ANTIMICROBIAL AGzNTS AND CHEMOTHERAPY, Jan. 1977, p. 74-79 Copyright X 1977 American Society for Microbiology
Vol. 11, No. 1 Printed in U.S.A.
Mezlocillin: In Vitro Studies of a New Broad-Spectrum Penicillin GERALD P. BODEY* AND THERESA PAN The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030
Received for publication 9 July 1976
Mezlocillin is a new semisynthetic penicillin that inhibited 71% of the isolates of Serratia marcescens, 67% of Escherichia coli, 50% of Enterobacter spp., and 49% ofKlebsiella spp. at a concentration of 12.5 ,ug/ml. It is also active against both indole-positive and -negative Proteus spp. and gram-positive cocci, except penicillin G-resistant Staphylococcus aureus. At a concentration of 100 ,mg/ml, it inhibited 94% of the isolates ofPseudomonas aeruginosa. It is more active than ampicillin, carbenicillin, and cephalothin against some gram-negative bacilli. gram-positive cocci, a 0.05-ml sample of a 10-2 dilution of this broth culture (approximately 106 CFU/ ml) was used as the inoculum for susceptibility testing. All gram-negative bacilli used in this study were cultured from blood specimens obtained from patients between 1967 and 1975. The patients were hospitalized at this institution and had underlying malignant diseases. A total of 100 isolates each of P. aeruginosa, Klebsiella spp., and Escherichia coli, 98 isolates of Proteus spp., 69 isolates of Serratia spp., and 89 isolates of Enterobacter spp. were used. All gram-positive cocci used in this study were cultured from specimens obtained from hospitalized patients, most of whom did not have cancer. A total of 22 isolates of Streptococcus pyogenes, 14 isolates of S. pneumoniae, and 62 isolates of Staphylococcus aureus were used. The susceptibility of isolates of S. aureus to penicillin G was determined by the broth dilution method. Isolates inhibited by less than 0.10 ,ug/ml were selected as penicillin G susceptible, and those isolates resistant to more than 25 jug/ml were selected as penicillin G resistant. Mezlocillin (BAY 1353) was supplied as a powder by Delbay Pharmaceuticals, Inc., Bloomfield, N.J. Ampicillin and BL-P1654, 6-[D-a-(3-guanylureido)phenylacetamido] penicillanic acid, were supplied by Bristol Laboratories, Syracuse, N.Y. Carbenicillin and ticarcillin were supplied by Beecham Pharmaceuticals, Bristol, Tenn. Twofold serial dilutions of the antibiotics were made with Mueller-Hinton broth, and the minimal inhibitory concentration (MIC) was determined after incubation at 37°C for 18 h. All wells containing trace growth or no discernible growth were subcultured on sheep blood agar. A calibrated pipette was used to transfer 0.01 ml of the inoculum. The minimum bactericidal concentration (MBC) was determined after incubation at 37C for 18 h. The MBC was defined as the lowest concentration of drug that yielded less than 50 colonies on subculture (less than 5 colonies/0.001 ml of inoculum). Comparison studies were conducted simultaneously.
Gram-negative bacilli continue to be a major cause of infection in hospitalized patients. This is especially true among patients with malignant diseases and patients receiving immunosuppressive therapy (3, 4). Often these patients do not respond, despite the in vitro susceptibility of the etiological organism to the antibiotic utilized (1). Consequently, there is a continuing need for the development of new antibiotics. The ureido-penicillins are an interesting group of semisynthetic penicillins that have broadspectrum activity against gram-negative bacilli, including Pseudomonas aeruginosa (2). Figure 1 shows the chemical structure of mezlocillin (r)-a[2-oxo-3-mesyl-imidazolidinyl)-carbonyll-aminobenzylpenicillin; BAY 1353), a new ureido-penicillin recently undergoing investigation (H. B. Konig, K. G. Metzger, H. A. Offe, and W. Schrock, Prog. Abstr. Intersci. Conf., Antimicrob. Agents Chemother., 14th, San Francisco, Calif., Abstr. 372, 1974; K. Metzger, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., 332, 1975). This report presents the results of in vitro studies of mezlocillin, which indicate that it may be a potentially useful antibiotic. MATERIALS AND METHODS Susceptibility tests were conducted on 556 clinical isolates of gram-negative bacilli and 88 clinical isolates of gram-positive cocci, using the dilution technique with an automatic microtiter system (Canalco, Autotiter instruction manual). All organisms were inoculated into Mueller-Hinton broth (Difco) and incubated at 37°C for 18 h. For gram-negative bacilli, a 0.05-ml sample of a 10-3 dilution of this broth culture (approximately 105 colony-forming units [CFU]/ml) was used as the inoculum. For 74
VOL. 11, 1977
IN VITRO STUDIES OF MEZLOCILLIN
RESULTS also the MBC, but this was not true for P. The in vitro activity of mezlocillin against aeruginosa. The MBC for most isolates of P. gram-positive cocci and gram-negative bacilli is aeruginosa was twice as high as the MIC. The effect of inoculum size on the MIC and shown in Fig. 2. Although the majority of isolates of S. pneumoniae were quite susceptible MBC was determined for 10 isolates each of K. to mezlocillin, 14% required concentrations of pneumoniae, E. coli, and P. aeruginosa (Fig. 6.25 to 25 ,ug/ml to inhibit their growth. Like- 3). For all isolates of K. pneumoniae and most wise, the majority of penicillin G-susceptible S. aureus were inhibited by 0.20 ug or less of In Vitro Activity of Meziocillin mezlocillin per ml, whereas penicillin G-resistant S. aureus were also resistant to mezlocillin. Isolates of S. pyogenes were quite susceptible to mezlocillin. Over 95% of the isolates of P. mirabilis and 70% of the isolates of indole-positive Proteus spp. were susceptible to 1.56 u.g or less of mezlocillin per ml. A concentration of 12.5 ,ug/ml inhibited 71% of the isolates of S. marCL cescens, 67% of E. coli, 50%o of Enterobacter spp., and 49% ofKlebsiella spp., but only 27% of 0~c0 P. aeruginosa. However, at a concentration of 100 ,g/ml, mezlocillin inhibited 94% of the isoE lates of P. aeruginosa. Generally, the MIC was Q 40 O
... CNH .....
I c-o /
N S02CH3 FIG. 1. Chemical structure of mezlocillin. -
7006 .025 .1 .39 1.56 6.25 25 loo 400 Minimum Inhibitory Concentration ( pg/ml ) FIG. 2. In vitro activity of mezlocillin against gram-negative bacilli and gram-positive cocci. Numbers in parentheses indicate the number of isolates tested.
Effect of Inoculum Size on Activity of Meziocillin Klebsiello pneumonioe
1.56 6.25 25 100 400 .39 1.56 6.25 25 400 39 1.56 6.25 25 100 400 Minimum Inhibitory Concentration (pg /lml)
FIG. 3. Effect of inoculum size on MIC and MBC of mezlocillin using 10 isolates each ofK. pneumoniae, E. coli, and P. aeruginosa.
76 BODEY AND PAN isolates ofE. coli, the MIC and MBC were the same. Inoculum size had the greatest effect on the activity of mezlocillin against K. pneumoniae. Using an inoculum of 105 CFU/ml, all isolates were inhibited at a concentration of 6.25 zg/ml, whereas using an inoculum of 107 CFU/ml, none was inhibited at a concentration of 400 jg/ml. The MIC and MBC for isolates of E. coli increased sixfold or more when the inoculum size was increased from 105 to 107 CFU/ ml. All of the isolates of P. aeruginosa were inhibited by 100 ug or less of mezlocillin per ml when the inoculum was 105 CFU/ml, but only
ANTIMICROB. AGENTS CHEMOTHER.
30% were inhibited by 400 pAg/ml when the inoculum was 107 CFU/ml. The effect of pH on the susceptibility of 10 isolates each of E. coli, K. pneumoniae, and P. aeruginosa is shown in Fig. 4. The pH was adjusted by the addition of phosphate buffer to Mueller-Hinton broth. The greatest effect was observed against isolates of E. coli, which were more susceptible at alkaline pH. The pH had no appreciable effect on the activity of mezlocillin against isolates of P. aeruginosa. Media had little effect on the activity of mezlocillin (Fig. 5). However, isolates ofK. pneumoniae, E. coli,
-pH 64 ~60 2pH72 BC ~~~~~~~~~~~~~~~~pH
Kiebsiteiasedmoo Psdoos ~~~~~~~~Escherichia
312 125 5039 1.56 6.25 25 100 625 25 Minimum Inhibitory Concentration (pg/ml)
FIG. 4. Effect of pH on the activity of mezlocillin against K. pneumoniae, E. coli, and P. aeruginosa. Ten isolates of each gram-negative bacillus were tested. 100.//
40 j 0
,X--- Tr pt,cse 'it fi
| i |
HNutrient Broth /ofusioBroinHert
1.56 6.25 25 .39 1.56 6.25 25 100 6.25 25 100 Minimum Inhibitory Concentration (pg/ml) FIG. 5. Effect of different media on the activity of mezlocillin. Ten isolates each ofK. pneumoniae, E. coli, and P. aeruginosa were tested.
IN VITRO STUDIES OF MEZLOCILLIN
VOL. 11, 1977
and P. aeruginosa were slightly more resistant cillin and cephalothin and one isolate that was when tested in nutrient broth, with the excep- relatively resistant to carbenicillin were intion of one isolate of E. coli which was consider- hibited by 6.25 jg of mezlocillin per ml. Mezloably more susceptible to mezlocillin when cillin was also the most active antibiotic tested in nutrient broth. against indole-positive Proteus spp. and was The activity of mezlocillin against 50 isolates consistently four times more active than careach of various gram-negative bacilli was com- benicillin. Ampicillin and cephalothin had minpared with ampicillin, carbenicillin, and cepha- imal activity against these isolates. The activity of mezlocillin was compared lothin (Fig. 6 to 10). Figure 10 shows similar data for 21 isolates of indole-positive Proteus with that of BL-P1654, carbenicillin, and ticarspp. Mezlocillin was the most active drug cillin against 50 isolates of P. aeruginosa (Fig. against isolates of E. coli, although about 30% 11). The most active antibiotic was BL-P1654 of the isolates were resistant to 25 ,ug/ml and and the least active was carbenicillin. Mezlocilsome of these isolates were more susceptible to lin and ticarcillin had comparable activity. cephalothin. Cephalothin was the most active antibiotic against K. pneumoniae, although DISCUSSION mezlocillin was the most active penicillin. Mezis a broad-spectrum semisynMezlocillin locillin was substantially more active than any of the other antibiotics against S. marces- thetic penicillin with activity against both cens. Cephalothin was virtually inactive, gram-positive cocci and gram-negative bacilli. whereas carbenicillin was active against nearly It is of particular interest because it is margin50% of these isolates. Mezlocillin was the most ally active against S. marcescens, K. pneumoactive antibiotic against Enterobacter spp. al- niae, and P. aeruginosa. It is more active in though 56% of the isolates were resistant to 25 vitro than ampicillin or carbenicillin againstE. ,ug/ml. Carbenicillin was the second most ac- coli and as active as these two penicillins tive antibiotic, but 74% of the isolates were against P. mirabilis. It is also more active in resistant to 25 ,ug of this drug per ml. All three vitro than carbenicillin against indole-positive penicillins were equally active againstP. mira- Proteus spp. and Enterobacter spp. It is as acbilis. Two isolates that were resistant to ampi- tive as cephalothin against 80% of the isolates
- - Meziocillin I --- Ampicillin I - Carbenicillin
- Meziocillin -- Ampicillin * * Carbenicillin o--o Cepholothin
a) 0 0
Comparative Activity of Antibiotics Against Klebsiella spp.
Comparative Activity of Antibiotics Against Escherichia coli
c 0~ 0
C-I 201 01
.025 .1 .39 1.56 6.25 25 100 400 Minimum Inhibitory Concentration (pg/mi) FIG. 6. Comparative activity of antibiotics against 50 isolates of E. coli. An inoculum of 10' CFUlml was used.
-025 .1 .39 1.55 6.25 25 100 400 Minimum Inhibitory Concentration (pg/ml)
FIG. 7. Comparative activity of antibiotics against K. pneumoniae. An inoculum of 105 CFUlml was used.
ANTIMICROB. AGENTS CHEMOTHER.
BODEY AND PAN
100 * Meziocillin Ampicillin .-. Carbenicillin o----o Cepholothin
.025 .1 .39 1[56 6.25 25 100 400 Minimum Inhibitory Concentration (pg/ml) FIG. 8. Comparative activity of antibiotics against S. marcescens. An inoculum of 105 CFUlml was used.
-025 .1 .39 1.56 6.25 25 100 400 Minimum Inhibitory Concentration (pg/ml) FIG. 9. Comparative activity of antibiotics against Enterobacter spp. An inoculum of 105 CFUlml was used.
.1 .39 1.56 625 25 100 400 Minimum Inhibitory Concentration (pg/mil) FIG. 10. Comparative activity of antibiotics against Proteus spp. Fifty isolates ofP. mirabilis (a) and 21 isolates of indole-positive Proteus spp (b) were tested. An inoculum of 105 CFUlml was used. .025
1.56 6.25 25
Minimum Inhibitory Concentration (pg/ml)
IN VITRO STUDIES OF MEZLOCILLIN
VOL. 11, 1977
*Meziocillin BL-P 1654
*.-.* Corbenicillin TirIcrIcIIIii nrr-il1l1in I _-u---'o.
.025 .1 39 1.56 6.25 25 100 400 Minimum Inhibitory Concentration (pg/mi ) FIG. 11. Comparative activity of penicillins against 50 isolates of P. aeruginosa. An inoculum of 105 CFU/ml was used.
of Klebsiella spp. and is the most active of the four antibiotics against S. marcescens. However, another ureido-penicillin, BP-P1654, is more active than mezlocillin in vitro against P. aeruginosa. The ureido-penicillins have been shown to be relatively resistant to destruction by the p-lactamase of isolates of E. coli and S. marcescens resistant to ampicillin, which may account for the better in vitro results with mezlocillin (B. Wiedemann, V. Kremery, and H. Knothe, Int. Kongr. Chemother., 9th, London, Abstr. M366, 1975). There has been controversy in the past over
the bactericidal activity of ureido-penicillins. Generally, mezlocillin was bactericidal at the MIC, but this was not true for isolates of P. aeruginosa. Inoculum size had a major effect on the activity of mezlocillin. It was ineffective against all isolates of K. pneumoniae and most isolates of P. aeruginosa when a large inoculum was used. This suggests that these cells are not uniformly susceptible to mezlocillin, and a large inoculum includes more cells that are inherently resistant to the antibiotic. This adverse effect of inoculum size has been observed with other penicillins (5, 6). Mezlocillin is an interesting new penicillin because of its broad-spectrum activity. The drug deserves further evaluation because it is potentially more active than ampicillin, carbenicillin, and cephalothin against some gramnegative bacilli. ACKNOWLEDGMENTS This work was supported by Public Health Service grant CA-10042 from the National Cancer Institute and a grantin-aid from Delbay Pharmaceuticals, Inc., Bloomfield, N.J. LITERATURE CITED 1. Bodey, G. P., E. Middleman, T. Umsawasdi, and V. Rodriguez. 1972. Infections in cancer patients-results with gentamicin sulfate therapy. Cancer 29:1697-1701. 2. Bodey, G. P., and D. Stewart. 1971. In vitro studies of semisynthetic a-(substituted-ureido) penicillins. Appl. Microbiol. 21:710-717. 3. Eickhoff, T. C. 1973. Infectious complications in renal transplant patients. Transplant. Proc. 5:1233-1238. 4. Inagaki, J., V. Rodriguez, and G. P. Bodey. 1974. Causes of death in cancer patients. Cancer 33:568573. 5. Neu, H. C., and E. B. Winshell. 1971. In vitro studies of
semisynthetic penicillin, 6-[D(-)-a-carboxy-3-thienylacetamido] penicillanic acid (BRL 2288), active against Pseudomonas, p. 385-389. Antimicrob.
Agents Chemother. 1970. 6. Standiford, H. C., A. C. Kind, and W. M. Kirby. 1969. Laboratory and clinical studies of carbenicillin against gram-negative bacilli, p. 286-291. Antimicrob. Agents Chemother. 1968.