In Vitro Studies with Cefazolin

Susceptibilities of 259 isolates of pathogenic bacteria to cefazolin were measured by broth and agar dilution procedures. Beta-hemolytic streptococci were inhibited by 0.25 μg/ml, whereas Staphylococcus aureus and alphahemolytic streptococci were inhibited by 2.0 μg/ml. Enterococci were resistant to less than 32 μg/ml. Wide variation was seen with gram-negative species. Most isolates of Klebsiella species and Proteus mirabilis were inhibited by 4.0 or 8.0 μg/ml. Escherichia coli were less susceptible, and most isolates of Pseudomonas aeruginosa, Serratia species, and Enterobacter species were resistant to 128 μg/ml.

Cefazolin is a new cephalosporin C derivative which has been shown to be broad spectrum, bactericidal and resistant to penicillinase produced by Staphylococcus aureus (5). In vitro, it is equivalent in activity to cephalothin against Diplococcus pneumoniae, S. aureus, group A streptococci, and Proteus mirabilis and four to eight times more active against Escherichia coli (7). Pharmacological studies have shown that intramuscular doses of cefazolin are well tolerated (5) and that higher and more prolonged serum levels are obtainable as compared to those obtained with comparable doses of cephaloridine (2,5,7). It has been shown to be effective in the treatment of a variety of bacterial infections including surgical infections (9), bacterial pneumonias (11), bacterial endocarditis (6), and urinary tract infections (3).
The studies reported here were undertaken to provide additional data regarding the in vitro activity of cefazolin against several species of pathogenic bacteria and also to determine if there were significant differences in susceptibility data obtained with routine agar and broth dilution procedures.
Two hundred and fifty-nine recent clinical isolates of bacteria were tested. These included 25 isolates each of Enterobacter species, E. coli, Klebsiella pneumoniae, P. mirabilis, Pseudomonas aeruginosa, Serratia species, betahemolytic streptococci, enterococci, and S. aureus. Ten isolates of indole-positive Proteus species and 24 isolates of alpha-hemolytic streptococci also were tested. E. coli ATCC 25922 and S. aureus ATCC 25923 were used as controls.
Minimal inhibitory concentrations (MICs) were determined in Mueller-Hinton broth (Difco) by a twofold dilution procedure (1). Concentrations of cefazolin ranged from 128 to 0.063 jsg/ml. Inocula containing approximately 10" cells were prepared from 1:1,000 dilutions of overnight broth cultures. The MIC was defined as the lowest concentration of drug which inhibited growth as determined visually by the absence of turbidity after incubation at 37 C for 24 h. Minimal bactericidal concentrations (MBC) were determined by subculture, with a calibrated loop, of all negative tubes to drug-free media with subsequent incubation at 37 C for 24 h. The MBC was defined as the lowest concentration of drug which yielded less than five colonies on subculture. MICs also were termined using the International Collaborative Study agar dilution procedure (4) and Mueller-Hinton agar (BBL). Cefazolin was serially diluted in twofold increments so that final concentrations of drug would range from 128 to 0.063 ug/ml. Five percent sheep blood was added in tests with alphaand beta-hemolytic streptococci and enterococci. Tests were performed with square, disposable petri dishes (100 by 15 mm). Test inocula prepared from overnight broth cultures, containing approximately 10' cells, were applied to the agar surfaces with an inoculating device similar to the Steer replicator (10). The MIC was defined as the lowest concentration of drug which inhibited colony formation after incubation at 37 C for 24 h. Sterile cefazolin standard (Eli Lilly and Co., lot 51-63-2B) was used in both studies. Generally, there was good agreement between MIC values obtained with cefazolin in this study and those reported by previous investiga-  tors (2,5,8). Klebsiella species were the most susceptible of the gram-negative organisms; 64 and 68%, respectively, were inhibited by 4 gg/ ml in the broth and agar dilution studies (Tables  1 and 2 studies. The enterococci generally were resistant to less than 32 ,g/ml. MBCs were determined as part of the broth dilution studies ( Table 3). The results of these latter tests indicated that MICs also were bactericidal for the beta-hemolytic streptococci and nearly so for the alpha-hemolytic streptococci. Bactericidal levels were generally twice the MIC value or less for the enterococci, S. aureus, Klebsiella species, E. coli, and P. mirabilis. MICs as determined by the broth and agar dilution procedures generally were in good agreement. Most differences were only twofold or one dilution factor. Statistical analyses (paired t test) revealed no significant differences in inhibitory concentrations measured by the two procedures (P > 0.05) for the following genera: Klebsiella, Proteus, alpha-hemolytic streptococci, beta-hemolytic streptococci, and enterococci. Differences in MIC values were significant in the case of S. aureus (P < 0.001) and of borderline significance in the case of E. coli (P < 0.05). Data for Enterobacter, Pseudomonas, and Serratia were excluded from analyses. This agreement also is demonstrated in Fig.  1, which depicts least-square regression lines for cefazolin MIC values as determined by the agar and broth dilution procedures plotted against paired zones of inhibition obtained with an experimental 30-,gg cefazolin disk. This degree of agreement suggests, at least for the cephalosporin antibiotics, that the agar dilution proce- dure may be used in lieu of broth dilution procedures for testing of large numbers of clinical isolates where the ease, rapidity, and economic advantages of the former method is desired.
As already reported by others, cefazolin appears to equivalent to cephalothin in terms of overall in vitro activity. Cefazolin offers apparent advantages over cephalothin and cephaloridine both in rates of adsorption and in peak serum levels obtainable in man via parenteral administration (5,7). Cefazolin may also have an advantage over cephaloridine because of its resistance to staphylococcal penicillinase (7). This was observed in a series of experiments using inocula of varying densities. These data, not reported here, showed that with E. coli tenfold differences in inoculum size often resulted in fourfold differences or greater in agar dilution end points. Similar changes in inoculum size had a moderate effect on MIC values for penicillin-susceptible and penicillin-resistant strains of S. aureus; a tenfold reduction in inoculum size generally was associated with a twofold reduction in the MIC. This effect of inoculum size may explain the differences seen in MIC values obtained with the two dilution methods for S. aureus and E. coli.