Vol. 17, No.6ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUne 1980, p. 993-10000066-4804/80/00-0993/08$02.00/0

MK0787 (N-Formiimidoyl Thienamycin): Evaluation of InVitro and In Vivo Activities

HELMUT KROPP,* JON G. SUNDELOF, JEAN S. KAHAN, FREDERICK M. KAHAN,AND JEROME BIRNBAUM

Merck Institute for Therapeutic Research, Rahway, New Jersey 07065

The practical application of thienamycin, a novel ,8-lactam antibiotic with abroad activity spectrum, was compromised by problems of instability. MK0787,N-formimidoyl thienamycin, does not have this liability. As reported, bacterialspecies resistant to most ,8-lactam antibiotics, such as Pseudomonas aeurginosa,Serratia, Enterobacter, Enterococcus, and Bacteroides spp., are uniformly sus-ceptible to MK0787, usually at one-half the inhibitory level of thienamycin.Bactericidal ativityusuallv occurs at the minimal inhibitory concentrationendpornt. Activity was reduced ony at the highest mocuum densities tested andby a lessor factor than was observed with reference 8l-lactam antibiotics activeagainst P. aeruginosa and ,f-lactamase-bearing strains. MK0787 exhibits a broadspectrum of in vivo actvity when evaluated parenterally for efficacy againstsystemic infections in mice. The order of potency in vivo, 0.03 to 0.06 mg/kg forgram-positive species and 0.65 to 3.8 mg/kg for gram-negative infections includingPseudomonas, exceeded that of thienamycin and was at least 10-fold superior toreference ,B-lactam antibiotics including two recently developed agents withantipseudomonal activity, cefotaxime and LY127935.

Thienamycin, a novel ,B-lactam antibiotic (1)(Fig. 1A) discovered in fermentation broths ofthe soil actinomycete Streptomyces cattleya (5)has an unusually broad spectrum of antibacterialactivity, with a potency comparable to or greaterthan the penicillins against gram-positive speciesand to the aminocyclitols against gram-negativespecies including Pseudomonas aeruginosa.tFurthermore, tiuenamycin isence of B-lactamase. and thereby exhibits vir-tually no cross-resistance with penicillins orcephalosporins (H. Kropp, J. S. Kahan, F. M.Kahan, J. Sundelof, G. Darland, and J. Birn-baum, Program Abstr. Intersci. Conf. Antimi-crob. Agents Chemother. 16th, Chicago, Ill.,abstr. no. 228, 1976).

However, the practical utility of thienamycinwas hindered by its unacceptable degree of in-stability both in concentrated solutions (5) andas an amorphous solid. These problems havebeen overcome by the synthesis of a series ofamidine derivatives (K. J. Wildonger, W. J.Leanza, T. W. Miller, and B. G. ChristensenProgram Abstr. Intersci. Conf. Antimicrob.Agents Chemother. 19th, Boston, Mass., abstr.no. 232, 1979) from among which the lowesthomolog, N-formimidoyl thienamycin (Fig. 1B),was isolated as crystalline monohydrate(MK0787) and selected for further development.The following report will show that MK0787retains and indeed significantly improves upon

the excellent in vitro and in vivo antibacterialactivity of the parent antibiotic. It also showsthe generally superior activity of the thienamy-cin antibiotics in comparison both with clinicallyestablished antibiotics and with recently devel-oped cephalosporin and cephamycin antibioticswith expanded gram-negative antibacterial spec-trum.

(This report was presented at the 19th Inter-science Conference on Antimicrobial Agents andChemotherapy, Boston, Mass., 1-5 October,1979.)

MATERIALS AND METHODSAntibiotics. Thienamycin used in these studies

was of 85% purity. The concentration of this antibioticin prepared solutions was measured by the differentialspectrophotometric assay procedure described by Ka-han et al. (5). Crystalline monohydrate preparationsof MK0787 were found to have an E%!W = 305.Solutions of thienamycin or MK0787 were prepared ineither 10 mM MOPS [3-(N-morpholino)propanesul-fonic acid, Calbiochem] (pH 7.1) or 10 mM sodiumphosphate (pH 7.1). To avoid the instability of thien-amycin in concentrated solutions, concentrations werelimited to 2.0 mg/ml. Reference antibiotics were ce-foxitin sodium (Mefoxin, Merck & Co., Inc.); cephalo-thin, cephaloridine, and cefazolin sodium (Keflin, Lor-idine, and Kefzol, respectively; Eli Lilly & Co.); com-pound LY127935 also known as Shionogi No. 6059-S(P. Demarco, Lilly Research Labs); piperacillin (A. C.Dornbush, Lederle Labs); HR-756 (cefotaxime,

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ANTIMICROB. AGENTS CHEMOTHER.

Thienomycin

MK 0787N-Formimidoyl Thienomycin

FIG. 1. Structural configuration of thienamycin(A) and N-formimidoyl thienamycin (MK0787) (B).

Hoechst); carbenicillin disodium (Pyopen, Beecham-Massengill); gentamicin sulfate (Garamycin, Scher-ing); amikacin (Dr. K. Price, Bristol Labs); clindamy-cin (Cleocin, UpJohn); and ampicillin sodium (Poly-cillin-N, Bristol). All test solutions were freshly pre-pared immediately before use according to productinformation inserts or special recommendations sup-plied.

Bacterial strains. The organisms used in all invivo studies and in a portion of the in vitro evaluationswere drawn from a panel of 40 laboratory strainsrepresenting 12 species. For each species, an isolate oftypical antibiotic susceptibility is represented. Theremaining isolates were selected for their resistance to,8-lactam antibiotics currently in use. Included in thisselection was Enterobacter cloacae 2647 (GoldnerHSC 18410/M62), a spontaneous fi-lactamase-nega-tive mutant of the parent strain 2646 (Goldner HSC18410/64) obtained from M. Goldner (4), ResearchInstitute Hospital for Sick Children, Toronto, Canada,and plasmid-mediated gentamicin- and carbenicillin-resistant P. aeruginosa 3350 (RPL11), a clinical isolatereceived from J. Loper at the University of Cincinnati,Ohio. Susceptibility tests were conducted on 119 clin-ical isolates of hospital origin. Ninety were isolates ofProteus, Pseudomonas, Serratia, and Enterococci re-sistant to one or more antibiotics, and 29 were clinicalisolates of Bacteroides fragilis. Taxonomic analysis of

the anaerobe collection by G. Darland of these labo-ratories showed the B. fragilis isolates to include 11strains of B. fragilis subsp. fragilis, 3 of B. fragilissubsp. distasonis, 3 of B. fragilis subsp. ovatus, and 1of B. fragilis subsp. vulgatus.

Susceptibility studies. Media used for aerobicspecies were Muller-Hint roth and agar. For Bac-teroides isolates, heart infsion agar was supple-mented after autoclaving with 0.5% yeast extract, 10jig of hemin per ml, and 0.5 ug of menadione per ml.Inocula were prepared from overnight stationary cul-tures grown in their respective media. Inoculum den-sity was verified in all studies by count of colony-forming units (CFU).

For agar dilution titrations, a Steers-Foltz replicator(10) was employed delivering 3.0 pd per application,corresponding to approximatelv 103 1 14 14cui±u~os and 107 CFU from the undiluted sus-pensions. The minimal inhibitory concentration (MIC)was defined as the lowest antibiotic concentration ina twofold serially diluted sequence of which growth ofno more than a single colony was found after 24 hof incubation at 370C, (anaerobically in the case ofBacteroides spp.) For the determination of MICs andminimal bactericidal concentrations (MBCs), brothdilution studies were conducted by the microtiter tech-nique. Volumes of 1.5 pl of diluted or undiluted culturewere transferred by using a Dynatech MIC-2000 in-oculator to microtiter wells containing 0.1 ml of atwofold serial dilution of the antibiotics in Mueller-Hinton broth. The lowest concentration showing anabsence of visible turbidity or sediment after 20 h ofincubation at 370C was designated the MIC. In thecase of wells inoculated with 103 or 105 CFU, the MBCwas defined as the lowest concentration yielding nogrowth after subculture onto antibiotic-free Mueller-Hinton agar, after 20 h of exposure to antibiotic. Atthe highest inoculum level, 107 CFU per well, fiveisolated colonies on subculture were allowed.

Disk diffusion studies employed the Kirby-Bauermethod (2) modified only by the use of an agar thick-ness of 0.2 cm.

In vivo efficacy studies. Cultures were grown inshake-flasks containing brain heart infusion for 10 hat 370C, and were diluted either in broth or in a 5%gastric mucin vehicle to provide, as determined in eachstudy, from 3 to 100 50% lethal doses. The challengedose was administered in 0.5 ml by the intraperitonealroute to CD, Swiss albino female mice, whose averagebody weight was 20 g. Five mice were allocated toeach treatment level, each treatment level being sep-arated by a fourfold factor in dose rate. Antibioticswere administered in all cases by the subcutaneousroute. Treatment was initiated immediately after in-oculation. Where two treatments were given, the sec-ond dose was administered at 6 h. Where three treat-ments were employed, the second was at 2 h and thethird was at 4 h postinfection. Fifty percent effectivedose rates were calculated (6) after a 7-day observationperiod.

RESULTSThe antibacterial spectra of MK0787, thiena-

mycin, and several reference antibiotics are com-pared in Tables 1 and 2. The potencies of the

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IN VITRO AND IN VIVO ACTIVITY OF MK0787 995

Strain (106 CFU)

1. Staphylococcus aureus2. Staphylococcus aureus3. Staphylococcus aureus4. Staphylococcus aureus5. Staphylococcus aureus6. Staphylococcus aureus7. Enterococcus8. Enterococcus9. Enterococcus

10. Escherichia coli11. Escherichia coli12. Escherichia coli13. Escherichia coli14. Escherichia coli15. Shigella spp.16. Salmonella typhimurium17. Enterobacter cloacae18. Enterobacter cloacae19. Enterobacter cloacae20. Enterobacter spp.21. Enterobacterspp.22. Enterobacter aerogenes23. Klebsiella pneumoniae24. Klebsiella pneumoniae25. Klebsiella spp.26. Klebsiella app.27. Klebsiella spp.28. Serratia spp.29. Serratia spp.30. Proteus mirabilis31. Proteus mirabilis32. Proteus mirabilis33. Proteus morganii34. Proteus morganii35. Providencia spp.

TABLE 1. Agar dilution susceptibility testsMIC (pg/ml) of:

2985210

2874231428674428b286428622863248228842964289128952880826

2647c264628282903290229062921292228882890288928402855312528312830283328342851

MK0787a THM

0.01 0.020.02 0.040.02 0.040.04 0.040.02 0.04

20.0 40.00.63 1.31.3 2.5

40.0 40.00.32 0.630.08 0.160.32 0.320.16 0.160.32 0.630.16 0.320.32 0.320.16 0.320.63 0.631.3 2.50.63 1.31.3 2.52.5 5.00.63 0.630.63 0.630.63 1.31.3 1.32.5 5.00.63 2.50.63 2.55.0 10.02.5 5.05.0 10.05.0 5.02.5 10.01.3 2.5

CFX CEF CAB

3.2 0.2 0.633.2 0.4 1.33.2 0.2 1.36.3 0.8 20.06.3 0.8 20.0

>100 >100 80.0>100 25.0 40.0>100 25.0 40.0>100 >100 >806.3 12.5 5.06.3 6.3 5.06.3 100 >80

50.0 >100 40.025.0 >100 >803.2 6.3 5.03.2 6.3 20.0

12.5 12.5 20.0>100 >100 >80.0>100 >100 20.0>100 >100 5.0>100 >100 20.0>100 >100 5.06.3 12.5 >806.3 100 >80

>100 >100 10.0>100 >100 10.0>100 >100 10.025.0 >100 10.012.5 >100 >803.2 6.3 1.3

25.0 50.0 2.56.3 >100 >80

12.5 >100 >8012.5 >100 1.31.6 >100 1.3

a Antibiotic Abbreviations: THM, thienamycin; CFX, cefoxitin; CEF, cephalothin; CAB, carbenicillin; GEN,gentamicin.

b Methicillin resistant.c Beta-lactamase-negative mutant (HSC 18410/M62) derived from parent strain 2646 (HSC 18410/62).

TABLE 2. Agar dilution susceptibility ofpseudomonas isolatesP. aeruginosa MIC (pg/mi) of:strain no. (10'

CFU) MK0787a THM CAB GEN AMK 6059-S HR-756 PIP

40 1.6 3.1 50 50 6.3 12.5 12.5 3.12824 0.63 0.63 80 2.52835 2.5 5 80 5.03350b 10 20 >80 >203286 2.5 10 80 5.0 6.3 50 25 6.33287C 2.5 10 >80 5.03288d 20 40 80 5.04293 6.3 25 100 12.5 25 25 25 6.34294 12.5 50 100 25 50 25 25 6.3

a Antibiotic Abbreviations: THM, thienamycin; CAB, carbenicillin; GEN, gentamicin; AMK, amikacin; PIP,piperacillin.

b Strain 3350 bears plasmid-mediated carbenicillin and gentamicin resistance.c Laboratory isolate from strain 3286, showing spontaneous resistance to carbenicillin.d Laboratory isolate from strain 3286, showing spontaneous resistance to thienamycin.

GEN

0.320.320.160.630.635.0

10.0>2020.00.321.3

>202.51.31.32.51.31.30.631.31.31.31.3

>201.31.31.32.51.35.0

10.02.52.50.320.63

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thienamycin antibiotics far exceed those of theother ,B-lactam antibiotics against both gram-positive species including Streptococcus (Enter-ococci) and gram-negative species includingPseudomonas. On average, MK0787 shows a

twofold advantage over thienamycin for the en-

tire range of species. Isolates exhibiting f6-lacta-mase-mediated resistance to one or more peni-cillins and cephalosporins remained inhibited bythe thienamycins at concentrations close to theMICs found for strains susceptible to fi-lactamsfrom the same species. An example of the ab-sence of cross-resistance is provided by the sim-ilar susceptibilities of Enterobacter 2646 and itsf,-lactamase-negative derivative 2647. All other/3-lactam agents tested, including the extendedspectrum cephalosporins, show much reducedactivity on Enterobacter 2646. A single excep-tion to the general absence of cross-resistancewas obtained with a methicillin-resistant Staph-ylococcus isolate (4428), whose intrinsic resist-ance to ,B-lactam antibiotics is known to be un-related to ,B-lactamase activity (11). Cross-resist-ance did not occur between the thienamycinsand carbenicillin for a P. aeruginosa isolate withheterotypic resistance to that antibiotic (e.g., P.aeruginosa 3287), nor was carbenicillin resist-ance found for a thienamycin-resistant variantof P. aeruginosa (3288). Both variants were

isolated in vitro from the susceptible parentspecies 3286.A survey of 29 Bacteroides isolates showed

them all to have a very high susceptibility toMK0787 (Table 3). The median MIC forMK0787 (0.25 ,g/ml) was comparable to clin-damycin, 4-fold superior to thienamycin, and 64-fold superior to that of cefoxitin.Agar dilution susceptibility tests were con-

ducted with 13 selected hospital isolates of P.aeruginosa, of which 6 were judged resistant tocarbenicillin (Table 4). Two inoculum levels, 105

and 107 CFU, were employed. Comparisons weremade with two anti-pseudomonal cephalospo-rins, 6059-S (LY127935) and HR-756 (cefotax-ime), and to gentamicin, amikacin, piperacillin,and carbenicillin. These isolates were uniformlysusceptible to MK0787 at both inoculum levels.Isolates resistant to carbenicillin were not sig-nificantly different in their susceptibility to thethienamycins. The median MICs for MK0787were superior both to thienamycin and to thereference antibiotics. At the higher inoculumlevel, the greater activity of MK0787 was moremarked. For example, it was 16-fold more activethan the ,B-lactam reference antibiotics.An additional comparative study of the effect

of inoculum on both bacteriostatic and bacteri-cidal endpoints in broth is presented in Table 5.A penicillin-susceptible Staphylococcus, a fl-lac-tam-resistant enteric species, and two carbeni-cillin-susceptible isolates of P. aeruginosa wereused. The two anti-pseudomonal cephalosporinswere again included among the reference anti-biotics. At the low and internediate inoculumlevels (103 and 105 CFU per microtiter well),MK0787 was bactericidal at its MIC. At the highinoculum level (107 CFU), MICs and MBCs wereelevated, generally fourfold. This increase wasless than that found for the reference agents,

TABLE 3. Comparative susceptibility studies on 29B. fragilis strains to MKO787 and three reference

antibioticsMIC (jg/mi)

Compound' For 50% For 90% ofRange of strains strains

MK0787 0.13-1.0 0.25 1.0Thienamycin 0.25-2.0 1.0 2.0Clindamycin <0.06-4.0 0.13 2.0Cefoxitin 8.0->32 16 32

a Agar dilution, 105 CFU.

TABLE 4. Effect of inoculum levels on susceptibility of 13 clinical P. aeruginosa isolates and seven referenceantibiotics

MIC (jig/ml) at:

i05 CFU inoculum 107 CFU inoculumCompound

For % strains For % strainsRange Range

50 90 50 90

MKO787 0.8-12.5 3.1 6.3 3.1-12.5 6.3 12.5Thienamycin 1.6-50 6.3 25 3.1-50 12.5 256059-S 12.5->50 25 50 50->50 >50 >50Cefotaxime HR-756 12.5->100 25 100 50->100 >100 >100Carbeniciflin 50->400 100 400 100->400 200 >400Piperacillin 3.1->100 6.3 25 25->100 100 >100Gentamicin 1.6->50 6.3 50 6.3->50 6.3 >50Amikacin 1.6-50 6.3 25 6.3-50 12.5 50

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IN VITRO AND IN VIVO ACTIVITY OF MK0787 997

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ANTIMICROB. AGENTS CHEMOTHER.

which in several instances lost their bactericidaleffect or were overgrown at the highest levelstested, despite high potency apparent at the lowinoculum levels.A survey by the disk diffusion technique of

the distribution of susceptibilities to MK0787 inselected hospital isolates from four importantantibiotic-resistant species is shown in Table 6.MICs were determined for zone intervals bylinear regression of zone sizes versus MIC as

shown for MK0787 in Fig. 2. Of the 90 strainssurveyed, only 3% had zone diameters corre-sponding to MICs greater than 10 ,ug of MK0787per ml, and over 80% were judged to have MICsequal to or less than 2 ,ug/ml. This is in contrastto a sizable population with resistance or inter-mediate susceptibility found for the comparisonantibiotics, which attest again to minimal cross-resistance.In vivo studies. The comparative efficacy of

TABLE 6. MICs for selected hospital isolates estinated from disk diffusion studieseMIC pg/mi

Organism (No.) Antibiotic For 90% ofRangeb For 50% of strains

Proteus spp. (24) MK0787 0.6-5.2 2.6 5.2Cefoxitin 8.0-.64 16.0 32.0Carbenicillin 1.0..256 8.0 2256Gentamicin 0.4-50 1.6 6.4

Pseudomonas aeruginosa MK0787 <.64-20 0.64 2.6(28) Carbeniciflin 4.0-2256 128.0 2256

Gentamicin 0.4-50 3.1 25.0Enterococcus (23) MK0787 <0.64-100 0.64 1.3

Carbenicillin <0.5-2256 16.0 32.0Serratia spp. (15) MK0787 <0.64-10 0.64 2.6

Cefoxitin 8.0-264 32.0 264.0Carbenicillin 8.0--256 2256 -256Gentamicin 0.4-50 6.2 50.0

Disk contents were: MK0787, 25 Lg; cefoxitin, 30 Lg; carbenicillin, 50,ug; and gentamicin, 10 Mg.b The range of MICs for comparison antibiotics was estimated from regressions found in the literature

(cefoxitin [13], gentamicin [8], carbenicillin [9]).

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Zone Diameter, mmFIG. 2. Regression analysis of zone diameter versus MIC. A disk content of 25 pg of MK0787 was used.

Symbols: 0, S. aureus; 0, Enterococcus; E. coli, Shigella, Salmonella; E: Enterobacter; A, Klebsiella; A,Serratia; V, Proteus, indole + and -; V, Pseudomonas; X, thienamycin-resistant Pseudomonas. The latter isa laboratory isolate deliberately included to extend the range of the regression. The resistant S. aureus strainis methicillin resistant. MIC data were obtained by the agar dilution technique at a point inoculum densityof iO5.

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IN VITRO AND IN VIVO ACTIVITY OF MK0787 999

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MK0787, thienamycin, and appropriate refer-ence antibiotics in protecting the mouse againstacute experimental infections is shown in Tables7 and 8. Treatment was in every case parenteral,since MK0787 (like thienamycin) showed littleactivity by the oral route against systemic infec-tions. Wherever possible, choice was made of areference antibiotic to which the infection wasjudged susceptible by in vitro criteria. The effi-cacy of MK0787 exceeded that of thienamycinand, with but one exception, all of the referenceagents, often by a factor of 10 or more. Thisfinding, obtained both among the gram-positivecocci and gram-negative enteric strains, is re-ported in Table 7 and for P. aeruginosa isolatesin Table 8.

DISCUSSIONThe N-formimidoyl derivative ofthienamycin,

MK0787, overcomes the physicochemical prob-lems ofthe parent antibiotic without subtractingfrom its desirable antibacterial properties, in-cluding anti-pseudomonal activity and lack ofcross-resistance. The potency of MK0787 is atleast equal and, for several important generasuch as Pseudomonas, Bacteroides, and Enter-ococcus, at least twofold superior to thienamy-cin. Moreover, its in vivo activity in severalinstances (e.g., Proteus and Enterobacter spp.)exceeds that of thienamycin by a greater factorthan can be accounted for by relative in vitroactivity. The additional advantage probably re-sults from the increased systemic persistence ofMK0787, which in turn reflects a lower rate ofrenal clearance of antibiotic. MK0787 has beenfound to have a longer half-life and greater areaunder the plasma-concentration time-course inmice, dogs, rhesus monkeys, and chimpanzees(H. Kropp, J. G. Sundelof, J. S. Kahan, F. M.Kahan, and J. Birnbaum, Program Abstr. Inter-sci. Conf. Antimicrob. Agents Chemother. 19th,Boston, Mass., abstr. no. 231, 1979).Newer fi-lactam antibiotics with resistance to

,8-lactamases and an extended gram-negativespectrum have generally shown decreased po-tency against the gram-positive sector. In thisrespect, MK0787 is an outstanding exception. Itenjoys an order of activity against gram-positivestrains including Enterococci comparable to thepenicillins. The consistent potency of MK0787is exemplified by efficacy of dose rates c4.0 mg/kg over the entire antibacterial spectrum in ex-perimental mice infections. By comparison, theextended-spectrum cephalosorins, HR-756 andLY127935, required, even in the most favorableinstances, a range of dose rates between 3.0 and30 mg/kg to protect against susceptible infec-tions. An additional advantage found for

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TABLE 8. Comparative efficacy ofMK0787 against experimental infections in mice by selected clinical strainsofP. aeruginosaa

Infection strain of P. Challenge dose ED80 (mg/kg per dose)baeruginosa (CFU/mouse) MK0787 THM GEN AMK 6059-S HR-756 CAB PIP

40C 1.8 x 105 0.74 1.56 85 15 13.2 118 133 29.43286c 3.1 x 10' 0.95 4.65 10.3 10.3 33.4 >500 334 245.02835 4.7 x 100 1.2 2.4 9.4 104293 1.2 x 107 0.78 50 504294 6.9 x 106 1.9 50 503350 R-GEN, CAB 1.7 x 106 1.2 2.5 >200 25

a Therapy: a three-dose regimen, with treatment at 0, 2 and 4 h after challenge, was applied.b ED5o, 50% effective dose. Antibiotic was administered subcutaneously. Abbreviations: THM, Thienamycin;

GEN, gentamicin; AMK, amikacin; CAB, carbenicillin; PIP, piperacillin.C For infections designated with footnote c, 5% gastric mucin was utilized as dilution vehicle; brain heat

infusion broth was used in all other cases.

MK0787 in comparison with these agents was amuch reduced inoculum effect, with gram-neg-ative enteric strains showing a high order ofresistance to #-lactamase and a retention of bac-tericidal effect of high inoculum densities ofPseudomonas strains.By the above criteria of laboratory evalua-

tions, MK0787 compares very favorably withestablished agents and with extended spectrumB-lactam antibiotics undergoing clinical investi-gation. Therapeutic evaluation of MK0787 inhumans is expected in the near future.

ACKNOWLEDGMENTWe acknowledge the competent technical assistance of

Hannah C. Remeta in portions of the above studies.

LITERATURE CITED

1. Albers-Schonberg, G., B. H. Arison, 0. D. Hensens,J. Hirshfield, K. Hoogsteen, E. A. Kaczka, R. E.Rhodes, J. S. Kahan, F. M. Kahan, R. W. Ratcliffe,E. Walton, L J. Ruswinkle, R. B. Morin,and B. G.Christensen. 1978. The structure and absolute config-uration of thienamycin. J. Am. Chem. Soc. 100:6491-6499.

2. Bauer, A. W., W. M. Kirby, J. C. Sherris, and M.Turck. 1966. Antibiotic susceptibility testing by a stan-dard single-disc method. Am. J. Clin. Pathol. 45:493-496.

3. Bodey, G. P., S. S. Weaver, and B. M. LeBlanc. 1979.Thienamycin: new beta-lactam antibiotic with potentbroad-spectrum activity. Antimicrob. Agents Chemo-ther. 15:518-521.

4: Goldner, M., D. G. Glass, and P. C. Fleming. 1969.Spontaneous mutant with loss of beta-lactamase inAerobacter cloacae. J. Bacteriol. 97:961.

5. Kahan, J. S., F. M. Kahan, R. Goegelman, S. A.Currie, M. Jackson, E. 0. Stapley, T. W. Miller, A.K. Miller, D. Hendlin, S. Mochales, S. Hernandez,H. B. Woodruff, and J. Birnbaum. 1979. Thiena-mycin, a new beta-lactam antibiotic. I. Discovery, tax-onomy, isolation and physical properties. J. Antibiot.32:1-12.

6. Knudson, L F., and J. M. Curtis. 1947. The use ofangular transformation in biological assay. J. Am. Stat.Assoc. 42:282-296.

7. Leanza, W. J., K. J. Wildonger, T. W. Miller, and B.G. Christensen. 1979. N-acetimidoyl and N-formimi-doyl thienamycin derivatives antipseudomonal beta-lactam antibiotics. J. Med. Chem. 22:1435.

8. Matsen, J. M., M. J. H. Koepke, and P. G. Quie. 1969.Evaluation of the Bauer-Kirby-Sherris-Turck single-disc diffusion method of antibiotic susceptibility testing,p. 445-453. Antimicrob. Agents Chemother. 1969.

9. Schoenknecht, F. D. 1973. The Kirby-Bauer techniquein clinical medicine and its application to carbenecillin.J. Infect. Dis. 127 (Suppl.):S111-S115

10. Steers, E., E. L. Foltz, B. S. Graves, and J. Riders.1959. An inocula replicating apparatus for routine test-ing of bacteria susceptibility to antibiotics. Antibiot.Chemother. 9:307-311.

11. Sutherland, R., and G. N. Rolinson. 1964. Character-istics of methicillin-resistant staphylococci. J. Bacteriol.87:887.

12. Tally, F. P., N. V. Jacobus, and S. L Gorbach. 1978.In vitro activity of thienamycin. Antimicrob. AgentsChemother. 14:436-438.

13. Wallick, H., and D. Hendlin. 1974. Cefoxitin, a semisyn-thetic cephamycin antibiotic: susceptibility studies.Antimicrob. Agents Chemother. 5:25-32.

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