In vitro activity of mecillinam against anaerobic bacteria.

A microtiter broth dilution method was employed to determine the in vitro activity of mecillinam against 201 recent clinical isolates of anaerobic bacteria. Both the anerobic gram-positive and anaerobic gram-negative bacilli displayed a wide range of minimal inhibitory concentrations of mecillinam; most strains were resistant to the antibiotic. The anaerobic cocci exhibited a narrower range of minimal inhibitory concentrations than were observed with other anaerobes, but also exhibited mecillinam resistance. As a single durg, mecillinam does not appear to be an effective antimicrobial agent against anaerobic bacteria.

Mecillinam, a new beta-lactam antibiotic, was first described by Lund and Tybring (4). This antibiotic belongs to a new and novel class of penicillins, the amidino penicillins. In general, penicillins, whether natural or semisynthetic, are acyl derivatives of 6-amino-penicillanic acid.
Mecillinam is a 6,8-amidinopenicillanic acid derivative (4). In contrast to most penicillins, which generally are more active against grampositive organisms, mecillinam possesses a remarkably high activity against many members of the Enterobacteriaceae. Mecillinam especially is active against Escherichia coli, Klebsiella pneumoniae, Enterobacter species, Citrobacter species, Salmonella, and Shigella, but is not active against Pseudomonas aeruginosa (5,11). Mecillinam is relatively inactive against gram-positive bacteria such as staphylococci and streptococci (5,6,13). The activity of mecillinam against anaerobic bacteria has been tested on only a limited basis (5,10). The purpose of this study was to further investigate the activity of mecillinam against a wide range of anaerobic bacteria.

MATERLALS AND METHODS
All anaerobic bacteria employed in this study were recent clinical isolates recovered from wound, lower respiratory, and genital tract specimens submitted to the Clinical Microbiology Laboratories of the North Carolina Memorial Hospital, Chapel Hill. All anaerobic bacteria employed for testing were first identified through the use of gas-liquid chromatography and specialized anaerobic biochemical tests (3 The in vitro activity of mecillinam against anerobic bacteria was investigated by employing a microtiter broth dilution method (8). By testing reference strains of B. fragilis ATCC 25285 and C. perfringens ATCC 13124 against penicillin G, cefoxitin, and clindamycin, this method was shown to yield results similar to those obtained with the reference agar dilution method (9). Mecillinam powder was supplied by Hoffmann-La Roche, Inc., Nutley, N.J. The other antibiotic powders were supplied as follows: penicillin G, Eli Lilly and Co., Indianapolis, Ind.; cefoxitin, Merck Sharp and Dohme, Inc., West Point, Pa.; and clindamycin, The Upjohn Co., Kalamazoo, Mich. The mecillinam powder initially was dissolved in sterile distilled water to yield a concentration of 1,280 pg/ml. Twelve serial twofold dilutions ranging between 128 and 0.06 pg/ml were then prepared in sterile NIH broth supplemented with 5 pug of hemin and 0.1 pug of menadione per ml. NIH broth was prepared by dissolving 5 g of yeast extract (Difco Laboratories, Detroit, Mich.), 15 g of Trypticase-peptone (BBL Microbiology Systems, Cockeysville, Md.), 1.0 g of dextrose, 2.5 g of sodium chloride, and 0.05 g of L-cysteine in 1 liter of distilled water. The medium was sterilized by autoclaving for 15 min at 121°C. The sterilized medium was then supplemented with hemin and menadione to yield final concentrations of 5 and 0.1 pg/ml, respectively. The osmolality of the supplemented NIH broth was determined to be 210 msmol/kg through use of an Advanced Digimatic Osmoter, model 3D (Advanced Instruments Co., Inc., Needham Heights, Mass.).
Portions (50 p1) of the mecillinam dilutions were aseptically dispensed into sterile U-bottom microtiter trays (Cooke Laboratories, Inc., Alexandria, Va.) with a Dynatech MIC-2000 dispenser (Cooke Laboratories, Inc.). Prepared microtiter trays not intended for use within 24 h of preparation were sealed in polyethylene bags and frozen at -70°C for a maximum of 2 weeks. Microtiter plates were prepared for inoculation by first incubating them at room temperature for 24 h in an anaerobic glove box (Coy Laboratory Products, Ann Arbor, Mich.) containing an atmosphere of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide.
The stability of mecillinam in the prepared microtiter trays was monitored by testing a strain of B. fragilis (for which the minimal inhibitory concentration [MIC] of mecillinam was known) on each day tests were performed. This quality control testing indicated that the antibiotic was stable for at least 2 weeks under the storage, prereduction, and incubation conditions employed. It also indicated that the antibiotic was not inactivated by the L-cysteine in the test medium.
Organisms tested were first grown in the glove box environment at 350C for 24 to 48 h on an agar medium which consisted of the previously described NIH broth supplemented with 1.5% agar and 5% defibrinated sheep blood (Brown Laboratories, Topeka, Kans.). Inocula were prepared by suspending well-isolated colonies of each test strain from NIH blood agar plates in 2 ml of supplemented Schaedler broth. The turbidity of the organism suspension was adjusted to equal that of a McFarland no. 0.5 turbidity standard. This standardized suspension then was diluted 1:100 in supplemented NIH broth. Volumes (50 pl) of the diluted suspension were added to each antibiotic-containing well and to a growth control well containing 50 pl of supplemented broth without antibiotics. With inoculation, the prepared antibiotic concentrations were further diluted to yield concentrations of mecillinam that ranged between 64 and 0.03 ,ug/ml. Inoculated plates were sealed in polyethylene bags and incubated in a glove box at 35°C for 48 h. The incubated plates were examined with an illuminated viewer (Cooke Laboratories, Inc.), and the MIC was determined as the lowest concentration of antibiotic that inhibited visible growth. Table 1 presents the results obtained for the 201 strains of anaerobic bacteria tested. C. bifermentans and E. lentum were the most susceptible of the anaerobic gram-positive bacilli tested. MICs for C. bifermentans ranged between 0.25 and 4.0 ,ug/ml, and MICs for E. lentum ranged between 1.0 and 4.0 ,g/ml. More elevated MICs were observed for P. acnes and P. granulosum: between 2.0 and 16.0,ug/ml and between 0.5 and 16,ug/ml, respectively. C. tertium, C. innocuum, and C. perfringens were the least susceptible of the anaerobic gram-positive bacilli. MICs for C. perfringens ranged between 8.0 and 64 ,g/ml, whereas C. innocuum displayed MICs that ranged between 16 and 64 ,ug/ ml. C. tertium demonstrated MICs that ranged between 8  V. parvula was the only anaerobic gram-negative coccus tested and displayed MICs ranging between 0.5 and 4.0 ,ug/ml. Of the anaerobic gram-positive cocci, all strains of P. micros, P. prevotii, and P. asaccharolyticus demonstrated MICs of s8.0 ,ug of mecillinam per ml. P. anaerobius displayed a wider range of MICs, with values ranging between 0.125 and 16 ,g/ml. Approximately 93% of the P. anaerobius strains were susceptible to 8.0 ,ug/ml, whereas approximately 7% required 16 ,tg/ml for inhibition. DISCUSSION Since mecillinam is poorly absorbed from the gastrointestinal tract, effective oral therapy originally was not possible, and the antibiotic was administered parenterally (7). Recently, an effective oral preparation has been developed. Pivmecillinam, the pivaloyloxymethyl ester of mecillinam, possesses no antibacterial activity per se but is well absorbed from the gastrointestinal tract. During or immediately after absorption, pivmecillinam is enzymatically hydrolyzed to mecillinam (7).

RESULTS
Roholt and co-workers, in a study of 9 patients, found peak serum levels after intramuscular administration of either 250 or 500 mg of mecillinam to be approximately 8 and 10 lOg/ml, respectively (7). The serum half-life of mecillinam intramuscularly administered was approximately 1 h (7). In a study of 10 fasting volunteers, Roholt and co-workers found mean peak serum levels after the oral administration of 100 mg of pivmecillinam to be approximately 5 jig/ ml, with a serum half-life of approximately 1 h (7).
Assuming a susceptible breakpoint for mecillinam to equal 4.0 jig/ml, this antibiotic appears relatively inactive against anaerobic bacteria. B. fragilis and C. perfringens, two of the most commonly isolated anaerobic bacteria, are highly resistant to mecillinam; none of the strains of C. perfringens tested, and only about 34% of the strains of B. fragilis, were susceptible to c4.0 ,ug of mecillinam per ml. C. bifermentans, E. lentum, and V. parvula were the only anaerobic bacteria that demonstrated consistent susceptibility to mecillinam. Resistance among other anaerobic bacteria was frequently encountered. Of all the anaerobic strains tested, for only 52.7% was the MIC <4.0,tg/ml. It is, therefore, extremely difficult to predict susceptibility to mecillinam. Since the susceptibility of the ma-jority of anaerobic bacteria tested cannot be predicted, susceptibility testing is mandatory if mecillinam is ever to be considered for treatment of anaerobic infections.
In vitro susceptibility testing of mecillinam requires a medium with the proper osmolality and conductivity. Susceptibility testing media with high osmolality and conductivity have been demonstrated to significantly reduce the activity of mecillinam (1,2,12). The osmolahlity and conductivity of the medium we employed were sufficiently low to insure accurate measurements of antibiotic activity.
Although mecillinam is active against many aerobic gram-negative bacilli, its potential use for the treatment of anaerobic infections appears limited. Should consideration be given to the clinical use of mecillinam for the treatment of anaerobic infections, in vitro susceptibility testing is strongly indicated.