In Vitro Inhibitory and Bactericidal Activity of Cefpiramide and Seven Antipseudomonal Agents Against Pseudomonas aeruginosa Michael Pfaller, Martha Bale, Theresa Preston, Wanita Howard, and Franklin Koontz
Cefpiramide was tested against 493 clinical isolates of Pseudomonas aeruginosa and the results of minimal inhibitory concentration, minimal bactericidal concentration, bactericidal rate, and time-kill synergy studies were compared with those obtained with seven other antipseudomonal agents. The minimal inhibitory concentrations of cefpiramide for P. aeruginosa were comparable to all of the agents tested. Minimal bactericidal cancentration results were generally within one twofold dilution of the minimal inhibitory concentration values for all agents tested. Bactericidal rate studies showed that at concentrations of four times the minimal inhibitory concentration, all of the agents produced rapid killing. Results of time-kill synergy studies showed a marked synergistic interaction between cefpiramide and each of three aminoglycosides, gentamicin, tobramycin, and amikacin. These results suggest that cefpiramide may be useful in the therapy of infections due to P_. aeruginosa.
INTRODUCTION Cefpiramide is a n e w semisynthetic c e p h a l o s p o r i n w h i c h is similar in structure to cefoperazone. Recently several groups have c o m p a r e d the in vitro activity of cefpiramide against a broad range of gram-negative and gram-positive organisms (Allan et al., 1985; F u k a s a w a et al., 1983; Kato et al., 1983; Pfaller et al., 1984). In general, cefpiramide was found to be s o m e w h a t more active than several of the newer cephalosporins against clinical isolates of Pseudomonas aeruginosa and several grampositive organisms i n c l u d i n g Staphylococcus aureus, S. epidermidis, and enterococci (Allan et al., 1985; F u k a s a w a et al., 1983; Kato et al., 1983; Pfaller et al., 1984). Cefpiramide, however, was the least active c e p h a l o s p o r i n tested against members of the family Enterobocteriaceae (Allan et al., 1985; Pfaller et al., 1984). Because cefpiramide a p p e a r e d to be particularly p r o m i s i n g as an a n t i p s e u d o m o n a l agent, we recently c o m p a r e d the in vitro activity of cefpiramide with that of several of the newer a n t i p s e u d o m o n a l ~3-1actam antibiotics against 493 clinical isolates of P s e u d o m o n a s aeruginosa obtained from the Clinical Microbiology Laboratory of the University of Iowa Hospitals and Clinics.
From the Clinical Microbiology Laboratory, Department of Pathology, University of Iowa
Hospitals and Clinics, and The Veterans Administration Medical Center, Iowa City, IA. Address reprint requests to: Michael A. Pfaller, M.D., Department of Pathology, 273 MRC,
University of Iowa College of Medicine, Iowa City, IA 52242. Received July 22, 1985; revised and accepted September 25, 1985. © 1986 Elsevier Science Publishing Co., Inc 52 Vanderbilt Avenue, New York, NY 10017
M. Pfaller et al.
MATERIALS AND METHODS Bacterial Strains Isolates of P. aeruginosa used in this study were routine clinical isolates collected from individual patients hospitalized at the University of Iowa HOspitals and Clinics from 1982 to 1984. The organisms collected were isolated from multiple body sites including blood, sterile body fluids other than blood, sputum, urine, stool, and wounds. Whenever possible duplicate isolates from individual patients were excluded. All isolates were identified by standard methods (Hugh and Gilardi, 1980) and were stored at room temperature on trypticase soy agar slants (BBL).
Antimicrobial Agents Pharmaceutical grade powders of amikacin (Bristol Laboratories, Syracuse, NY), azloci]lin (Miles Laboratories, Inc., West Haven, CT), cefoperazone (Pfizer Inc., New York, NY), cefpiramide (Wyeth Laboratories, Philadelphia, PA), cefsulodin (Abbott Laboratories, North Chicago, IL), ceftazidime (Glaxo Laboratories, Research Triangle Park, NC), gentamicin (Schering Corp., Kenilworth, NJ), imipenem (Merck Sharp & Dohme, Rahway, NJ), piperacillin (Lederle Laboratories, Pearl River, NY), and tobramycin (Eli Lilly and Co., Indianapolis, IN) were all obtained from the manufacturers. All antibiotic solutions were prepared according to manufacturers instructions, diluted in cation-supplemented Mueller-Hinton broth (Gibco Laboratories, Madison, WI), and dispensed into the wells of plastic microdilution plates using a Dynatech MIC 2000 system (Dynatech Laboratories, Inc., Alexandria, VA). The plates were made in two batches, sealed in plastic bags, and stored at - 70°C until used in the study. All plates were used within 4 wk of preparation.
Susceptibility Studies Minimal inhibitory concentrations (MIC) were determined on all isolates by the microdilution method as described by Jones et al. (1985). The plates were inoculated using the Dynatech MIC 2000 system. Verification of inoculum size was determined using quantitative plating techniques. Minimal bactericidal concentrations (MBC) were determined on 50 randomly selected isolates (every 10th isolate tested for MIC determination). After the MIC determination was made, the contents of each clear well were aspirated and a 0.01 ml aliquot was spread onto the surface of a blood agar plate (GIBCO). Plates were incubated at 35°C for 18-24 hr and the number of visible colonies was recorded. The MBC was recorded as the lowest concentration of antibiotic that produced killing of the initial inoculum of 99.9% or greater (less than or equal to five colonies). The effect of inoculum size on MIC and MBC determinations with cefpiramide was examined by testing five isolates by the microbroth dilution method described previously. Each isolate was tested in cation-supplemented Mueller-Hinton broth with inoculum sizes of 103,105, and 107 CFU/ml as determined by quantitative plating techniques. Bactericidal rate determinations were performed on three different isolates using the time-kill technique described by Schoenknecht et al. (1985). Each isolate was tested against cefpiramide, cefoperazone, ceftazidime, and imipenem at concentrations equal to the MIC and four. times the MIC of each antimicrobial agent, respectively.
In Vitro Activity of Cefpiramide
To d e t e r m i n e w h e t h e r synergistic killing occurred w i t h combinations of cefpiramide and aminoglycosides, we performed time-kill studies on three different isolates with cefpiramide, gentamicin, tobramycin, and a m i k a c i n alone and in combination. In each case the concentrations tested were selected to c o r r e s p o n d to the MIC of the organism to c e f p i r a m i d e and one-third the MIC of each aminoglycoside. Synergistic killing was defined as at least a 2-log greater decrease in colony counts at 24 hr with the c o m b i n a t i o n as c o m p a r e d w i t h the most active single agent (Schoenknecht et al., 1985).
Statistical Calculations Statistical analysis was performed by ×2 testing with Yates' correction (Colton, 1974). RESULTS The comparative a n t i p s e u d o m o n a l activity of cefpiramide versus that of three cephalosporins (cefoperazone, cefsulodin, and ceftazidime), two p e n i c i l l i n s (azlocillin and piperacillin), one c a r b a p e n e m (imipenem), and one a m i n o g l y c o s i d e (gentamicin) is s u m m a r i z e d in Table 1. The MICs of cefpiramide for P. aeruginosa were c o m p a r a b l e to all of the agents tested. At a concentration of 16 ~g/ml cefpiramide inhibited 91.9% of all strains tested and was significantly more active than azlocillin (84.4%, p < 0.01) and less active than i m i p e n e m (98.2%, p < 0.01) and cefsulodin (95.4%, p < 0.05). There was no significant difference b e t w e e n the activity of cefpiramide and cefoperazone (88.9%, p > 0.05), piperacillin (89.5%, p > 0.05), gentamicin (92.9%, p > 0.05), and ceftazidime (94.1%, p > 0.05). The effect of i n o c u l u m size on the MIC and MBC values of cefpiramide was also evaluated (Table 2). A n increase in i n o c u l u m from 10 a to 107 CFU/ml resulted in a twofold to fourfold increase in the MIC and MBC values for the five isolates tested. The results of bactericidal rate studies s h o w e d significant regrowth at 24 hr for each of the three P. a e r u g i n o s a strains tested w h e n incubated in the presence of cefpiramide, ceftazidime, cefoperazone, or i m i p e n e m at concentrations equal to the MIC for each antimicrobial agent (data not shown). Each of the three test strains were r a p i d l y killed at concentrations equal to four times the MIC for each a n t i p s e u d o m o n a l agent (Figure 1). In general, i m i p e n e m at four times the MIC, p r o d u c e d the most r a p i d killing. Cefpiramide, cefoperazone, and ceftazidime demonstrated equivalent rates of killing of the isolates tested in this s t u d y (Figure 1).
TABLE 1. In Vitro Activity of the New A n t i p s e u d o m o n a l Agents Versus 493 Clinical Isolates of P s e u d o m o n a s aeruginosa Cumulative percent of strains inhibited by (p.g/ml): Antimicrobial <0.25
Azlocillin Piperacillin Cefoperazone Cefpiramide Cefsulodin Ceftazidime Gentamicin Imipenem
0.8 0.6 0.6 0.6 0.8 0.4 0.6 1.4
1.0 1.4 0.8 1.2 3.4 2.6 0.8 14.1
1.4 1.4 1.8 1.6 12.5 34.1 2.4 53.2
3.2 13.6 6.7 38.3 61.5 74.6 14.8 88.0
36.8 64.3 60.5 74.8 81.3 88.6 59.6 94.5
70.4 80.5 77.1 85.4 91.1 92.5 84.2 95.5
84.4 89.5 88.9 91.9 95.4 94.1 92.9 98.2
89.7 92.3 93.1 93.5 97.0 98.0 94.5 99..6
91.9 93.7 94.3 96.8 98.4 98.6 96.3 99.6
93.3 95.7 98.0 98.4 99.8 99.6 97.0 99.6
96.6 97.8 99.0 99.4 99.8 99.8 98.6 99.8
100 100 100 100 100 100 100 100
0.8 0.6 0.6 0.6 0.8 0.4 0.6 0.8
M. Pfaller et al.
TABLE 2. Effect of I n o c u l u m Size on the A n t i p s e u d o m o n a l Activity of Cefpiramide MIC (MBC) with inoculum of: Strain
107-8 107-12 107-13 107-14 107-15
4 (8)° 2 (4) 4 (4) 2 (4) 2 (4)
105 CFU/ml 8 4 4 4 4
(16) (4) (8) (8) (8)
107 CFU/ml 8 2 4 4 4
(16) (8) (4) (16) (4)
°MIC and MBC values in micrograms per milliliter.
The results of testing cefpiramide in c o m b i n a t i o n with three different aminoglycosides (gentamicin, tobramycin, and amikacin) is presented in Figure 2. The combination of c e f p i r a m i d e and either gentamicin, tobramycin, or a m i k a c i n resulted in synergistic killing of each of the three strains of P. aeruginosa tested. In general, the c o m b i n a t i o n of c e f p i r a m i d e plus a m i k a c i n p r o d u c e d the most r a p i d killing; however, killing was virtually c o m p l e t e w i t h all three cefpiramide to aminoglycoside combinations b y 4 - 6 hr of incubation•
FIGURE 1. Killing kinetics of cefpiramide (O---O),ceftazidime (A--A), cefoperazone (C~-~), and imipenem (~---C]). Concentrations tested were four times the MIC value for each agent: cefpiramide (16 ~g/ml), ceftazidime (8 ~g/ml), cefoperazone (16 ~,g/ml), imipenem (4 ~g/ml). The results shown are those obtained with strain 34-6. Control growth curve (no antibiotic) is indicated by ( ~ - - ~ ) .
6 Time (hr)
In Vitro Activity of Cefpiramide
8 7 6
6 E t.t.
3 2 I 0
FIGURE 2. Killing kinetics of P. aernginosa strain 34-6 by cefpiramide and aminoglycosides alone and in combination at concentrations equal to the MIC cefpiramide (4 p.g/ml) and onethird the MIC of (A) gentamicin (1 p.g/ml), (B) tobramycin (0.3 ttg/ml), and (C) amikacin (2.5 p~g/ml). Drug-free control (O---4t), cefpiramide alone (0--0), gentamicin alone (o--o), tobramycin alone (O--4H, amikacin alone (,--*), cefpiramide plus gentamicin, tobramycin or amikacin ( . - - . ) . DISCUSSION
The results of this study both confirm and extend those of four previously published studies on the in vitro activity of cefpiramide (Allan et al., 1985; Fukasawa et al., 1983; Kato et al., 1982; Pfaller et al., 1984). Cefpiramide was shown to have potent
M. Pfaller et al.
inhibitory and bactericidal activity against a large number of clinical isolates of P. aeruginosa. The antipseudomonal activity of cefpiramide compared favorably with that of several of the newer ~-lactam agents with well-documented antipseudomonal activity, including azlocillin, piperacillin, cefsulodin, ceftazidime, and cefoperazone (Allan et al., 1985; Fass, 1983; Pfaller et al., 1984; Rolston et al., 1984). Of the antipseudomonal agents tested in this study only imipenem and cefsulodin were signficantly more active than cefpiramide. These data are consistent with those reported in two large Japanese studies (Fukasawa et al., 1983; Kato et al., 1982) and two recent U.S. studies (Allan et al., 1985; Pfaller et al., 1984). Although the previous work of Pfaller et al., (1984), using clinical isolates of P. aeruginosa obtained from patients hospitalized at Barnes Hospital in St. Louis, MO, reported considerably higher MIC values (MIC90 of 128 ~g/ml) than observed in either the present study or the three additional published studies (Fukasawa et al., 1983; Kato et al., 1982; Allan et al., 1985), the relative activity of cefpiramide compared with the other antipseudomonal agents tested was similar to that reported in these studies. Thus, although the actual MIC values of cefpiramide versus P. aeruginosa may vary from one institution to another its relative position as one of the more active antipseudomonal agents should remain constant. In contrast to the previously reported studies noted above, we also examined the rate of in vitro killing of P. aeruginosa produced by cefpiramide both alone and in combination with three commonly used aminoglycosides (Figures 1 and 2). Rapid bactericidal activity was seen with cefpiramide, as well as with the three additional ~-lactam agents tested including ceftazidime, cefoperazone, and imipenem (Figure 1). In addition, we found impressive synergistic killing with cefpiramide in combination with either gentamicin, tobramycin, or amikacin (Figure 2). These findings are consistent with those of Allan et al. (1985) who reported that the combination of cefpiramide plus gentamicin produced synergistic inhibition of 80% of P. aeruginosa strains tested using an agar dilution checkerboard technique. Based on these in vitro results we believe that cefpiramide may be a useful addition to the growing list of antipseudomonal agents. Its rapid bactericidal activity in combination with aminoglycosides and its prolonged half-life of 4-5 hr (Nakagawa et al., 1984) make it potentially useful in serious pseudomonal infections. Clinical evaluation of the efficacy of cefpiramide in the treatment of infections due to P. aeruginosa is warranted based on these in vitro findings. REFERENCES
Allan JD, Elipoulos GM, Ferraro MJ, Moellering RC Jr (1985) Comparative in vitro activities of cefpiramide and apalcillin individually and in combination. Antimicrob Agents Chemother 27:782. Colton T (1974) Inference on Proportions. In Statistics in Medicine. Ed., T Colton. Boston: Little, Brown and Company, pp 174-177. Fukasawa M, Noguchi H, Okuda T, Komatsu T, Yano K (1983) In vitro antibacterial activity of SM-1652, a new broad-spectrum cephalosporin with antipseudomonal activity. Antimicrob Agents Chemother 23:195. Hugh R, Gilardi GL (1980) Pseudomonas. In Manual of Clinical Microbiology, 3rd ed. Eds., EH Lennette, A Balows, WJ Hausler Jr, JP Truant. Washington, DC:American Society for Microbiology, pp 288-317. Jones RN, Barry AL, Gavan TL, Washington JAII (1985) Susceptibility tests: microdilution and macrodilution broth procedures. In Manual of Clinical Microbiology, 4th ed. Eds., EH Lennette, A Balows, WJ Hausler Jr, HJ Shadomy. Washington, DC: American Society for Microbiology, pp 972-977. Kato M, Inoue M, Mitsuhashi S (1982) Antibacterial activities of SM-1652 compared with those of other broad-spectrum cephalosporins. Antimicrob Agents Chemother 22:721.
In Vitro Activity of Cefpiramide
Nakagawa K, Koyama M, Matsue H, Ikeda C, Yano K, Nakatsuru N, Yoshinaga K, Noguchi T (1984) Pharmacokinetics of cefpiramide (SM-1652) in humans. Antimicrob Agents Chemother 25:221. Pfaller MA, Niles AC, Murray PR (1984) In vitro antibacterial activity of cefpiramide. Antimicrob Agents Chemother 25:368. Rolston KVI, Chandrasekar PH, LeFrock JL, Schell RF (1984) The activity of ceftazidime, other 13-1actams, and aminoglycosides against Pseudomonas aeruginosa. Chemotherapy 30:31. Schoenknecht FD, Sabath LD, Thornsberry C (1985) Susceptibility tests: Special Tests. In Manual of Clinical Microbiology, 4th ed Eds., EH Lennette, A Balows, W] Hausler It, HI Shadomy. Washington, DC: American Society for Microbiology, pp 1000-1008