JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1985, p. 133-134 0095-1137/85/010133-02$02.

00/0

Vol. 21, No. 1

Copyright 1985, American Society for Microbiology

Effect of Refrigerated Storage

on

Cefaclor in Mueller-Hinton Agar

A. MICHELE SURPRENANT* AND DAVID A. PRESTON Lilly Research I;aboratories, Indianapolis, Indiana 46285
Received 2 August 1984/Accepted 25 September 1984

Cefaclor is less stable than most cephalosporins in media at 35C. We demonstrated that the activity of cefaclor in Mueller-Hinton agar diminishes continuously at 4C, resulting in a loss of two-thirds of the activity within 21 days. We recommend that agar dilution plates for this cephalosporin be prepared on the day of their
use.

Chemical instability of ,B-lactam antibiotics in solution is well known (1, 8). The specific reactions that lead to the breakdowp of these antibiotics are variable according to the chemical structure of the antibiotic in question and its physical environment. Cefaclor is a ephalosporin.derivative having' a phenylglycyl moiety in the C-7 position' and a chlorine atom in the C-3 position of the cephalosporin nucleus. Under certain conditions, this molecule readily undergoes hydrolysis by intramolecular nucleophilic attack on the ,3-lactam bond by the C-7 side chain amino group (5). This self-destructive event appears to be responsible for the relatively rapid loss of biological activity of cefaclor solutions in bacteriological media at 35C (4, 7). Because the rate of degradation of cefaclor in media at 35C is somewhat faster than that of most other cephalosporins, we compared the rates of loss of biological activity of cefaclor and a comparison cephalosporin, cefamandole, dissolved in Mueller-Hinton agar at 4C. We prepared agar dilution plates as described previously (6) with cefaclor and cefamandol in concentrations ranging from 128 to 0.03 ,ug/ml. The plates were sealed in plastic bags and stored at 4C. On days 1, 7, 14, and 21, duplicate sets of plates containing each antibiotic were removed from refrigeration, brought to ambient temperature, and inoculated according to previously described standards (6) with a group of bacterial isolates representing a diversity of genera and species for which testing on unsupplemented Mueller-Hinton agar is appropriate. The isolates included Escherichia coli (3), Enterobacter spp. (3), Klebsiella pneumoniae (4), Providencia stuartii (3), Serratia marcescens (3), and Staphylococcus aureus (9). After overnight incubation at 350C, the lowest concentration of each antibiotic preventing macroscopic growth of each culture was recorded as the MIC. A barely visible haze or a single colony at the inoculum site was ignored. For each antibiotic, geometric mean MICs were calculated for each test day for only the group of isolates that had definitive endpoints at all four assay times. By day 21 of storage at 4C, the geometric mean MIC of cefaclor had increased approximately sevenfold (Table 1). By contrast, the geometric mean MIC for cefamandole remained close to the initial value. The relatively rapid loss of cefaclor activity suggested by the shift in the mean MIC is consistent with the findings of Foglesong et al. (3), who reported that cefaclor dissolved in 0.1 M phosphate buffer (pH 7) loses over 50% of its biological activity within 72 h at 4C.
*

We also determined the rate of loss of cefaclor in Mueller-Hinton agar by a direct microbiological quantitative assay similar'to that used by Ryan et al. (Bacteriol. Proc. p. 74, abstr. no. M41, 1969) and by Baron and Hindler (2) in the study of other antibiotics. Liquefied and cooled (52C) antibiotic medium 1 (Difo Laboratories) seeded with Bacillus subtilis ATCC 6633 spores (final concentration, 2.5 x 106 spores per ml) was pipetted in 6-ml volumes to 90-mm flat-bottomed plastic disposable petri dishes and allowed to solidify. Assay' plates were prepared and used immediately on each test day. Frshly prepared Mueller-Hinton agar dilution plates containing cefaclor at concentrations of 16, 8, 4, 2, and 1 ,ug/ml were prepared and stored at 4C. On days 1, 7, 14, and 21, triplicate plug samples were removed from each cefaclor plate by using slight suction through a thin-walled brass tube (4.5-mm inside diameter). Each plug was transferred to the surface of an assay plate. Plates incubated overnight at 35C developed zones of'inhibition around the agar plugs. Mean zone diameters for the various cefaclor concentrations sampled on day 1 were plotted on semilogarithmic graph paper against cefaclor concentrations (logarithmic). The resulting dose-response curve was used as the standard against which subsequent weekly samples were compared. Potencies of samples taken at 7, 14, and 21 days were expressed as percentages of the initial sample values (day 1) (Table 2). All concentrations of cefaclor lost potency at approximately the same rate. By the end of week 3, the biological activity in each plate was slightly less than one-third of its initial concentration. This loss is sufficient to explain a shift of two dilutions in MIC endpoints.
TABLE 1. Performance of agar dilution plates after storage at 4C
Storage (wk)
Cefaclor (18)a

Geometric mean MIC (pig/ml) Cefamandole

(24)a

0 1 2 3

3.3 4.1 11.8

1.2 1.5

22,6
6.8

1.5 1.6
1.3

3/0 ratio

Corresponding author.
133

parentheses. Because MIC tests were performed in duplicate, the number of values for the geometric means was twice the number of isolates.

a Number of isolates considered in the geometric means is shown within

134

NOTES

J. CLIN. MICROBIOL. LITERATURE CITED Anhalt, J. P., and J. A. Washington II. 1980. Preparation and storage of antimicrobic solutions, p. 495-496. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and J. P. Truant (ed.), Manual of clinical microbiology, 3rd ed. American Society for Microbiology, Washington, D.C. Baron, E. J., and J. A. Hindler. 1984. Bioactivity of imipenem as a function of medium, time, and temperature. Antimicrob. Agents Chemother. 25:781-782. Foglesong, M. A., J. W. Lamb, and J. V. Dietz. 1978. Stability and blood level determinations of cefaclor, a new oral cephalosporin antibiotic. Antimicrob. Agents Chemother. 13:49-52. Gillett, A. P., J. M. Andrews, and R. Wise. 1979. Comparative in vitro microbiological activity and stability of cefaclor. Postgrad. Med. J. 55(Suppl. 4):9-11. Indelicato, J. M., A. Dinner, L. R. Peters, and W. L. Wilham. 1977. Hydrolysis of 3-chloro-3-cephems. Intramolecular nucleophilic attack in cefaclor. J. Med. Chem. 20:961-963. National Committee for Clinical Laboratory Standards. 1983. Standard methods for dilution antimicrobial susceptibility tests of bacteria which grow aerobically. Tentative standard M7-T. National Committee for Clinical Laboratory Standards, Villanova, Pa. Preston, D. A. 1977. Summary of laboratory studies on the antibacterial activity of cefaclor. Postgrad. Med. J. 55(Suppl. 4):22-29. Wick, W. E. 1964. Influence of antibiotic stability on the results of in vitro testing procedures. J. Bacteriol. 87:1162-1170.

TABLE 2. Stability of cefaclor in Mueller-Hinton agar stored at 40C

No. of days at 4'C

1.

% of initial concn (,ug/ml):

16 8 4 2
1

7 14

60
44

21
a

31

65 48 33

65 45 25

74 48 31

64 50 <50a

2.

Minimum detection limit, 0.5

p.g/ml.

3.
4.

According to the tentative standard on dilution testing (6), dilution plates may be used immediately or stored in plastic bags at 4C for up to 4 weeks. The standard also states with emphasis that appropriate reference control strains should be tested each time tests are performed to ensure that no deterioration of antimicrobial agents has occurred. Our results indicate that, in addition to regular quality control testing to monitor for loss of potency, precautions should be taken to avoid degradation of cefaclor in agar dilution plates. For routine work, cefaclor agar dilution plates should be prepared within 48 h of the test. For reference work, plates should be prepared on the day of the
agar

5. 6.

7. 8.

test.