ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1979, p. 346-350 0066-4804/79/03-0346/05$02.00/0
Vol. 15, No.3
In Vitro Activity of Piperacllin Compared with That of Carbenicillin, Ticarcllin, Ampicillin, Cephalothin, and Cefamandole Against Pseudomonas aeruginosa and Enterobacteriaceae PRAMOD P. SHAH, DALIUS J. BRIEDIS,t HUGH G. ROBSON,* AND JAMES P. CONTERATO Division of Infectious Diseases, Department of Medicine, Royal Victoria Hospital, Montreal, Quebec, Canada H3A lAl Received for publication 14 November 1978
Piperacillin (T-1220), a semisynthetic derivative of aminobenzylpenicillin, was than either carbenicillin or ticarcillin against Pseudomonas aerugi60% of isolates were inhibited at a concentration of 6.3 ug/ml. Piperacillin was bactericidal for 84% of Pseudomonas strains at 100 ,ug/ml, carbenicillin killed 60%, and ticarcillin killed 68% at that concentration. Piperacilhin was also more active than the other penicillins against isolates of Escherichia coli, Enterobacter, and Proteus mirabilis. The combination of piperacillin and tobramycin, demonstrating synergistic inhibition of 87% of strains of P. aeruginosa, was the most active of the penicillin-aminoglycoside combinations tested for synergism. more active nosa; over
Piperacillin sodium (T-1220) is a new semisynthetic penicillin with a broad spectrum of activity against gram-negative bacteria. In recent studies, many isolates of Pseudomonas aeruginosa and members of the Enterobacteriaceae were noted to be more susceptible to piperacillin than to carbenicillin (6, 14, 15). In the current study, the in vitro activity of piperacillin was compared with that of carbenicillin and ticarcillin against P. aeruginosa, and with that of carbenicillin, ticarcillin, ampicillin, cephalothin, and cefamandole against various Enterobacteriaceae. In addition, we compared combinations of piperacillin or carbenicillin with gentamicin or tobramycin for synergistic activity against P. aeruginosa.
6.0 mg/dl, and the magnesium concentration was 2.2 to 2.5 mg/dl in the various batches used.
Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). A microtitration broth dilution technique similar to that described by Harwick et al. was employed (8). Stock solutions of piperacillin, carbenicillin, and ticarcillin were prepared in Mueller-Hinton broth at a concentration of 533 ,g/ml. After appropriate dilutions in Mueller-Hinton broth and addition of the bacterial inoculum, the antibiotic concentrations ranged from 400 to 0.8 ,ug/ml, in a final volume of 200 ,ul. For ampicillin, cephalothin, and cefamandole, stock solutions were prepared at a concentration of 266 ,ug/ml, yielding antibiotic concentrations of 200 to 0.4
'Ug/ml.
The bacterial inoculum consisted of 50 pd of an overnight broth culture diluted 10-3 in Mueller-Hinton broth. Inoculated plates were capped with sterile lids, MATERIALS AND METHODS Bacterial isolates. Fifty isolates each of Esche- stacked, and incubated aerobically at 37°C for 18 h. richia coli, Klebsiellapneumoniae, Proteus mirabilis, The antibiotic concentration of the first clear well in and P. aeruginosa, 37 of Enterobacter spp., 28 indole- each twofold dilution series was taken as the MIC. positive Proteus, 13 Providencia stuartii, and 5 Ser- The MBC of the three penicillins was determined for ratia marcescens constituted the 283 bacterial isolates isolates of P. aeruginosa. A 0.001-ml aliquot from each obtained from the diagnostic microbiology laboratory well showing no visible growth was subcultured to a of the Royal Victoria Hospital. The identity of all segment of a sheep blood agar plate. The MBC was isolates was confirmed by standard methods (3, 9). defined as the lowest antibiotic concentration showing Not more than one isolate of a given species per no growth after 48 h of incubation at 37°C. Pencillin-aminoglycoside synergy against patient was included. A microtiter modification of the Culture medium. Mueller-Hinton broth was sup- Pseudomonas. plemented with calcium chloride and magnesium chlo- checkerboard broth dilution technique was used (13). ride (12). The final calcium concentration was 5.4 to Stock solutions of piperacillin and carbenicillin were prepared in supplemented Mueller-Hinton broth at a t Present address: Division of Virology, New Adden- concentration of 800 pg/ml, and gentamicin and tobrabrooke's Hospital, Cambridge, England. mycin stock solutions were prepared at 50 ,g/ml. After 346
VOL. 15, 1979
PIPERACILLIN ACTIVITY IN VITRO
appropriate dilutions and addition of the bacterial inoculum, final concentrations of the peniciUins and aminoglycosides were 400 to 0.4 jLg/ml and 12.5 to 0.2 ,tg/ml, respectively. The bacterial inocula were as described previously. The plates were read after 18 h of incubation at 37°C. Fifteen isolates of P. aeruginosa were studied for evidence of synergistic inhibition by each potential combination of piperacillin or carbenicillin and gentamicin or tobramycin. Each isolate was tested simultaneously against all the drug combinations. Isobolograms were constructed describing the MIC of each antibiotic concentration (13). Results were interpreted according to the criteria described by Weinstein et al. (16) and include the category described as marked synergism. RESULTS
Comparative antibacterial activity. The MIC and MBC of piperacillin, carbenicillin, and ticarcillin of 50 isolates of P. aeruginosa is shown in Fig. 1. Of the three penicillins, piperacillin was the most active, inhibiting over 60% of isolates at a concentration of 6.3 jig/ml compared with 4% inhibited by ticarcillin and none inhibited by carbenicillin. At a concentration of 100 jig/ml, piperacilhin inhibited 92%, carbenicillin inhibited 78%, and ticarcillin inhibited 86% of the isolates. Piperacillin was bactericidal against 60% of the strains of P. aeruginosa at 25 ,ug/ml, compared with none for carbenicllin and 16% for ticarcillin. The percentage of Pseudomonas strains killed at a concentration of 100 jg/ml was as follows: piperacillin, 84%; carbenicillin 60%; and ticarcillin, 68%. However, the spread between MIC and MBC of piperacillin was widest. The percentage of isolates for which the difference between the MIC and MBC was not greater than fourfold was 74% for piperacillin, 98% for carbenicillin, and 92% for ticarcillin. Four of the six isolates with carbenicillin and ticarcillin MIC ; 400 jLg/ml also had piperacillin PSEUDOMONAS AERUGINOSA *_0-*
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FIG. 1. MICs and MBCs ofpiperacillin, ticarcillin, and carbenicillin for 50 strains of P. aeruginosa.
347
MIC 5 400 ,ug/ml, but one each was inhibited by piperacillin at 25 and 50 ,ug/ml. The MICs of the six agents for the Enterobacteriaceae are shown in Table 1. For the 50 E. coli isolates, piperacillin was the most active penicillin, inhibiting the growth of 76% at 3.1 ,Ig/ ml. However, cefamandole was the most active agent of those tested against E. coli, inhibiting 50% of the isolates at 0.4 ,ig/ml and more than 90% at 12.5 ,ug/ml. At a concentration of 100 jig/ml, piperacillin inhibited 30% of K. pneumoniae compared with 10% or fewer by carbenicillin, ticarcillin, and ampicillin. Cefamandole was very much more active than any of the penicillins or cephalothin, inhibiting 64% of isolates at a concentration of 3.1 jig/ml. Piperacillin showed somewhat greater activity against Enterobacter spp. than the other penicillins tested. At 50 jig/ml, it inhibited 86% of the isolates. Cefamandole was again considerably more active than cephalothin against Enterobacter spp. but less active than the penicillins. Piperacillin was the most active agent tested against P. mirabilis, inhibiting 90% at 0.8 ,jg/ml. Cefamandole was more active than cephalothin at lower concentrations. Cefamandole inhibited 80% and cephalothin inhibited 30% of isolates at 3.1 ,ig/ml. At concentrations of 25 jig/ml and above, the activities of cefamandole and cephalothin were similar. Both piperacillin and carbenicillin inhibited over 75% of indole-positive Proteus isolates at 12.5 jig/ml. Ticarcillin, although somewhat less active than carbenicillin and piperacillin at the lower concentrations, showed similar activity to those agents at concentrations above 6.3 ,ug/ml. Neither ampicillin nor cephalothin demonstrated useful activity. Cefamandole on the other hand inhibited 32% of the isolates at 12.5 jig/ml and 70% at 50 jig/ml. Ticarcillin showed the best in vitro activity of the four penicillins against P. stuartii, inhibiting 61% of the isolates at 25 jig/ml. Cefamandole was even more active, inhibiting 85% of P. stuartii at 25 jig/ml. Neither ampicillin nor cephalothin showed a potentially useful degree of antibacterial activity. Of the five isolates of S. marcescens, piperacillin inhibited three, carbenicillin inhibited five, and ticarcillin inhibited five at a concentration of 100 jig/ml. All isolates grew in the presence of 100 jig of ampicillin, cephalothin, and cefamandole per ml. Synergistic inhibition of Pseudomwnas by penicillin-aminoglycoside combinations. The Pseudomonas isolates tested were chosen from among the 50 whose MICs had previously been determined. For 11 the MIC of carbenicillin
348
SHAH ET AL.
ANTIMICROB. AGENTS CHEMOTHER
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VOL, 15, 1979
PIPERACILLIN ACTIVITY IN VITRO
349
TABLE 2. Synergistic activity of antibiotic combinations against Pseudomonas Antibiotic combination
Carbenicillin + gentamic'm CarbeniciUin + tobramycin Piperacillin + gentamicin Piperacillin + tobramycin
No. of isolates
Synergism (no)*
Marked
Additive or
synergism (no.)
indifferent (no.)
15 15 15 15
9 5 6 4
3 5 7 9
3 5 2 2
was 100 ,ug or more per ml, and for 11 the piperacillin MIC did not exceed 25 Ag/ml. Synergistic inhibition of a majority of the isolates was observed with all antibiotic combinations, as shown in Table 2. The combination that produced the largest number of markedly synergistic inhibitory results was that of piperacillin plus tobramycin, followed closely by piperacilhin plus gentamicin. Both combinations showed synergism or marked synergism against 13 of 15 isolates. In no case was an antagonistic result observed.
DISCUSSION The increasing occurrence of serious infections caused by gram-negative aerobic bacilli, the acquisition of antibiotic resistance by the causal organisms, frequently poor therapeutic results, and the occurrence of adverse drug reactions all serve to stimulate the search for more effective chemotherapeutic agents (2, 4, 7). Therapy of Pseudomonas infections, in particulari is frequently unsuccessful, although the presence and severity of an underlying disease are a major determinant of the outcome (5). Carbenicillin, the first penicillin with significant anti-pseudomonas activity to be clinically available, is now widely used. Despite its broad spectrum of activity against aerobic gram-negative bacilli, carbenicilhin is not an ideal antimicrobial agent. Its intrinsic activity against isolates of P. aeruginosa is frequently low. When given in the very large amounts necessary for successful therapy, adverse effects such as excessive sodium load, hypokalemia (10), and interference with platelet function leading to hemorrhage (1) may result. Piperacillin appears to be a very promising anti-pseudomonas agent. In the present study piperacillin was much more active in vitro than carbenicillin against P. aeruginosa. There was approximately an eightfold difference between the amounts of piperacillin and carbenicillin required to inhibit 50% of P. aeruginosa isolates. This is in close agreement with the observations of Ueo et al. (14). Ninety percent of isolates were inhibited at a concentration of 50 ,ug/ml, a concentration readily achieved in human serum after intravenous administration of doses of 1 g or more (T. B. Tjandramaga, A. Muflie, R. Ver-
besselt, P. J. de Schepper, and L. Verbist, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 17th, New York, N.Y., Abstr. no. 323, 1977). Piperacillin also possessed bactericidal activity against a majority of P. aeruginosa isolates in contrast to other new agents such as pirbenicillin and BL-P 1654 (11). Although its intrinsic bactericidal activity may be less than that of carbenicillin or ticarcillin, many more isolates were killed by piperacillin than by the other agents at all concentrations in the 12.5- to 100-,g/ml range. Fu and Neu noted that most carbenicillin-resistant P. aeruginosa were susceptible to piperacillin (6). We tested only six P. aeruginosa isolates with high-level resistance to carbenicillin or ticarcillin (MIC > 400 jig/ml). Only two were susceptible to piperacillin. As with carbenicillin, the addition of gentamicin or tobramycin enhanced the anti-Pseudomonas activity of piperacillin. The method employed measured synergistic inhibitory activity of piperacillin in combination with either aminoglycoside; there is, however, a close correlation between such results and the documentation of a synergistic bactericidal effect of the antibiotic combination as determined by subcultures from clear wells to solid media or by in vitro killing curves (16). Although the number of isolates of P. aeruginosa so tested was only 15, the appearance of synergy between piperacillin and either aminoglycoside in 87% of instances was impressive. Piperacillin exhibited superior inhibitory activity to the other penicillins against many Enterobacteriaceae, especially P. mirabilis and to a lesser extent E. coli and Enterobacter spp. As with the other penicillins, piperacillin possessed little activity against K. pneumoniae strains. Compared with the new cephalosporin agent cefamandole, piperacillin was more active only against most isolates of P. mirabilis, indole-positive Proteus, and Enterobacter spp. Based on its in vitro spectrum of activity, piperacillin may be useful in therapy of infection caused by P. aeruginosa and some Enterobacteriaceae such as Proteus and Enterobacter spp. ACKNOWLEDGEMENT This work was supported by a grant from Canada Limited.
Cyananiid of
350
SHAH ET AL.
LITERATURE CITED 1. Brown, C. H., E. A. Natelson, M. W. Bradshaw, T. W. Williams, and C. P. Alfrey. 1974. The hemostatic defect produced by carbenicillin. N. Engl. J. Med. 291: 265-270. 2. Du Pont, H. L., and W. W. Spink. 1969. Infections due to gram-negative organisms: an analysis of 860 patients with bacteremia at the University of Minnesota Medical Center, 1958-1966. Medicine (Baltimore) 48:307-332. 3. Ewing, W. H., and W. J. Martin. 1974. Enterobacteriaceae, p. 189-221. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 4. Finland, M. 1972. Changing pattems of susceptibility of common bacterial pathogens to antimicrobial agents. Ann. Intem. Med. 76:1009-1036. 5. Flick, M. R., and L E. Cluff. 1976. Pseudomonas bacteremia. Am. J. Med. 60:501-508. 6. Fu, K. P., and H. C. Neu. 1978. Piperacillin, a new penicillin active against many bacteria resistant to other penicillins. Antimicrob. Agents Chemother. 13:358-367. 7. Greene, W. H., M. Moody, S. Schimpff, V. M. Young, and P. H. Wiernik. 1973. Pseudomonas aeruginosa resistant to carbenicillin and gentamicin. Ann. Intem. Med. 79:684-689. 8. Harwick, H. J., P. Weiss, and F. R. Fekety. 1968. Application of microtitration techniques to bacteriostatic and bactericidal antibiotic susceptibility testing. J. Lab. Clin. Med. 72:511-516. 9. Hugh, R., and G. L Gilardi. 1974. Pseudomonas, p. 250-269. In E. H. Lennette, E. H. Spaulding, and J. P.
ANTIMICROB. AGENTS CHEMOTHER. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 10. Klastersky, J., B. Vanderkelen, D. Daneau, and M. Mathieu. 1973. Carbenicilhin and hypokalemia (letter). Ann. Intern. Med. 78:774-775. 11. Lopez, C. E., H. C. Standiford, B. A. Tatem, F. M. Calia, S. C. Schimpff, M. J. Snyder, and R. B. Hornick. 1977. Pirbenicillin: comparison with carbenicillin and BL-P1654, alone and with gentamicin, against Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 11:441-448. 12. Reller, L B., F. D. Schoenknecht, M. A. Kenny, and J. C. Sherris. 1974. Antibiotic susceptibility testing of Pseudomonas aeruginosa: selection of a control strain and criteria for magnesium and calcium content in media. J. Infect. Dis. 130:454-463. 13. Sabath, L D., C. E. McCall, N. H. Steigbigel, and M. Finland. 1967. Synergistic penicillin combinations for treatment of human urinary-tract infections, p. 149-155. Antimicrob. Agents Chemother. 1966. 14. Ueo, K., Y. Fukuoka, T. Hayashi, T. Yasuda, H. Taki, M. Tai, Y. Watanabe, L. Saikawa, and S. Mitsuhashi. 1977. In vitro and in vivo antibacterial activity of T-1220, a hew semisynthetic penicillin. Antimicrob. Agents Chemother. 12:455-460. 15. Verbist, L. 1978. In vitro activity of piperacillin, a new semisynthetic penicillin with an unusually broad spectrum of activity. Antimicrob. Agents Chemother. 13: 349-357. 16. Weinstein, R. J., L S. Young, and W. L Hewitt. 1975. Comparison of methods for assessing in vitro antibiotic synergism against Pseudomonas and Serratia. J. Lab. Clin. Med. 86:853-862.
Vol. 15, No.3
In Vitro Activity of Piperacllin Compared with That of Carbenicillin, Ticarcllin, Ampicillin, Cephalothin, and Cefamandole Against Pseudomonas aeruginosa and Enterobacteriaceae PRAMOD P. SHAH, DALIUS J. BRIEDIS,t HUGH G. ROBSON,* AND JAMES P. CONTERATO Division of Infectious Diseases, Department of Medicine, Royal Victoria Hospital, Montreal, Quebec, Canada H3A lAl Received for publication 14 November 1978
Piperacillin (T-1220), a semisynthetic derivative of aminobenzylpenicillin, was than either carbenicillin or ticarcillin against Pseudomonas aerugi60% of isolates were inhibited at a concentration of 6.3 ug/ml. Piperacillin was bactericidal for 84% of Pseudomonas strains at 100 ,ug/ml, carbenicillin killed 60%, and ticarcillin killed 68% at that concentration. Piperacilhin was also more active than the other penicillins against isolates of Escherichia coli, Enterobacter, and Proteus mirabilis. The combination of piperacillin and tobramycin, demonstrating synergistic inhibition of 87% of strains of P. aeruginosa, was the most active of the penicillin-aminoglycoside combinations tested for synergism. more active nosa; over
Piperacillin sodium (T-1220) is a new semisynthetic penicillin with a broad spectrum of activity against gram-negative bacteria. In recent studies, many isolates of Pseudomonas aeruginosa and members of the Enterobacteriaceae were noted to be more susceptible to piperacillin than to carbenicillin (6, 14, 15). In the current study, the in vitro activity of piperacillin was compared with that of carbenicillin and ticarcillin against P. aeruginosa, and with that of carbenicillin, ticarcillin, ampicillin, cephalothin, and cefamandole against various Enterobacteriaceae. In addition, we compared combinations of piperacillin or carbenicillin with gentamicin or tobramycin for synergistic activity against P. aeruginosa.
6.0 mg/dl, and the magnesium concentration was 2.2 to 2.5 mg/dl in the various batches used.
Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). A microtitration broth dilution technique similar to that described by Harwick et al. was employed (8). Stock solutions of piperacillin, carbenicillin, and ticarcillin were prepared in Mueller-Hinton broth at a concentration of 533 ,g/ml. After appropriate dilutions in Mueller-Hinton broth and addition of the bacterial inoculum, the antibiotic concentrations ranged from 400 to 0.8 ,ug/ml, in a final volume of 200 ,ul. For ampicillin, cephalothin, and cefamandole, stock solutions were prepared at a concentration of 266 ,ug/ml, yielding antibiotic concentrations of 200 to 0.4
'Ug/ml.
The bacterial inoculum consisted of 50 pd of an overnight broth culture diluted 10-3 in Mueller-Hinton broth. Inoculated plates were capped with sterile lids, MATERIALS AND METHODS Bacterial isolates. Fifty isolates each of Esche- stacked, and incubated aerobically at 37°C for 18 h. richia coli, Klebsiellapneumoniae, Proteus mirabilis, The antibiotic concentration of the first clear well in and P. aeruginosa, 37 of Enterobacter spp., 28 indole- each twofold dilution series was taken as the MIC. positive Proteus, 13 Providencia stuartii, and 5 Ser- The MBC of the three penicillins was determined for ratia marcescens constituted the 283 bacterial isolates isolates of P. aeruginosa. A 0.001-ml aliquot from each obtained from the diagnostic microbiology laboratory well showing no visible growth was subcultured to a of the Royal Victoria Hospital. The identity of all segment of a sheep blood agar plate. The MBC was isolates was confirmed by standard methods (3, 9). defined as the lowest antibiotic concentration showing Not more than one isolate of a given species per no growth after 48 h of incubation at 37°C. Pencillin-aminoglycoside synergy against patient was included. A microtiter modification of the Culture medium. Mueller-Hinton broth was sup- Pseudomonas. plemented with calcium chloride and magnesium chlo- checkerboard broth dilution technique was used (13). ride (12). The final calcium concentration was 5.4 to Stock solutions of piperacillin and carbenicillin were prepared in supplemented Mueller-Hinton broth at a t Present address: Division of Virology, New Adden- concentration of 800 pg/ml, and gentamicin and tobrabrooke's Hospital, Cambridge, England. mycin stock solutions were prepared at 50 ,g/ml. After 346
VOL. 15, 1979
PIPERACILLIN ACTIVITY IN VITRO
appropriate dilutions and addition of the bacterial inoculum, final concentrations of the peniciUins and aminoglycosides were 400 to 0.4 jLg/ml and 12.5 to 0.2 ,tg/ml, respectively. The bacterial inocula were as described previously. The plates were read after 18 h of incubation at 37°C. Fifteen isolates of P. aeruginosa were studied for evidence of synergistic inhibition by each potential combination of piperacillin or carbenicillin and gentamicin or tobramycin. Each isolate was tested simultaneously against all the drug combinations. Isobolograms were constructed describing the MIC of each antibiotic concentration (13). Results were interpreted according to the criteria described by Weinstein et al. (16) and include the category described as marked synergism. RESULTS
Comparative antibacterial activity. The MIC and MBC of piperacillin, carbenicillin, and ticarcillin of 50 isolates of P. aeruginosa is shown in Fig. 1. Of the three penicillins, piperacillin was the most active, inhibiting over 60% of isolates at a concentration of 6.3 jig/ml compared with 4% inhibited by ticarcillin and none inhibited by carbenicillin. At a concentration of 100 jig/ml, piperacilhin inhibited 92%, carbenicillin inhibited 78%, and ticarcillin inhibited 86% of the isolates. Piperacillin was bactericidal against 60% of the strains of P. aeruginosa at 25 ,ug/ml, compared with none for carbenicllin and 16% for ticarcillin. The percentage of Pseudomonas strains killed at a concentration of 100 jg/ml was as follows: piperacillin, 84%; carbenicillin 60%; and ticarcillin, 68%. However, the spread between MIC and MBC of piperacillin was widest. The percentage of isolates for which the difference between the MIC and MBC was not greater than fourfold was 74% for piperacillin, 98% for carbenicillin, and 92% for ticarcillin. Four of the six isolates with carbenicillin and ticarcillin MIC ; 400 jLg/ml also had piperacillin PSEUDOMONAS AERUGINOSA *_0-*
MIC-PIPERACILLIN MBC-PIPERACILLIN
MIC- TICARCILLIN *-*
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347
MIC 5 400 ,ug/ml, but one each was inhibited by piperacillin at 25 and 50 ,ug/ml. The MICs of the six agents for the Enterobacteriaceae are shown in Table 1. For the 50 E. coli isolates, piperacillin was the most active penicillin, inhibiting the growth of 76% at 3.1 ,Ig/ ml. However, cefamandole was the most active agent of those tested against E. coli, inhibiting 50% of the isolates at 0.4 ,ig/ml and more than 90% at 12.5 ,ug/ml. At a concentration of 100 jig/ml, piperacillin inhibited 30% of K. pneumoniae compared with 10% or fewer by carbenicillin, ticarcillin, and ampicillin. Cefamandole was very much more active than any of the penicillins or cephalothin, inhibiting 64% of isolates at a concentration of 3.1 jig/ml. Piperacillin showed somewhat greater activity against Enterobacter spp. than the other penicillins tested. At 50 jig/ml, it inhibited 86% of the isolates. Cefamandole was again considerably more active than cephalothin against Enterobacter spp. but less active than the penicillins. Piperacillin was the most active agent tested against P. mirabilis, inhibiting 90% at 0.8 ,jg/ml. Cefamandole was more active than cephalothin at lower concentrations. Cefamandole inhibited 80% and cephalothin inhibited 30% of isolates at 3.1 ,ig/ml. At concentrations of 25 jig/ml and above, the activities of cefamandole and cephalothin were similar. Both piperacillin and carbenicillin inhibited over 75% of indole-positive Proteus isolates at 12.5 jig/ml. Ticarcillin, although somewhat less active than carbenicillin and piperacillin at the lower concentrations, showed similar activity to those agents at concentrations above 6.3 ,ug/ml. Neither ampicillin nor cephalothin demonstrated useful activity. Cefamandole on the other hand inhibited 32% of the isolates at 12.5 jig/ml and 70% at 50 jig/ml. Ticarcillin showed the best in vitro activity of the four penicillins against P. stuartii, inhibiting 61% of the isolates at 25 jig/ml. Cefamandole was even more active, inhibiting 85% of P. stuartii at 25 jig/ml. Neither ampicillin nor cephalothin showed a potentially useful degree of antibacterial activity. Of the five isolates of S. marcescens, piperacillin inhibited three, carbenicillin inhibited five, and ticarcillin inhibited five at a concentration of 100 jig/ml. All isolates grew in the presence of 100 jig of ampicillin, cephalothin, and cefamandole per ml. Synergistic inhibition of Pseudomwnas by penicillin-aminoglycoside combinations. The Pseudomonas isolates tested were chosen from among the 50 whose MICs had previously been determined. For 11 the MIC of carbenicillin
348
SHAH ET AL.
ANTIMICROB. AGENTS CHEMOTHER
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VOL, 15, 1979
PIPERACILLIN ACTIVITY IN VITRO
349
TABLE 2. Synergistic activity of antibiotic combinations against Pseudomonas Antibiotic combination
Carbenicillin + gentamic'm CarbeniciUin + tobramycin Piperacillin + gentamicin Piperacillin + tobramycin
No. of isolates
Synergism (no)*
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indifferent (no.)
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9 5 6 4
3 5 7 9
3 5 2 2
was 100 ,ug or more per ml, and for 11 the piperacillin MIC did not exceed 25 Ag/ml. Synergistic inhibition of a majority of the isolates was observed with all antibiotic combinations, as shown in Table 2. The combination that produced the largest number of markedly synergistic inhibitory results was that of piperacillin plus tobramycin, followed closely by piperacilhin plus gentamicin. Both combinations showed synergism or marked synergism against 13 of 15 isolates. In no case was an antagonistic result observed.
DISCUSSION The increasing occurrence of serious infections caused by gram-negative aerobic bacilli, the acquisition of antibiotic resistance by the causal organisms, frequently poor therapeutic results, and the occurrence of adverse drug reactions all serve to stimulate the search for more effective chemotherapeutic agents (2, 4, 7). Therapy of Pseudomonas infections, in particulari is frequently unsuccessful, although the presence and severity of an underlying disease are a major determinant of the outcome (5). Carbenicillin, the first penicillin with significant anti-pseudomonas activity to be clinically available, is now widely used. Despite its broad spectrum of activity against aerobic gram-negative bacilli, carbenicilhin is not an ideal antimicrobial agent. Its intrinsic activity against isolates of P. aeruginosa is frequently low. When given in the very large amounts necessary for successful therapy, adverse effects such as excessive sodium load, hypokalemia (10), and interference with platelet function leading to hemorrhage (1) may result. Piperacillin appears to be a very promising anti-pseudomonas agent. In the present study piperacillin was much more active in vitro than carbenicillin against P. aeruginosa. There was approximately an eightfold difference between the amounts of piperacillin and carbenicillin required to inhibit 50% of P. aeruginosa isolates. This is in close agreement with the observations of Ueo et al. (14). Ninety percent of isolates were inhibited at a concentration of 50 ,ug/ml, a concentration readily achieved in human serum after intravenous administration of doses of 1 g or more (T. B. Tjandramaga, A. Muflie, R. Ver-
besselt, P. J. de Schepper, and L. Verbist, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 17th, New York, N.Y., Abstr. no. 323, 1977). Piperacillin also possessed bactericidal activity against a majority of P. aeruginosa isolates in contrast to other new agents such as pirbenicillin and BL-P 1654 (11). Although its intrinsic bactericidal activity may be less than that of carbenicillin or ticarcillin, many more isolates were killed by piperacillin than by the other agents at all concentrations in the 12.5- to 100-,g/ml range. Fu and Neu noted that most carbenicillin-resistant P. aeruginosa were susceptible to piperacillin (6). We tested only six P. aeruginosa isolates with high-level resistance to carbenicillin or ticarcillin (MIC > 400 jig/ml). Only two were susceptible to piperacillin. As with carbenicillin, the addition of gentamicin or tobramycin enhanced the anti-Pseudomonas activity of piperacillin. The method employed measured synergistic inhibitory activity of piperacillin in combination with either aminoglycoside; there is, however, a close correlation between such results and the documentation of a synergistic bactericidal effect of the antibiotic combination as determined by subcultures from clear wells to solid media or by in vitro killing curves (16). Although the number of isolates of P. aeruginosa so tested was only 15, the appearance of synergy between piperacillin and either aminoglycoside in 87% of instances was impressive. Piperacillin exhibited superior inhibitory activity to the other penicillins against many Enterobacteriaceae, especially P. mirabilis and to a lesser extent E. coli and Enterobacter spp. As with the other penicillins, piperacillin possessed little activity against K. pneumoniae strains. Compared with the new cephalosporin agent cefamandole, piperacillin was more active only against most isolates of P. mirabilis, indole-positive Proteus, and Enterobacter spp. Based on its in vitro spectrum of activity, piperacillin may be useful in therapy of infection caused by P. aeruginosa and some Enterobacteriaceae such as Proteus and Enterobacter spp. ACKNOWLEDGEMENT This work was supported by a grant from Canada Limited.
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