16(3):341. DOI: 10.1128/AAC.16.3.341.

1979,Antimicrob. Agents Chemother. R Wise, J M Andrews and K A Bedford beta-lactam antibiotics.comparison with otheroxa-beta-lactam: an in vitro LY127935, a novel

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Vol. 16, No. 3ANTimICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1979, p. 341-3450066-4804/79/09-0341/05$02.00/0

LY127935, a Novel Oxa-,B-Lactam: an In Vitro Comparisonwith Other f8-Lactam AntibioticsR. WISE,* J. M. ANDREWS, AND K. A. BEDFORD

Department ofMedical Microbiology, Dudley Road Hospital, Birningham B18 7QH, England

Received for publication 29 June 1979

The in vitro activities of LY127935 (LY) were compared with those of otherB8-lactam antibiotics. LY was highly active (minimal inhibitory concentration[MIC] range 0.06 to 0.25 jig/ml) against the common Enterobacteriaceae (includ-ing Providencia stuartiia, Enterobacter, and Serrati, marcescens), 8 to 16 timesmore active than cefoxitin, cefuroxime, or cefazolin, and from one-half to one-

eighth as active as cefotaxime (HR756). The activity ofLY against Pseudomonasaeruginosa (with MICs of 4 and 64 ug/ml for 50 and 90% of test strains,respectively) was essentially similar to that of cefotaxime, but was only one-halfas active as CGP 7174/E. LY, cefoxitin, and cefotaxime were essentially equallyactive against Bacteroides fragilis-each was more active than cefuroxime andcefazolin. Against Staphylococcus aureus, LY (50% MIC and 90% MIC of 4 and16 ,ug/ml, respectively) was less active than cefotaximne, cefoxitin, or cefuroximeand one-eighth as active as cefazolin. The composition and pH of the culturemedium had little effect on the activity of LY, although 7.2 appeared to be theoptimum pH.

Cephalosporin research has produced com-pounds of considerable interest such as the f,-lactamase-stable cefuroxime and the cephamy-cin cefoxitin. Cefotaxime (HR756), introducedmore recently, has a much broader spectrum ofactivity and greater potency than other relatedcephalosporins (3, 8). LY127937 (LY), a noveloxa-,B-lactam derivative developed by the Shion-ogi Co. Osaka, Japan (6059-S), also promises tobe of considerable interest (S. Matsuura, T.Yoshida, and S. Kuwahara, Program Abstr. In-tersci. Conf. Antimicrob. Agents Chemother.18th, Atlanta, Ga., abstr. no. 152, 1978). Thiscompound, 7-[[carboxy(4-hydroxyphenyl), ace-tyl]amino] - 7-methoxy-3-[ [(1-methyl- 1H-tet-raxol -5 - yl)thio]methyl] - 8- oxo - 5 -oxa - 1 - azabi-cyclo[4.2.0]oct-2-ene-2-carboxylic acid, struc-turally similar to cefoxitin, has a dihydro-oxazinering in place of the dihydro-thiazine ring com-mon to cephalosporins and cephamycins. In thecurrent study the activities of LY and other ,8-lactam antibiotics were compared against a widerange of recent clinical isolates. The novel ceph-alosporin CGP 7174/E was included in the in-vestigation of the strains of Pseudomonasaeruginosa (5).

MATERIALS AND METHODSStrains and antimicrobial agents. The strains

examined in this study are listed in Table 1 accordingto classification and number. Of the total of 239, 234were recent clinical isolates from patients in Dudley

Road Hospital. Of the remaining five, three were /?-lactamase-positive Neisseria gonorrhoeae strains ob-tained from A. Percival (Liverpool, England) and W.A. Ashford (U. S. Air Force). Two strains of P. aerugi-nosa known to be Tem' were obtained from E. Low-bury (Birmingham, England). All strains were identi-fied by the API (API Laboratory Products Ltd., Farn-borough, England) method. The production of ,B-lac-tamase by some strains was verified using the chro-mogenic cephalosporin substrate nitrocefin (4).The antibiotics were obtained from the following

sources: LY and cefazolin from Lilly Research,Windlesham, England; benzylpenicillin, ampicillin,and carbenicillin from Beecham Research Laborato-ries, Brentford, Middlesex, England, cefoxitin fromMerck Sharp & Dohme, Hoddesdon, England; cefu-roxime from Glaxo Laboratories, Greenford, Middle-sex, England; CGP 7174/E from Ciba Geigy, Horsham,England; and cefotaxime from Roussel Laboratories,Wembley, England.

Methods. The activities of the compounds werestudied by a routine agar plate dilution method usingIsosensitest agar, pH 7.2 (Oxoid, Basingstoke, Eng-land). The Isosensitest agar was supplemented as fol-lows: with 5% lysed human blood to support thegrowth of Bacteroides fragilis, a Levinthal prepara-tion to support the growth of Haemophilus influ-enzae, and a chocolate agar to support the growth ofN. gonorrhoeae.

Inocula were prepared as follows. For all strainsexcept those of B. fragilis, streptococci (includingpneumococci), N. gonorrhoeae, and H. influenzae, theorganisms were grown overnight in nutrient brothyielding a viable count of about iOW colony-formingunits (CFU) per ml. Strains of B. fragilis were grown

341

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TABLE 1. MIC inhibiting cumulative percentage of isolates'No. of .n.itc MIC range 50% MIC 90% MIC

Species isolates Antibiotic (ig/l) (L/Im) (Ag/ml)Escherichia coli 25 LY 0.06-0.25 0.12 0.25

CefotaximeCefoxitinCefuroximeCefazolin

0.015-1.01-81-81-8

0.06211

0.12444

Klebsiella pneumoniae

Proteus mirabilis

Indole-positive Proteus spp.

Enterobacter spp.

Salmonella spp.(including 2 S. typhi)

Providencia stuartii

Serratia marcescens

Pseudomonas aeruginosa

Haemophilus influenzae

25 LYCefotaximeCefoxitinCefuroximeCefazolin

10 LYCefotaximeCefoxitinCefuroximeCefazolin

10 LYCefotaximeCefoxitinCefuroximeCefazolin

10 LYCefotaximeCefoxitinCefuroximeCefazolin

5 LYCefotaximeCefoxitinCefuroximeCefazolin

10 LYCefotaximeCefoxitinCefuroximeCefazolin

5 LYCefotaximeCefoxitinCefuroximeCefazolin

35 LYCefotazimeCarbenicillinCGP 7174/E

25 LYCefotaximeAmpicillinCefoxitinCefuroxime

0.06-20.008-0.250.5-40.25-161-128

0.06-0.250.015-0.121-161-161-32

0.12-0.250.015-11-44->12864->128

0.06-0.250.06-0.2564->1281-41-32

0.03-0.120.03-0.061-21-21

0.03-0.060.06-0.121-41-42-16

0.250.06-0.254-88-32>128

1-1284-12816->10281-64

0.015-0.250.004-0.030.12-41-40.25-1

0.120.03111

0.120.015112

0.120.061

1664

0.120.06

>12824

0.060.06111

0.060.06128

0.250.128

32>128

48

642

0.060.0040.2520.5

0.250.12248

0.250.03224

0.120.12464

>128

0.120.12

>12828

0.120.06221

0.060.124416

0.250.258

32>128

6464

102432

0.060.015441

342 WISE, ANDREWS, AND BEDFORD ANTIMICROB. AGENTS CHEMOTHER.

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LY127935, A NOVEL OXA-(i-LACTAM 343

TABLE 1-Continued

Species No. of Antibiotic MIC range 50% MIC 90% MICisolates (ILg/ml) (jig/ml) (,ug/ml)

Neisseria gonorrhoeae 25 LY 0.008-1 0.03 0.25Penicillin 0.008-1 0.12 1Cefoxitin 0.12-0.5 0.25 0.5Cefuroxime 0.008-1 0.06 0.5Cefazolin 0.12-2 0.5 1

Bacteroides fragilis subsp. 25 LY 0.25->4 0.5 4fragilis Cefotaxime 1-64 2 2

Penicillin 2-512 8 16Cefoxitin 2-16 4 8Cefuroxime 1-128 4 128Cefazolin 2->128 16 128

Staphylococcus aureus 24 LY 4-16 4 16(including 2 methicillin- Cefotaxime 0.5-8 1 8resistant strains) Cefoxitin 2-8 2 8

Cefuroxime 0.5-8 1 4Cefazolin 0.12-4 0.25 2

a Inoculum, 103 CFU.

overnight in thioglycolate broth, streptococci weregrown in Todd-Hewitt broth, and R. influenzae strainswere grown in Levinthal broth yielding about the sameviable counts. Strains of N. gonorrhoeae were grownovernight on a chocolate agar plate, the growth beingremoved just before use and resuspended in peptonewater so as to yield a viable count of 106 to 107 CFU/ml. Two inocula, 103 and 106 CFU/ml, were employedin the sensitivity tests of all organuism except N.gonorrhoeae. These were obtained by transferring 1pi undiluted and a 1:1,000 dilution of the overnightculture to the surface of the antibiotic-containing agarby a Denley (Denley-Tech Ltd., Billingshurst, Eng-land) multipoint inoculating device. Strains of N. gon-orrhoeae were tested undiluted and at a 1:10 dilution,thus yielding final inocula of 103 to 104 and 102 to 103CFU.

All plates were incubated in air at 370C, with theexception of the anaerobes which were incubated in aGasPak jar (BBL Microbiology Systems, Cockeysville,Md.) and H. influenzae and N. gonorrhoeae whichwere incubated in a 10% C02 incubator. The period ofincubation was 24 h. The minimal inhibitory concen-tration (MIC) of the antibiotic was defined as themicrograms per milliliter of medium at which therewas an estimated 99% reduction (by counting) in theoriginal inoculum.The influence of different media upon the MIC of

LY was evaluated using five strains each of Esche-richia coli, Klebsiella spp., Enterobacter spp., Provi-dencia stuartii, Staphylococcus aureus, indole-posi-tive and -negative Proteus spp., P. aeruginosa, Ser-ratia marcescens, and Lancefield group D strepto-cocci. The media chosen were as follows: Isosensitest,pH 7.2, 6.0, and 8.0; Wellcotest, pH 7.2 (WeilcomeResearch Laboratory, Beckenham, England); SAF, pH7.2 (Mast Laboratory, Liverpool, England); Penassayno. 1., pH 6.6 (Oxoid Ltd.), Penassay no. 5, pH 8.0; andMeuller-Hinton, pH 7.3 (Difco Laboratories, Detroit,Mich.). The two inocula and incubation conditionsdescribed above were employed.

RESULTS

Table 1 summarizes the results obtained fromthe 234 isolates tested at an inoculum of 103CFU. LY had a high degree of activity againstthe Enterobacteriaceae, including known 8-lac-tamase-producing strains, showing about one-half the activity of cefotaxime but being at least8- to 16-fold more active than cefoxitin, cefurox-ime, and cefazolin. The clinically important gen-era tested, Providencia and Serratia, also werehighly susceptible to LY and cefotaxime. It wasnoted that, when a strain showed decreasedsusceptibility to cefotaxime, this was reflected indecreased susceptibility to LY and cefoxitin, forexample, an E. coli with an MIC to cefotaximeof 1 Atg/ml, to LY of 0.25 ,ug/ml, and to cefoxitinof 8 ,g/ml.Among the Enterobacteriaceae, an increase

in inoculum to 106 CFU resulted in at most atwo-fold decrease in susceptibility to LY, cefo-taxime, cefoxitin, and cefuroximne.LY and cefotaxime were essentially equally

active against P. aeruginosa but only one-halfas active as CGP 7174/E. Tem+ and Tem-strains were equally susceptible to LY and ce-fotaxime.LY was clearly more active than ampicillin,

cefoxitin, and cefuroxime against H. influenzaebut was only one-fourth to one-sixteenth as ac-tive as cefotaxime. The eight fi-lactamase-pro-ducing strains were as susceptible to LY andcefotaxime as were the non-f-lactamase pro-ducers. Whereas an increase in inoculum of ,B-lactamase producers had little effect on the ac-tivity of LY, it decreased the activity of ampicil-lin markedly.

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344 WISE, ANDREWS, AND BEDFORD

The activity of LY against N. gonorrhoeaewas similar to that of cefuroxime, non-f8-lacta-mase producers being equally susceptible to LYand penicillin. The f8-lactamase producers werepenicillin resistant (MIC > 1 ILg/ml), and two ofthese three strains were susceptible to LY andcefuroxime (MIC < 1 ,g/ml). One strain (fromLiverpool) had an MIC ofLY and cefuroxime of1 .g/Ii.The B. fragilis strains were four- to eight-fold

more susceptible to LY than to cefoxitin orcefotaxime at the 50% MIC endpoint. Four ofthe 25 strains had an MIC to LY of 24 ,ug/ml.An eightfold decrease in susceptibility to cefo-taxime was noted on increasing the inoculum to106 CFU, but only a twofold decrease was en-countered with LY and cefoxitin.LY was less active against S. aureus than the

three other cephalosporins, whereas Lancefieldgroup A streptococci (two strains tested, notshown in the table) and Streptococcus pneumo-niae (three strains tested) were susceptible (LYMIC of 0.25 to 0.5 ug/ml). Lancefield group Dstreptococci (five strains tested) were highly re-sistant, having an MIC > 16,ug/ml.A study of the effects of culture medium upon

the MIC of LY, involving work with 45 isolates,revealed only nil or one discrepant result (de-fined as an MIC of greater than twofold com-pared with the reference medium, Isosensitest,pH 7.2) with the following media: Wellcotest,SAF, Meuller-Hinton, and Isosensitest (pH 8.0).Penassay no. 5 (pH 8) and Isosensitest (pH 6.0)showed 4 and 5 discrepant results, respectively,and Penassay no. 1 (pH 6.6) showed 10 discrep-ant results. In all cases the MICs ofLY obtainedin the discrepant results were higher than thoseon the reference media. It appeared that theoptimum pH for the in vitro activity of LY was7.2.

DISCUSSIONLY, a structurally novel /-lactam, shares

many properties with the cephalosporins. Thisstudy shows that, with the exception of S. au-reus, LY is more active than cefoxitin or cefu-roxime and hence more active than cefamandole(2). LY shares with cefotaxime a high degree ofactivity against the Enterobacteriaceae (8) in-cluding the "problem" organisms of hospitalpractice such as Serratia, P. stuartii, and theEnterobacter spp. The activity of LY against P.aeruginosa is similar to that of cefotaxime butless than the antipseudomonal cephalosporinCGP 7174/E. It would appear that LY is stableto 18-lactamase hydrolysis as little decrease insusceptibility was noted on increasing the inoc-ulum.

ANTIMICROB. AGENTS CHEMOTHER.

Against B. fragilis, LY was four to eight timesmore active than cefoxitin, an agent of provenclinical usefulness in anaerobic sepsis (6). Fourstrains of decreased susceptibility (two of whichwere highly penicillin resistant) were encoun-tered, but little inoculum effect was noted.Whether this resistance is related to poor cellpenetration or ,8-lactam hydrolysis is not known.In the case of cefotaxime, both mechanisms areprobably involved (1, 7).Against gram-positive pathogens, LY had ac-

tivity similar to that of cefotaxime, namely, S.aureus strains were relatively less susceptible,fecal streptococci were highly resistant, andgroup A streptococci and pneumococci were sus-ceptible. N. gonorrhoeae and H. influenzae, in-cluding the clinically important ,t-lactamase-producing strains, were highly susceptible.

Studies in experimental animals suggest thatLY will need to be administered parenterally.The compound is excreted in a form microbio-logically indistinguishable from the parent com-pound, unlike cefotaxime which undergoes con-siderable metabolism to the desacetyl form (P.Johnson, R. Glomot, and M. Kramer, ProgramAbstr. Intersci. Conf. Antimicrob. Agents Chem-other. 18th, Atlanta, Ga., abstr. no. 81, 1978).Preliminary data (R. Wild, personal communi-cation) suggest that high serum levels areachieved and the serum half-life is relativelylong. Thus, administration of relatively smalldoses ofLY might be efficacious in the treatmentof the common gram-negative infections; higherdoses would probably be needed for treatmentof S. aureus or P. aeruginosa infections.LY has a novel structure, an extremely inter-

esting antimicrobial spectrum, and a wide poten-tial demanding clinical study.

ACKNOWLEDGMENT

We thank R. Wild of Lilly Research for his help and advice.

LITERATURE CITED1. Drasar, F. A., W. Farrell, A. J. Howard, C. Hince, T.

Leung, and J. D. Williams. 1978. Activity of HR756against H. influenzae and B. fragilis and Gram-nega-tive rods. J. Antimicrob. Chemother. 4:445-450.

2. Eykyn, S., C. Jenkins, A. King, and L Phillips. 1976.Antibacterial activity of cefuroxime, a new cephalospo-rin antibiotic, compared with that of cephaloridine,cephalothin, or cefamandole. Antimicrob. AgentsChemother. 9:690-695.

3. Fu, K. P., and H. C. Neu. 1978. Beta-lactamase stabilityof HR756, a novel cephalosporin, compared to that ofcefuroxime and cefoxitin. Antimicrob. Agents Chemo-ther. 14:322-326.

4. O'Callaghan, C. H., A. Morris, S. M., Kirby, andA. M. Shingler. 1972. Novel method for the detectionof fi-lactamase by using a chromogenic cephalosporinsubstrate. Antimicrob. Agents Chemother. 1:283-288.

5. Tosch, W., F. Kradolfer, E. A. Konopka, J. Regos, W.Zimmerman, and 0. Zak. 1978. In vitro characteri-zation of CGP 7174/E, a cephalosporin active against

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LY127935, A NOVEL OXA-f,-LACTAM 345

Pseudomonas, p. 843-844. In W. Siegenthaler and R.Luthy (ed.), Current chemotherapy, vol. 2. AmericanSociety for Microbiology, Washington, D.C..

6. Wilson, P., T. Leung, and J. D. Williams. 1978. Anti-bacterial activity, pharmacokinetics and efficacy of ce-foxitin in patients with abdominal sepsis and otherinfections. J. Antimicrob. Chemother. 4(Suppl. B):127-

141.7. Wise, R. 1979. Bacteroides fragilis and HR756. J. Anti-

microb. Chemother. 5:115-116.8. Wise, R., T. Rollason, M. N. Logan, J. M. Andrews,

and K. A. Bedford. 1978. HR756, a highly activecephalosporin: comparison with cefazolin and carbeni-cillin. Antimicrob. Agents Chemother. 14:807-811.

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