a r d , a n d approximately 0.003 ~1 (equivalent to 105 cfu/spot) w a s placed on each plate. All plates w e r e i n c u b a t e d at 35°C for 48 hr in an anaerobic c h a m b e r . The e n d p o i n t was either no g r o w t h or one colony or slight h a z e on the spot. Bacteroides fragilis A T C C 25285 a n d B. thetaiotaomicron A T C C 29741 w e r e r u n concurrently for m e t h o d quality control.
Antimicrobial Agents The following antimicrobials w e r e u s e d in the study: cefotaxime (CTX), desacetylcefotaxime (dCTX), cefotaxime + desacetylcefotaxime (CTX/dCTX), cefoxitin (CFOX), cefotetan (CTAN), ceftizoxime (CTIZ), clindamycin (CLIND), c h l o r a m p h e n i c o l (CLOR), m e t r o n i d a z o l e (METR), ampicillin-sulbactam (A/S). The c o m b i n a t i o n of CTX a n d dCTX w a s in a 1:1 ratio, the combination of ampicillin a n d sulbactam w a s in a 2:1 ratio.
Synergy Studies Drug interaction studies w e r e categorized according to the definition b y N e u [1982]. Briefly, these effects are described as follows: s y n e r g y w a s defined as a four-fold or greater reduction in the MIC of b o t h agents. Partial s y n e r g y w a s seen with a four-fold reduction in the MIC of one a g e n t a n d a two-fold or less reduction in the MIC of the other agent. W h e n no change or a two-fold increase or decrease in the MIC of either or b o t h agents w a s o b s e r v e d , it w a s classified as indifference. A n t a g o n i s m w a s defined as a four-fold or greater increase in the MIC of b o t h agents.
B.L. Wasilauskas
TABLE 1.
C o m p a r i s o n of Ten Antimicrobial A g e n t s Tested Against 62 Clinical Isolates of Bacteroides fragilis
Antimicrobic~ CTX dCTX CTX/dCTX CFOX CTAN CTIZ CLIND CLOR METR A/S
MICs0b 4 8 4 8 8 2 <1 2 <1 1
MIC90b 64 >64 64 32 64 16 4 4 1 4
MODE 4 4 <1 8 8 <1 <1 2 <1 1
85 82 85 87 84 97 92 100 97 100
(32) (32) (32) (16) (32) (32) (4) (16) (16) (16)
"CTX = cefotaxime, dCTX = desacetylcefotaxime, CTX/dCTX = cefotaxime + desacetylcefotaxime,CFOX = cefoxitin, CTAN = cefotetan, CTIZ = ceftizoxime, CLIND = clindamycin, CLOR = chloramphenical, METR = metronidazole, A/S = arnpicillin + sulbactam. VMICs0and MICg0refer to concentration of antibiotic that inhibited 50% and 90%, respectively. c% susceptible at NCCLS. Breakpoint in ~g/ml.
was at least fourfold lower t h a n either d r u g alone, e.g. synergy. The B. thetaiotaomicron isolates w e r e m o r e resistant to these agents t h a n B. fragilis. CLOR, METR, and A/S w e r e the m o s t effective at 100% of strains inhibited. H o w e v e r , of the o t h e r drugs, only CTIZ h a d activity >90%. C T A N w a s the least effective at 32%. METR h a d the best overall MIC values of ~<1. The CLIND-resistance rate w a s 14%. The eight isolates of B. distasonis w e r e also m o r e
TABLE 2.
C o m p a r i s o n of Ten Antimicrobial Agents Tested Against 28 Clinical Isolates of B. thetaiotaomicron
RESULTS The results of the susceptibility studies are s h o w n in Tables 1-3. Of the 10 antimicrobial agents that w e r e tested, only C L O R a n d A/S inhibited all of the isolates. METR a n d CTIZ w e r e also v e r y effective (>90%) susceptible. However, the activity of the other agents varied with the species tested. METR, CTIZ a n d C L I N D w e r e v e r y active against the B. fragilis strains. The CTX/dCTX, CTX, and dCTX were similar in their effectiveness a n d c o m p a r a b l e to CFOX a n d CTAN. A l t h o u g h the CTX/dCTX s h o w e d the s a m e n u m b e r of isolates in the susceptible range as CTX and dCTX, its modal MIC of ~ = 1
% Susc. (BKPT)c
Antimicrobic~ CTX dCTX CTX/dCTX CFOX CTAN CTIZ CLIND CLOR METR A/S
MIC50b
MICgob
MODE
32 64 16 16 64 8 2 4 <1
~64 >64 >64 32 >64 32 8 4 <1
32 64 16 16 64 8 2 4 <1
1
8
1
aSee foonotes a-~, Table 1.
% Susc. (BKPT)c 71 46 82 68 32 96 86 100 100 100
(32) (32) (32) (16) (32) (32) (4) (16) (16) (16)
CTX/dCTX Against B. fragilis gr.
T A B L E 3.
sonis strains d e m o n s t r a t e d 63% resistance to CFOX a n d
C o m p a r i s o n of Ten Antimicrobial A g e n t s Tested Against Eight Clinical Isolates of B. distasonis
AntimicrobiU CTX dCTX CTX/dCTX CFOX CTAN CTIZ CLIND CLOR METR A/S
41
MIC50a 2 4 <1 32 64 <1 <1 4 <1 4
MIC90 a
MODE
% Susc. (BKFF) a
<1 <1 <1 64 >64 <1 <1 4 <1 16
75 (32) 75 (32) 75 (32) 37 (16) 38 (32) 100 (32) 100 (4) 100 (16) 100 (16) 100 (16)
>64 >64 >64 >64 >64 32 4 8 1 16
~See footnotes ~, Table 1.
resistant, a l t h o u g h t h e y did not d e m o n s t r a t e quite the d e g r e e of resistance as B. thetaiotaomicron. All isolates w e r e susceptible to CTIZ, CLIND, CLOR, METR a n d A/S. There w a s no difference in susceptibility b e t w e e n CTX, dCTX, or CTX/dCTX, all of w h i c h inhibited 75% of the isolates. H o w e v e r , all three d r u g s h a d m o d a l MIC values of ~<1. This species w a s the m o s t resistant to CFOX with only 37% of the isolates susceptible. As seen in Table 4, m o r e t h a n half of the B. fragilis group isolates that were resistant to either CFOX, CTX, dCTX, CTX/dCTX w e r e also resistant to CTAN. All of the isolates resistant to CTIZ were also resistant to CTX, dCTX, and CTX/dCTX. A high degree (>65%) of crossresistance w a s seen b e t w e e n CTX/dCTX a n d CTX, CFOX, a n d dCTX. The c e p h a l o s p o r i n s s h o w e d an exp e c t e d low (<25%) cross-resistance w i t h METR a n d CLIND. An analysis of the resistance detected a m o n g the individual species is s h o w n in Table 5. The B. distaTABLE 4.
CTAN, b u t all isolates w e r e susceptible to CTIZ a n d CLIND. A relatively low level of resistance (3-18%) for all antimicrobics w a s o b s e r v e d w i t h the B. fragilis isolates. H o w e v e r , t w o of these isolates w e r e resistant to METR with MIC values of >16 btg/ml. The t w o isolates of B. bivius w e r e susceptible to all d r u g s tested. Of the four B. distasonis isolates that w e r e beta-lact a m a s e negative, two w e r e susceptible to all d r u g s tested, one w a s resistant to C T A N at 64 ~tg/ml, a n d one isolate was resistant to C T A N at 64 ~tg/ml a n d CFOX at 32 ~tg/ml. Within the g r o u p of six B. fragilis isolates that w e r e beta-lactamase negative, one w a s resistant to C T A N at 64 ~g/ml, w h e r e a s the o t h e r five w e r e susceptible to all the drugs tested. Of the 90 B. fragilis g r o u p isolates that w e r e beta-lactamase positive, 29% h a d dCTX MICs that w e r e less t h a n that of for CTX alone. The synergistic effects of CTX a n d dCTX c o m p a r e d to the activity of each d r u g alone are s h o w n in Figure 1. Most of the isolates w e r e indifferent to the combination. O n l y 40% d e m o n s t r a t e d a n y t y p e of s y n e r g y and eight isolates exhibited s o m e degree of antagonism.
DISCUSSION
The results of this s t u d y s h o w that traditional antimicrobial agents such as CLOR, CLIND, a n d METR used against a n a e r o b e s are still v e r y active agents against isolates of the B. fragilis group. This is in agreem e n t with the findings of others (Jones 1984; Cuchural et al., 1988). N e w e r agents such as CTIZ a n d A/S are similarly effective against these o r g a n i s m s a n d provide less toxic alternatives to conventional therapies. The metabolite, dCTX, w a s as effective as secondgeneration cephalosporins. C T A N w a s d i s a p p o i n t ing in its activity against species other t h a n B. fragilis, which is in accord with the findings of Cuchural et al. (1988). CTX/dCTX w a s superior to either CTX or dCTX
N u m b e r of B. fragilis G r o u p Isolates Resistant to P r i m a r y D r u g a n d D e m o n s t r a t i n g Resistance to O t h e r D r u g s % Cross-resistant to
Primary Drug ~
No. of Isolates Resistant to Primary Drug
CFOX
CFOX CTX dCTX CTX/dCTX CTIZ CTAN CLIND METR
22 19 28 16 3 34 9 2
-53 54 69 33 56 44 50
aSee footnote a, Table 1.
CTX
dCTX
CTX/dCTX
45
68 84 -94 100 59 33 50
50 63 54 -100 29 11 50
--
5Y 75 100 35 44 0
CTIZ
CTAN
CLIND
METR
5
86 63 71 63 33 -44 0
14 21 11 6 0 12 -0
5
16
11 19 -3 0 0
0
4 6 0 0 11 --
42
B.L. Wasilauskas
TABLE 5.
Prevalence of Resistance Among Bacteroides Species Number of Isolates (%) from Each Species That are Resistant
Drug"
Total No. Resistant
B. distasonis
B. fragilis
B. thetaiotaomicron
CFOX CTX dCTX CTX/dCTX CTIZ CTAN CLIND METR
22 19 28 16 3 34 9 2
5 (63) 2 (25) 2 (25) 2 (25) 0 5 (63) 0 0
8 (13) 9 (15) 11 (18) 9 (15) 2 (3) 10 (16) 5 (8) 2 (3)
9 (32) 8 (29) 15 (54) 5 (18) 1 (4) 19 (68) 4 (14) 0
"See footnotea, Table 1.
alone and was moderately effective against these organisms. However, synergistic activity was demonstrated at only 40% with the combination of drugs. In fact, the combination proved to be antagonistic to eight of the isolates. This is contrary to nearly all other studies (Jones, 1984; Jones et al., 1984; Chin and Neu, 1984), but this finding may reflect a regional difference in the isolates tested. Regional variation of anaerobic organism resistance to various antimicrobial agents is becoming a common finding (Chuchural et al., 1988; Jones et al., 1984). Limbert et al. in (1982) had postulated that increased stability of dCTX to beta-lactamases might be responsible for the synergistic activity of the metabolite with CTX. Our findings do not support this since only 29% of the 90 ~-lactamase-positive isolates had DTAX MIC values that were lower than CTAX. It appears that additional mechanisms are responsible for synergistic activity with Bacteroides strains. The results of the beta-lactamase studies seem to indicate that some of these isolates, especially among the B. distasonis species, possess mechanisms of re-
sistance to CTAN (MIC = 64 t~g/ml) other than the production of beta-lactamases. However, since only a small sample was tested, this needs to be studied with a larger number of isolates. Analysis of cross-resistance among these organisms shows that CTAN and CFOX possess substantial cross-resistance with the other cephalosporins except CTIZ. This is similar to the findings of Cucheral et al. (1988), although the rates in the present study were not as high. CTX, dCTX, and CTX/dCTX all had 100% cross-resistance with the three isolates that were resistant to CTIZ. In summary, the traditional antimicrobial agents still appear to be quite effective against the B. fragilis group. Newer third-generation cephalosporins and A/S offer reasonable and perhaps less-toxic therapeutic alternatives. CTX/dCTX appears to possess some synergistic activity although not as much as has been previously reported (Jones, 1984). CTAN and CFOX seem to be losing activity against this group of clinically important microorganisms.
Number of isolates 40 30 20 10 0 Synergy
Partial
Indiff
Antag
Antimicrobial Interaction •
B. frag
FIGURE 1.
[ ] B. theta
[ ] B. dist
Bacteroides fragilis groul>-100 clinical isolates.
[ ] B. biv
CTX/dCTX Against B. fragilis gr.
43
REFERENCES Borobio MV, Aznar J, Jimenez R, Garcia F, Perea EJ (1980) Comparative in vitro activity of 1-oxa-beta lactam (LY127935) and cefoperazone with other beta-lactam antibiotics against anaerobic bacteria. Antimicrob Agents Chemother 17:129. Chin N, Neu HC (1984) Cefotaxime and desacetylcefotaxime: An example of advantageous antimicrobial metabolism. Diagn Microbiol Infect Dis 2:21S. Cuchural GJ Jr, Tally FP, Jacobus NV, Aldridge K, Cleary T, Finegold SM, Hill G, Iannini P, O'Keefe JP, Pierson C, Crook D, Russo T, Hecht D (1988) Susceptibility of the Bacteroides fragilis group in the United States: Analysis by site of isolation. Antimicrob Agents Chemother 32:717. Jacobus NV, Tally FP, Barza M, Gorbach SL (1980) Susceptibility of anaerobic bacteria to cefoperazone and other beta-lactam antibiotics.. Clin Ther 3:34. Jones RN, Barry AL, Thornsberry C (1982) Antimicrobial activity of desacetylcefotaxime alone and in combination with cefotaxime; Evidence of synergy. Rev Infect Dis 4:$366. Jones RN (1984) The activity of cefotaxime and desacetylcefotaxime against Bacteroides species compared to 7methoxy cephems and other anti-anaerobic drugs. J Antimicrob Chemother 14(Suppl B):39.
Jones RN, Barry AL, Packer PR (1984) The activity of cefotaxime and desacetylcefotaxime alone and in combination against anaerobes and staphylococci. Diagn Microbiol Infect Dis 2:37S. Jorgensen JH, Crawford SA, Alexander GA (1980) Comparison of moxalactam (LY127935) and cefotaxime against anaerobic bacteria. Antimicrob Agents Chemother 17:901. Limbert M, Seibert G, Schrinner E (1982) The cooperation of cefotaxime and desacetylcefotaxime with respect to antibacterial activity and beta-lactamase stability. Infection 10:97. National Committee for Clinical Laboratory Standards (1985) Reference agar dilution procedure for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard : MII-A. NCCLS. Villanova, PA. Neu HC (1982) Antibacterial activity of desactylcefotaxime alone and in combination with cefotaxime. Rev. Infect Dis 4:$374. Schrinner E, Limbert M, Seeger K, Seibert G, Novick WJ, Jr (1984) The in vitro antimicrobial activity of desacetylcefotaxime compared to other related beta-lactams. Diagn Microbiol Infect Dis 2:13S.
DIAGN MICROBIOLINFECTDIS 1989;12:39-43
Effectiveness of Cefotaxime Alone and in Combination with Desacetylcefotaxime Against
Bacteroides fragilis Benedict L. Wasilauskas
The synergistic activity of the combination cefotaxime-desacetylcefotaxime (CTX/dCTX) was compared to the effectiveness of seven other antimicrobial agents: cefoxitin (CFOX), cefotetan (CTAN), ceftizoxime (CTIZ), chloramphenicol (CLOR), clindamycin (CLIND), metronidazole (METR), and ampicillin-sulbactam (A/S) tested against 100 clinical isolates belonging to the Bacteroides fragilis group. All tests were performed using the NCCLS reference agar-dilution method. The overall susceptibility of these organisms to CTX/dCTX was 84% compared to CFOX at 78% or CTAN at 66%. The other
antimicrobials inhibited greater than 90% of these isolates. There was no difference between the susceptibility rates of CTX/dCTX and CTX with the B fragilis (85%) or B. distasonis (75%) strains. Bacteroides thetaiotaomicron showed a 11% greater susceptibility to CTX/dCTX than to CTX. Of the 100 isolates tested, 40% showed either synergy or partial synergistic interactions between CTX and dCTX. Most of the isolates showed indifference (52%), while 8% demonstrated antagonism; a relatively unique finding to date.
INTRODUCTION
eration cephalosporins and combinations of betalactamase inhibitors with penicillins, our laboratory was interested in evaluating the efficacy of CTX and dCTX alone and in combination (CTX/dCTX) in a comparative study with other antimicrobial agents against strains of Bacteroides.
The antimicrobial activity of cefotaxime (CTX) against various anaerobes and Bacteroides is well known (Jorgensen et al., 1980; Borobio et al., 1980; Jacobus et al., 1980). Since CTX is metabolized to a desacetyl form similar to other cephalosporins (cephalothin and cephapirim), the activity of this metabolite has also been studied (Schrinner et al., 1984; Limbert et al., 1982). In vitro aerobic activity similar to second-generation cephalosporins has been ascribed to desacetylcefotaxime (dCTX) (Schrinner et al., 1984; Neu, 1982). More recently, significant synergistic activity using both compounds against various microorganisms has also been observed (Jones et al., 1982; Chin and Neu 1984). With the introduction of newer third-gen-
From the Department of Pathology,BowmanGray School of Medicine, WakeForest University, Winston-Salem,North Carolina Address reprint requests to: Dr. BenedictL. Wasilauskas,Department of PathologyWake Forest University:BowmanGray School of Medicine, Winston-Salem,NC 27103. Received August 30, 1988; revised and accepted September 1, 1988. © 1989 Elsevier Science PublishingCo., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/88/$3.50
MATERIALS AND METHODS Organisms One hundred isolates of the Bacteroides fragilis group were tested. The following species were included: 62 B. fragilis, 28 B. thetaiotaomicron, 8 B. distasonis, and 2 B. bivius. Of this group, 90 were beta-lactamase positive by the chromogenic cephalosporin assay (Cefinase, BBL, Cockeysville, MD). The remaining ten beta-lactamase negative isolates consisted of six B. fragilis and four B. distasonis strains.
Susceptibility Testing The National Committee for Clinical Laboratory Standards (NCCLS, 1985) reference agar dilution method as outlined in their approved standard M11A was used for all testing. Briefly, this consisted of using Wilkins-Chalgren agar without additional
40
s u p p l e m e n t s . Serial twofold dilutions of the antimicrobial agents from 64 ~g/ml through 1 ~g/ml were used. The i n o c u l u m w a s p r e p a r e d by g r o w i n g each isolate in thioglycollate b r o t h s u p p l e m e n t e d with vitamin K a n d hemin. The turbidity of g r o w t h w a s adjusted to a 0.5 b a r i u m sulfate s t a n d a r d , a n d approximately 0.003 ~1 (equivalent to 105 cfu/spot) w a s placed on each plate. All plates w e r e i n c u b a t e d at 35°C for 48 hr in an anaerobic c h a m b e r . The e n d p o i n t was either no g r o w t h or one colony or slight h a z e on the spot. Bacteroides fragilis A T C C 25285 a n d B. thetaiotaomicron A T C C 29741 w e r e r u n concurrently for m e t h o d quality control.
Antimicrobial Agents The following antimicrobials w e r e u s e d in the study: cefotaxime (CTX), desacetylcefotaxime (dCTX), cefotaxime + desacetylcefotaxime (CTX/dCTX), cefoxitin (CFOX), cefotetan (CTAN), ceftizoxime (CTIZ), clindamycin (CLIND), c h l o r a m p h e n i c o l (CLOR), m e t r o n i d a z o l e (METR), ampicillin-sulbactam (A/S). The c o m b i n a t i o n of CTX a n d dCTX w a s in a 1:1 ratio, the combination of ampicillin a n d sulbactam w a s in a 2:1 ratio.
Synergy Studies Drug interaction studies w e r e categorized according to the definition b y N e u [1982]. Briefly, these effects are described as follows: s y n e r g y w a s defined as a four-fold or greater reduction in the MIC of b o t h agents. Partial s y n e r g y w a s seen with a four-fold reduction in the MIC of one a g e n t a n d a two-fold or less reduction in the MIC of the other agent. W h e n no change or a two-fold increase or decrease in the MIC of either or b o t h agents w a s o b s e r v e d , it w a s classified as indifference. A n t a g o n i s m w a s defined as a four-fold or greater increase in the MIC of b o t h agents.
B.L. Wasilauskas
TABLE 1.
C o m p a r i s o n of Ten Antimicrobial A g e n t s Tested Against 62 Clinical Isolates of Bacteroides fragilis
Antimicrobic~ CTX dCTX CTX/dCTX CFOX CTAN CTIZ CLIND CLOR METR A/S
MICs0b 4 8 4 8 8 2 <1 2 <1 1
MIC90b 64 >64 64 32 64 16 4 4 1 4
MODE 4 4 <1 8 8 <1 <1 2 <1 1
85 82 85 87 84 97 92 100 97 100
(32) (32) (32) (16) (32) (32) (4) (16) (16) (16)
"CTX = cefotaxime, dCTX = desacetylcefotaxime, CTX/dCTX = cefotaxime + desacetylcefotaxime,CFOX = cefoxitin, CTAN = cefotetan, CTIZ = ceftizoxime, CLIND = clindamycin, CLOR = chloramphenical, METR = metronidazole, A/S = arnpicillin + sulbactam. VMICs0and MICg0refer to concentration of antibiotic that inhibited 50% and 90%, respectively. c% susceptible at NCCLS. Breakpoint in ~g/ml.
was at least fourfold lower t h a n either d r u g alone, e.g. synergy. The B. thetaiotaomicron isolates w e r e m o r e resistant to these agents t h a n B. fragilis. CLOR, METR, and A/S w e r e the m o s t effective at 100% of strains inhibited. H o w e v e r , of the o t h e r drugs, only CTIZ h a d activity >90%. C T A N w a s the least effective at 32%. METR h a d the best overall MIC values of ~<1. The CLIND-resistance rate w a s 14%. The eight isolates of B. distasonis w e r e also m o r e
TABLE 2.
C o m p a r i s o n of Ten Antimicrobial Agents Tested Against 28 Clinical Isolates of B. thetaiotaomicron
RESULTS The results of the susceptibility studies are s h o w n in Tables 1-3. Of the 10 antimicrobial agents that w e r e tested, only C L O R a n d A/S inhibited all of the isolates. METR a n d CTIZ w e r e also v e r y effective (>90%) susceptible. However, the activity of the other agents varied with the species tested. METR, CTIZ a n d C L I N D w e r e v e r y active against the B. fragilis strains. The CTX/dCTX, CTX, and dCTX were similar in their effectiveness a n d c o m p a r a b l e to CFOX a n d CTAN. A l t h o u g h the CTX/dCTX s h o w e d the s a m e n u m b e r of isolates in the susceptible range as CTX and dCTX, its modal MIC of ~ = 1
% Susc. (BKPT)c
Antimicrobic~ CTX dCTX CTX/dCTX CFOX CTAN CTIZ CLIND CLOR METR A/S
MIC50b
MICgob
MODE
32 64 16 16 64 8 2 4 <1
~64 >64 >64 32 >64 32 8 4 <1
32 64 16 16 64 8 2 4 <1
1
8
1
aSee foonotes a-~, Table 1.
% Susc. (BKPT)c 71 46 82 68 32 96 86 100 100 100
(32) (32) (32) (16) (32) (32) (4) (16) (16) (16)
CTX/dCTX Against B. fragilis gr.
T A B L E 3.
sonis strains d e m o n s t r a t e d 63% resistance to CFOX a n d
C o m p a r i s o n of Ten Antimicrobial A g e n t s Tested Against Eight Clinical Isolates of B. distasonis
AntimicrobiU CTX dCTX CTX/dCTX CFOX CTAN CTIZ CLIND CLOR METR A/S
41
MIC50a 2 4 <1 32 64 <1 <1 4 <1 4
MIC90 a
MODE
% Susc. (BKFF) a
<1 <1 <1 64 >64 <1 <1 4 <1 16
75 (32) 75 (32) 75 (32) 37 (16) 38 (32) 100 (32) 100 (4) 100 (16) 100 (16) 100 (16)
>64 >64 >64 >64 >64 32 4 8 1 16
~See footnotes ~, Table 1.
resistant, a l t h o u g h t h e y did not d e m o n s t r a t e quite the d e g r e e of resistance as B. thetaiotaomicron. All isolates w e r e susceptible to CTIZ, CLIND, CLOR, METR a n d A/S. There w a s no difference in susceptibility b e t w e e n CTX, dCTX, or CTX/dCTX, all of w h i c h inhibited 75% of the isolates. H o w e v e r , all three d r u g s h a d m o d a l MIC values of ~<1. This species w a s the m o s t resistant to CFOX with only 37% of the isolates susceptible. As seen in Table 4, m o r e t h a n half of the B. fragilis group isolates that were resistant to either CFOX, CTX, dCTX, CTX/dCTX w e r e also resistant to CTAN. All of the isolates resistant to CTIZ were also resistant to CTX, dCTX, and CTX/dCTX. A high degree (>65%) of crossresistance w a s seen b e t w e e n CTX/dCTX a n d CTX, CFOX, a n d dCTX. The c e p h a l o s p o r i n s s h o w e d an exp e c t e d low (<25%) cross-resistance w i t h METR a n d CLIND. An analysis of the resistance detected a m o n g the individual species is s h o w n in Table 5. The B. distaTABLE 4.
CTAN, b u t all isolates w e r e susceptible to CTIZ a n d CLIND. A relatively low level of resistance (3-18%) for all antimicrobics w a s o b s e r v e d w i t h the B. fragilis isolates. H o w e v e r , t w o of these isolates w e r e resistant to METR with MIC values of >16 btg/ml. The t w o isolates of B. bivius w e r e susceptible to all d r u g s tested. Of the four B. distasonis isolates that w e r e beta-lact a m a s e negative, two w e r e susceptible to all d r u g s tested, one w a s resistant to C T A N at 64 ~tg/ml, a n d one isolate was resistant to C T A N at 64 ~tg/ml a n d CFOX at 32 ~tg/ml. Within the g r o u p of six B. fragilis isolates that w e r e beta-lactamase negative, one w a s resistant to C T A N at 64 ~g/ml, w h e r e a s the o t h e r five w e r e susceptible to all the drugs tested. Of the 90 B. fragilis g r o u p isolates that w e r e beta-lactamase positive, 29% h a d dCTX MICs that w e r e less t h a n that of for CTX alone. The synergistic effects of CTX a n d dCTX c o m p a r e d to the activity of each d r u g alone are s h o w n in Figure 1. Most of the isolates w e r e indifferent to the combination. O n l y 40% d e m o n s t r a t e d a n y t y p e of s y n e r g y and eight isolates exhibited s o m e degree of antagonism.
DISCUSSION
The results of this s t u d y s h o w that traditional antimicrobial agents such as CLOR, CLIND, a n d METR used against a n a e r o b e s are still v e r y active agents against isolates of the B. fragilis group. This is in agreem e n t with the findings of others (Jones 1984; Cuchural et al., 1988). N e w e r agents such as CTIZ a n d A/S are similarly effective against these o r g a n i s m s a n d provide less toxic alternatives to conventional therapies. The metabolite, dCTX, w a s as effective as secondgeneration cephalosporins. C T A N w a s d i s a p p o i n t ing in its activity against species other t h a n B. fragilis, which is in accord with the findings of Cuchural et al. (1988). CTX/dCTX w a s superior to either CTX or dCTX
N u m b e r of B. fragilis G r o u p Isolates Resistant to P r i m a r y D r u g a n d D e m o n s t r a t i n g Resistance to O t h e r D r u g s % Cross-resistant to
Primary Drug ~
No. of Isolates Resistant to Primary Drug
CFOX
CFOX CTX dCTX CTX/dCTX CTIZ CTAN CLIND METR
22 19 28 16 3 34 9 2
-53 54 69 33 56 44 50
aSee footnote a, Table 1.
CTX
dCTX
CTX/dCTX
45
68 84 -94 100 59 33 50
50 63 54 -100 29 11 50
--
5Y 75 100 35 44 0
CTIZ
CTAN
CLIND
METR
5
86 63 71 63 33 -44 0
14 21 11 6 0 12 -0
5
16
11 19 -3 0 0
0
4 6 0 0 11 --
42
B.L. Wasilauskas
TABLE 5.
Prevalence of Resistance Among Bacteroides Species Number of Isolates (%) from Each Species That are Resistant
Drug"
Total No. Resistant
B. distasonis
B. fragilis
B. thetaiotaomicron
CFOX CTX dCTX CTX/dCTX CTIZ CTAN CLIND METR
22 19 28 16 3 34 9 2
5 (63) 2 (25) 2 (25) 2 (25) 0 5 (63) 0 0
8 (13) 9 (15) 11 (18) 9 (15) 2 (3) 10 (16) 5 (8) 2 (3)
9 (32) 8 (29) 15 (54) 5 (18) 1 (4) 19 (68) 4 (14) 0
"See footnotea, Table 1.
alone and was moderately effective against these organisms. However, synergistic activity was demonstrated at only 40% with the combination of drugs. In fact, the combination proved to be antagonistic to eight of the isolates. This is contrary to nearly all other studies (Jones, 1984; Jones et al., 1984; Chin and Neu, 1984), but this finding may reflect a regional difference in the isolates tested. Regional variation of anaerobic organism resistance to various antimicrobial agents is becoming a common finding (Chuchural et al., 1988; Jones et al., 1984). Limbert et al. in (1982) had postulated that increased stability of dCTX to beta-lactamases might be responsible for the synergistic activity of the metabolite with CTX. Our findings do not support this since only 29% of the 90 ~-lactamase-positive isolates had DTAX MIC values that were lower than CTAX. It appears that additional mechanisms are responsible for synergistic activity with Bacteroides strains. The results of the beta-lactamase studies seem to indicate that some of these isolates, especially among the B. distasonis species, possess mechanisms of re-
sistance to CTAN (MIC = 64 t~g/ml) other than the production of beta-lactamases. However, since only a small sample was tested, this needs to be studied with a larger number of isolates. Analysis of cross-resistance among these organisms shows that CTAN and CFOX possess substantial cross-resistance with the other cephalosporins except CTIZ. This is similar to the findings of Cucheral et al. (1988), although the rates in the present study were not as high. CTX, dCTX, and CTX/dCTX all had 100% cross-resistance with the three isolates that were resistant to CTIZ. In summary, the traditional antimicrobial agents still appear to be quite effective against the B. fragilis group. Newer third-generation cephalosporins and A/S offer reasonable and perhaps less-toxic therapeutic alternatives. CTX/dCTX appears to possess some synergistic activity although not as much as has been previously reported (Jones, 1984). CTAN and CFOX seem to be losing activity against this group of clinically important microorganisms.
Number of isolates 40 30 20 10 0 Synergy
Partial
Indiff
Antag
Antimicrobial Interaction •
B. frag
FIGURE 1.
[ ] B. theta
[ ] B. dist
Bacteroides fragilis groul>-100 clinical isolates.
[ ] B. biv
CTX/dCTX Against B. fragilis gr.
43
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