with Stability to Dehydropeptidase I

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 99, p. 5- Vol. 33, No. 0066-0/9/005-0$0.00/0 Copyright C 99, American Society for Microbiology In Vitro Antibacterial Activity of, a Carbapenem Antibiotic with Stability to Dehydropeptidase I JEFFREY R. EDWARDS,* PHILIP J. TURNER, CHRISTINE WANNOP, ERICA S. WITHNELL, AMANDA J. GRINDEY, AND KEVIN NAIRN Antibiotic Development Group, ICI Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, SKIO TG, England Received July 9/Accepted November 9, a new carbapenem antibiotic, was demonstrated to have potent antibacterial activity against a broad spectrum of aerobes, including Staphylococcus aureus, beta-hemolytic streptococci, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria spp., members of the family Enterobacternaceae, Pseudomonas spp., and gram-positive and gram-negative anaerobes in a collection of,0 unselected clinical isolates. At a concentration of,lg/ml, inhibited 90% of these strains. The spectkrum of activity of ceftazidiine and cefotaxime was more limited, and many of the Enterobacteriaceae and Pseudomonas spp. were resistant to these agentsv, piperacillin, or gentamncin. A collection of ofloxacin-resistant strains was inhibited by or imipenem at,ug/ml. was more active than metronidazole and clindamycin against anaerobes. Of the carbapenems, inipenem had greater activity against staphylococci, but was much more active against Haemophius, BranhameUa, and Neisseria spp. and all genera of Enterobacteriaceae tested. The MIC of for 90% of these strains ranged from <0.00 to,ug/ml. When tested against strains of Pseudomonas aeruginosa, inhibited 76% at,ug/ml but imipenem inhibited only 5% at this concentration. Both carbapenems exhibited similar activities against Bacteroides spp., but was more active than hinipenem against Clostridium spp. The MBC of was most commonly the same as or twice the MIC. and imipenem showed excellent activities against bacteria elaborating chromosome- or plasmid-mediated,b-lactamases, including those conferring resistance to broad-spectrum cephalosporins. The activity of was generally unaffected by the culture nmedium used, ph, 5% human serum, and inoculum size, but the susceptibility of Xanthomonas maltophilia was medium dependent., (R,5S,6S)-3-[(3S,5S)-5-dimethylcarbamoylpyrrolidin-3-ylthio] -6-[(R)--hydroxyethyl]--methyl-7-oxo-- azabicyclo[3..0]hept--ene--carboxylic acid, is a new carbapenem antibiotic. The chemical structure is presented in Fig.. Published information on is limited to structureactivity relationships (M. Sunagawa, H. Matsumura, T. Inoue, M. Fukasawa, and M. Kato, Program Abstr. 7th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 75, 97), activity against clinical isolates from Japan (M. Fukasawa, Y. Sumita, E. Tada, and T. Okuda, 7th ICAAC, abstr. no. 753, 97), activity against imipenem-resistant Pseudomonas aeruginosa (J. R. Edwards and C. Wannop, 7th ICAAC, abstr. no. 75, 97), affinities for penicillinbinding proteins and morphological changes (Y. Sumita, M. Fukasawa, and T. Okuda, 7th ICAAC, abstr. no. 756, 97), activity in vivo (T. Okuda, M. Fukasawa, T. Tanio, Y. Sumita, E. Tada, and T. Yukimatsu, 7th ICAAC, abstr. no. 757, 97), and animal pharmacokinetics and stability in vitro to renal dehydropeptidase I (T. Tanio, H. Nouda, E. Tada, T. Kohzuki, M. Kato, M. Fukasawa, T. Okuda, and S. Kamidono, 7th ICAAC, abstr. no. 75, 97). These data differentiated from imipenem on the basis of stability to human renal dehydropeptidase I, greater activity against gram-negative aerobes, and activity against imipenem-resistant P. aeruginosa. This investigation was made to compare the in vitro activity of with that of imipenemn and other selected agents (results were presented in part previously [J. R. Edwards, P. J. Turner, E. S. Withnell, and K. Nairn, 7th ICAAC, abstr. no. 755, 97]). The influence of miscella- * Corresponding author. 5 neous factors on antibacterial activity, the MBC, and the activity of against bacterial strains elaborating chromosome- or plasmid-mediated,b-lactamases were also examined. MATERIALS AND METHODS Antibiotics. was supplied by Sumitomo Pharmaceuticals Co. Ltd., Japan. All other antibiotics were obtained from commercial sources. The compounds included were imipenem, ceftazidime, cefotaxime, piperacillin, and gentamicin. and clindamycin were also tested against anaerobes, and methicillin and vancomycin were included when testing methicillin-resistant staphylococci. A commercially available combination of imipenem and cilastatin (Zienam, MSD) was used as the source of imipenem. Great care was taken to establish equivalence with a reference sample of imipenem and to show that the presence of cilastatin had no effect on the antibacterial activity of imipenem (3; unpublished observations). Bacterial strains. The clinical isolates tested were obtained mostly from hospitals in the United Kingdom since 93. This collection of,0 strains was unselected but contained many which were resistant to P-lactam antibiotics or gentamicin. MICs for members of the family Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp. of gentamicin (>,ug/ml), cefotaxi-me or ceftazidime (>,ug/ml), or piperacillin (>6 jxg/ml) were identified, and results were also analyzed separately. Further clinical isolates, elaborating plasmidmediated resistance to broad-spectrum cephalosporins or resistance to ofloxacin, were supplied by Danielle Sirot and G. Seibert, respectively. Laboratory strains elaborating characterized,-lactamases and derepresssed mutants overproducing their type I,B-

EDWARDS ET AL. OHCH3 H H CON ' < ~ ~ _/>P CH3 0 N COOH NH FIG.. Chemical structure of. lactamase () were tested for susceptibilities to, imipenem, and ceftazidime. A collection of 30 laboratory and reference strains of aerobic and anaerobic bacteria was used in tests to assess the influence of miscellaneous factors on the antibacterial activity of. This number was increased to 97 by the addition of clinical isolates when the MBC was determined. These strains were distributed as follows: 6 Staphylococcus aureus (methicillin susceptible), 5 coagulase-negative staphylococci (methicillin susceptible), 5 Enterococcus faecalis, 57 members of the Enterobacteriaceae ( species), 5 Acinetobacter spp., 7 P. aeruginosa, 5 Pseudomonas cepacia, Xanthomonas maltophilia, and 6 Bacteroides spp. Antimicrobial susceptibility tests. The majority of MICs were determined by the agar dilution technique by using Iso-Sensitest agar (IST) (Oxoid Ltd., London, England) with 5% horse blood added for fastidious organisms or anaerobes. Each antibiotic was prepared freshly in phosphate buffer ( M, ph 7.0) prior to use, and all MICs, with the exception of studies of inoculum effect, were determined by using an inoculum estimated by reference to an opacity standard to be 0 CFU per inoculum spot. Results were noted after overnight incubation at 37 C in an appropriate environment. Tests that included nonenteric streptococci or Haemophilus, Neisseria, or Branhamella spp. were incubated in 5% C0, and anaerobes were incubated in an atmosphere of 0% carbon dioxide and 90% hydrogen. The MIC was defined as the smallest concentration of antibiotic that prevented visible growth; a haze at the point of inoculation or one discrete colony was ignored. MBCs. MBCs were determined for 97 strains. MICs were first defined by the broth dilution technique with IST broth for aerobes and Wilkins-Chalgren broth for anaerobes. The bacterial inoculum (final) was 05 CFU/ml for both aerobes and anaerobes, judged by reference to an opacity standard. The MIC (micrograms per milliliter) was recorded after overnight incubation in an appropriate environment; the endpoint was no visible growth. The MBC was then determined by placing subculture from tubes with no growth, from the MIC range, onto agar plates and observing for growth after overnight incubation. The subculture included a dilution of /0, as an attempt to minimize carry-over of antibacterial activity, and the MBC was defined as the lowest compound concentration which reduced the starting inoculum by.3 logo; reduction in numbers was assessed by viable counts. ANTIMICROB. AGENTS CHEMOTHER. Studies of effects of miscellaneous factors on activity. The influence of human serum on MICs was assessed by using IST agar to which 0 or 5% of heat-inactivated pooled human serum had been added, and the effect of ph was assessed by using IST agar which had been adjusted during preparation to ph 6, 7, or. The influence of inoculum size was assessed by using a bacterial challenge of 0, 05, or 06 CFU per inoculum spot, again on IST agar. The following agar media were compared for use in MIC tests. Iso- Sensitest (Oxoid), Mueller-Hinton (Difco Laboratories, Detroit, Mich.), and Diagnostic Sensitivity Test (Oxoid) agars, each with and without 5% horse blood, were used in tests including both aerobes and anaerobes, and Wilkins-Chalgren and Brucella Base agars (Oxoid) plus 5% horse blood were used for anaerobes only. RESULTS AND DISCUSSION Activity against clinical isolates. The susceptibilities of the,0 unselected clinical isolates to and comparator agents are presented in Table. At,ug/ml, inhibited 90% of the strains of methicillin-susceptible S. aureus, and at,ug/ml it inhibited 90% of the methicillinresistant strains tested. Coagulase-negative staphylococci were a little less susceptible. was more active against staphylococci, the difference being greatest with methicillin-resistant strains. However, the therapeutic usefulness of any P-lactam antibiotic against methicillin-resistant strains of staphylococci may be questioned even if in vitro MICs are low (6). Nonenteric streptococci were very susceptible to all of the antibiotics tested; at,ug/ml inhibited 00% of 0 strains tested. E. faecalis was less susceptible; or imipenem at,ug/ml inhibited 00% of the strains, and piperacillin was the most active comparator. The nutritionally fastidious bacteria Haemophilus influenzae, Branhamella catarrhalis, and Neisseria spp. were more susceptible to than to imipenem. at,ug/ml was -fold more potent than imipenem against H. influenzae and at.0.00,ug/ml was -fold more potent than imipenem against B. catarrhalis and Neisseria meningitidis. At,ug/ml, inhibited most isolates of Escherichia coli, Citrobacter diversus, Citrobacterfreundii, Klebsiella pneumoniae, Klebsiella aerogenes, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, Proteus mirabilis, Proteus vulgaris, Morganella morganii, and Providencia spp. It was generally - to 3-fold more potent than imipenem. and gentamicin were the least potent of the comparator agents. All of the strains of Pseudomonas spp. tested were inhibited by at,ug/ml. The strains of P. aeruginosa were more susceptible to than to imipenem; 76% were inhibited by at pug/ml, but only 5% were inhibited by this concentration of imipenem. The numbers of the other Pseudomonas spp. tested were small; within the group of 7 strains tested, 9 strains of P. cepacia were inhibited by at or,ug of imipenem per ml. at,ug/ml inhibited 90% of the strains, but cefotaxime, piperacillin, and gentamicin were much less potent. All strains of Acinetobacter calcoaceticus were inhibited by at,ug/ml. (,Lg/ml) inhibited these strains, but the comparator agents were nmuch less active. was more active than metronidazole or clindamycin against Bacteroides fragilis subsp. fragilis and other Bacteroides spp. Against these species, and imipenem exhibited similar activites, but at.,ug/ml was at least eightfold more active than imipenem against Clostridium perfringens. at,ug/ml inhibited all strains of Clostridium difficile and at,ug/ml inhibited all Peptostreptococcus strains tested. This collection of,0 strains proved to contain many which were resistant to one or more of the comparator agents. Among members of the Enterobacteriaceae, there were 3 strains which showed resistance to broad-spectrum

VOL. 33, 99 IN VITRO ACTIVITY OF 7 Organism (no. tested) TABLE. Staphylococcus aureus (9) Staphylococcus aureus, methicillin resistant (0) Staphylococci, coagulase negative (73) Staphylococci, coagulase negative, methicillin resistant () Streptococcus pneumoniae (30) Streptococcus group A () Streptococcus agalactiae () Streptococcus equi () Streptococcus group G (5) Enterococcus faecalis () Activities of and comparator agents against,0 unselected clinical isolates Antibiotic Vancomycin Methicillin Vancomycin Methicillin Cefotazime MIC (Lg/ml)a Range 50% 90% - 0.0- -3 - - 0.0-> - - - - - - -6-0.0- so0.00- -3 - -3-6 - - -( - - - -> 0.0-3 s-0.00- - - - - - - <0.00- - - - - - - - - 0.0- - - -> -> - _0.00 _<0.00 0.0 _0.00 _0.00 _0.00 0.0 _0.00 _0.00 0.0 _0.00 _0.00 0.0 3 3 3 3 0.0 _0.00 _0.00 0.0 0.0 0.0 0.0 _0.00-0.00 0.0 > > 3 > Continued on following page

EDWARDS ET AL. Organism (no. tested) Haemophilus influenzae () Branhamella catarrhalis (3) Neisseria gonorrhoeae () Neisseria meningitidis (3) Escherichia coli () Citrobacter diversus (0) Citrobacterfreundii () Klebsiella pneumoniae (9) Klebsiella aerogenes (33) Klebsiella oxytoca (35) TABLE -Continued Antibiotic Ceftotaxime ANTIMICROB. AGENTS CHEMOTHER. MIC (WLg/ml)a Range 50% 90% <0.00- --0.00- - <0.00- <0.00- <0.00- - <0.00-0.0- <0.00- <0.00- <0.00- <0.00- <0.00- <0.00- <0.00- - <0.00- <0.00 0.0- <0.00- <0.00- <0.00-> <0.00-6 -> -> - - - - -.0.00- - -> - -> -> - - - - - - - - -> ->.0.00- - -> 0.0-6 -> - 0.0 0.0 0.0 <0.00 0.0.0.00 0.0 0.0 0.0 0.0 0.0 0.0 3 0.0.0.00.0.00 0.0 3 0.0 6 6 > 6 Continued on following page

VOL. 33, 99 Organism (no. tested) Enterobacter cloacae (70) Enterobacter aerogenes () Serratia marcescens (59) Proteus mirabilis (9) Proteus vulgaris (0) Morganella morganii (0) Providencia spp. (5)b Pseudomonas aeruginosa () Pseudomonas spp. (7)C TABLE -Continued Antibiotic Ceftotaxime IN VITRO ACTIVITY OF 9 MIC (,Lg/ml)a Range. 50% 90% - - -> -> -> - - - - -3 -> -6 - - - - -> -6 0.0- - 0.0- - - - - 0.0- - - - 0.0- - - a<0.00--6 - - - - - - -3 - - -6 _0.00-> -> -> - -> - '0.00- -6-0.0 _0.00 0.0 0.0 6 > 6 6 6 > 6 6 3 6 6 3 3 Continued on following page

0 EDWARDS ET AL. ANTIMICROB. AGENTS CHEMOTHER. Organism (no. tested) Xanthomonas maltophilia (5) Acinetobacter calcoaceticus subsp. anitratus (39) Bacteroides fragilis (56) Bacteroides spp. ()d Clostridium perfringens (5) Clostridium difficile () Peptostreptococcus spp. (0) TABLE -Continued AniitcMIC (,ug/mly' AntRb5otic Range 50%o 90% <0.00- -> <0.00-3 <0.00-6 <0.00- - - - -3 0.0-6 -> -6 <- <- -> -6 -> - ->.-.- -> <-6.-6 - <- < -.- '- s-.- <- - - 3-6 3- - - -3.- - <- <-.- s- - a 50% and 90%, MIC for 50 and 90%o of isolates, respectively. b Providencia alcalifaciens (n = ) and P. stuartii (n = 3). c Pseudomonas putida (n = 3), P. cepacia (n = 9), P. fluorescens (n = ), P. stutzeri (n = ), and P. acidivorans (n = ). d Bacteroides vulgatus (n = ), B. distasonis (n = 9), B. thetaiotaomicron (n = 7), and B. ovatus (n = ). 3 > s s. s s 3 6 3 S s > 6 > 6 > 3 6 > 3 s 6 3 cephalosporins, 66 which were resistant to piperacillin, and 5 which were resistant to gentamicin, all of which were inhibited by or imipenem at,ug/ml. Resistance to the other P-lactam antibiotics and gentamicin was seen most commonly among Enterobacter cloacae; K. oxytoca was also frequently resistant to gentamicin. There were, 3, and 9 strains of Acinetobacter spp. which were resistant to the cephalosporins, piperacillin, and gentamicin, respectively; all were inhibited by or imipenem at,ug/ml. Among the strains of P. aeruginosa tested, resistance to cefotaxime was recorded frequently. Seven ceftazidimeresistant strains were inhibited by at,ug/ml, piperacillin-resistant strains were inhibited by at,ug/ml, and 9 gentamicin-resistant strains were inhibited by at,ug/ml. The comparable results for imipenem were,, and,ug/ml, respectively. A further collection of

VOL. 33, 99 IN VITRO ACTIVITY OF TABLE. Activities of and imipenem against strains encoding plasmid-mediated resistance to broad-spectrum cephalosporins Strain 3-Lactamase MIC (jig/ml) specified Escherichia coli 90 TEM-3 (CTX-) 0.0 Klebsiella aerogenes 076 TEM-3 (CTX-) 0.0 Enterobacter aerogenes 07 TEM-3 (CTX-) 3 Serratia marcescens 0 TEM-3 (CTX-) 3 Klebsiella aerogenes 077 TEM-5 (CAZ-) Escherichia coli ATCC 59 0.0 clinical isolates resistant to broad-spectrum cephalosporins or ofloxacin was tested also. Four strains harboring plasmids encoding the TEM-3 (CTX-) P-lactamase and one encoding TEM-5 (CAZ-) were all inhibited by at jig/ml or by imipenem at,ug/ml (Table ). Members of the Enterobacteriaceae elaborating plasmid-mediated resistance to broad-spectrum cephalosporins are being reported with increasing frequency in Europe (, 5). Twenty ofloxacinresistant strains (five S. aureus, three Klebsiella spp., five Serratia spp., two Acinetobacter spp., and five P. aeruginosa) were inhibited by or imipenem at jig/ml. Activity against,l-lactamase-producing strains. and imipenem showed excellent activities against S. aureus, Enterobacteriaceae, and P. aeruginosa strains elaborating plasmid- or chromosome-mediated P-lactamases. was active against many of these strains but showed reduced activity against the Escherichia coli elaborating the OXA- enzyme and very poor activity (MIC,.3,ug/ml) against Enterobacteriaceae or P. aeruginosa strains derepressed for their chromosomally encoded type I P-lactamases (Table 3). Bactericidal activity. The MBC was defined for and imipenem against a collection of 97 strains comprising aerobic and anaerobic bacteria. The results, expressed as a multiple of the MIC, show that for most strains the MBC was the same as or twice the MIC of each compound (Table ). Values which were greater than twice the MIC were randomly distributed throughout the genera tested. Effects of miscellaneous factors on activity. The presence of 0 or 5% of human serum, a medium ph of 6, 7, or, or a bacterial challenge of 0, 05, or 06 CFU per inoculum spot did not significantly alter MICs of. Results were generally within the range of experimental error, and no trends were noted. An exception was seen in tests including X. maltophilia, the susceptibility of which was influenced by the choice of culture medium. When tested on Diagnostic TABLE 3. Activities of, imipenem, and ceftazidime against bacteria elaborating plasmid- or chromosome-encoded 3-lactamases Strain,-Lactamase specified MIC (,ug/ml) Staphylococcus aureus 7N PC 0.0 Escherichia coli J53. 0.0 TEM- TEM- OXA- OXA- OXA-3 SHV- HMS- 0.0 Pseudomonas aeruginosa PU PSE- PSE- PSE-3 PSE- Pseudomonas aeruginosa S (I+) I Pseudomonas aeruginosa SH (DR)' I 6 Citrobacterfreundii 00 (I') I 0.0 Citrobacterfreundii 03 (DR) I Enterobacter cloacae 09 (I+) I Enterobacter cloacae 0 (DR) I 3 Klebsiella oxytoca 055 Klebsiella oxytoca 056 IVc a Spontaneous mutants with derepressed (DR) expression of type I (I+),-lactamase, derived from corresponding wild-type inducible parents.

EDWARDS ET AL. Antibiotic TABLE. MBCs of and imipenem expressed as multiples of the MIC No. of strains for which MBC is (multiple of MIC): 57 30 9 7 3 Sensitivity Test agar, most strains were inhibited by SM- 733 at,ug/ml; on Mueller-Hinton agar, typical MICs were > jig/ml; and on IST agar, results ranged from.0.00 to,ug/ml. was inactive against of the 5 strains irrespective of the medium used. The initial report () showing the activity of imipenem against almost all clinically significant aerobic and anaerobic bacteria has been confirmed in numerous other publications. Results presented here for are in close agreement with those of Fukasawa et al. (7th ICAAC) and show that this new carbapenem has a spectrum of activity comparable to that of imipenem but with notable differences in activity against H. influenzae, B. catarrhalis, N. meningitidis, and members of the Enterobacteriaceae. These differences, primarily in potency, could assume significance if the pharmacokinetics of the two compounds are similar. The enhanced ANTIMICROB. AGENTS CHEMOTHER. activity against P. aeruginosa is the subject of further studies. ACKNOWLEDGMENTS We are indebted to T. D. Hennessey for critical reading of the manuscript and to Bev Hoult for the final presentation. LITERATURE CITED. Bauernfeind, A., and G. Horl. 97. Novel R-factor borne,blactamase of Escherichia coli conferring resistance to cephalosporins. Infection 5:57-59.. Curtis, N. A. C., R. L. Eisenstadt, C. Rudd, and A. J. White. 96. Inducible type I P-lactamases of Gram-negative bacteria and resistance to,-lactam antibiotics. J. Antimicrob. Chemother. 7:5-6. 3. Kahan, F. M., H. Kropp, J. G. Sundelof, and J. Birnbaum. 93. Thienamycin: development of imipenem-cilastatin. J. Antimicrob. Chemother. (Suppl D):-35.. Kropp, H., J. S. Kahan, F. M. Kahan, and J. Birnbaum. 90. MK07 (N-formimidoyl thienamycin): evaluation of in vitro and in vivo activities. Antimicrob. Agents Chemother. 7:993-000. 5. Labia, R., A Morand, M. Guionie, M. Heitz, and J. S. Pitton. 96. P-lactamases de Klebsiella oxytoca: etude de leur action sur les cephalosporins de troisieme generation. Pathol. Biol. 3:6-65. 6. Thornsbury, C. 9. The development of antimicrobial resistance in staphylococci. J. Antimicrob. Chemother. (Suppl. C):9-. Downloaded from http://aac.asm.org/ on April 0, 09 by guest