Abstract

1. F1-1169 Tazobactam Piperacillin BLI-489 In Vitro Activities of Piperacillin in Combination with the Penem β-lactamase Inhibitor BLI-489 by Time-Kill Kinetic Studies P. J. Petersen, C. H. Jones, A. M. Venkatesan, and P. A. Bradford. Infectious Disease Research, Wyeth Research, Pearl River, N.Y. Abstract Background: The β-lactamase inhibitor, BLI-489 (BLI), has shown in vitro activity against molecular Class A, D and C enzymes and extended spectrum β-lactamases (ESBLs). In this study the in vitro activities of the combination of piperacillin (PIP) and BLI or tazobactam (TZB) were determined by time kill kinetics against ten well-characterized β-lactamase producing organisms. Methods: Time-kill assays were performed by the broth macrodilution method, as suggested by the CLSI guidelines. Flasks containing 50 mL of MHB with the appropriate antimicrobial agent were inoculated with 50 mL of the test organism to a density of approximately 106 CFU/mL. Aliquots were removed, diluted, and spiral plated to determine viable counts. Results: The combination of PIP:BLI-489, at 4 x MIC, demonstrated a 6 hour reduction in inoculum of 1.7 to 2.5 log10 CFU/ml. PIP:BLI-489 with an average decrease of 2.4 log10 CFU/ml, significantly reduced the initial inoculum of a Class A producing E. coli (TEM-1) and K. pneumoniae (SHV-11). PIP:BLI-489 also demonstrated a average reduction of 2.2 log10 CFU/ml against strains producing either TEM-10 or SHV-5 or CTX-M-5 ESBLs. In contrast, PIP:TZB at the same concentration failed to reduce the original inoculum for SHV-5 and CTX-M-5 producing pathogens. In addition, an average reduction of 2.3 log10 CFU/ml of viable bacterial counts were shown by the PIP:BLI-489 combination against Class C (AmpC or ACT-1) or Class D (OXA-1) enzyme producing strains. The PIP:TZB combination was not effective in maintaining the original inoculum concentration of the Class C producing isolates. Although regrowth was observed for many isolates tested by the 24 hour time point, the clinical relevance of this phenomenon is unknown. Conclusion: The PIP:BLI-489 combinations demonstrated an average 2.2 log10CFU/ml decrease in the initial inoculum by the 6 hour time point against the ten β-lactamase producing trains tested. The PIP:BLI-489 combination offers an advantage over the current commercial inhibitors and warrants further development. Antibacterial Activity of Piperacillin Alone and in Combination with Tazobactam (TZB) or BLI-489 Against K. pneumoniae GAR 7693 (SHV-11) Antibacterial Activity of Piperacillin Alone and in Combination with Tazobactam (TZB) or BLI-489 (BLI) Against E. coli GC 2814 (ACT-1) Antibacterial Activity of Piperacillin Alone and in Combination with Tazobactam (TZB) or BLI-489 (BLI) Against E. coli GAR 5946 (OXA-1) Materials and Methods Strains and Drugs All organisms used in this study were taken from the Wyeth Research culture collection: Escherichia coli GC 6265 (TEM-1, ClassA) Escherichia coli GC 2022(TEM-10, ClassA, ESBL) Klebsiella pneumoniae GAR 7693 (SHV-11) Klebsiella pneumoniae GAR 7978 (SHV-1, Class A and SHV-5, A (ESBL)) Salmonella entrica ser Typhimurium GC 4195 (CTX-M-5) Staphylococcus aureus GC 6468 ( β-lactamase positive, MSSA). Enterobacter cloacae GC 4142 (AmpC, ClassC) Pseudomonas aeruginosa GC 1764 (AmpC, Class C) Escherichia coli GAR 2814 (ACT-1, Class C) Escherichia coli GAR 5946 (OXA-1, Class D) Piperacillin (PIP) and tazobactam (TZB) powder were obtained from Wyeth Research, Pearl River, NY. The 6-methylidene penem molecule, BLI-489, is a novel compounds synthesized as a joint effort between Chemical & Screening Sciences, Wyeth Research (Pearl River, NY) and Wyeth-Lederle Japan (Saitama, Japan) MIC Determinations Susceptibility testing was performed according to the CLSI recommended broth microdilution test method (4,5). Inocula were prepared by direct colony suspension yielding a final well concentration of 1 – 5 x105 CFU/ml. Standard quality control strains E. coli ATCC 25922, E. coli ATCC 35218, P aeruginosa ATCC27853 and S. aureus ATCC29213 were included in each experiment. Time-kill Kinetic Assays Time-kill assays were performed by the broth macrodilution method, as suggested by the CLSI guidelines (9). The time-kill curves, against β-lactamase producing gram-negative and gram-positive bacteria were conducted with a final concentration of piperacillin, PIP:BLI-489 at four times the MIC for each test organism. The PIP:TZB combination was tested at the same concentrations used for PIP:BLI-489. Flasks containing 50 mL of MHB with the appropriate antimicrobial agent were inoculated with 50 mL of the test organism in logarithmic growth phase adjusted to a density of approximately 106 CFU/mL. Test flasks were incubated in a shaking water bath (150 rpm) at 35ºC in ambient air. Aliquots were removed at 0, 2, 4, 6 and 24 hours for the determination of viable counts. Serial dilutions were prepared in sterile 0.85% sodium chloride solution and diluted samples (0.05 ml) were plated in duplicate onto trypticase soy agar plates (Remel, Lenexa KS.) with a spiral plater (Microbiology International, Frederick MD). Using the Protocol plate reader (Microbiology International, Frederick MD) the total bacterial colony forming units/ml (log10CFU/ml) were determined after 18 hours incubation at 35ºC in ambient air. Killing curves were constructed by plotting the log10 CFU/ml versus time over 24 h, and the change in bacterial concentration was determined. Bactericidal activity was defined as a reduction of 99.9% ( 3 log10) of the total count of CFU/ml in the original inoculum. Bacteriostatic activity was defined as maintenance of the original inoculum concentration or a reduction of less than 99.9% ( 3 log10) of the total count of CFU/ml in the original inoculum. Introduction The increased resistance to -lactam antibacterial agents due to -lactamase enzymes in the hospital and community setting has been acknowledged as a global medical problem (3, 6, 7). The extensive use of third generation cephalosporin antibacterial agents has resulted in the emergence of diverse ESBL variants (Class A and Class D) as well as AmpC (Class C) enzymes. Further adding to the problem is the frequency of ESBL-producing isolates that are multiply-resistant to other classes of antimicrobial agents (i.e. aminoglycosides and quinolones) (6). The current -lactamase inhibitor combinations fail to target the majority of ESBL-producing strains and are marginally effective against Class C producing strains (8). In addition, cefepime, the most effective fourth generation cephalosporin, has been associated with clinical failures against a variety of pathogens (1, 2, 7, 10, 11). Recently, we have reported that a bicyclic substitution via a methylidene linkage to the 6-position of the penem molecule conferred activity as an inhibitor of Class A, Class C and Class D β-lactamase enzymes (12-14). The enhancement of the activity and efficacy of piperacillin was exhibited both in vitro and in vivo when administered in combination (14). These new -lactamase inhibitors offer a broader spectrum of activity compared to the current commercially available inhibitors. This study was performed to evaluate in vitro activities of the penem -lactamase inhibitor, BLI-489, in combination with piperacillin by Time-Kill Kinetic studies against β-lactamase producing organisms. Antibacterial Activity of Piperacillin Alone and in Combination with Tazobactam (TZB) or BLI-489 (BLI) Against E. coli GC 6265 (TEM-1) Antibacterial Activity of Piperacillin Alone and in Combination with Tazobactam (TZB) or BLI-489 (BLI) Against K. pneumoniae GAR 7978 (SHV-1, SHV-5) Antibacterial Activity of Piperacillin Alone and in Combination with Tazobactam (TZB) or BLI-489 Against E. cloacae GC 4142 (Amp-C) References 1. Barnaud, G., Y. Benzerara, J. Gravisse, L. Raskine, M. J. Sanson-Le Pors, R. Labia, and G. Arlet. 2004. Selection during cefepime treatment of a new cephalosporinase variant with extended-spectrum resistance to cefepime in an Enterobacter aerogenes clinical isolate. Antimicrob. Agents Chemother. 48:1040-2. 2. Barnaud, G., R. Labia, L. Raskine, M. J. Sanson-Le Pors, A. Philippon, and G. Arlet. 2001. Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate. FEMS Microb. Lett. 195:185-90. 3. Bradford, P. A. 2001. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev. 14:933-51. 4. CLSI. 2006. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standards -Seventh edition: M7-A7, vol. 26. Clinical and Laboratory Standards Institute, Wayne, PA. 5. CLSI. 2006. Performance standards for antimicrobial susceptibility testing, Sixteenth informational supplement M100-S16, vol. 26. Clinical Laboratory Standards Institute, Wayne, PA. 6. Gniadkowski, M. 2001. Evolution and epidemiology of extended-spectrum beta-lactamases (ESBLs) and ESBL-producing microorganisms. Clin. Microbiol. Infect. 7:597-608. 7. Kotapati, S., L. J. Kuti, C. H. Nightingale, and D. P. Nicolau. 2005. Clinical implications of extended spectrum b-lactamases (ESBL) producing Klebsiella species and Escherichia coli on cefepime effectiveness. J. Infect. 51:211-217. 8. Maiti, S. N., O. A. Phillips, R. G. Micetich, and D. M. Livermore. 1998. Beta-lactamase inhibitors: agents to overcome bacterial resistance. Curr. Med. Chem. 5:441-56. 9. NCCLS. 1999. Methods for determining bactericidal activity of antimicrobial agents: Approved guideline M26-A, vol. 19. National Committee for Clinical Laboratory Standards, Wayne, PA. 10. Song, W., E. S. Moland, N. D. Hanson, J. S. Lewis, J. H. Jorgensen, and K. S. Thomson. 2005. Failure of cefepime therapy in treatment of Klebsiella pneumoniae bacteremia. J. Clini. Microbiol. 43:4891-4. 11. Szabo, D., R. A. Bonomo, F. Silveira, A. W. Pasculle, C. Baxter, P. K. Linden, A. M. Hujer, K. M. Hujer, K. Deeley, and D. L. Paterson. 2005. SHV-type extended-spectrum beta-lactamase production is associated with Reduced cefepime susceptibility in Enterobacter cloacae. J. Clin. Microbiol. 43:5058-64. 12. Venkatesan, A. M., A. Agarwal, T. Abe, H. Ushirogochi, I. Yamamura, T. Kumagai, P. J. Petersen, W. J. Weiss, E. Lenoy, Y. Yang, D. M. Shlaes, J. L. Ryan, and T. S. Mansour. 2004. Novel imidazole substituted 6-methylidene-penems as broad-spectrum beta-lactamase inhibitors. Bioorg. Med. Chem. 12:5807-17. 13. Venkatesan, A. M., Y. Gu, O. Dos Santos, T. Abe, A. Agarwal, Y. Yang, P. J. Petersen, W. J. Weiss, T. S. Mansour, M. Nukaga, A. M. Hujer, R. A. Bonomo, and J. R. Knox. 2004. Structure-activity relationship of 6-methylidene penems bearing tricyclic heterocycles as broad-spectrum beta-lactamase inhibitors: crystallographic structures show unexpected binding of 1,4-thiazepine intermediates. J. Med. Chem. 47:6556-68. 14. Weiss, W. J., P. J. Petersen, T. M. Murphy, L. Tardio, Y. Yang, P. A. Bradford, A. M. Venkatesan, T. Abe, T. Isoda, A. Mihira, H. Ushirogochi, T. Takasake, S. Projan, J. O'Connell, and T. S. Mansour. 2004. In vitro and in vivo activities of novel 6-methylidene penems as beta-lactamase inhibitors. Antimicrob. Agents Chemother. 48:4589-96. Time Point Dilution Conclusions • The combination of PIP:BLI-489 demonstrated an average 2.2 log10 CFU/ml decrease in the initial inoculum concentration. • The PIP:BLI-489 combination demonstrated a significant reduction in CFU against bacteria that produced Class A ESBL and Class C enzymes. • The PIP:BLI showed a >2log10 reduction in 6 hours against a Class D producing pathogen. • When tested at the same concentration the PIP:BLI-489 showed a larger decrease in viable bacteria then PIP:TZB. Antibiotic and Pathogen Spiral Plating Antibacterial Activity of Piperacillin Alone and in Combination with Tazobactam (TZB) or BLI-489 (BLI) Against E. coli GC 2022 (TEM-10) Antibacterial Activity of Piperacillin Alone and in Combination with Tazobactam (TZB) or BLI-489 Against S. enterica ser. Typhimurium GC 4195 (CTX-M-5) Antibacterial Activity of Piperacillin Alone and in Combination with Tazobactam (TZB) or BLI-489 (BLI) Against P.aeruginosa GC 1764 (Amp-C) CFU Determination and Kill-Curve Construction Kill-Curve Kinetics