R. N. JONES Clinical Microbiology Laboratory, Kaiser Foundation Hospital Laboratories Clackamas, Oregon 97015, U.S.A.

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1 VOL. XXX NO. THE JOURNAL OF ANTBOTCS 7 ACLLN (T-), A NEW SEMSYNTHETC PENCLLN: N VTRO ANTMCROBAL ACTVTY COMPARSON WTH ENCLLN, CLLN, CLLN, LOTHN, ANDOLE AND TN R. N. JONES Clinical Microbiology Laboratory, Kaiser Foundation Hospital Laboratories Clackamas, Oregon 97, U.S.A. C. THORNSBERRY Antimicrobic nvestigation Section, Center for Disease Control, Atlanta, Georgia, U.S.A. A. L. BARRY Clinical Microbiology Laboratories, University of California (Davis) Sacramento Medical Center, Sacramento, California 7, U.S.A. P. C. FUCHS Department of Pathology, St. Vincent Hospital and Medical Center, Portland, Oregon 9, U.S.A. T. L. GAVAN Department of Microbiology, The Cleveland Clinic, Cleveland, Ohio 6, U.S.A. and E. H. GERLACH Microbiology Laboratory, St. Francis Hospital, Wichita, Kansas 6, U.S.A. (Received for Publication October, 977) (T-) is a new semisynthetic penicillin with an unusually broad spectrum of antimicrobial activity. n vitro comparisons of this drug with 6 other beta-lactam antimicrobics (ticarcillin, carbenicillin, ampicillin, cephalothin, cefamandole and cefoxitin) were conducted. These included minimal inhibitory concentrations (MC) against 9 bacterial isolates, the minimal lethal concentrations (MLC) against of those, as well as the effect of inoculum size on the MC and MLC of the drugs. had significantly greater activity than did the other penicillins against Pseudomonas species and Klebsiella pneumoniae. Against P. aeruginosa piperacillin was - and 6-fold more active than ticarcillin and carbenicillin, respectively. The MLC of piperacillin rarely differed from the MC by more than one loge dilutions except against P. aeruginosa in which the MLC was -fold greater or more than the MC of % of isolates tested., carbenicillin and cefoxitin showed minimal inoculum size effects. Cefamandole results showed the greatest inoculum size variation with % and 7 % of isolates showing an -fold increase in MC and MLC respectively by increasing inoculum from to 7 CFU/ml. was intermediately effected having % of strains > -fold increase in MC., sodium 6-[D(-)-a(-ethyl-,-dioxo-l-piperazinylcarbonylamino)-a- phenylacetamido] penicillinate, is a new semisynthetic penicillin derived from aminobenzyl-penicillin (Fig. ). This compound formerly known as T-, has potent antibacterial activity against bacterial species previously refractory to such related compounds as carbenicillin and ticarcillin. n addition, favorable pharmacokinetics and infection studies have been reported in humans and in experimental animals (abstracts 9 and, Sixteenth nterscience

2 THE JOURNAL OF ANTBOTCS DEC. 977 Conference on Antimicrobial Agents and Chemotherapy, Chicago, 976). This in vitro investigation directly compares the antimicrobial activity of piperacillin with that of related semisynthetic penicillins carbenicillin, ticarcillin, and ampicillin, plus three reference cephalosporins-cephamycins, cephalothin, cefamandole and cefoxitin. Materials and Methods Antibiotics: The beta-lactam antimicrobic laboratory standard powders were supplied by the following pharmaceutical companies: from Lederle Laboratories, Pearl River, New York; cefamandole and cephalothin from Eli Lilly & Company, ndianapolis, ndiana; cefoxitin from Merck Sharp & Dohme, Rahway, New Jersey; carbenicillin and ticarcillin from Beecham Laboratories, Bristol, Tennessee; and ampicillin from Bristol Laboratories, Syracuse, New York. Organisms: A total of 9 bacterial isolates were contributed by the collaborating laboratories for this study. These include 9 strains of the Enterobacteriaceae, 6 strains of non-enterobacteriaceae grain-negative bacilli, and strains of gram-positive cocci. They were further subdivided into the following genus and species categories: 9 Escherichia coli, Klebsiella pueumoniae, Enterobacter species, 7 indolepositive Proteus species, Proteus mirabilis, Serratia species, Pseudomonas aeruginosa, Pseudomonas cepacia, 6 Pseudomonas fluorescens-putida, Pseudomonas maltop/tilia, Pseudomonas stut_eri, Acinetobacter anitratus, Aeromonas hydrophila, Staphylococcus aureus ( methicillinresistant), Streptococcus faecalis, Streptococcus pueumoniae and Streptococcus pyogenes. Most of the isolates were tested in duplicate by two of the collaborating laboratories (Center for Disease Control and the Sacramento Medical Center) in a manner previously reported., A third laboratory, Kaiser Foundation, also tested a more limited number (MLCs and inoculum size studies). No significant variation in MC results were encountered between these participating laboratories. Antimicrobic Susceptibility Testing: Minimum inhibitory concentrations (MCs) were determined by the broth microdilution method. MUELLER-HNTON broth was commercially dispensed in a single lot of plastic trays (Micro Media Systems, San Jose, California) and distributed to the testing laboratories. The trays were stored at -6 C until inoculated. Prior to use the trays were thawed at room temperature (approximately - minutes) and inoculated with disposable inoculators delivering ul to each well. At all three laboratories, a logarithmic phase broth culture was diluted to match the turbidity of a. MAcFARLAND standard. The suspension was then diluted : in sterile water containing. Tween and dispensed as described earlier. Final inoculum achieved was x colony forming units (CFU) per ml. For the testing of the fastidious streptococci including S. pyogeues and S. pneumoniae, the inoculum was standardized in MUELLER-HNTON broth containing % lysed rabbit blood and. ml of this adjusted cell suspension was added to each microdilution well, giving a final concentration of x CFU/ml. The MC was recorded as the lowest concentration totally inhibiting bacterial growth (clear well), after approximately hours of incubation at C in a forced air incubator. Occasionally, visible growth occurred in concentrations - wells above the MC (the skipped-tube phenomenon). Minimum lethal concentrations were determined for organisms from seven genera by subculturing /rl from each microdilution well. The /tl subcultures were transferred to trypticase soy agar with %) sheep blood. The subcultures were made with multiple inocul un replicator onto a x nom plate. After hours of incubation, the endpoints were read as the lowest concentration yielding no more than. % survivors (99.9 % kill). The effect of varying the inoculum concentrations on MC-MLC endpoints was studied on rapid growing facultative anaerobes. Trays were inoculated to achieve final concentrations of,, and 7CFU/rnl. MCs and MLCs were interpreted as described above.

3 VOL. XXX NO. THE JOURNAL OF ANTBOTCS 9 Results MC Comparisons Table summarizes the cumulative percentage susceptibility results for five bacterial species to increasing concentrations of piperacillin and six other beta-lactam antibiotics. These species were grouped for tabulation because of the bimodal distribution of susceptible bacterial populations. is the most active semisynthetic penicillin against the four Enterobacteriaceae species tested. At clinically achievable serum levels piperacillin would inhibit 7% of the species tested compared to 6-% for carbenicillin or ticarcillin and -% for ampicillin. Most noteworthy is the piperacillin activity versus Klebsiella pneumoniae where piperacillin inhibits % at 6 /,cg/ml in contrast to 6% at,ug/ml for carbenicillin/ticarcillin and % at,ug/ml of ampicillin. This activity is comparable to that of reference cephalosporin cephalothin (% at,ug/ml) and second generation cephalosporins, cefamandole ( % at,ug/ml) and cefoxitin ( % at /cg/ml). The latter two compounds possess favorable in vitro characteristics as compared to currently available cephalosporin C derivatives,,). However, piperacillin also incorporates these features with broader coverage within both Enterobacter species and indole-positive Proteus species. Species variation in susceptibility was encountered among the indole-positive Proteus species., carbenicillin and ticarcillin had comparable inhibitory effects against this genus subgroup. Ampicillin and cephalothin are ineffective and cefoxitin is the most active against Pr. rettgeri and Pr. vulgaris. Cefamandole activity would rank Pr. rettgeri > Pr. morganii > Pr. vulgaris as previously reported.,,) Like the second generation cephalosporins, piperacillin had decreased activity against staphylococcal species `). All methicillin-sensitive S. aureus isolates were inhibited by leg/ml of carbenicillin and ticarcillin compared to % at 6,ug/ml of piperacillin. Of note is the overlap of MCs of piperacillin and ampicillin against penicillinase-producing and deficient strains in contrast to carbenicillin and ticarcillin which show a clear bimodal distribution of the two groups. Cephalothin was most active among the cephalosporins confirming prior reports,). The median MC values for piperacillin and six other beta-lactams against five other isolates are presented in Table. These five bacterial species have unimodal narrow susceptibility ranges to beta-lactam antibiotics. All seven antibiotics were effective against Proteus mirabilis, Streptococcus pneumoniae and Streptococcus pyogenes. was the most active against Serratia species with a median MC of leg/ml. Ampicillin and the three cephalosporins had median MCs _ 6 leg/ml. was -fold more active than carbenicillin and ticarcillin against S. faecalis. Only ampicillin was more active (median MC= ltg/ml) with the cephalosporins medians well above clinically achievable concentrations. The susceptibility expressed as median MCs of non-enterobacteriaceae gram-negative bacilli are shown in Table. Only piperacillin was active against all Pseudomonas species tested. was - 6 times more active than ticarcillin and carbenicillin against Ps. aeruginosa, - times against Ps. cepacia, times against Ps. fluorescence putida, - times against Ps. stutzeri and equally inhibitory versus Ps. maltophilia. was one log dilution more active than carbenicillin against Ps. aeruginosa confirming prior reports6,7). Acinetobacter anitratus isolates were only inhibited by clinically achievable concentrations of piperacillin, carbenicillin and ticarcillin. There was a marked susceptibility variation for Aeromonas

4 THE JOURNAL OF ANTBOTCS DEC. 977 Table. Cumulative percentage susceptibility of five bacterial species ( organisms) to increasing concentrations of piperacillin and six other beta-lactam antibiotics. Organism (#) Antibiotic Cumulative % MC (,ug/ml) of 6 6 E. coli (9) Enterobacter speciesa () Klebsiella pneunroniae () CEFA M Proteus species (7) ndole-positive Staphylococcus aureus () Methicillin-sensitive Staphylococcus aureus () methicillin-resistant i 7 a. ncludes Enterobacter cloacae (), Enterobacter aerogenes () and Enterobacter a,;glomerans (6). b. ncludes Proteus morganii (), Proteus retteeri (9), Proteus vuharis (6). rvdroplrila to the seven antimicrobics with cefamandole and piperacillin being most active. Among the Ps. aeruginosa isolates tested, several strains were resistant (>ltg!ml) to gentamicin and/or tobramycin. These isolates and the sensitive strains of Ps. aeruginosa are shown in Table. ncreased resistance to the aminoglycosides was accompanied by increased resistance to the semisynthetic penicillins e.g. piperacillin, carbenicillin and ticarcillin. Only the piperacillin MCs remained at or below clinically achievable concentrations for these gentamicin resistant and gentamicintobramycin resistant strains. MC-MLC Comparisons The MLC/MC ratios for piperacillin, ticarcillin and carbenicillin are shown in Table. Only

5 VOL. XXX NO. THE JOURNAL OF ANTBOTCS Table. Median MC concentrations (,ug/ml) of selected bacterial species having unimodal population distributions to piperacillin and six other beta-lactam antibiotics. Antibiotic Ampicillin Cephalothin Cefamandole Cefoxitin Proteus mirabilis ()-. Serratia species ()... > 6 faecalis ().. > Streptococcus pnecnnontae () <. <. <. <. <. <.. pyogenes () <. <_. <. <. <. <.. a. Number of isolates tested. Table. Median MC concentrations (µg/ml) for non-enterobacteriaceae gram-negative bacilli including isolates of Pseudonionas species to piperacillin and six other beta-lactam antibiotics. Antibiotic aerugmosa (), cepacta () Pseudomonas fluorescensputida (6) inaltophilia () stutzeri () Acinetobacter anitratus (L) Aeromonas hydrophila (L) Ampicillin Cephalothin Cefamandole Cefoxitin 6 > > > >. > > > 6. > > > >... > > >.. a. Number of isolates tested Table. Relationship of piperacillin, carbenicillin and ticarcillin MC values in Pseudomonas aerugiuosa isolates having aminoglycoside resistance. Aminoglycoside susceptibility Gentamicin / Tobramycin Sensitive / Sensitive Resistant / Sensitive Resistant / Resistant No. Tested 9 6 (- ) (-6) 6(6-.) Median(range) MC in jig/ml; 6( - > ) (6- >) >(- >) (<_.- >) (- > ) >(- >) a. Sensitive is = or less than pg/ml to both aminoglycosides tested in cation supplement MUELLER- HXTON broth. of organisms (%) had MLC values more than -fold higher than the piperacillin MC. These included single strains of Proteus morganii and Pseudoinonas aeruginosa. had the most with / and carbenicillin the least with /. MLC;MC ratios were also determined for ampicillin, cephalothin, cefamandole and cefoxitin. Cefamandole demonstrated the widest variation between inhibitory and cidal endpoints confirming previous reports', ). Seventeen percent of MLCs were >- fold greater than cefamandole MCs. Nearly all of these were Enterobacter species, indole-positive Proteus species and Serratia. Cefoxitin had ratios similar to piperacillin and superior to other cephalosporins,). noculum Size MC-MLC Comparisons The effects of inoculum size on MC and MLC results were studied on organisms at,,

6 THE JOURNAL OF ANTBOTCS DEC. 977 Table. MC-MLC comparison of piperacillin, carbenicillin and ticarcillin for CFU/ml inoculum size. E. coli () Organism (#) Klebsiella pneumoniae () Enterobacter speciesa () Proteus mirabilis () lndole-positive Protein speciesb () Serratia marcescens () Pseudomonas aeruginosa (9) Staphylococcus aureus () Antibiotic MLC/MC a. ncludes five strains each of Ent. aerogenes and Ent. cloacae. b. ncludes Proteus morganii (), Proteus rettgeri () and Proteus vulgaris () l ratios or more l and 7 colony forming units/ml (Table 6). Only those organisms (listed in Table ) not having off-scale MC values were tabulated. Lower numbers of cefamandole MC and MLC were noted due to a large incidence of tg/ml MC-MLC values among the Enterobacteriaceae in addition to >,ug/ml MC-MLC against Ps. aeruginosa, Serratia and some Proteus species., carbenicillin and cefoxitin showed minimal variation (increase) in MC or MLC when the inoculum was raised from to CFU/ml and to 7 CFU/ml. Ninety-two to % of the endpoints were < log dilutions. was similar to the above agents at - CFU/ml, but had - % of MC- MLC results markedly altered (> -fold increase) by inoculum increases to 7 CFU/ml. Nearly Table 6. Percent of isolates having MC or MLC results within loge dilutions (<_ fold increase) after increasing the inoculum concentration CFU/ml for piperacillin, ticarcillin, carbenicillin, cefamandole and cefoxitin. Antibiotic (#) () () () Cefamandole () Cefoxitin (6) CFU/mla all isolates having piperacillin susceptibility inoculum effect were Klebsiella pneurnoniae (mean MC- MLC increase= 6-fold). Cefamandole MCs and MLCs were markedly increased by raising inoculum MC MLC ' 7 CFU/mPb MC 76 MLC a. Comparison of MC and MLC results at CFU/ml inoculum concentration compared to those at CFU/ml. b. Comparison of MC and MLC results at ' CFU/ml inoculum concentration compared to those at CFU/ml.

7 VOL. XXX NO. THE JOURNAL OF ANTBOTCS concentrations. From to % of the strains tested had cefamandole MC-MLCs showing increases of > -fold with several isolates MLC - to -fold higher at 7 CFU/ml. Discussion (T-) belongs to the semisynthetic group of penicillins and is structurally similar to ampicillin. More extensive a-substitutions have provided this compound with a broad antimicrobial spectrum that includes gram-positive and gram-negative bacteria, especially Pseudomonas, Klebsiella, Serratia, Proteus and Enterococci. The compound is poorly absorbed orally, but high serum concentrations are achieved by parenteral routes. Other new semisynthetic penicillins that most resemble piperacillin are BL-P6 (Bristol), PC- (Sumitomo) and pirbenicillin (CP-,99-, Pfizer). All of these antimicrobics have antipseudomonas activity - to 6-fold that of carbenicillin-). However, each has one or more disadvantages when compared to piperacillin. BL-P6 is adversely affected by both serum and increased inoculum concentrations, has a limited spectrum versus all Proteus species and lastly is toxic to some animal species'). PC- also is less active than piperacillin against Proteus mirabilis, the indole-positive Proteus species and Serratia species9). Like PC-, pirbenicillin has little activity on Proteus species and is significantly more protein bound than piperacillin). compared to carbenicillin and ticarcillin had a wider antimicrobial spectrum and increased activity, particularly against Pseudomonas species, Serratia and Klebsiella pneumoniae. Like the new cephalosporins, piperacillin showed excellent inhibitory activity against Enterobacter species and indole-positive Proteus species, while adding the Enterococci, Serratia species, and K. pneumoniae to its spectrum. Only ampicillin was superior to piperacillin against S. faecalis. The only apparent drawbacks to piperacillin were the slight inoculum size effects on MC and MLCs at ' CFU/ml levels, particularly among K. pneumoniae isolates. Also piperacillin is susceptible to beta-lactamase activity similar to carbenicillin, ticarcillin and ampicillin. These features may limit its efficacy against beta-] actamase-producing S. aureus, H. influenzae, N. gonorrhoeae and some populations of Enterobacteriaceae including E. coli. The combination of excellent in vitro inhibiting effects on a wide spectrum of bacteria and in vivo pharmacology makes piperacillin (T-) a very promising parenteral antimicrobic. f dosages were used permitting piperacillin serum levels of 6 /cg/ml, this would inhibit % of the gram-positive and negative organisms in a clinical isolate study of, strains). Ninety-one percent would be covered by concentrations of 6 pg/ml. These in vitro data coupled with a serum protein binding, active urinary excretion (66%), high biliary concentrations, favorable human pharmacokinetics, and effective treatment of experimental animal infection favors further human in vivo investigations. Additional in vitro studies on anaerobic bacteria are also needed. Acknowledgements We gratefully acknowledge the expert technical assistance of CAROLYN BAKER, ROLLE PACKER and ROBERT BADAL. Also we thank BARBARA BEARDSLEY for her help in the preparation of this manuscript. References ) BARRY, A. L.; C. THORNSBERRY, R. N. JONES, P. C. FUCHS, T. L. GAVAN & E. H. GERLACH: Cefuroxime, in vitro comparison to six other parenteral cephalosporins. (in press), 977 ) JONES, R. N.; C. THORNSBERRY, A. L. BARRY, P. C. FUCHS, T. L. GAVAN & E. H. GERLACH: BL-S76, a new parenteral cephalosporin.. n vitro antimicrobial activity comparison with six related cephalosporins. J. Antibiotics : -, 977 ) JONES, R. N. & P. C. FUCHS: Comparison of in vitro antimicrobial activity of cefamandole and cefazolin with cephalothin against over, clinical bacterial isolates. Antimicr. Agents & Chemoth. 9: 66-69, 976 ) JONES, R. N.; P. C. FUCHS, T. L. GAVAN, E. H. GERLACH, A. L. BARRY & C. THORNSBERRY: BL-S76,

8 THE JOURNAL OF ANTBOTCS DEC. 977 a new parenteral cephalosporin.. A collaborative in vitro susceptibility comparison to cephalothin against,76 clinical bacterial isolates. J. Antibiotics : 76, 977 ) JONES, R. N.; P. C. FUCHS, T. L. GAVAN, E. H. GERLACH, A. L. BARRY & C. THORNSBERRY: Cefuroxime, a new parenteral cephalosporin: Collaborative in vitro susceptibility comparison with cephalothin against,7 clinical bacterial isolates. Antimicr. Agents & Chemoth. : 7, 977 6) FUCHS, P. C.; T. L. GAVAN, E. H. GERLACH, R. N. JONES, A. L. BARRY & C. THORNSBERRY: : a collaborative in vitro comparison with carbenicillin against over 9, clinical bacterial isolates. Am. J. Med. Sci. (in press) ) FUCHS, P. C; C. THORNSBERRY, A. L. BARRY, T. L. GAVAN, E. H. GERLACH & R. N. JONES:, carbenicillin and BL-P: n vitro comparison of three antipseudomonal semisynthetic penicillins. J. Antibiotics : 6-, 977 ) PRCE, K. E.; F. LETNER, M. MSEK, D. R. CHSHOLM &T. A. PURSANO: BL-P6, anew broad spectrum penicillin. Antimicr. Agents & Chemoth. -97: 7-9, 97 9) NOGUCH, H.; Y. EDA, H. ToBK, T. NAKAGOME & T. KOMATSU: PC-, a novel broad-spectrum semisynthetic penicillin with marked antipseudomonal activity: Microbiological evaluation. Antimicr. Agents & Chemoth. 9 : , 976 ) WSE, R.; J.M.ANDREWS & K.A.BEDORD: Pirbenicillin, a semisynthetic penicillin with antipseudomonal activity. J. Antimicr. Chemoth. : 7 -, 977 ) SCOY, R. E. V.; E. WARREN & J. A. WASHNGTON: in vitro antimicrobial activity of a new semisynthetic penicillin BL-P6. Antimicr. Agents & Chemoth. -97: -6,97 ) GERLACH, E. H.; P. C. FUCHS, R. N. JONES, T. L. GAVAN, A. L. BARRY & C. THORNSBERRY:, a new parenteral broad spectrum penicillin: Collaborative in vitro susceptibility comparison with carbenicillin against, clinical bacterial isolates. Antimicr. Agents & Chemoth. (in press), 97