ANTImICROBIAL AGaNTs AND CHDMoTHzRA&Y, Apr. 1976, p. 668-674 Copyright 0) 1976 American Society for Microbiology
Vol. 9, No. 4' Printed in U.SA.
Pirbenicillin, a New Semisynthetic Penicillin with BroadSpectrum Activity GERALD P. BODEY,* VICTORIO RODRIGUEZ, AnD SUZANNE WEAVER Department ofDevelopmental Therapeutics, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77025 Received for publication 24 November 1975
Pirbenicillin is a new semisynthetic penicillin which inhibited 67% of isolates of Proteus aeruginosa tested in our laboratory, 93% of P. mirabilis, 31% of Enterobacter spp., 41% of Serratia spp., and 58% of Escherichia coli at a concentration of 6.25 Mg/ml. Its activity appeared to be inoculum dependent and it was virtually inactive against 1O7 inocula of P. aeruginosa. It was more active than carbenicillin or ticarcillin, but less active than BL-P1654 againstP. aeruginosa. Carbenicillin and ticarcillin appeared to be more active than pirbenicillin against Proteus spp., but pirbenicillin was active against some isolates of Klebsiella spp. The synthesis of ampicillin represented an important advance in antibiotic research because this was the first truly broad-spectrum penicillin. The subsequent discovery of carbenicillin extended the spectrum of penicillins to include indole positive Proteus spp. and Pseudomonas aeruginosa (1). Clinical studies demonstrated that this antibiotic was efficacious for the treatment of Pseudomonas infections, even in patients with severely impaired host defenses (3). Because of its rather marginal activity, carbenicillin must be administered in high doses which are associated with undesirable side effects such as electrolyte imbalance and coagulation abnormalities (4). Consequently, research has been directed toward synthesizing new penicillin derivatives with greater antipseudomonal activity. One ofthese derivatives, ticarcillin, is more active than carbenicillin in vitro and has been used successfully at lower doses for the treatment of Pseudomonas infections (6). Pirbenicillin, 6-[D-2-phenyl-2-(N4-pyridyl-formimidyl aminoacetamido)-acetamidol-penicillanic acid (CP-33, 994) is a newly synthesized penicillin derivative which appears to have substantially greater activity than carbenicillin against P. aeruginosa. This report presents the results of in vitro studies of pirbenicillin which indicate that it may be a potentially useful antibiotic.
nisms were inoculated into Mueller-Hinton broth (Difco) and incubated at 37 C for 18 h. For gramnegative bacilli, a 0.05-ml sample of a 10-3 dilution of this broth culture (approximately 105 colonyforming units [CFU]I/ml) was used as the inoculum. For 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 ofP. aeruginosa and Escherichia coli, 119 isolates of Proteus spp., 70 isolates of Serratia spp., 86 isolates of Enterobacter spp., and 50 isolates ofKlebsiella spp. was 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 25 isolates of Streptococcus pyogenes, 14 isolates of Streptococcus pneumoniae and 100 isolates of Staphylococcus aureus was 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 ,ug/ml were selected as penicillin G resistant. Pirbenicillin was supplied as a powder by Pfizer Central Research, Groton, Conn. BL-P1654 6-[D-a(3-guanylureido)-phenylacetamido]penicillanic acid was supplied by Bristol Laboratories, Syracuse, N. Y. Carbenicillin and ticarcillin were supplied by Beecham Pharmaceuticals, Bristol, Tenn. Twofold MATERIALS AND METHODS serial dilutions of the antibiotics were made with Susceptibility tests were conducted on 525 clinical Mueller-Hinton broth, to which phosphate buffer isolates of gram-negative bacilli and 139 clinical was added to a pH of 7.2. The minimal inhibitory isolates of gram-positive cocci, using the dilution concentration (MIC) was determined after incubatechnique with an automatic microtiter system tion at 37 C for 18 h. All wells containing trace (Canalco: Autotiter Instruction Manual). All orga- growth or no discernible growth were subcultured 668
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on sheep blood agar. A calibrated pipette was used 100 to transfer 0.01 ml of the inoculum. The minimum bactericidal concentration (MBC) was determined after incubation at 37 C for 18 h. The MBC was defined as the lowest concentration of drug which 80 yielded less than 50 colonies on subculture (less than 5 colonies per 0.001 ml of inoculum). Comparison studies were conducted simultaneously. The concentration of pirbenicillin in media was determined by an agar well method with Sarcina 0- 60t lutea ATCC9341 as the test organism. This organism was prepared so that a 1:10 dilution had an absorbIance of 0.6 at a wavelength of 580 nm, and 4 ml of 0 E 40 this preparation was added to 1 liter of antibiotic medium no. 1 (Difco). After mixing thoroughly, 14 Cml of broth was poured in plates (100 by 15 mm). Wells (0.75-mm diameter and 0.75-mm depth) were cut into the agar and filled with 0.05 ml of each specimen. The plates were incubated at 37 C for 18 h. Zones of inhibition were measured with a caliper, and the concentration was determined from a standard curve. A standard curve was prepared every time samples were assayed. All studies were per.025 10 39 1.56 6.25 25 100 400 formed in triplicate. Minimum Inhibitory Concentrotion The effect of pirbenicillin on the growth of 10 isolates of P. aeruginosa was determined in the folFIG. 1. In activity of pirbenicillin against lowing fashion. All 10 isolates were inoculated into gram-negativevitro bacilli and gram-positive cocci. NumMueller-Hinton broth and incubated at 37 C for 18 bers in parentheses indicate number ofisolates tested. h. A 0.1-ml aliquot of broth containing each Pseudomonas isolate was inoculated into each of five tubes containing 9.9 ml of unbuffered Mueller-Hin- bacter spp., 41% of Serratia spp., 58% of E. coli, ton broth. Two ofthe five tubes contained pirbenicil- 67% ofP. aeruginosa, and 93% of P. mirabilis. lin at a concentration equal to the MIC for that At 50 ,ug/ml, the antibiotic inhibited 34% of Pseudomonas isolate. Two other tubes contained isolates of Klebsiella spp. and 28% of indolepirbenicillin at a concentration equal to four times positive Proteus spp. Generally, using the the MIC for that Pseudomonas isolate. The fifth buffered Mueller-Hinton broth, the MIC was tube served as a control. All of the tubes were incu- also the MBC. However, the MBC for isolates of bated at 37 C for 24 h. After vigorous agitation, a P. aeruginosa was usually two to four times 0.1-ml aliquot was removed from each tube at 0, 2, 4, 6, 8, 10, 12, 15, 18, 21, and 24 h and then subcultured higher than MIC. For example, 6.25 ,ug/ml was on Mueller-Hinton agar. The number of viable orga- the MIC and MBC for 58% of isolates of E. coli, nisms present was determined from these subcul- the MIC for 41%, and MBC for 34% of isolates of tures. At 8 h, additional pirbenicillin was added to Serratia spp., and the MIC for 67% of isolates of two tubes (one containing the MIC and one contain- P. aeruginosa, but the MBC for only 40% of ing four times the MIC) so that the concentration these latter isolates. was the same as at the onset of the experiment, The effect of inoculum size on the MIC and assuming that all of the original pirbenicillin had been destroyed. The concentration of pirbenicillin in MBC was determined for 10 isolates each ofE. each tube was assayed at 8 h and at 24 h. The MIC coli and P. aeruginosa (Fig. 2). For most isowas determined for all organisms still growing at 24 lates, at both inoculum sizes, the MIC and MBC were similar. However, the MIC and h. %
RESULTS The in vitro activity of pirbenicillin against gram-positive cocci and gram-negative bacilli is shown in Fig. 1. Nearly all isolates of S. pyogenes and S. pneumoniae were inhibited by 0.10 ,ug/ml or less. All isolates of penicillin Gsensitive S. aureus were inhibited by 1.56 ,g/ ml, whereas only 40% of penicillin G-resistant S. aureus was inhibited by 3.12 ,ug of pirbenicillin per ml. At a concentration of 6.25 pg/ml, pirbenicillin inhibited 31% of isolates of Entero-
MBC for isolates of E. coli increased fourfold when the inoculum was increased from 105 CFU/ml to 107 CFU/ml. Inoculum size had a major effect on the inhibitory activity of pirbenicillin against isolates of P. aeruginosa. Only one isolate was inhibited by 400 ,ug of pirbenicillin per ml when an inoculum of 107 CFU/ml was used. The effect of pH on susceptibility of 28 isolates of P. aeruginosa to pirbenicillin, BLP1654, carbenicillin, and ticarcillin was investigated (Fig. 3). Phosphate buffer was added to Mueller-Hinton broth to a final pH of 6.4 and
670 BODEY, RODRIGUEZ, AND WEAVER 7.2. BL-P1654 was the most active antibiotic at both H+ concentrations and carbenicillin was the least active. Pirbenicillin, carbenicillin, and ticarcillin were more active at pH 7.2, whereas BL-P1654 was more active 'at pH 6.4. The difference between the MIC and MBC was similar at both H+ concentrations for carbenicillin, BL-P1654, and pirbenicillin. The difference between MIC and MBC for ticarcillin was greater at pH 6.4 than at pH 7.2.
ANTIMICROB. AGENTS CHZMOTHER.
The activity of the antibiotics in different media was studied, using these same isolates of P. aeruginosa (Fig. 4). BL-P1654 was the most active antibiotic in every medium and was most affected by the composition of the media. All of the antibiotics were most active in M9 broth which does not contain preformed amino acids. They were least active in Mueller-Hinton broth buffered at pH 7.2. Except for BL-P1654, the greatest differences of MIC and MBC were observed with M9 broth. Interestingly, the MICs for carbenicillin and ticarcillin were the lowest with M9 broth, but the MBCs were the highest in these medium. The activity of pirbenicillin against 25 isolates each of various gram-negative bacilli was compared with ticarcillin and carbenicillin, using Mueller-Hinton broth buffered at pH 7.2 (Fig. 5-8). Carbenicillin was least active Z E against E. coli, whereas ticarcillin and pirbenicillin had similar activity. Pirbenicillin was substantially less active than ticarcillin and carbenicillin against indole-positive and -negative Proteus spp. The activity of all three antibiotics was similar against isolates of Enterobacter spp. and Serratia spp. Pirbenicillin was Minimum Inhibitory Concentration (yig/mi) substantially more active than carbenicillin FIG. 2. Effect of inoculum size on MIC and MBC against isolates of P. aeruginosa. Pirbenicillin of pirbenicillin. Ten isolates each of P. aeruginosa was the only antibiotic with activity against isolates of Klebsiella spp. and E. coli were tested. z
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6.25 25 100 400 6.25 25 Minimum Inhibitory Concentration (pLg/ml)
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FIG. 3. Effect ofpH on activity of penicillins against P. aeruginosa. Twenty-eight isolates were tested.
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100 80
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o
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. MBC
.
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Broth
MBC Imueller Hinton Brothi 6.25 25 100 400 6.25 25 Minimum Inhibitory Concentration (jig/ml) 13
100
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FIG. 4. Effect of media on activity of antipseudomonal penicillins against 28 isolates of P. aeruginosa.
The effect of pirbenicillin on the proliferation of 10 isolates of P. aeruginosa was studied. Figure 9 shows the median results with six isolates which were initially susceptible to 12.5 ,ug of pirbenicillin per ml. At 8 h, the concentration had decreased from 106/ml to 103/ml when exposed to 12.5 ,ug/ml (MIC) or 50 ,ug/ml (four times MIC) of pirbenicillin. About 50% of the antibiotic had been inactivated at this time. Hence, during a portion of this 8-h period, organisms incubated in media containing the MIC of pirbenicillin were exposed to a subinhibitory concentration. If no additional antibiotic was added at this time, the organisms proliferated so that the concentration was the same as the controls at 24 h. The MIC for these persistent organisms had increased from 12.5 ,ug/ml to 100 ,ug/ml. Even if additional drug was added at 8 h, the organisms were able to proliferate subsequently, but only after a 4-h delay. These persistent organisms also had developed increased resistance. Organisms exposed to 50 ,ug of pirbenicillin per ml were all killed eventually because the concentration of pirbenicillin remained above the MIC. Complete killing occurred sooner if additional drug was added at 8 h. Similar results were observed with four isolates of P. aeruginosa which had MICs of 6.25,
6.25, 25, and 50 ,ug/ml. When pirbenicillin was incubated alone, there was no appreciable destruction of drug at 8 h. However, at 24 h, 7 to 22% was inactivated. DISCUSSION Pirbenicillin is a broad-spectrum penicillin with activity against both gram-positive cocci and gram-negative bacilli. It is more active than carbenicillin or ticarcillin against P. aeruginosa, but less active than BL-P1654. Clinical studies of the latter antibiotic have been abandoned because of its potential nephrotoxicity. Pirbenicillin is less active than either carbenicillin or ticarcillin against Proteus spp. It is as active as carbenicillin against Serratia spp. and Enterobacter spp. These results are in agreement with those of Retsema et al. who also demonstrated that pirbenicillin was more active than carbenicillin against experimental Pseudomonas infections in mice (J. A. Retsema, A. R. English, and J. E. Lynch, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., Abstr. 252, 1975; Retsema and English, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., Abstr. 253, 1975).
ANnimacRoB. AGZWM CHEMOTHZR.
BODEY, RODRIGUEZ, AND WEAVER
672
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.20 .78 3.12 12.5 50 200 Minimum Inhibitory Concentration (ag/ml) FIG. 5. Comparative activity of semisynthetic penicillins against E. coli. Twenty-five isolates were tested.
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Ir * Pirbenicillin
A Ticarcillin There has been some controversy over the o Carbenicillin bactericidal activity of pirbenicillin. Lopez et 80[ t al. found considerable differences between the MIC and MBC against isolates ofP. aeruginosa 0 (C. Lopez, H. Standiford, B. Tatum, F. Calia, S. Shimpif, M. Synder, and R. Hornick, Prog. 10va Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., Abstr. 0 60 F 254, 1975). These differences appear to be due to the chemical instability of pirbenicillin in alkaline media (Retsema et al., Prog. Intersci. Conf. Antimicrob. Agents Chemother., Abstr. a40 [ 252, 1975). The pH of Mueller-Hinton broth increases to over 8 during growth of P. aerugi- 21 nosa causing degradation of pirbenicillin. This C-) problem can be circumvented by buffering the media. Consequently, we used only Mueller20 . Hinton broth buffered to pH 7.2 for our studies, whereas Lopez et al. used unbuffered media. I The difference between MIC and MBC tended ,-A / I0I1I to be minimal in buffered media. The efficacy of pirbenicillin was profoundly influenced by inoculum size. This was espe.10 .39 1.56 6.25 25 100 400 cially true for isolates of P. aeruginosa which Minimum Inhibitory Concentration (,.g/ml) became resistant to pirbenicillin when the inoculum was increased from 105 to 107 cells. The FIG. 7. Comparative activity of semisynthetic penactivity of other penicillins is also affected by icillins against indole-positive Proteus spp. -0
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/1
PIRBENICILLIN
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673
the size of inoculum (5, 7). Pirbenicillin was most effective in a basic medium such as M9 which contains no preformed amino acids. The effect of media on the activity of BL-P1654, a related semisynthetic penicillin, has been described previously (2). 80 When isolates ofP. aeruginosa with an inoc(A ulum size of 106 organisms/ml are exposed to the MIC for pirbenicillin in unbuffered Muel0 ler-Hinton broth, killing occurs during the first 70 60 I I 8 h. However, sufficient drug is destroyed, so that the concentration falls below the MIC. At VI this suboptimal concentration, persisting orga0 0 nisms develop resistance and begin to proliferC._ ate. This can be avoided by exposing the orga0 nisms to a higher concentration initially. IncJ creasing the concentration to above the MIC at C-) I I 8 h has little effect, indicating that resistance probably has already developed. Unfortunately, we only repeated MIC determinations on organisms that survived at 24 h. We have * Pirbenicillin observed similar results with a few isolates of * ricorcillin E. coli, and Lopez et al. reported similar results O Carbenicillin with P. aeruginosa (Prog. Intersci. Conf. AntiI microb. Agents Chemother., Abstr. 254, 1975). These observations suggest that it may be im. A . OW portant to maintain serum concentrations con1.56 6.25 25 100 400 tinuously above the MIC of infecting organisms Minimum Inhibitory Concentration (pg/ml) FIG. 8. Comparative activity of semisynthetic pen- when treating patients with this drug. Pirbenicillin is an interesting new semisynicillins against P. aeruginosa. 0
-0
-
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FIG. 9. Effect ofpirbenicillin on growth of six isolates ofP. aeruginosa, all of which were initially inhibited by 12.5 pg/ml. Tube A initially contained 12.5 pg ofpirbenicillin per ml and tube C initially contained 50 pg ofpirbenicillin per ml. No additional drug was added to these tubes at 8 h. Tube B initially contained 12.5 pgl ml and an additional 12.5 pg/ml was added at 8 h. Tube D initially contained 50 pgIml and an additional 50 pgIml was added at 8 h. For details of experiment, see text.
674 BODEY, RODRIGUEZ, AND WEAVER thetic penicillin with broad-spectrum activity. Although it is not as active against P. aeruginosa as BL-P1654 in vitro, it does not cause nephrotoxicity in animals. This drug deserves clinical investigation since it may be more effective than carbenicillin or ticarcillin. It may also prove efficacious against K. pneumoniae since it inhibits 70% of these isolates in vitro at 200 jug/ml. Although this represents only marginal activity, it may be possible to maintain adequate serum concentrations with high doses of drug. ACKNOWLEDGMENTS This investigation was supported by Public Health service grant CA 10042 from the National Cancer Institute and by a grant-in-aid from Pfizer Central Research, Groton, Conn. LITERATURE CITED 1. Acred, P., D. M. Brown, E. J. Knudsen, G. N. Robinson, and R. Sutherland. 1967. New semisynthetic
ANTIMaCROB. AGENTS CHEMOTHER. 2. 3.
4.
5.
6. 7.
penicillin active against Pseudomonas pyocyanea. Nature (London) 215:25-30. Bodey, G. P., and D. Stewart. In vitro studies on semisynthetic -(substituted-ureido) penicillins. Appl. Microbiol. 21:710-717. Bodey, G. P., J. P. Whitecar. Jr., E. Middleman, and V. Rodriguez. 1971. Carbenicillin therapy of Pseudomonas infections. J. Am. Med. Assoc. 218:62-66. Brown, C. H., III, E. A. Natelson, M. W. Bradhaw, T. W. Williams, Jr., and C. P. Alfrey, Jr. 1974. The hemostatic defect produced by carbenicillin. New Engl. J. Med. 219:265-270. Neu, H. C., and E. B. Winshell. 1871. In vitro studies of a semisynthetic penicillin, 6-[D(-)a-carboxy-3thienylacetamido] penicillanic acid (BRL 2288) active against Pseudomonas, p. 385-389. Antimicrob. Agents Chemother. 1970. Rodriguez, V., G. P. Bodey, N. Horikoshi, J. Inagaki, and K. B. McCredie. 1973. Ticarcidin therapy of infections. Antimicrob. Agents Chemother. 4:427-431. StandUford, H. C., A. C. Kind, and W. M. M. Kirby. 1969. Laboratory and clinical studies of carbenicillin against gram-negative bacilli, p. 286-291. Antimicrob. Agents Chemother. 1968.
Vol. 9, No. 4' Printed in U.SA.
Pirbenicillin, a New Semisynthetic Penicillin with BroadSpectrum Activity GERALD P. BODEY,* VICTORIO RODRIGUEZ, AnD SUZANNE WEAVER Department ofDevelopmental Therapeutics, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77025 Received for publication 24 November 1975
Pirbenicillin is a new semisynthetic penicillin which inhibited 67% of isolates of Proteus aeruginosa tested in our laboratory, 93% of P. mirabilis, 31% of Enterobacter spp., 41% of Serratia spp., and 58% of Escherichia coli at a concentration of 6.25 Mg/ml. Its activity appeared to be inoculum dependent and it was virtually inactive against 1O7 inocula of P. aeruginosa. It was more active than carbenicillin or ticarcillin, but less active than BL-P1654 againstP. aeruginosa. Carbenicillin and ticarcillin appeared to be more active than pirbenicillin against Proteus spp., but pirbenicillin was active against some isolates of Klebsiella spp. The synthesis of ampicillin represented an important advance in antibiotic research because this was the first truly broad-spectrum penicillin. The subsequent discovery of carbenicillin extended the spectrum of penicillins to include indole positive Proteus spp. and Pseudomonas aeruginosa (1). Clinical studies demonstrated that this antibiotic was efficacious for the treatment of Pseudomonas infections, even in patients with severely impaired host defenses (3). Because of its rather marginal activity, carbenicillin must be administered in high doses which are associated with undesirable side effects such as electrolyte imbalance and coagulation abnormalities (4). Consequently, research has been directed toward synthesizing new penicillin derivatives with greater antipseudomonal activity. One ofthese derivatives, ticarcillin, is more active than carbenicillin in vitro and has been used successfully at lower doses for the treatment of Pseudomonas infections (6). Pirbenicillin, 6-[D-2-phenyl-2-(N4-pyridyl-formimidyl aminoacetamido)-acetamidol-penicillanic acid (CP-33, 994) is a newly synthesized penicillin derivative which appears to have substantially greater activity than carbenicillin against P. aeruginosa. This report presents the results of in vitro studies of pirbenicillin which indicate that it may be a potentially useful antibiotic.
nisms were inoculated into Mueller-Hinton broth (Difco) and incubated at 37 C for 18 h. For gramnegative bacilli, a 0.05-ml sample of a 10-3 dilution of this broth culture (approximately 105 colonyforming units [CFU]I/ml) was used as the inoculum. For 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 ofP. aeruginosa and Escherichia coli, 119 isolates of Proteus spp., 70 isolates of Serratia spp., 86 isolates of Enterobacter spp., and 50 isolates ofKlebsiella spp. was 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 25 isolates of Streptococcus pyogenes, 14 isolates of Streptococcus pneumoniae and 100 isolates of Staphylococcus aureus was 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 ,ug/ml were selected as penicillin G resistant. Pirbenicillin was supplied as a powder by Pfizer Central Research, Groton, Conn. BL-P1654 6-[D-a(3-guanylureido)-phenylacetamido]penicillanic acid was supplied by Bristol Laboratories, Syracuse, N. Y. Carbenicillin and ticarcillin were supplied by Beecham Pharmaceuticals, Bristol, Tenn. Twofold MATERIALS AND METHODS serial dilutions of the antibiotics were made with Susceptibility tests were conducted on 525 clinical Mueller-Hinton broth, to which phosphate buffer isolates of gram-negative bacilli and 139 clinical was added to a pH of 7.2. The minimal inhibitory isolates of gram-positive cocci, using the dilution concentration (MIC) was determined after incubatechnique with an automatic microtiter system tion at 37 C for 18 h. All wells containing trace (Canalco: Autotiter Instruction Manual). All orga- growth or no discernible growth were subcultured 668
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on sheep blood agar. A calibrated pipette was used 100 to transfer 0.01 ml of the inoculum. The minimum bactericidal concentration (MBC) was determined after incubation at 37 C for 18 h. The MBC was defined as the lowest concentration of drug which 80 yielded less than 50 colonies on subculture (less than 5 colonies per 0.001 ml of inoculum). Comparison studies were conducted simultaneously. The concentration of pirbenicillin in media was determined by an agar well method with Sarcina 0- 60t lutea ATCC9341 as the test organism. This organism was prepared so that a 1:10 dilution had an absorbIance of 0.6 at a wavelength of 580 nm, and 4 ml of 0 E 40 this preparation was added to 1 liter of antibiotic medium no. 1 (Difco). After mixing thoroughly, 14 Cml of broth was poured in plates (100 by 15 mm). Wells (0.75-mm diameter and 0.75-mm depth) were cut into the agar and filled with 0.05 ml of each specimen. The plates were incubated at 37 C for 18 h. Zones of inhibition were measured with a caliper, and the concentration was determined from a standard curve. A standard curve was prepared every time samples were assayed. All studies were per.025 10 39 1.56 6.25 25 100 400 formed in triplicate. Minimum Inhibitory Concentrotion The effect of pirbenicillin on the growth of 10 isolates of P. aeruginosa was determined in the folFIG. 1. In activity of pirbenicillin against lowing fashion. All 10 isolates were inoculated into gram-negativevitro bacilli and gram-positive cocci. NumMueller-Hinton broth and incubated at 37 C for 18 bers in parentheses indicate number ofisolates tested. h. A 0.1-ml aliquot of broth containing each Pseudomonas isolate was inoculated into each of five tubes containing 9.9 ml of unbuffered Mueller-Hin- bacter spp., 41% of Serratia spp., 58% of E. coli, ton broth. Two ofthe five tubes contained pirbenicil- 67% ofP. aeruginosa, and 93% of P. mirabilis. lin at a concentration equal to the MIC for that At 50 ,ug/ml, the antibiotic inhibited 34% of Pseudomonas isolate. Two other tubes contained isolates of Klebsiella spp. and 28% of indolepirbenicillin at a concentration equal to four times positive Proteus spp. Generally, using the the MIC for that Pseudomonas isolate. The fifth buffered Mueller-Hinton broth, the MIC was tube served as a control. All of the tubes were incu- also the MBC. However, the MBC for isolates of bated at 37 C for 24 h. After vigorous agitation, a P. aeruginosa was usually two to four times 0.1-ml aliquot was removed from each tube at 0, 2, 4, 6, 8, 10, 12, 15, 18, 21, and 24 h and then subcultured higher than MIC. For example, 6.25 ,ug/ml was on Mueller-Hinton agar. The number of viable orga- the MIC and MBC for 58% of isolates of E. coli, nisms present was determined from these subcul- the MIC for 41%, and MBC for 34% of isolates of tures. At 8 h, additional pirbenicillin was added to Serratia spp., and the MIC for 67% of isolates of two tubes (one containing the MIC and one contain- P. aeruginosa, but the MBC for only 40% of ing four times the MIC) so that the concentration these latter isolates. was the same as at the onset of the experiment, The effect of inoculum size on the MIC and assuming that all of the original pirbenicillin had been destroyed. The concentration of pirbenicillin in MBC was determined for 10 isolates each ofE. each tube was assayed at 8 h and at 24 h. The MIC coli and P. aeruginosa (Fig. 2). For most isowas determined for all organisms still growing at 24 lates, at both inoculum sizes, the MIC and MBC were similar. However, the MIC and h. %
RESULTS The in vitro activity of pirbenicillin against gram-positive cocci and gram-negative bacilli is shown in Fig. 1. Nearly all isolates of S. pyogenes and S. pneumoniae were inhibited by 0.10 ,ug/ml or less. All isolates of penicillin Gsensitive S. aureus were inhibited by 1.56 ,g/ ml, whereas only 40% of penicillin G-resistant S. aureus was inhibited by 3.12 ,ug of pirbenicillin per ml. At a concentration of 6.25 pg/ml, pirbenicillin inhibited 31% of isolates of Entero-
MBC for isolates of E. coli increased fourfold when the inoculum was increased from 105 CFU/ml to 107 CFU/ml. Inoculum size had a major effect on the inhibitory activity of pirbenicillin against isolates of P. aeruginosa. Only one isolate was inhibited by 400 ,ug of pirbenicillin per ml when an inoculum of 107 CFU/ml was used. The effect of pH on susceptibility of 28 isolates of P. aeruginosa to pirbenicillin, BLP1654, carbenicillin, and ticarcillin was investigated (Fig. 3). Phosphate buffer was added to Mueller-Hinton broth to a final pH of 6.4 and
670 BODEY, RODRIGUEZ, AND WEAVER 7.2. BL-P1654 was the most active antibiotic at both H+ concentrations and carbenicillin was the least active. Pirbenicillin, carbenicillin, and ticarcillin were more active at pH 7.2, whereas BL-P1654 was more active 'at pH 6.4. The difference between the MIC and MBC was similar at both H+ concentrations for carbenicillin, BL-P1654, and pirbenicillin. The difference between MIC and MBC for ticarcillin was greater at pH 6.4 than at pH 7.2.
ANTIMICROB. AGENTS CHZMOTHER.
The activity of the antibiotics in different media was studied, using these same isolates of P. aeruginosa (Fig. 4). BL-P1654 was the most active antibiotic in every medium and was most affected by the composition of the media. All of the antibiotics were most active in M9 broth which does not contain preformed amino acids. They were least active in Mueller-Hinton broth buffered at pH 7.2. Except for BL-P1654, the greatest differences of MIC and MBC were observed with M9 broth. Interestingly, the MICs for carbenicillin and ticarcillin were the lowest with M9 broth, but the MBCs were the highest in these medium. The activity of pirbenicillin against 25 isolates each of various gram-negative bacilli was compared with ticarcillin and carbenicillin, using Mueller-Hinton broth buffered at pH 7.2 (Fig. 5-8). Carbenicillin was least active Z E against E. coli, whereas ticarcillin and pirbenicillin had similar activity. Pirbenicillin was substantially less active than ticarcillin and carbenicillin against indole-positive and -negative Proteus spp. The activity of all three antibiotics was similar against isolates of Enterobacter spp. and Serratia spp. Pirbenicillin was Minimum Inhibitory Concentration (yig/mi) substantially more active than carbenicillin FIG. 2. Effect of inoculum size on MIC and MBC against isolates of P. aeruginosa. Pirbenicillin of pirbenicillin. Ten isolates each of P. aeruginosa was the only antibiotic with activity against isolates of Klebsiella spp. and E. coli were tested. z
I.4
100 80 60 (1)
a) 0 0 4-
(,)
0
0 0 0. 0
0L
40 20 0 100 80 60 40 20
o pH 6.4 - MIC * pH 6.4-MBC a pH 7. 2-MIC -
O '-k
0.39
1.56
pH72-MBC
6.25 25 100 400 6.25 25 Minimum Inhibitory Concentration (pLg/ml)
100
400
FIG. 3. Effect ofpH on activity of penicillins against P. aeruginosa. Twenty-eight isolates were tested.
VOL. 9, 1976
PIRBENICILLIN
671
100 80
60 O 40 .0
20 %+.cn
o
0
0
40 / 20 ,,' 0 0.39 1.56
Broth MIC IM-9
. MBC
.
*
MBC Broin Heort Infusion
o MBC
Broth
MBC Imueller Hinton Brothi 6.25 25 100 400 6.25 25 Minimum Inhibitory Concentration (jig/ml) 13
100
400
FIG. 4. Effect of media on activity of antipseudomonal penicillins against 28 isolates of P. aeruginosa.
The effect of pirbenicillin on the proliferation of 10 isolates of P. aeruginosa was studied. Figure 9 shows the median results with six isolates which were initially susceptible to 12.5 ,ug of pirbenicillin per ml. At 8 h, the concentration had decreased from 106/ml to 103/ml when exposed to 12.5 ,ug/ml (MIC) or 50 ,ug/ml (four times MIC) of pirbenicillin. About 50% of the antibiotic had been inactivated at this time. Hence, during a portion of this 8-h period, organisms incubated in media containing the MIC of pirbenicillin were exposed to a subinhibitory concentration. If no additional antibiotic was added at this time, the organisms proliferated so that the concentration was the same as the controls at 24 h. The MIC for these persistent organisms had increased from 12.5 ,ug/ml to 100 ,ug/ml. Even if additional drug was added at 8 h, the organisms were able to proliferate subsequently, but only after a 4-h delay. These persistent organisms also had developed increased resistance. Organisms exposed to 50 ,ug of pirbenicillin per ml were all killed eventually because the concentration of pirbenicillin remained above the MIC. Complete killing occurred sooner if additional drug was added at 8 h. Similar results were observed with four isolates of P. aeruginosa which had MICs of 6.25,
6.25, 25, and 50 ,ug/ml. When pirbenicillin was incubated alone, there was no appreciable destruction of drug at 8 h. However, at 24 h, 7 to 22% was inactivated. DISCUSSION Pirbenicillin is a broad-spectrum penicillin with activity against both gram-positive cocci and gram-negative bacilli. It is more active than carbenicillin or ticarcillin against P. aeruginosa, but less active than BL-P1654. Clinical studies of the latter antibiotic have been abandoned because of its potential nephrotoxicity. Pirbenicillin is less active than either carbenicillin or ticarcillin against Proteus spp. It is as active as carbenicillin against Serratia spp. and Enterobacter spp. These results are in agreement with those of Retsema et al. who also demonstrated that pirbenicillin was more active than carbenicillin against experimental Pseudomonas infections in mice (J. A. Retsema, A. R. English, and J. E. Lynch, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., Abstr. 252, 1975; Retsema and English, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., Abstr. 253, 1975).
ANnimacRoB. AGZWM CHEMOTHZR.
BODEY, RODRIGUEZ, AND WEAVER
672
100
* Pirbenicillin A Ticorcillin o Corbenicllin
80
U,
-
0
-
60
0
0
0c 0-
40
a*
.' 40 a
t E - , # ii
E
O
I |
"
20
|
lil
I
.39 1.56 6.25 25 1 00 400 Minimum Inhibitory Concentration (jig/ml) FIG. 6. Comparative activity ofsemisynthetic penicillins against P. mirabilis. .10
I
0
o~~~~~
-
o L-,
* Pirbenicillin Ticarcillin *~~~~ Carbenicillin
l
|
t
.20 .78 3.12 12.5 50 200 Minimum Inhibitory Concentration (ag/ml) FIG. 5. Comparative activity of semisynthetic penicillins against E. coli. Twenty-five isolates were tested.
2
Ir * Pirbenicillin
A Ticarcillin There has been some controversy over the o Carbenicillin bactericidal activity of pirbenicillin. Lopez et 80[ t al. found considerable differences between the MIC and MBC against isolates ofP. aeruginosa 0 (C. Lopez, H. Standiford, B. Tatum, F. Calia, S. Shimpif, M. Synder, and R. Hornick, Prog. 10va Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., Abstr. 0 60 F 254, 1975). These differences appear to be due to the chemical instability of pirbenicillin in alkaline media (Retsema et al., Prog. Intersci. Conf. Antimicrob. Agents Chemother., Abstr. a40 [ 252, 1975). The pH of Mueller-Hinton broth increases to over 8 during growth of P. aerugi- 21 nosa causing degradation of pirbenicillin. This C-) problem can be circumvented by buffering the media. Consequently, we used only Mueller20 . Hinton broth buffered to pH 7.2 for our studies, whereas Lopez et al. used unbuffered media. I The difference between MIC and MBC tended ,-A / I0I1I to be minimal in buffered media. The efficacy of pirbenicillin was profoundly influenced by inoculum size. This was espe.10 .39 1.56 6.25 25 100 400 cially true for isolates of P. aeruginosa which Minimum Inhibitory Concentration (,.g/ml) became resistant to pirbenicillin when the inoculum was increased from 105 to 107 cells. The FIG. 7. Comparative activity of semisynthetic penactivity of other penicillins is also affected by icillins against indole-positive Proteus spp. -0
0-
4f.i
/1
PIRBENICILLIN
VOL. 9, 1976
673
the size of inoculum (5, 7). Pirbenicillin was most effective in a basic medium such as M9 which contains no preformed amino acids. The effect of media on the activity of BL-P1654, a related semisynthetic penicillin, has been described previously (2). 80 When isolates ofP. aeruginosa with an inoc(A ulum size of 106 organisms/ml are exposed to the MIC for pirbenicillin in unbuffered Muel0 ler-Hinton broth, killing occurs during the first 70 60 I I 8 h. However, sufficient drug is destroyed, so that the concentration falls below the MIC. At VI this suboptimal concentration, persisting orga0 0 nisms develop resistance and begin to proliferC._ ate. This can be avoided by exposing the orga0 nisms to a higher concentration initially. IncJ creasing the concentration to above the MIC at C-) I I 8 h has little effect, indicating that resistance probably has already developed. Unfortunately, we only repeated MIC determinations on organisms that survived at 24 h. We have * Pirbenicillin observed similar results with a few isolates of * ricorcillin E. coli, and Lopez et al. reported similar results O Carbenicillin with P. aeruginosa (Prog. Intersci. Conf. AntiI microb. Agents Chemother., Abstr. 254, 1975). These observations suggest that it may be im. A . OW portant to maintain serum concentrations con1.56 6.25 25 100 400 tinuously above the MIC of infecting organisms Minimum Inhibitory Concentration (pg/ml) FIG. 8. Comparative activity of semisynthetic pen- when treating patients with this drug. Pirbenicillin is an interesting new semisynicillins against P. aeruginosa. 0
-0
-
4-
~I
/
Drug Concentrotion (#g/ml) 6.1 6.1 16.9 16.9
7.1 19.9 22.7 58.3
,12.5,ag/ml
10 EU, a
a
zOOig/ml
I
8 12.5 MIC -0OOg/ml
0 %I-
0
6
/
a
0
0._ -4 O4 u
Control Tube A TubeB A Tube C O TubeD *
O
2
c71
0
1-.
1
4
8
'Iw
,
12 15 Hours
18
21
24
FIG. 9. Effect ofpirbenicillin on growth of six isolates ofP. aeruginosa, all of which were initially inhibited by 12.5 pg/ml. Tube A initially contained 12.5 pg ofpirbenicillin per ml and tube C initially contained 50 pg ofpirbenicillin per ml. No additional drug was added to these tubes at 8 h. Tube B initially contained 12.5 pgl ml and an additional 12.5 pg/ml was added at 8 h. Tube D initially contained 50 pgIml and an additional 50 pgIml was added at 8 h. For details of experiment, see text.
674 BODEY, RODRIGUEZ, AND WEAVER thetic penicillin with broad-spectrum activity. Although it is not as active against P. aeruginosa as BL-P1654 in vitro, it does not cause nephrotoxicity in animals. This drug deserves clinical investigation since it may be more effective than carbenicillin or ticarcillin. It may also prove efficacious against K. pneumoniae since it inhibits 70% of these isolates in vitro at 200 jug/ml. Although this represents only marginal activity, it may be possible to maintain adequate serum concentrations with high doses of drug. ACKNOWLEDGMENTS This investigation was supported by Public Health service grant CA 10042 from the National Cancer Institute and by a grant-in-aid from Pfizer Central Research, Groton, Conn. LITERATURE CITED 1. Acred, P., D. M. Brown, E. J. Knudsen, G. N. Robinson, and R. Sutherland. 1967. New semisynthetic
ANTIMaCROB. AGENTS CHEMOTHER. 2. 3.
4.
5.
6. 7.
penicillin active against Pseudomonas pyocyanea. Nature (London) 215:25-30. Bodey, G. P., and D. Stewart. In vitro studies on semisynthetic -(substituted-ureido) penicillins. Appl. Microbiol. 21:710-717. Bodey, G. P., J. P. Whitecar. Jr., E. Middleman, and V. Rodriguez. 1971. Carbenicillin therapy of Pseudomonas infections. J. Am. Med. Assoc. 218:62-66. Brown, C. H., III, E. A. Natelson, M. W. Bradhaw, T. W. Williams, Jr., and C. P. Alfrey, Jr. 1974. The hemostatic defect produced by carbenicillin. New Engl. J. Med. 219:265-270. Neu, H. C., and E. B. Winshell. 1871. In vitro studies of a semisynthetic penicillin, 6-[D(-)a-carboxy-3thienylacetamido] penicillanic acid (BRL 2288) active against Pseudomonas, p. 385-389. Antimicrob. Agents Chemother. 1970. Rodriguez, V., G. P. Bodey, N. Horikoshi, J. Inagaki, and K. B. McCredie. 1973. Ticarcidin therapy of infections. Antimicrob. Agents Chemother. 4:427-431. StandUford, H. C., A. C. Kind, and W. M. M. Kirby. 1969. Laboratory and clinical studies of carbenicillin against gram-negative bacilli, p. 286-291. Antimicrob. Agents Chemother. 1968.
