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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1979, p. 315-317 Vol. 15, No. 2 0066-4804/79/0015-0315/03$02.00/0 Effect of on the Minimum Inhibitory Concentration of , , Carbenicillin, or Cephalothin Against Clinical Isolates Resistant to Beta- Lactam L. DUMON, P. ADRIAENS, J. ANNI9, AND H. EYSSEN* The Rega Institute, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium Received for publication 6 November 1978 The effect of clavulanic acid on the minimum inhibitory concentration of benzylpenicillin, ampicillin, carbenicillin, or cephalothin against 353 clinical iso- lates of - and/or cephalothin-resistant strains was estimated. Resistance to ,B-lactam antibiotics, caused by using a diffusion method on agar plates con- mainly by ,8-lactamase-producing bacteria, is a taining ampicillin at a concentration of 10 ,ug/ continuing obstacle to effective antimicrobial ml. Due to the inactivation of the ,B-lactamase therapy. New semisynthetic and of the Klebsiella by clavulanic acid, the ampi- , resistant to f,-lactamases, have cillin was protected, and inhibition zones were been and are being developed to cope with this formed. Sodium clavulanate reference standard problem. An alternate approach of relatively was kindly supplied by M. Cole, Beecham Phar- recent interest is the use of ,B-lactamase inhibi- maceuticals, England. The presence of clavu- tors, such as clavulanic acid (1), in combination lanic acid in the extract was confirmed by high- with the above antimicrobial agents. To date performance liquid chromatography of the ben- studies of these combinations have been quite zyl ester, which was prepared from the sodium limited, involving work with a single bacterial salt by reaction with benzyl bromide in dime- species (3, 7; C. Thornsberry, C. N. Baker, and thylformamide. Chromatography was per- L. A. Kirven, Program Abstr. Intersci. Conf. formed on a silica gel (10 ,m) column (0.46 by Antimicrob. Agents Chemother. 17th, New 25 cm) with dichloromethane-acetonitrile-t-bu- York, N.Y., Abstr. no. 61, 1977) or a limited tyl alcohol (97:2:1) at 2 ml/min. In this system, number of species (4, 6). These studies have not the product had the same retention time (45 dealt definitively with the critical issue of what min) as the benzyl ester of reference clavulanic such combinations add to the activities of the acid; in addition, both products gave identical penicillins and cephalosporins when applied to mass spectra (M.' = 289). No other antibiotics the mix ofpenicillin-resistant pathogens encoun- were detected in the crude sodium clavulanate tered clinically. This issue has been examined in powder by paper chromatography (n-butyl al- our laboratories with the results summarized in cohol-acetic acid-water, 12:3:5) and bioassay of this report. Specifically, we have assessed the the paper strips against K. edwardsii or Staph- impact of combining clavulanic acid with ben- ylococcus aureus. The impurities in the prepa- zylpenicillin, ampicillin, carbenicillin, or cepha- ration consisted mainly of sodium chloride, fatty lothin on the activities of these agents against acids, and other unidentified compounds, but 353 penicillin-resistant clinical isolates, selected they did not interfere with the activity of cla- without regard for /3-lactamase activity. vulanic acid. Clavulanic acid used in the assays was pro- We have studied the influence of clavulanic duced by Streptomyces clavuligerus ATCC acid on the resistance of S. aureus to benzylpen- 27064 in DAS culture medium (5). After incu- icillin, of Serratia, Proteus, and Pseudomonas bation for 96 h at 270C, the culture filtrate was spp. to carbenicillin, and of Escherichia coli, acidified to pH 2.5 with 4 N HCl, extracted twice Klebsiella spp., Enterobacter spp., and Serratia with n-butyl alcohol-ethyl acetate (3:1), and spp. to ampicillin. In addition, the gram-negative reextracted into an aqueous solution neutralized bacilli, except Proteus and Pseudomonas spp., to pH 7.0 with 1 N NaOH. The aqueous phase were tested for susceptibility to combinations of was lyophilized, yielding a powder which con- cephalothin and clavulanic acid. Sodium clavu- tained 30% (wt/wt) of sodium clavulanate as lanate and the penicillin or cephalothin, each biologically estimated with Klebsiella edwardsii dissolved in 1 ml of sterile 0.05 M phosphate 315 316 NOTES ANTIMICROB. AGENTS CHEMOTHER.

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C) .> ~ ~ ~ ~ 4) 02. cjn 5>2~ i VOL. 15, 1979 NOTES 317 buffer at pH 6.5, were dispensed in petri dishes gonorrhoeae (6), and Bacteroides fragilis (6, 7) and mixed with 8 ml of tryptic soy agar (Difco, were susceptible to the action of clavulanic acid Detroit, Mich.). Sodium clavulanate was tested plus f8-lactam antibiotics. at final concentrations of 5 or 20 ,tg/ml. These The large differences among various bacterial concentrations were chosen since serum levels species and different strains of the same species of 5 ,ug/ml can be reached after oral or parenteral in their response to clavulanic acid are not en- administration (2); higher levels may be ob- tirely unexpected. Hunter et al. (2) reported that tained, e.g., in the urine. The antibiotics were the effect of the 8-lactamase inhibitor depends tested in twofold dilutions from 100 to 0.1 ,ug/ml. on the type and amount of,B-lactamase produced Control plates without penicillin, cephalothin, (if formed), the location of the /1-lactamase (in- or clavulanic acid were included in each assay. tra- or extracellular), the penetration of the ,B- Plates were inoculated with approximately 105 lactam into the cell, the stability of colony-forming units and incubated at 370C the f-lactam antibiotic to /?-lactamases, and the overnight. Minimum inhibitory concentration activity of the /8-lactam antibiotic used. There- (MIC) was defined as the lowest antibiotic con- fore, in vitro testing of the effect of clavulanic centration inhibiting visual growth on the agar acid against ,B-lactam-resistant clinical isolates plates. might be indicated before therapeutical appli- The activity of clavulanic acid on penicillin- cation of this compound. or cephalothin-resistant strains varied largely between the species tested, but also differed We thank E. Mathijs, S. Asselberghs, and G. Hamelryck between strains of the same species (Table 1). for their technical assistance. We acknowledge X. Delacourt, More than 50% of the penicillin-resistant S. au- St. Michiels-Clinic, Brussels, and J. Bogaerts and J. Vandev- reus strains became susceptible to 0.2 ug of enne, Academic Hospital St. Rafael, Leuven, for the isolation benzylpenicillin in the presence of 5 ,ug of cla- and identification of the clinical isolates. L.D. is a fellow of the Belgian I.W.O.N.L. (Instituut tot vulanic acid per ml; moreover, these strains were Aanmoediging van het Wetenschappelijk Onderzoek in susceptible to 20 ,tg of clavulanic acid alone. Nijverheid en Landbouw). Klebsiella spp. also responded well to clavulanic acid plus penicillin; clavulanic acid at 5,tg/ml LITERATURE CITED reduced the MIC of ampicillin to c6.2 ,ug/ml for 1. Brown, A. G., D. Butterworth, M. Cole, G. Hanscomb, 50% of the strains. After raising the concentra- J. D. Hood, C. Reading, and G. N. Rolinson. 1976. Naturally-occuring ,B-lactamase inhibitors with antibac- tion of the fl-lactamase inhibitor to 20 ,ug/ml, terial activity. J. Antibiot. 29:668-669. the 75% inhibition level was reached at 6.2 ,tg of 2. Hunter, P. A., C. Reading, and D. A. Witting. 1978. In ampicillin or cephalothin. Furthermore, the as- vitro and in vivo properties of BRL 14151, a novel beta- sociation of 20 ,ug of clavulanic acid with 6.2 ,g lactam with beta-lactamase-inhibitory properties, p. 478-480. In W. Siegenthaler and R. Luthy (ed.), Current of ampicillin per ml also inhibited 75% of the E. Chemotherapy; Proc. 10th Int. Congress Chemother., coli strains. At 5 ,tg of clavulanic acid, however, vol. I. American Society for Microbiology, Washington, only 25% of E. coli strains were sensitive to 6.2 D.C. ,ug of cephalothin per ml. Although most strains 3. Jackson, R. T., L. R. F. Harris, and R. H. Alford. 1978. Sodium clavulanate potentiation ofcephalosporin activ- of S. aureus, Klebsiella, and E. coli responded ity against clinical isolates of cephalothin-resistant to clavulanic acid plus penicillins or cephalothin, KlebsielUa pneumoniae. Antimicrob. Agents Chemo- in no case was the 90% inhibition level reached. ther. 14:118-125. Proteus mirabilis and indole-positive Proteus 4. Paisley, J. W., and J. A. Washington II. 1978. Com- bined activity of clavulanic acid and against sp. were only moderately susceptible to clavu- ticarcillin-resistant, gram-negative bacilli. Antimicrob. lanic acid plus carbenicillin; no major differences Agents Chemother. 14:224-227. between indole-negative and indole-positive 5. Reading, C., and M. Cole. 1977. Clavulanic acid: a beta- strains were observed. Almost no effect of cla- lactamase-inhibiting beta-lactam from Streptomyces clavuligerus. Antimicrob. Agents Chemother. 11: vulanic acid was noticed against Pseudomonas, 852-857. Enterobacter, and Serratia spp. On the other 6. Wise, R., J. M. Andrews, and K. A. Bedford. 1978. In hand, it should be mentioned that previous pa- vitro study of clavulanic acid in combination with pen- pers on species not included in this study re- icillin, amoxycillin, and carbenicillin. Antimicrob. ported that strains of Agents Chemother. 13:389-393. fl-lactamase-producing 7. Wust, J., and T. D. Wilkins. 1978. Effect of clavulanic Haemophilus influenzae (Thornsberry et al., acid on anaerobic bacteria resistant to beta-lactam anti- 17th ICAAC, Abstr. no. 61, 1977), Neisseria biotics. Antimicrob. Agents Chemother. 13:130-133.