Comparison of the Inoculum Effect of Cefoxitin And

Home , Cefotetan, Ceftizoxime

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1991, p. 1868-1874 Vol. 35, No. 9 0066-4804/91/091868-07$02.00/0 Copyright C 1991, American Society for Microbiology Comparison of the Inoculum Effect of Cefoxitin and Other Cephalosporins and of P-Lactamase Inhibitors and Their Penicillin- Derived Components on the Bacteroides fragilis Group ELLIE J. C. GOLDSTEIN,l.2* DIANE M. CITRON,1 AND CHARLES E. CHERUBIN3 R. M. Alden Research Laboratory, Santa Monica Hospital Medical Center, Santa Monica, California 904041; Department of Medicine, UCLA School of Medicine, Los Angeles, California 900242; and Department of Medicine, Mercy Hospital & Medical Center, Chicago, Illinois 606163 Received 12 March 1991/Accepted 12 July 1991

We compared the inoculum effects for 109 recent clinical isolates of the Bacteroidesfragilis group of cefoxitin, cefotetan, ceftizoxime, ceftriaxone, and three (3-lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) and their penicillin-derived components. Bactericidal activity was assayed and morphologic changes were monitored for selected strains exhibiting a large inoculum effect. Ceftizoxime demonstrated the largest inoculum effect, followed by cefotetan and ceftriaxone. The large inoculum effect of ceftizoxime and ceftriaxone was correlated with filamentous transformation at the high inoculum (108 CFU/ml) and lack of bactericidal activity suggesting drug destruction or inactivation. Cefotetan was bactericidal for B. fragilis isolates but not for other members of the B. fragilis group. Cefoxitin showed the least inoculum effect and was consistently bactericidal at high (108 CFU/ml), standard (106 CFU/ml), and low (104 CFU/ml) inocula, followed by ampicillin-sulbactam. Piperacillin-tazobactam and ticarcillin-clavulanic acid showed an intermediate inoculum effect. The degree of inoculum effect observed generally correlated with bactericidal activity at all inocula.

The inoculum effect, a large increase in the MIC occurring tus, 20 strains; B. distasonis, 20 strains; and B. vulgatus, 20 with an increase in the inoculum size of a microorganism, is strains). Isolates were identified by standard criteria (9, 15, one of the variables that influence susceptibility testing and 20). B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC occurs primarily with beta-lactam antibiotics (7, 8, 11, 16). A 29741 were used as controls. Isolates were stored at -70°C number of studies have shown that the Bacteroides fragilis in 10% skim milk and transferred at least twice on prere- group species produce 1-lactamases and may exhibit an duced anaerobically sterilized brucella agar supplemented inoculum effect (3, 5, 7, 16, 23, 24). Sutter et al. (21) have with vitamin K1, hemin, and 5% sheep blood (BBA) (Anaer- suggested that both large and small inocula should be used to obe Systems, San Jose, Calif.) prior to testing to assure detect resistance of anaerobic bacteria to beta-lactam purity and viability. agents. In a recent review on the inoculum effect, Brook (7) Antimicrobial agents. Standard powders were supplied as summarized the laboratory experience to date with B. fragi- follows: cefoxitin, Merck Sharp & Dohme, West Point, Pa.; lis group species and noted that while some of the cephalo- cefotetan,' Stuart Pharmaceuticals, Wilmington, Del.; cefti- sporins had been studied, there was scant data on the zoxime, SmithKline & French Laboratories, Philadelphia, P-lactamase inhibitor combinations and their penicillin-de- Pa.; ceftriaxone, Roche Laboratories, Nutley, N.J.; ampi- rived components (1, 3, 17, 18). It seems important to cillin and sulbactam, Pfizer Inc., New York, N.Y.; ticarcillin evaluate the 1-lactamase inhibitor combinations since these and clavulanic acid, Beecham Pharmaceuticals Inc, Bristol, compounds are used in clinical situations where B. fragilis Tenn.; and piperacillin and tazobactam, Lederle Laborato- group species are 'potential pathogens and an increased ries, Pearl River, N.Y. The antimicrobial powders were density' of inoculum might cause increased enzymatic de- reconstituted according to the manufacturers' instructions struction. and used on the same day to prepare microdilution plates, Consequently, we have compared the inoculum effect for which were then stored at -70°C until used. For the kill clinical isolate-s of B. fragilis group species and the bacteri- curves, stock solutions were prepared and stored at -70°C cidal activity of cefoxitin, ceftizoxime, ceftriaxone, andI until used. three P-lactamase inhibitors and their penicillin-derivedI Microdilution testing. Serial dilutions of antibiotics ranging components. from 128 to 0.06 ,ug/ml' (final concentration) were prepared in Anaerobe Broth MIC (Difco Laboratories, Inc., Detroit, Mich.). The P-lactamase inhibitor combinations were pre- MATERIALS AND METHODS pared according to the manufacturers' instructions with a Organisms. A total of 109 recent, sequentially collected ratio of 8:1 for piperacillin and tazobactam and 2:1 for clinical isolates of the B. fragilis group were studied (B. ampicillin and sulbactam, and for ticarcillin and clavulanic fragilis, 26 strains; B. thetaiotaomicron, 23 strains; B. ova- acid, the clavulanate was held constant -at 2 ,ug/ml. Mi- crodilution plates were prepared by using the Quick Spense II (Sandy Spring Instrument Co. Inc., Germantown, Md.). * Corresponding author. Organisms were grown on BBA for 48 h. The 108-CFU/ml 1868 VOL. 35, 1991 INOCULUM EFFECT FOR B. FRAGILIS GROUP 1869 inoculum was prepared by suspending a sufficient number of RESULTS colonies in Anaerobe Broth MIC to achieve a visual turbidity equal to McFarland standard no. 1. This was diluted 1:100 to The inoculum effect of the B. fragilis group, as measured achieve the 106-CFU/ml inoculum. The 106-CFU/ml inocu- by MICs for 90% of the organisms (MIC90s), geometric mean lum was diluted 1:100 to prepare the 104-CFU/ml inoculum. MICs, the ratios of the geometric mean MICs obtained at Inoculum densities were verified by quantitative plating onto 108- versus 104-CFU/ml inocula, and percent susceptibility BBA for several strains each day of the test. The microdi- at the breakpoint, is presented in Table 1. The inoculum lution plates were thawed and conditioned in the anaerobic effect was greater for B. fragilis than for the other members chamber (Anaerobe Systems) for 4 h prior to inoculation. of the B. fragilis group. The addition of ,B-lactamase inhibi- Fifty microliters of the inoculum was added to each well tors (sulbactam, clavulanic acid, and tazobactam) did not containing 50 microliters of the diluted antibiotics. alter the inoculum effect but did decrease the MIC9J and the Growth geometric mean MIC values. Of the P-lactamase inhibitor controls containing no antibiotic and broth controls contain- combinations tested, ampicillin-sulbactam showed the least ing no organism were included in each run. Additionally, an inoculum effect, though not the lowest geometric mean inoculum control containing 1 drop of 10% formalin was MICs, at all inocula. In general, geometric mean MICs were included because 108 CFU/ml produces a small button in the lowest for ticarcillin-clavulanic acid. Cefoxitin showed the wells. Trays were inoculated with an automatic pipet (Rainin least inoculum effect of all the agents tested. The MIC90s of Instruments Co. Inc., Woburn, Mass.), sealed, and incu- the 3-lactamase inhibitor combinations were somewhat bated for 48 h. All manipulations were carried out within an lower at the 104- and 106-CFU/ml inocula than those noted anaerobic chamber. For the trays containing 106- and 104- for cefoxitin. However, at the 108-CFU/ml inoculum, the CFU/ml inocula, the MIC was defined as the lowest concen- percent susceptibility and the MIC90s of cefoxitin and the tration of antibiotic that caused complete inhibition of P-lactamase inhibitor combinations were similar. growth. For trays containing the 108-CFU/ml inoculum, With the cephalosporins, the largest inoculum effect oc- inhibition of growth was defined as wells that showed no curred with ceftizoxime, followed by ceftriaxone and ce- growth or a button less than twice the diameter of the fotetan. Most strains of B. thetaiotaomicron, B. distasonis, inoculum control. and B. ovatus were resistant to cefotetan even at the Kill curves. Selected strains that showed a large inoculum 104-CFU/ml inoculum. Cefotetan exhibited little inoculum effect were selected for further study. B. fragilis ATCC effect and relatively low MIC90s and geometric mean MICs 25285 was chosen as a control. Prereduced anaerobically against B. fragilis and B. vulgatus strains. The MICs of sterilized Anaerobe Broth MIC with resazurin Eh indicator ceftizoxime and ceftriaxone against most members of the B. (1 mg/liter) was prepared by combining ingredients in the fragilis group were >128 jig/ml at both the 106- and 108- anaerobic chamber. The broth was boiled until the resazurin CFU/ml inocula. Overall, MIC90s, geometric mean MICs, became colorless, and then 45 ml was dispensed into 100-ml and the inoculum effect were higher with ceftizoxime and serum vials. The vials were capped with butyl rubber septa ceftriaxone and the parent penicillin compounds (ampicillin, and aluminum seals, and the seals were crimped to create an piperacillin, ticarcillin) than with cefoxitin, cefotetan, and airtight cap. The vials were then removed from the anaerobic the P-lactamase inhibitor combinations. chamber and sterilized at 250°C for 15 min. All broths were Killing kinetics. Against B. fragilis ATCC 25289 (Fig. 1), colorless prior to use. Antibiotics were added at the follow- ceftizoxime showed bacteriostatic activity and ampicillin ing concentrations (in micrograms per milliliter): ampicillin, and ceftriaxone showed regrowth at 24 h with the 108- 16; ampicillin-sulbactam, 16/8; ticarcillin, 64; ticarcillin-cla- CFU/ml inoculum. Examination of the broth cultures con- vulanic acid, 64/2; piperacillin, 64; piperacillin-tazobactam, taining ceftizoxime and ceftriaxone showed gross turbidity 64/8; and cefoxitin, cefotetan, ceftizoxime, and ceftriaxone, at the 108-CFU/ml inoculum and tangled, elongated forms on 32. wet mount (Table 2). The other agents tested were bacteri- Inocula were prepared by incubating the test strain over- cidal at 24 h at all inocula. Our clinical strain, B. fragilis 4147 (data not shown) showed killing kinetics similar to those of night in a vial containing the prereduced anaerobically B. fragilis ATCC 25289. sterilized Anaerobe Broth MIC on a shaker at 36°C. Depend- B. fragilis 4347 (Fig. 2), a strain exhibiting a large inocu- ing on the turbidity, the overnight culture was diluted 1:2 to lum effect, was not killed by ampicillin, ticarcillin, or piper- 1:5 and 5 ml was added to the prewarmed test vials for a acillin, but ampicillin-sulbactam, piperacillin-tazobactam, 108-CFU/ml inoculum. A 1:100 dilution was prepared, and 5 and ticarcillin-clavulanic acid were bactericidal. Results for ml was added to the test vials to obtain the 106-CFU/ml the cephalosporins tested are not shown in Fig. 2 since the inoculum. Antibiotics and inoculum were added to the organism was resistant to them even at the low inoculum. anaerobic chamber by using plastic syringes that had been B. thetaiotaomicron 3118 (Fig. 3) shows a pattern similar preconditioned in the anaerobic chamber for at least 24 h to that of B.fragilis 4347 with no or poor bactericidal activity prior to use to avoid oxidizing the cultures. Incubation was by ampicillin, piperacillin, and ticarcillin but good bacteri- on a shaker in an aerobic incubator. The resazurin remained cidal activity at both inocula by these agents when combined colorless throughout the incubation. Growth was quantified with their ,B-lactamase inhibitors. Elongated forms were at the time of inoculation and at 3, 6, 9, and 24 h by removing present at the 108-CFU/ml inoculum on the wet mounts of aliquots from the vials by using a syringe equipped with a ampicillin, ticarcillin, and piperacillin. Again, the data for 25-gauge needle. The aliquots were transferred into the the cephalosporins are not shown as the isolate was resistant anaerobic chamber, 10-fold dilutions were prepared, and 0.1 to them even at the low inoculum. ml of the appropriate dilutions was plated onto BBA by B. ovatus 3119 (Fig. 4) was susceptible to all the 1-lacta- means of a Rotaplate (Fisher Scientific, Indiana, Pa.). The mase inhibitor combinations, and therefore they are not plates were incubated for 48 to 72 h, and colonies were shown in Fig. 4. Cefoxitin was bactericidal at both inocula. counted. Wet mounts from the 108-CFU/ml bottles were Ceftizoxime, ceftriaxone, and cefotetan showed large inoc- examined for morphologic changes at 3 and 6 h. ulum effects. Ceftizoxime was clearly bactericidal at the 1870 GOLDSTEIN ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1. Inoculum effect of antimicrobial agents on B. fragilis group strains Result at inoculum: Organism 104 106 CFU/ml 108 CFU/ml Ratio GM (no. of strains) CFU/ml 1081/104 and antibiotic" MICgo GM MIC % MIC GM MIC MIC%)GM MIC (,g/ml) (pg/ml)' suscept.d 0 suscept. ' suscept. B. fragilis (26) Ampicillin >128 23.2 65 >128 38.4 50 >128 89.6 0 >3.9 Ampicillin-sulbactam 4 1.5 100 8 1.9 100 64 6.8 88 4.5 Ticarcillin >128 15.2 85 >128 41.6 69 >128 96.0 50 >6.3 Ticarcillin-clavulanate 2 0.3 100 4 0.7 100 128 3.8 81 12.7 Piperacillin 64 5.2 96 >128 13.6 85 > 128 54.4 62 >10.5 Piperacillin-tazobactam 4 1.4 100 8 2.7 100 64 12.0 92 8.6 Cefoxitin 32 9.6 96 32 13.6 96 64 20.2 88 2.1 Cefotetan 64 7.2 81 >128 13.6 77 > 128 22.4 73 3.1 Ceftizoxime 16 1.8 100 64 13.6 85 >128 153.6 4 >85.3 Ceftriaxone 64 6.0 84 >128 27.2 62 > 128 166.4 4 >27.7 B. thetaiotaomicron (23) Ampicillin > 128 54.4 35 >128 83.2 13 >128 108.8 4 >2.0 Ampicillin-sulbactam 8 2.6 96 16 3.6 96 16 5.2 91 2.0 Ticarcillin > 128 54.4 74 >128 89.6 70 >128 115.2 61 >2.1 Ticarcillin-clavulanate 4 1.2 100 8 2.8 100 16 6.8 91 5.7 Piperacillin 128 22.4 83 >128 44.8 74 >128 128.0 43 >5.7 Piperacillin-tazobactam 16 8.0 100 32 14.4 100 > 128 41.6 83 5.2 Cefoxitin 32 25.0 91 64 47.0 78 > 128 54.4 65 2.2 Cefotetan >128 64.0 52 >128 128 17 > 128 230.4 0 >3.6 Ceftizoxime 16 5.2 96 128 83.2 35 >128 243.2 0 >46.8 Ceftriaxone > 128 70.4 35 >128 153.6 9 >128 243.2 0 >3.5 B. distasonis (20) Ampicillin > 128 8.0 75 >128 13.6 75 > 128 30.4 60 3.8 Ampicillin-sulbactam 8 3.2 95 8 5.8 95 32 12.8 80 4.0 Ticarcillin > 128 11.2 80 >128 18.4 80 >128 26.4 75 2.4 Ticarcillin-clavulanate 8 3.6 100 16 7.2 100 32 8.4 100 2.3 Piperacillin 32 3.9 95 >128 12.8 85 >128 121.6 35 >31.2 Piperacillin-tazobactam 8 2.4 100 32 6.4 95 128 66.4 65 27.7 Cefoxitin 16 7.8 95 64 18.4 85 64 19.2 85 2.5 Cefotetan 128 25.6 80 >128 59.2 55 >128 64.0 45 2.5 Ceftizoxime 16 1.2 95 >128 4.6 85 > 128 29.6 75 >24.7 Ceftriaxone 32 3.0 90 >128 15.2 70 > 128 86.4 50 >28.8 B. ovatus (20) Ampicillin 64 25.6 45 64 38.4 25 > 128 86.4 0 3.4 Ampicillin-sulbactam 4 1.6 100 4 2.0 100 8 3.0 95 1.9 Ticarcillin 128 36.8 85 >128 62.4 60 >128 118.4 35 >3.2 Ticarcillin-clavulanate 1 0.6 100 2 1.1 100 4 2.3 100 4.0 Piperacillin 16 10.4 90 64 17.6 90 >128 54.4 75 5.2 Piperacillin-tazobactam 8 3.3 100 8 4.8 100 16 12.4 100 3.8 Cefoxitin 32 13.2 95 32 14.8 95 32 22.4 95 1.7 Cefotetan 64 26.4 75 128 48.0 55 128 121.6 5 4.6 Ceftizoxime 16 6.8 100 64 26.4 75 >128 128.0 10 >18.8 Ceftriaxone > 128 59.2 45 >128 83.2 30 >128 211.2 0 >3.6 B. vulgatus (20) Ampicillin Nle NI NI >128 13.6 70 >128 41.6 35 >3.1 Ampicillin-sulbactam NI NI NI 4 1.7 100 8 3.6 95 2.1 Ticarcillin NI NI NI 32 8.8 90 >128 44.8 65 >5.1 Ticarcillin-clavulanate NI NI NI 1 0.4 100 4 1.9 95 5.0 Piperacillin NI NI NI >128 8.8 85 >128 27.2 75 3.1 Piperacillin-tazobactam NI NI NI 8 2.9 100 16 8.0 100 2.8 Cefoxitin NI NI NI 8 4.4 95 16 6.4 90 1.5 Cefotetan NI NI NI 32 10.0 90 32 12.0 90 1.2 Ceftizoxime NI NI NI 16 6.0 95 >128 102.4 20 >17.1 Ceftriaxone NI NI NI >128 6.8 85 >128 70.4 35 >10.4 a Breakpoints (in micrograms per milliliter): ampicillin, 16; ampicillin-sulbactam, 16/8; ticarcillin, 64; ticarcillin-clavulanate, 64/2; piperacillin, 64; piperacillin- tazobactam, 64/8; and cefoxitin, cefotetan, ceftizoxime, and ceftriaxone, 32. 6 Ratio of the geometric mean MICs at 108- and 104-CFU/ml inocula. ' For strains exhibiting off-scale values of >128, 256 was used for the calculations. d Percentage of strains susceptible at breakpoint concentrations of the antibiotics. e NI, noninterpretable. Growth of most strains was poor at this inoculum, resulting in many skipped wells and uninterpretable results. VOL. 35, 1991 INOCULUM EFFECT FOR B. FRAGILIS GROUP 1871

A A/S T T/C P P/T FOX TET ZOX TRX GC

cli0 cs (D 106 at 6h a [] 108 at 6h 0 C * 24h -3 C = Clear 0 H -4 =Hazy 0 T =Turbid -5 -6 HT CH CH CC CH CC CH CH HT HT TT C (D OD \ O C O O OD 0 C MIC t \ , OD NoCD~ ~ r' 7- _n c

FIG. 1. Killing kinetics for B. fragilis ATCC 25285. Abbreviations: A, ampicillin; A/S, ampicillin-sulbactam; t, ticarcillin; T/C, ticarcillin-clavulanic acid; P, piperacillin; P/T, piperacillin-tazobactam; FOX, cefoxitin; TET, cefotetan; ZOX, ceftizoxime; TRX, ceftriaxone; GC, growth control. Where the black bar is higher than the shaded or open bars, regrowth from 6 to 24 h was seen. Clear (C), hazy (H), and turbid (T) indicate the appearance of the broth after 48 h of incubation.

106-CFU/ml inoculum but bacteriostatic with regrowth oc- DISCUSSION curring at 24 h for the 108-CFU/ml inoculum. B. distasonis 3568 (Fig. 5) showed bactericidal activity at In a review of the inoculum effect, Brook (7). noted that both inocula for the ,-lactamase inhibitor combinations but there was little inoculum effect observed for the B. fragilis not for their penicillin-derived components. B. vulgatus 2269 group with cefoxitin but that most of the other beta-lactams (Fig. 6) showed bactericidal activity at both inocula only tested, especially ceftizoxime, exhibited a marked inoculum for piperacillin-tazobactam. Ticarcillin-clavulanic acid was effect. In our study, ceftizoxime exhibited the largest and somewhat active at the low inoculum, and ampicillin-sulbac- cefoxitin the least inoculum effect. Inoculum concentration tam showed little activity. MICs of ceftizoxime, ceftriaxone, showed little or no influence on kill kinetics of cefoxitin. and cefotetan, but not cefoxitin, were above the breakpoint The studies noted by Brook (7) either tested all the at the 106-CFU/ml inoculum for these two organisms, and combined B. fragilis group species without identification by therefore these data are not displayed. species or listed only the species B. fragilis as tested. The Morphological changes. Table 2 describes the morpholog- studies cited also used a variety of methods and; inocula. Our ical changes with a 108-CFU/ml inoculum after exposure to data indicate that variation in inoculum effect is-also species various antimicrobial agents for 3 to 6 h of incubation. The dependent. In general, B. fragilis and then B. thetaiotaomi- penicillins, ceftizoxime, and ceftriaxone showed elongated cron showed the greatest and B. ovatus and B. vulgatus forms. B. distasonis showed elongated forms with exposure showed the least inoculum effect. With piperacillin-tazobac- to piperacillin and piperacillin-tazobactam which were still tam, B. distasonis showed the greatest inoculum effect, with bactericidal. Elongated forms were not noted with the other B. fragilis second. With ceftizoxime and ceftriaxone, the 3-lactamase inhibitor combinations. Spheroplast formation order of inoculum effect was B. fragilis > B. thetaiotaomi- was most notable for cefoxitin and the 1-lactamase inhibitor cron > B. distasonis > B. ovatus = B. vulgatus (Table 1). combinations. Recent in vivo studies (19) note that the inoculum effect may

TABLE 2. Appearance of bacterial cells at 108-CFU/ml inoculum examined after 3 to 6 h of exposure to antibiotics in Wilkins-Chalgren broth on a shaker Appearance of exposed cellsa Antibiotic B. fragilis B. fragilis B. ovatus B. fragilis B. thetaiotaomicron B. distasonis B. vulgatus B. fragilis ATCC 25285 4806 3119 4147 3118 3568 2269 4347 Ampicillin NT NT NT S, L L, N N N N, L Ampicillin-sulbactam NT NT NT S, L S N, S N, S S, N Ticarcillin NT NT NT S, L L, N N N, L L Ticarcillin-clavulanate NT NT NT S, L S, L S N, S S, N Piperacillin NT NT NT S, L N, L L N L Piperacillin-tazobactam NT NT NT S, L S, N L N, S S, N Cefoxitin S S S S, N NT NT NT NT Cefotetan S S L L, N, S NT NT NT NT Ceftizoxime L L L, S L NT NT NT NT Ceftriaxone L, N L, S L L NT NT NT NT a N, normal appearance; L, elongated; S, spheroplasts; NT, not tested. 1872 GOLDSTEIN ET AL. ANTIMICROB. AGENTS CHEMOTHER.

A A/S T T/C P P/T GC FOX TET ZOX TRX GC

~0 cq 'I!' M lO- at 6h Cc } 106 at 6h O 10s at 6h a J 108 at 6h 0 0 * 24h * 24h C = Clear C = Clear 0 -3- H =Hazy 9 H =Hazy -4- - T T =Turbid -JSb T = Turbid -5-

X00¢z 0 0 ¢ (D 00aatN N W - JNW - NWN If TT TT

NWN NO NO NO FIG. 2. Killing kinetics for B.fragilis 4347. See the legend to Fig. IK) IMN Io w ON 1 other details. for FIG. 4. Killing kinetics for B. ovatus 3119. See the legend to Fig. 1 for other details. have clinical significance. If this is the case, it would for clinical laboratories to consider therefore seem important more clinically relevant remains to be determined. to species level of the B. fragilis group mem- method is identification Other cephalosporins, such as cefoxitin, have not exhibited bers in assisting clinicians to help choose therapeutic regi- such marked variations in MICs with the two methods. Until mens. clinical correlation of patient outcome with ceftizoxime Ceftizoxime and ceftriaxone had a marked inoculum effect methods is established, it would seem that was substantially underestimated by the geometric susceptibility testing the geo- prudent for clinicians to closely observe patients treated for mean calculations. For the 108-CFU/ml inoculum, infections with ceftizoxime to assess their be considered incalculable since MICs for mixed anaerobic metric mean could clinical response. >20 to 50% of the strains were !128 jig/ml. For these to that of cefoxitin as the number in the calcula- Cefotetan showed activity equivalent strains, 256 p,g/ml was taken against B. fragilis at both inocula, but geometric mean MICs tions of the geometric mean. However high they may ap- ovatus, and B. thetaio- artificially low. Inactivation or drug and MIC90s for B. distasonis, B. pear, they could be taomicron were higher with cefotetan than with cefoxitin. destruction are possible explanations for the inoculum effect This is in accord with prior studies (10, 13) that note seen with ceftizoxime and ceftriaxone. The hypothesis of against indi- of Aldridge et variability in the in vitro activity of cefotetan drug destruction is supported by the findings vidual members of the B. fragilis group species. al. (1). Studying cefoxitin-resistant B. fragilis group isolates, poorer in vitro activity to ceftizoxime Ceftriaxone generally showed they noted that the addition of sulbactam against all member species of the B. fragilis group than markedly reduced the MICs of ceftizoxime. ceftriaxone also ex- regarding the susceptibility test- cefoxitin or ceftizoxime. Additionally, Additional concerns exist a inoculum effect. Filamentous trans- B. fragilis in that marked differ- hibited relatively large ing of ceftizoxime against formation was seen at the high inoculum and correlated with ences in MICs have been noted when the agar dilution and activity. broth dilution methods have been used (2, 4, 23). Which bacteriostatic

A A/S T T/C P P/T GC A A/S T T/C P P/T GC 4 3- a 0 1- 2- z 1 0 0 WtI 1- r- 0 106 at6h 0 l06 at 6 acw W -1- 106 at 6h 108 at 6h aO- * * 24h & -2- 24h ar- = = C =Clear C Clear 0 -3- H -Hazy 0 H =Hazy 0 -4- T =Turbid -j T =Turbid 9 -5- -6- TT CH TT CT TT CH TT TT CH TT CH TT CH TT

I 28OD W O OD WCJ MIC N OD CD O a N -- NW i) NWX n g ax , 8 N t FIG. 3. Killing kinetics for B. thetaiotaomicron 3118. See the FIG. 5. Killing kinetics for B. distasonis 3568. See the legend to legend to Fig. 1 for other details. Fig. 1 for other details. VOL. 35, 1991 INOCULUM EFFECT FOR B. FRAGILIS GROUP 1873

A A/S T T/C P P/T GC ACKNOWLEDGMENTS We thank Margareta I. Ostovari, Alice E. Goldstein, Gregory Ferguson, and Judee H. Knight for various forms of assistance. This study was supported, in part, by a grant from Merck Sharp & Dohme, West Point, Pa.

o4-4 REFERENCES EO 106 at 6h 1. 5- 108 at 6h Aldridge, K. E., A. Hendererberg, and C. V. Sanders. 1990. El Enhanced antimicrobial activity of old and new beta-lactams * 24h against cefoxitin resistant Bacteroides fragilis group isolates by C = Clear beta-lactamase inhibitors. J. Antimicrob. Chemother. 25:873- 883. -J- H =Hazy 2. Aldridge, K. E., and C. V. Sanders. 1987. Antibiotic- and T =Turbid method-dependent variation in the susceptibility testing results of Bacteroides fragilis group isolates. J. Clin. Microbiol. 25: M6C TT TT TT HT TT CH TT 2317-2321. 3. Aldridge, K. E., C. V. Sanders, and R. L. Marrier. 1986. OD CM CD CD CD Co CD MIC a)\ 0 IT N to K) N Ni N N N Ca) (D Variations in the potentiation of beta-lactam antibiotic activity by clavulanic acid and sulbactam against multiply antibiotic- FIG. 6. Killing kinetics for B. vulgatus 2269. See the legend to resistant bacteria. J. Antimicrob. Chemother. 17:463-469. Fig. 1 for other details. 4. Aldridge, K. E., H. M. Wexler, C. 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