Stability. Received for publication 1 August to be fl-lactamase-producing strains.

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1978, p /78/ $02.00/0 Copyright X) 1978 American Society for Microbiology Vol. 13, No. 4 Printed in U.S.A. Cefaclor: In Vitro Spectrum of Activity and Beta-Lactamase Stability HAROLD C. NEU* AND KWUNG P. FU Departments ofmedicine and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York Received for publication 1 August 1977 The in vitro activity of cefaclor against 556 clinical isolates of gram-positive and gram-negative bacteria was compared with that of other cephalosporins. Cefaclor had activity similar to that of cephalexin against gram-positive bacteria. It showed greater activity against Haemophilus strains than did cephalexin and inhibited 8)-lactamase-producing Haemophilus isolates. Cefaclor was more active than cephalexin or cephalothin against Escherichia coli, Salmonella, and Shigella isolates but did not act against Serratia, Acinetobacter, indole-positive Proteus, or Bacteroides isolates. Cefaclor was resistant to type III (TEM),Blactamases but was destroyed by type I,8-lactamases and, to a lesser degree, by type IV and type V,8-lactamases. The use of oral cephalosporins as alternative agents to penicillins for outpatient therapy has increased in recent years because of concern over allergic reactions to penicillins and because of the increased number of 18-lactamase-producing staphylococci. Currently, in excess of 50 to 75% of Staphylococcus aureus isolates from outpatients are resistant to penicillin. Recently,,8- lactamase-producing Haemophilus influenzae, constituting 2 to 12% of the H. influenzae isolated, have been found in most areas of the world. Neisseria gonorrhoeae have also been shown to contain plasmids mediating /8-lactamase production. Of equal importance is the increased resistance of Escherichia coli and other members of the Enterobacteriaceae to penicillins and to some cephalosporins (3, 4). To date, only two oral cephalosporins, cephalexin and cephradine, have been used extensively in the United States. These two agents have similar in vitro activities. We wished to evaluate the in vitro activity of cefaclor, a new cephalosporin that has a chloro group substituted for the methyl group of cephalexin at position 3 of the dihydrothiazine ring. We also wished to compare its activity with those of available oral agents and the major new /-lactamase-resistant cephalosporins we studied earlier (2, 3). (This material was presented at the 16th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, 1976.) MATERIALS AND METHODS Cefaclor was obtained from Eli Lilly & Co. All other agents were gifts from their manufacturers. Bacterial isolates came from urine, sputum, or wounds of out- 584 patients from the Columbia-Presbyterian Medical Center. There was an effort to select isolates known to be fl-lactamase-producing strains. Susceptibility tests were performed with both agar and broth (Mueller-Hinton; BBL), using i colonyforming units as the final inoculum, as detailed previously (2). Broth dilutions were performed in tubes. Minimum bactericidal concentrations (MBCs) were defined as the lowest concentrations of antibiotic that yielded less than five colonies when 0.01 ml was subcultured onto blood agar containing no antibiotic.,b- Lactamase assays were done by both microiodometric and spectrophotometric methods, as described previously (5, 6). RESULTS The overall activity of cefaclor against common gram-positive organisms and certain gramnegative species is given in Table 1. Among the streptococci, isolates of all Streptococcus viridans, Strep. bovis, and Strep. pneumoniae were inhibited by a concentration of 0.8,ug/ml. However, the Strep. pyogenes and Strep. agalactiae were susceptible to concentrations of 0.2 to 3.1,ug/ml. Strep. faecalis, Strep. durans, and Strep. faecium, all true enterococci, required for inhibition minimal inhibitory concentrations (MICs) equivalent to jig or greater per ml. All of our isolates of H. influenzae and Neisseria, which included five 8?-lactamase-producing strains of H. influenzae and two,8-lactamase-producing strains of N. gonorrhoeae, were inhibited by 1.6,ug of cefaclor per ml. Cefaclor inhibited the gram-negative bacteria shown in Table 1. At a level of 12.5,ug/ml, 73% of the E. coli, 90% of the Salmonella, 55% of the Shigella, 77% of the Citrobacter, 91% of the

2 VOL. 13, 1978 CEFACLOR ACTIVITY 585 No. of TABLE I. In vitro activity of cefaclor Cumulative % inhibition at MIC ( ug/ml) of: Organism strains < >400 Strep. viridans Strep. pneumoniae Strep. pyogenes Strep. agalactiae Strep. bovis Strep. faecalis Strep. faecium Strep. durans Staph. aureus Staph. epidermidis N. gonorrhoeae H. influenzae N. meningitidis E. coli Salmonella Shigella Citrobacter Klebsiella Enterobacter Serratia P. mirabilis P. morganii P. rettgeri P. vulgaris Providencia Acinetobacter Pseudomonas Bacteroides Klebsiella, and 75% of the P. mirabilis isolates were inhibited. However, only 18% of the Enterobacter, 3% of the Serratia, 9% of the Proteus morganii, and 16% of the Providencia strains, and less than % of the Proteus vulgaris, Proteus rettgeri, and Bacteroides fragilis isolates, were inhibited. All Pseudomonas strains were as resistant to cefaclor as they were to available cephalosporins. The effects of the medium and of the inoculum size, as well as the differences between MICs and MBCs, vary among different cephalosporins. These factors were examined for cefaclor. Table 2 illustrates the differences between the MICs and MBCs of representative bacterial isolates. There was a two- to eightfold increase in MBCs over MICs for Staph. aureus and members of the Enterobacteriaceae, but the increase varied within a species (except for Enterobacter and Proteus, in which there was usually an eightfold increase). Size of inoculum in the test had a significant effect on MICs (Table 3). Against an inoculum of 7 colony-forming units, the cefaclor MIC was two- to eightfold greater than against an inoculum of 3 colony-forming units for both Staph. aureus and members of the Enterobacteriaceae. Enterobacter cloacae was resistant to cefaclor irrespective of inoculum TABLE 2. Comparison ofmics and MBCs of cefaclor No. of No. of strains strains with MBC > MIC No. with ofiden- Organism strains tical tested MIC 2a > and MBC Staph. aureus Strep. faecalis E. coli K. pneumoniae E. cloacae P. mirabilis P. morganii S. marcescens S. typhimurium a Fold greater. size, whereas another isolate was susceptible to an MIC of 50 ytg/ml when the inoculum was 3 CFU, and to an MIC of >400 plg/ml when the inoculum was 7 colony-forming units. In all of the media, MBCs were 2- to 16-fold greater than MICs. MICs for cefaclor, when measured in human serum, were two- to eight-

3 586 NEU AND FU ANTIMICROB. AGENTS CHEMOTHER. fold greater than those observed in tests conducted in broth. Table 4 demonstrates that the cefaclor MICs for both gram-positive and gram-negative bacteria were similar in many different media. In TABLE 3. Organism Inoculum effect on cefaclor MICs MIC (pg/mi) 1o7a 5 3 Staph. aureus E. coli... > S. marcescens... > K. pneumoniae P. morganii E. cloacae... > E. cloacae... > a Colony-forming units. TABLE 4. the presence of serum, cefaclor MICs were consistently greater than in any of the media tested. It is unclear whether this effect was due to increased decay of cefaclor in serum because, after a 3-h incubation in serum, 60% of the drug was destroyed (compared with 30% when incubated in broth at 370C). The comparative activity of cefaclor and other agents is given in Tables 5 and 6. Cefaclor was less active than either cephalexin or cephalothin against Strep. pyogenes. In contrast, it was more active than cephalexin but less active than cephalothin against Staph. aureus and Staph. epidermidis. Although cefaclor, like cefazolin, inhibited Strep. faecalis isolates when at,ug/ml, its activity against these organisms was minimal relative to that of ampicillin. The activities of cefaclor and other cephalo- Effect ofgrowth medium on cefaclor MICs MIC (pg/mil) Organism MH Serum NBa COP1 BHIC TSBd (95%) ph 6 ph 7 ph 8 ( Staph. aureus Strep. faecalis E. coli E. cloacae S. marcescens 800 K. pneumoniae P. morganii > a NB, Nutrient broth. 0Col, Columbia broth. ebhi, Brain-heart infusion. d TSB, Trypticase soy broth. ' MH, Mueller-Hinton medium. TABLE 5. Comparative activity of cefaclor and other known f8-lactam antibiotics against gram-positive organisms No. of Cumulative % inhibition at MIC (pg/mi) of: Organism Antibiotic strains tested Strep. pyogenes8... Cefaclor Penicillin G Staph. aureusi... Cefaclor Staph. epidermidis I... Cefaclor Oxacillin Strep. faecalisb... Cefaclor Cefazolin 14 Ampicillin adetermined with horse serum in brain-heart infusion agar. b Determined in Mueller-Hinton agar at ph 7.0.

4 VOL. 13, 1978 CEFACLOR ACTIVITY 587 TABLE 6. Comparative activity of cefaclor and other known cephalosporins against gram-negative organisms No. of Cumulative % inhibition at MIC (,ug/ml) of: Organism Antibiotic strains tested E. coli... Cefaclor E. coli (cephalothin-resistant)... Cefaclor Salmonella..... Cefaclor Shigella..... Cefaclor Citrobacter..... Cefaclor Klebsiella..... Cefaclor Enterobacter..... Cefaclor P. mirabilis... Cefaclor Proteus, indole positive Providencia... Cefaclor... Cefaclor H. influenzaea... Cefaclor Amoxicillin a Determined in chocolate agar sporins against gram-negative bacilli are shown in Table 6. Cefaclor was twice as active as cephalexin against cephalothin-susceptible E. coli and had activity equal to that of cefamandole. Against cephalothin-resistant E. coli, which contained primarily type ld /-lactamases (6), the activity was similar to that of cefamandole and cephalexin. Against E. coli containing type II and type IV 18-lactamases, cefaclor had activity similar to that of cephalexin. Against E. coli with type III fi-lactamases, cefaclor and cephalexin had very similar activities. Against Salmonella isolates (S. typhimurium, S. enteritidis, and S. heidelberg), which possess primarily type III,B-lactamases, cefaclor was no more active than other compounds. The same was true of Shigella sonnei isolates, which were f.-lactamase producers. Cefaclor was the most active agent tested against Salmonella isolates and was as active as cefamandole against Shigella isolates, which did not produce 8.i-lactamases. Cefaclor had greater activity than cephalexin against Citrobacter strains. Against Klebsiella isolates, the activity of cefaclor w4s comparable to that of the newer parenteral agents, but cefaclor was less active than cefamandole against 97 97

5 588 NEU AND FU Enterobacter isolates. Although cefaclor was more active than cephalexin against P. mirabilis, cefaclor showed low activity against indolepositive Proteus strains when compared with cefoxitin. and cefaclor acted equally poorly against Serratia, Acinetobacter, and Bacteroides fragilis isolates, and cefaclor failed to provide oral cephalosporin coverage against these organisms. Cefaclor was active against both ampicillin-susceptible H. influenzae and,8-lactamase-producing isolates. Cefaclor was markedly more active against Haemophilus isolates than was cephalexin. The instability of cefaclor in the presence of various gram-negative,8-lactamases is shown in Table 7. Although the compound was resistant to the most prevalent plasmid-mediated /8-lactamase (type III or TEM), which is present in E. coli, H. influenzae, and N. gonorrhoeae, it was much less stable than cefoxitin and no more stable than cephalexin. Its greater stability as compared with that of cephalothin may have contributed to its increased activity against some Enterobacter and E. coli isolates. Table 8 demonstrates that the MICs of cefaclor were consistently lower than those of cephalexin, although the compound was destroyed to a similar or greater extent by the /3-lactamases present in the bacteria. This would seem to indicate that factors other than 18-lactamase stability play a role in the activity of this compound. DISCUSSION On the basis of this and another comparative study (1), cefaclor has been shown to have activ- TABLE 7. Relative hydrolysis of cephalosporins by /3-lactamasesa Hydrolysis (%) Source of Type of enzyme enzymeb Cefa- Cepha- Cefoxclor lexin itin E. coli... III E. coli... II Klebsiella I... İ 0 Citrobacter Serratia... I 0 Serratia... I Enterobacter... V 50 0 P. mirabilis... IV 17 0 a fi-latamase assays were performed with partially purified or purified enzymes in a spectrophotometric assay. Hydrolysis of cephalothin was set at % for all organisms listed. Type III f,-lactamase had minimal activity against cephalothin, but that activity was set at %. b Classified according to the Richmond classification. ANTIMICROB. AGENTS CHEMOTHER. TABLE 8. Comparison of the hydrolysis of cefaclor, cephalexin, and cephalothina MIC (Ag/ml) Organism Cefaclor E. coli ()b 6.3 (0) 12.5 (50) P. mirabilis (157) 12.5 (26) 3.1 (5) Klebsiella (0) 3.1 (0) 3.1 (19) Acinetobacter (167) 50 (0) (2) Entrobacter (31) (64) 200 (0) amics were determined by the agar method. Hydrolysis of the compounds was determined by a sonic extract of the organisms. bnumbers in parentheses indicate micromoles per minute per milligram. ity similar to that of cephalexin against a number of gram-positive species and to be more active than cephalexin against Haemophilus isolates. This compound was resistant to the same f8- lactamases that cephalexin resisted and was more active than cephalexin against cephalothin-susceptible members of the Enterobacteriaceae, such as E. coli, Salmonella, and Shigella. Cefaclor was active against some cephalothinresistant strains, but this activity could not be reliably predicted. This study has shown that inoculum size has a significant effect on the MICs of cefaclor determined in broth. There was a difference in MICs and MBCs when cefaclor was tested against some gram-negative bacilli such as Enterobacter and Proteus. Such differences have also been encountered with cefamandole and, to a degree, with other cephalosporins (4). The relevance of these observations to clinical situations is unknown. Further clinical studies are necessary to determine the relative role of cefaclor compared with that of the cephalosporins now available. LITERATURE CITED 1. Bill, N. J., and J. A. Washington II Comparison of in vitro activity of cephalexin, cephradine, and cefaclor. Antimicrob. Agents Chemother. 11: Fu, K. P., and H. C. Neu In vitro study of netilmicin compared with other aminoglycosides. Antimicrob. Agents Chemother. : Neu, H. C , a semisynthetic cephamycin antibiotic: antibacterial spectrum and resistance to hydrolysis by gram-negative beta-lactamases. Antimicrob. Agents Chemother. 6: Neu, H. C , a cephalosporin antibiotic with an unusually wide spectrum of activity. Antimicrob. Agents Chemother. 6: Novick, R. P Microiodometric assay for penicillinase. J. Biochem. 83: Richmond, M. H., and R. B. Sykes The fl-lactamases of gram-negative bacteria and their possible physiological role. Adv. Microb. Physiol. 9:31-88.