The antibacterial activity of a new penem antibiotic, Sch 29482 (SCH), was examined in comparison with appropriate cephalosporins and penicillins. The drug inhibited penicillinase-positive and negative staphylococci equally well, being 2-5 times more active than cephalothin or cefamandole and 10-20 times more active than methicillin. Staphylococci resistant to methicillin were susceptible to SCH in agar dilution tests. Staphylococci tolerant to methicillin were also tolerant to SCH. Streptococci and pneumococci were highly susceptible to the drug. The agent was of only moderate activity against enterococci, especially Streptococcus faecium strains. MICs of ampicillin and penicillin G against enterococci were 4-8 times lower than those of SCH. SCH was bactericidal. Neither the choice of the method used for susceptibility testing, nor the size of the inoculum nor various test media influenced the in-vitro activity of this drug against a representative collection of Gram-negative and Gram-positive bacteri

Kayser, F. H.

Kuhn, I.

The antibacterial activity of a new penem antibiotic, Sch 29482 (SCH), was examined in comparison with appropriate cephalosporins and penicillins. The drug inhibited penicillinase-positive and negative staphylococci equally well, being 2-5 times more active than cephalothin or cefamandole and 10-20 times more active than methicillin. Staphylococci resistant to methicillin were susceptible to SCH in agar dilution tests. Staphylococci tolerant to methicillin were also tolerant to SCH. Streptococci and pneumococci were highly susceptible to the drug. The agent was of only moderate activity against enterococci, especially Streptococcus faecium strains. MICs of ampicillin and penicillin G against enterococci were 4-8 times lower than those of SCH. SCH was bactericidal. Neither the choice of the method used for susceptibility testing, nor the size of the inoculum nor various test media influenced the in-vitro activity of this drug against a representative collection of Gram-negative and Gram-positive bacteria

F. H. Kayser

I. Kuhn

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Sch 29482, laboratory evaluation of a new penem antibiotic

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Journal of Antimicrobial Chemotherapy (1982) 9, Suppl. C. 181-188Sch 29482, laboratory evaluation of a new penem antibioticF. H. Kayser and I. KuhnInstitute of Medical Microbiology, University of Zurich, 8028 Zurich, SwitzerlandThe antibacterial activity of a new penem antibiotic, Sch 29482 (SCH), was examinedin comparison with appropriate cephalosporins and penicillins. The drug inhibitedpenicillinase-positive and negative staphylococci equally well, being 2-5 times moreactive than cephalothin or cefamandole and 10-20 times more active than methicillin.Staphylococci resistant to methicillin were susceptible to SCH in agar dilution tests.Staphylococci tolerant to methicillin were also tolerant to SCH. Streptococci andpneumococci were highly susceptible to the drug. The agent was of only moderateactivity against enterococci, especially Streptococcus faecium strains. MICs ofampicillin and penicillin G against enterococci were 4-8 times lower than those ofSCH. SCH was bactericidal. Neither the choice of the method used for susceptibilitytesting, nor the size of the inoculum nor various test media influenced the in-vitroactivity of this drug against a representative collection of Gram-negative and Gram-positive bacteria.IntroductionSch 29482 (SCH) is a new penem antibiotic which is chemically related to the carbapenemantibiotic thienamycin. The present study was undertaken to compare the in-vitroefficacy of this drug with various other /Mactam antibiotics. Some 250 clinical isolates ofGram-positive cocci were included in the study to provide a good indication of thepotential efficacy of SCH in clinical application for Gram-positive infections. With arepresentative number of Gram-negative and Gram-positive bacteria, the influence ofvarious in-vitro parameters, such as method of susceptibility testing, test medium andinoculum size, on results obtained with SCH was examined. All our data emphasize thefact that this penem antibiotic possesses promising in-vitro characteristics and,therefore, warrants further laboratory and clinical evaluation.MethodsBacteriaThe micro-organisms used were isolated from clinical material and identified accordingto standard procedures. About 250 Gram-positive organisms were examined. Thecollection of Gram-negative bacteria included: Escherichia coli (16 strains); Klebsiellapneumoniae (8 strains) ; Enterobacter aerogenes (4 strains); Enterobacter cloacae (4strains); Serratia marcescens (5 strains); Proteus mirabilis (4 strains); Proteus vulgaris (4strains); Providentia rettgeri (4 strains); Morganella morganii (4 strains) andAcinetobacter calcoaceticus (4 strains).1810305-7453/82/9C0181 +08 $01.00/0 © 1982 The British Society for Antimicrobial Chemotherapy182 F. H. Kayser and I. KuhnAntibioticsStock solutions of SCH, methicillin, penicillin G, ampicillin, cephalothin, cefamandole,cefaclor, cephalexin, cefoperazone and N-formimidoyl thienamycin were prepared insterile distilled water and either used immediately or stored at -80°C, if necessary. Alldrugs were kindly provided by the pharmaceutical companies.Susceptibility testsMinimal inhibitory concentrations (MICs) were determined by serial twofold dilutionsof antibiotics, usually in Mueller-Hinton agar (BBL), or in other broth or agar media asindicated in the text. Pneumococci and haemolytic streptococci were tested on agarcontaining 1% (w/v) Isovitalex (BBL) and 1% (w/v) haemoglobin (BBL). The inoculumin agar dilution was about 104 per spot, and in micro-broth dilution 5 x 10s to 106 perml MICs were read after incubation at 37°C for 18 to 24 h. The MIC techniquescorresponded to the procedures proposed by the National Committee of ClinicalLaboratory Standards (1980).Minimal bactericidal concentrations (MBCs) were determined by subculturingapproximately 5 y\ of the medium in each well of an MIC plate on Mueller-Hintonagar. The MBC was the lowest concentration of antibiotic that caused a 99-9%reduction in the number of viable organisms.Populations of methicillin-resistant staphylococci were analysed by disaggregation ofovernight broth cultures (brief, controlled exposure to 20-kilocycle sound) and surfaceinoculation of appropriate dilutions on drug-containing Mueller-Hinton agar plates.Colony counts after 48 h of incubation at 30 or 37°C allowed calculation of the numberof viable units among 108 colony forming units (cfu) resistant to each concentration ofantimicrobial agent.Tolerance in staphylococci was examined by plating 20 ^ 1 of each well of an MICplate on Mueller-Hinton agar and counting the number of cfu. Kinetics of killing wasinvestigated in 20 ml Mueller-Hinton broth, to which 100 times the MIC of drug wasadded. At appropriate time intervals, 0-4 ml samples were withdrawn, and incubated for10 to 15 min at 20°C with 01 ml of a crude Bacillus cereus enzyme preparation (Sabathet al., 1971). This served to destroy any drug and, thus to avoid carry-over ofbacteriostatic activity. Destruction of drug in the broth was examined by a plate assay,using Bacillus subtilis ATCC 6633 as test organism (Bennet et al., 1966).ResultsActivity against Gram-positive organismsTable I summarizes MIC data of SCH against Gram-positive bacteria. As can be seen,the drug showed excellent activity against staphylococci and streptococci including thepneumococci. The drug showed activity against methicillin-resistant staphylococci also.Enterococci, especially Str. faecium, were not very susceptible to the agent. However,the incidence of Str. faecium among enterococci is only 1-8% in our area.Table II compares the activity of various /J-lactam antibiotics against Staphylococcusaureus (N = 35), Staph. epidermidis (N = 48) and methicillin-resistant Staph. aureus(N = 40). Staph. epidermidis includes coagulase-negative organisms. No furtherspeciation was done. SCH proved to be highly active against penicillinase-positiveIn-vitro evaluation of Sch 29482 183Table I. Activity of Sch 29482 against Gram-positive bacteriaOrganismStaphylococcusMethicillin-resistantPenicillinase-positivePenicillinase-negativeStreptococcus faecalisStreptococcus faeciumStreptococcus duransHaemolytic streptococciGroup AGroup BGroup CPneumococciNo.ofstrains405132311432597Minimal inhibitoryconcentration (mg/1)Mean0-260080087-619-55 00030-12014Mode0-25012500680wide range8 0003012012MIC90-46014019-3786-50040130-7130 001 003 007•Concentration required to inhibit 90% of the strains examined.Table II. Comparative activity of /Mactam antibiotics against Staph. aureus (N = 35), Staph.epidermidis (N = 48), and methicillin-resistant Staph. aureus (N = 40)AntibioticSCHMethicillinPenicillin GCephalothinCefamandoleCefaclorCefoperazoneN-FormimidoylthienamycinSCH(30°C)PenicillinasepositiveMIC (mean)008200-20-344-221-33003—PenicillinasenegativeMIC (mean)0081-20020160-241 96102003—Methicillin-resistantMIC (mean) MIC 90*0-264-80-91-933-78-70081-60-467-22-54-87318-7014•Concentration required to inhibit 90% of the examined strains.Incubation was carried out at 37°C, unless otherwise indicated, for 24 h.and negative strains. Only the thienamycin compound had similar activity. SCH andthienamycin also showed activity against methicillin-resistant organisms, when tested inagar dilution with an inoculum of 10* per spot and incubated for 24 h at 37°C. Thesetwo drugs were more active than cefamandole or cephalothin, which were shownelsewhere to be the most active /Mactam drugs against methicillin-resistantstaphylococci (Kayser, 1980). Incubation of plates at 30°C increased MICs of SCHapproximately 10 times.184 F. H. Kayser and I. Kuhn8§~ 6(J_ 5o1 «•5 3sF lz~izHZSCH^(37°C;48h)1 -1 r' L_^SCH(30°C;48 h)1Oxocillin<"(37°C;48 h)1—11<-Cefomondole 1,(37°C; 461 11h)-0 03 0-06 0125 025 0-5 1 2 4 B 16 32 64 128 256Antibiotic (mg/l)Figure 1. Composition of highly methicillin-resistant Staph. aureus strain EK 695 of cells with differentlevels of resistance to SCH, cefamandole and oxacillinIt is well known that populations of methicillin-resistant staphylococci are composedof cells differing widely in their resistance to /Mactam antibiotics. This heterogeneity wasalso observed in case of SCH (see Figure 1). However, the resistant minorities inpopulations are less resistant to SCH than to oxacillin or cefamandole.Penicillin-tolerant staphylococci are known to be susceptible to penicillins in MICtests, but highly resistant to the killing effect of these drugs (Sabath et al., 1977). Weexamined the tolerance phenomenon in 32 Staph. aureus and epidermidis strains (16 ofeach species) against methicillin and SCH. Only one Staph. aureus culture was found tobe tolerant to the two drugs. Figure 2 demonstrates the kinetics of killing of the toleranticrStaph. aureus,tolerant8 12 16Incubation ( h )20 24Figure 2. Rates of killing of Staph. aureus strain FK. 484 and FK 485 by methicillin and SCH. StrainFK 484: • methicillin 60 mg/l, A SCH 6 mg/l; strain FK 485: O methicillin 60 mg/l, A SCH 6 mg/l.In-vitro evaluation of Sch 29482 185Table HI. Comparative activity of /J-lactam antibiotics against enterococci (N = 48)Minimal inhibitory concentration (mg/1)Antibiotic MeanSCHPenicillin GAmpicillinMethicillinCephalothinCefamandoleCefaclorCefoperazoneN-Formimidoylthienamycin9-8210-734-934-429-8119028-54-3lode82132323212832MIC90*3001002-9157013001000220090080Concentration required to inhibit 90% of the examined strains.and a non-tolerant strain by methicillin and SCH. Destruction of drugs during growthof the tolerant culture in comparison with the non-tolerant culture did not occur.Table HI summarizes MIC data of /?-lactam antibiotics against enterococci. AlthoughSCH was not as active as penicillin or ampicillin against enterococci, it showed someactivity, which was about 4 times greater than the activity of cephalosporins.Thienamycin also proved to have a moderate activity against these problem bacteria.All drugs were active against /J-haemolytic streptococci and pneumococci (seeTable IV). Penicillin G and thienamycin were most active, closely followed by SCH,ampicillin and cefamandole.Examination of bactericidal activity of SCH gave MBC values which were identicalto MICs against most of 80 Gram-positive strains examined. MBCs for methicillin-resistant staphylococci were 4-16 times, and for the tolerant Staph. aureus culture, moreTable IV. Comparative activity of /Mactam antibiotics against streptococci and pneumococciAntibioticSCHPenicillin GAmpicillinMethicillinCephalothinCefaclorCefalexinCefamandoleCefoperazoneTV-Form imidoylthienamycinGroup A(W =25)00300040010130-250-250-5003013< 0 0 3ModeGroup B(N = 9)013003010-50-25808 00130-25sSO-03MIC (mg/1)Group C(JV=7)0130010030-250-25202 00060-25«=003Pneumococci(N = 30)00300080010060060-2510006006<003186 F. H. Kayser and I. KuhnTable V. Effect of methods and media on in-vitro activity of Sch 29482Method andmediumAgar dilutionprocedure (NCCLS)Mueller-Hinton-Agar(Oxoid)Isosensitest-Agar(Oxoid)Diagnostic SensitivityTest-Agar (Oxoid)Micro-broth dilutionprocedure (NCCLS)Mueller-Hinton broth(Oxoid)Mueller-Hinton brothplus cation supplement(see NCCLS)MinimalEnterobacteru(N = 67)0910-850-931081 37inhibitory concentrationi Staphylococci(N = 32)0120100070-25016(mean) (mg/1)Enterococci(tf = 8)5-74-46-26-89-8than 100 times higher than MICs. Killing of Staph. aureus and enterococci was often lesseffective at higher drug concentrations.In-vitro factors and activity of SCH/Mactam antibiotics are known to be of varying stability to /J-lactamases. Instability of adrug to a /Mactamase can result in wide differences in MIC results according to themethod used. Instability also can result in a significant inoculum effect. Activity ofantimicrobials in vitro can also be influenced by the composition of the test medium. We,therefore, examined the influence of some in-vitro parameters on results obtained withSCH. The enterobacteria used for these tests were a representative collection oforganisms as described in the 'Methods' section. Table V demonstrates that neither themethod nor various media influenced MIC results of SCH. Table VI shows that the sizeof the inoculum also had no effects on test results. The only exceptions were methicillin-resistant staphylococci. Because of the typical heterogeneity (see Figure 1), MICs formethicillin-resistant staphylococci were greater at higher inoculum sizes.DiscussionThe penem antibiotic SCH has been shown in this study to be an excellent antibioticagainst most species of Gram-positive organisms. In particular it exhibited some activityagainst methicillin-resistant staphylococci, although the typical characteristics of thesebacteria—heterogeneity and influence of incubation temperature on phenotypicexpression of resistance—were also observed with SCH. The drug was as active aspenicillin G against penicillinase-negative variants of methicillin-resistant strains (datawere not shown here) and, thus can be considered as a possible choice in the treatmentof infections caused by such bacteria. This might be important in the future, becauseIn-vitro evaluation of Sch 29482 187Table VI. Effect of inoculum size on activity of Sch 29482OrganismEnterobacteria(N= 10)StaphylococciStaph. aureus(methicillin-resistant)(AT = 2 )Enterococci(AT = 5 )EnterobacteriaStaphylococciStaph. aureus(methicillin-resistant)Enterococci103/spot0-340030-503-5104/ml0-280090-704-60Mean MIC (mg/1)107spot0-450130-704-6Mean MIC (mg/1)105/ml0-230082-80610in agar dilution105/spot1100161661in broth dilution106/ml0-450174005-30106/spot1-400-25327 0107/ml0-370-2590001210there is a world-wide increase of epidemic infections in hospitals caused bystaphylococci, simultaneously resistant to /?-lactam antibiotics, aminoglycosides andother antimicrobials (Shanson, Kensit & Duke, 1976; Crossley et al., 1979; Graham etal., 1980; Amirak et al, 1981. Kayser, F. H., 1981 personal observation). SCH has alsobeen shown to be highly active against streptococci and pneumococci. Enterococci,however, were not as susceptible to SCH as to ampicillin or penicillin G, although theywere more sensitive to this agent than to cephalosporin antibiotics. The good activity ofSCH against the small collection of Gram-negative bacteria suggests that this drug has abroad antibacterial spectrum of activity.Neither the size of the inoculum, the method used for susceptibility testing nor varioustest media affected the in-vitro activity of SCH against Gram-negative and Gram-positive bacteria. Such parameters can influence susceptibility testing of bacteria against/Mactam antibiotics that have a reduced stability to /J-lactamases. It was shown, forinstance, that the in-vitro activity of cefoperazone, cefamandole and cephalothin againstGram-negative bacteria producing large amounts of TEM-/Mactamase, is highlydependent on the test method and the size of the inoculum used (Kayser, Huf &Homberger, 1981). Since such TEM-producers were among the strains examined andsince neither the inoculum nor the method influenced the activity of SCH against thesestrains, it can be concluded that this drug is remarkably stable against the most frequentlyoccurring /Mactamase of Gram-negative bacteria (Mathew, 1979).ReferencesAmirak, I. D., Li, A. K. C , Williams, R. J. & Noone, P. (1981). A fatal infection caused bymethicillin-resistant Staphylococcus aureus acquiring resistance to gentamicin and fusidicacid during therapy. Journal of Infection 3, 50-8.188 F. H. Kayser and I. KuhnBennett, J. V., Brodie, J. L., Benner, E. J. & Kirby, W. M. M. (1966). Simplified, accurate methodfor antibiotic assay of clinical specimens. Applied Microbiology 14, 170-7.Crossley, K., Loesch, D., Landesman B., Mead, K., Chern, M. & Strate R. (1979). An outbreak ofinfections caused by strains of Siaphylococcus aureus resistant to methicillin andaminoglycosides. II. Epidemiological studies. Journal of Infectious Diseases 139, 273-87.Graham, D. R., Correavillasenor, A., Anderson, R. L., Vollman, J. H. & Baine, W. B. (1980).Epidemic neonatal gentamicin-methicillin-resistant Staphylococcus aureus infectionassociated with nonspecific topical use of gentamicin. Journal of Pediatrics 97, 972-8.Kayers, F. H. (1980). Die Resistenz methicillin-resistenter Staphylokokken gegenuber neuenCephalosporin-Antibiotika. Infection 8, 165-70Kayser, F. H., Huf, E. & Homberger, F. (1981). The microbiology of cefoperazone. Infection 9,Suppl. 1, 6-12.Mathew, M. (1979). Plasmid-mediated /?-lactamases of Gram-negative bacteria: properties anddistribution. Journal of Antimicrobial Chemotherapy 5, 349-58.National Committee of Clinical Laboratory Standards (NCCLS). (1980). Standard methods fordilution antimicrobial susceptibility tests for bacteria which grow aerobically. NCCLSProposed Standard: PSM-7.Sabath, L. D., Casey, J. I., Ruck, P. A., Stumpf, L. L. & Finland, M. (1971). Rapid microassay ofgentamicin, kanamycin, neomycin, streptomycin, and vancomycin in serum or plasma.Journal of Laboratory and Clinical Medicine 78, 457-63.Sabath, L. D., Wheeler, N., Laverdiere, M., Blazevic, D. & Wilkinson, B. J. (1977). A new type ofpenicillin resistance of Staphylococcus aureus. Lancet i, 443-6.Shanson, D. C , Kensit, J. G. & Duke, R. (1976). Outbreak of hospital infections with a strain ofStaphylococcus aureus resistant to gentamicin and methicillin. Lancet ii, 1347-8.

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Sch 29482, laboratory evaluation of a new penem antibiotic

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