ANTIMICROBiAL AGENTS AND CHEMOTHERAPY, Feb. 1980, p.129-131 Vol. 17, No. 2 0066-4804/80/02-0129/03$02.00/0 Comparative In Vitro Activity of 1-Oxa-,8-Lactam (LY127935) and with Other fl-Lactam Against Anaerobic Bacteria M. VICTORIA BOROBIO, JAVIER AZNAR, RUFINO JIMENEZ, FERNANDO GARCIA, AND EVELIO J. PEREA Department ofMicrobiology, University ofSeville Medical School, Seville 9, Spain The in vitro activity of 1-oxa-f8-lactam (LY127935), cefoperazone (T-1551), , cefsulodin, , , and on 85 anaerobic clinical isolates (30 Bacteroides, 30 Clostridium, 25 Peptococcaceae) wa simultaneously determined by the agar dilution test in two different media, Brucella Agar (Difco Laboratories) and Wilkins-Chalgren agar. In Wilkins-Chalgren agar, 90% of Bac- teroides were inhibited by (micrograms per milliliter): LY127935, 0.5; T-1551, 64; cefoxitin or cefuroxime, 8; cefsulodin or cefotaxime, 32; and cefaclor, 128. All Clostridia were inhibited in Wilkins-Chalgren by (micrograms per milliliter): LY127935, 4; T-1551, 2; cefoxitin, 6; cefuroxime, 0.12; cefsulodin, 0.5; cefaclor, 1; and cefotaxime, 8. All Peptococcaceae were inhibited by T-1551, cefsulodin or cefotaxime at 4 ug/ml and by cefoxitin or cefuroxime at 1 to 2 ,tg/ml. With cefaclor at 8 ,ug/ml, 92% of strains were inhibited, and LY127935 at 16 ,ug/ml only inhibited 64% of strains. LY127935 was the most active of the antibiotics tested against Bacteroides, showing good activity against Clostridia and poor activity on Peptococcaceae, whereas T-1551 was more active against Peptococcaceae and had similar activity against Clostridia and poor activity on Bacteroides. There are no significant differences between minimal inhibitory concentrations obtained in Brucella Agar and those obtained in Wilkins-Chalgren. A great number of new have Agar was the most commonly used medium for been discovered in recent years. Some of them anaerobic susceptibility testing (9, 12, 13), and have been tested against anaerobes, either Bac- Wilkins-Chalgren agar was suggested by the Na- teroides, Clostridia, or Peptococcaceae, but in tional Committee for Clinical Laboratory Stan- general these antibiotics have not been tested dards (V. L. Sutter, A. L. Barry, W. J. Martin, J. simultaneously with all the three bacterial E. Rosenblatt, V. R. Dowell, Jr., C. Thornsberry, groups. The most studied is cefoxitin, T. D. Wilkins, and R. J. Zabransky, Progr. Abstr. whose range of activity presently is well known Intersci. Conf. Antimicrob. Agents Chemother., (6, 9, 11). 16th, Chicago, Ill. abstr. no. 453, 1976). The purpose of the present study was to com- pare the in vitro activity of two new antibiotics, 1-oxa 8i-lactam (LY127935) (6R, 7R)-7-[(car- MATERIALS AND METHODS boxy(4-hidroxyphenyl)acetyl]amino)-7-meth- Bacterial strains. A total of 85 isolates including oxy-3-[[(1-methyl-1H-tetrazol-5-yl)thio]methyl] 30 Bacteroides (20 B. fragilis, 4 B. distasonis, 3 B. -8-oxo-5-oxa-1-azabicyclo[4.2.0]oct-2-ene-2 car- vulgatus, 3 B. thetaiotaomicron), 30 Clostridium (13 boxylic acid, disodium salt, and cefoperazone (T- B. perfringens, 11 B. bifermentans, 2 B. sordellii, 2 B. 1551)[(sodium 7-D(-)-a-(4-ethyl-2,3-dioxo-1-pi- septicum, 2 B. beijerinckii), and 25 Peptococcaceae perazinecarboxamido)-a-(4-hidroxyphenyl acet- (14 P.peptococcus spp., 11 P.peptostreptococcus spp.) amido]-3-[1-methyl-lH-tetrazol-5yl)-thiome- were examined in this study. All were obtained from thyl]-3--4-carboxylate)] with cefoxitin, in-patients of the University Hospital of Seville during cefuroxime, cefsulodin, cefotaxime, and cefaclor 1978. Identification to species level was done by the bacteria: Bacteroides system of Holdeman and Moore (4). against anaerobic (fragilis Five reference strains recommended by the Na- group), Clostridium spp., and Peptococcaceae tional Committee for Clinical Laboratory Standards spp. isolated from clinical material. (Sutter et al., 16th ICAAC, abstr. no. 453) were used: We determined simultaneously the miniimal B. fragilis ATCC 25285, B. vulgatus ATCC 29327, C. inhibitory concentrations (MICs) in two differ- perfringens ATCC 13124, P. magnus ATCC 29328, ent culture media, Brucella Agar (Difco Labo- and P. assacharolyticus MAYO P-227, all kindly sup- ratories) and Wilkins-Chalgren agar. Brucella plied by J. E. Rosenblatt. 129 130 BOROBIO ET AL. ANTmICROB. AGENTs CHEMOTHER. Antibiotics. Laboratory standards were kindly (3.8 pg/ml), cefuroxime (7.7 jig/ml), cefsulodin supplied as follows: LY127935 and cefaclor from Eli (8 pug/ml), and cefotaxime (14.5 ,ug/ml). The least Lilly & Co., cefoperazone from Pfizer Inc., cefoxitin active were cefoperazone (73.1 pg/ml) and cefa- from Merck Sharp & Dohme, cefuroxime from Glaxo clor (91.4 pig/ml). In Brucella Agar, the results Co., cefsulodin from Ciba-Geigy, and cefotaxime from Roussel-Uclaf. are similar, with the exception of cefuroxime, which seems to more active in Susceptibility testing method. The MIC was de- be Wilkins-Chal- termined by the agar dilution method with a replicator gren, having a median value of 3.5 ,g/ml. apparatus (10). Inoculum was prepared as described Among the 30 Bacteroides strains examined, by Sutter and Washington (12). Antibiotics were in- only four of them failed to produce ,8-lactamase. corporated into the media in twofold dilutions. There were no significant differences between Media. Two different culture media were used: the median MICs of the producers and those of Brucella Agar and Wilkins-Chalgren agar. Brucella the nonproducers with most of the antibiotics Agar was supplemented with vitamin K1 and enriched tested. With cefoperazone, those values were 64 with 5% laked sheep blood (11). was Wilkins-Chalgren ,ug/ml in the producers group and 8 pg/ml in the prepared as described by Wilkins and Chaen and supplemented with vitamin K1 and hemin (14). nonproducers. No conclusion can be obtained Detection of 84-lactamases. ,B-Lactamase produc- because of the small number of the nonpro- tion was determined in all Bacteroides strains by the ducers. plate method as descibed by O'Callaghan et al. (8). Against Clostridia all antibiotics showed ex- cellent activity. LY127935 and cefoperazone re- RESULTS AND DISCUSSION quired concentrations of 2 to 4 pg/ml to inhibit 100% of strains. 0.06 pAg of cefoxitin per ml and Table 1 shows the MICs of the seven antibi- 0.12 pug of cefuroxime per ml inhibited all strains. otics needed to inhibit 50 and 90% of the anaer- These results with cefoxitin are similar to those obic bacteria groups in Wilkins-Chalgren. previously reported by Perea et al. (9) and lower Against the Bacteroides species examined, than those obtained by Chow and Bednorz (2). LY127935, the most active antibiotic, inhibited Cefuroxime yielded MICs lower than those 90% at 0.5 pg/ml. Cefoperazone showed less ac- obtained by Jones et al. (5) and Neu and Fu (7) tivity, requiring 64 ug/ml to inhibit 90% of in Mueller-Hinton agar. Cefsulodin and cefaclor strains. The other agents tested, cefoxitin and required concentrations of 0.5 to 1 pug/ml to cefuroxime, inhibited 90% at 8 pig/ml, whereas inhibit all strains. MICs with cefaclor were cefotaxime and cefsulodin required 32 pug/ml. slightly lower than those reported by others (1, These results are similar to those previously 2). Cefotaxime was the least active, inhibiting reported (6, 9), but other authors testing cefu- 90% of strains at 4 pug/ml. roxime in supplemented brain heart infusion Against Peptococcaceae, LY127935 of all anti- obtained only 21% of strains inhibited at 32 ug/ biotics tested showed the least activity, requiring ml (5). Our results with cefsulodin and cefotax- more than 16 pug/ml to inhibit 90% of strains. All ime show MICs higher than those obtained in other cephalosporins inhibited all strains at 2 the Iso-Sensitest media by Hamilton-Miller et pug/ml, with the exception of cefaclor which in- al. (3) and lower than those obtained in supple- hibited only 80% of strains at this concentration. mented Mueller-Hinton agar by Neu and Fu (6). These results are similar to those obtained by Cefaclor was the least active, inhibiting 90% of other authors (2, 5). strains at 128 pg/ml; these results are similar to Differences in species susceptibility were not those from Bach et al. (1). All these results are found among the Bacteroides fragilis group, nor reflected in the median values; LY127935 was among the Clostridium species or Peptococca- the most active (0.7 ug/ml), followed by cefoxitin ceae. TABLE 1. MICs at which 50 and 90% ofanaerobes were inhibited by cephalosporinsa B. fragilis Clostridium Peptococcaceae Antibiotic (30 isolates) (30 isolates) (25 isolates) 50% 90% 50% 90% 50% 90% LY127935 0.5 0.5 1 2 16 >16 Cefoperazone 32 64 1 2 0.25 2 Cefoxitin 2 8 0.06 0.06 0.12 0.5 Cefuroxime 4 8 0.06 0.12 0.25 1 Cefsulodin 4 32 0.06 0.12 0.06 0.25 Cefotaxime 8 32 2 4 0.25 1 Cefaclor 64 128 0.5 0.5 0.5 4 a Each value represents MIC in micrograms per milliliter. VOL. 17, 1980 LY127935 VS. CEFOPERAZONE 131 In general there are no significant differences 2. Chow, A. W., and D. Bednorz. 1978. Comparative in between the MICs in vitro activity of new cephalosporins against anaerobic obtained Brucella Agar bacteria. Antimicrob. Agents Chemother. 14:668-671. and those obtained in Wilkins-Chalgren; how- 3. Hamilton-Miller, J. M. T., W. Brumfftt, and A. V. ever in Wilkins-Chalgren these values are Reynolds. 1978. Cefotaxime (HR 756) a new cephalo- slightly lower than in Brucella Agar with most sporin with exceptional broad-spectrum activity in vi- antibiotics. tro. J. Antimicrob. Chemother. 4:437-444. 4. Holdeman, L. V., and W. E. C. Moore (ed.). 1977. Considering the achievable serum levels after Anaerobe laboratory manual, 4th ed. Virginia Polytech- intramuscular administration of 1 g of each anti- nic Institute and State University, Blacksburg, Va. biotic, we can conclude theoretically that 5. Jones, R. N., P. C. Fuchs., T. L Gavan., E. H. Ger- LY127935 would inhibit all Bacteroides and lach., A. L Barry, and C. Thormsberry. 1977. Cefu- roxime, a new parenteral : collaborative Clostridia strains tested and possibly half of the in vitro susceptibility comparison with cephalotin Peptococcaceae. Cefoperazone would inhibit al- against 5,887 clinical bacterial isolates. Antimicrob. most half of Bacteroides and all Clostridia and Agents Chemother. 13:47-50. Peptococcaceae. 6. Neu, H. C., N. Aswapokee, P. Aswapokee, and K. P. Fu. 1979. HR 756 a new cephalosporin active against Two-thirds of Bacteroides and all Clostridia gram-positive and gram-negative aerobic and anaerobic and Peptococcaceae would be inhibited by ce- bacteria. Antimicrob. Agents Chemother. 15:273-281. furoxime, cefsulodin, or cefotaxime. Cefoxitin 7. Neu, H. C., and K. P. Fu. 1978. Cefuroxime, a beta- would inhibit all the three groups tested. Finally, lactamase-resistant cephalosporin with a broad spec- trum of gram-positive and -negative activity. Antimi- cefaclor would not be useful in treatment of crob. Agents Chemother. 13:657-664. Bacteroides infections, but would appear to be 8. O'Callaghan, C. H., A. Morris, S. M. Kirby, and A. H. useful in infections produced by Peptococcaceae Shingler. 1972. Novel method for detection of ,-lac- and Clostridia. tamases by using a chromogenic cephalosporin sub- strate. Antimicrob. Agents Chemother. 1:283-288. 9. Perea, E. J., J. Aznar, AL C. Garcia-Iglesias, and M. ADDENDUM V. Borobio. 1978. Cefoxitin sodium activity against anaerobes: effect of the inoculum size, pH variation and Compound LY127935 is described in this issue as an oxa- different culture media. J. Antimicrob. Chemother. B8-lactam. In past isues it was called 1-oxacephalosporin. The 4(Suppl. B):55-60. name cephem was proposed in 1962 (J. Am. Chem. Soc. 84: 10. Steers, E. E., F. L Foltz., B. S. Graves, and J. Rider. 3401) as a generic designation for the fused ,-lactam dihydro- 1959. An inocula replicating apparatus for routine test- thiazine ring system found in the cephalosporins. This system ing of bacterial susceptibility of antibiotics. Antibiot. has been generally accepted. The oxa-,B-lactam compound Chemother. 9:307-311. LY127935 has a ,-lactam dihydrooxazine ring system and thus 11. Sutter, V. L, and S. M. Finegold. 1976. Susceptibility cannot qualify as a cephem or cephalosporin by definition. To test of anaerobic bacteria to 23 antimicrobial agents. avoid confusion, the nonspecific term oxa-,B-lactam is used, Antimicrob. Agents Chemother. 10:736-752. pending approval of a generic name. 12. Sutter, V. L., and J. A. Washington II. 1974. Suscep- tibility testing ofanaerobes, p 436-438. In E. H. Lenette, E. H. Spaulding, and J. P. Truant (ed.), Manual of LITERATURE CITED clinical microbiology. Amencan Society for Microbiol- 1. Bach, V. T., M. M. Khurana, and H. Thadepalli. 1978. ogy, Washington, D.C. In vitro activity of cefacolor against aerobic and anaer- 13. Wilkins, T. D., and S. Chalgren. 1976. Medium for use obic bacteria. Antimicrob. Agents Chemother. 13:210- in antibiotic susceptibility testing of anaerobic bacteria. 213. Antimicrob. Agents Chemother. 10:926-928.