[Reviewer1]Please see comment 21 above. Please confirm if the difference between this table and the table in the abstract are correct

Contact information: Activity of -avibactam versus , including multiple-drug IHMA, Inc. 2122 Palmer Drive resistant isolates in Europe - the 2012 INFORM programme Schaumburg, IL 60173 P1782 USA Sam Bouchillon1, Daryl Hoban1, Meredith Hackel1, Robert Badal1, Ian Morrissey2, and Wright W. Nichols3 Phone: +1.847.303.5003 1International Health Management Associates, Schaumburg, IL, USA Fax: +1.847.303.5601 2 IHMA Europe Sàrl, Epalinges, Switzerland www.ihmainc.com 3AstraZeneca Pharmaceuticals, Waltham, MA, USA

Revised Abstract Results Conclusions

Objectives: Avibactam is a novel investigational non-β-lactam β-lactamase inhibitor that is being developed for use in  Activity of various antimicrobials used to treat P. aeruginosa infections combination with ceftazidime (CAZ). Avibactam does not have any clinically meaningful intrinsic antibacterial activity, Table 1. S/I/R, MIC50s, and MIC90s of agents with antipseudomonal activity from 707 P. Table 2. S/I/R, MIC50s, and MIC90s of agents when tested against 110 ceftazidime non-susceptible but inhibits Ambler class A β-lactamases including extended-spectrum enzymes (ESBLs) and KPCs, class C β- aeruginosa clinical isolates collected from Europe in 2012. P. aeruginosa clinical isolates collected from Europe in 2012. varied greatly, with amikacin showing the lowest non-susceptible rate lactamases, and some class D enzymes. It thus restores the activity of CAZ against difficult to treat Gram-negative Antimicrobial Susceptible Intermediate Resistant MIC MIC Minimum Maximum pathogens such as Pseudomonas aeruginosa (PA) including multiple-drug resistant (MDR) isolates. 50 90 of 11.6% and at the highest non-susceptible rate of 96.5% Ceftazidime-avibactam na* na na 4 16 1 > 128 Methods: 707 clinically relevant PA from multiple sources were collected in 2012 at 62 centres in 17 European Antimicrobial Susceptible Intermediate Resistant MIC50 MIC90 Minimum Maximum Amikacin 60.9 11.8 27.3 8 > 32 1 > 32 from all isolates evaluated, indicating that current therapeutic choices countries. MICs were measured as specified by CLSI broth microdilution and interpreted following EUCAST 2013 Ceftazidime-avibactam na* na na 2 8 0.06 > 128 Aztreonam 0 39.1 60.9 32 128 8 > 128 guidelines. Amikacin 88.5 3.7 7.80 4 16 < 0.25 > 32 for this pathogen continue to diminish. Results: The in vitro activity of ceftazidime-avibactam and 9 comparators are presented in the following table for all PA, Aztreonam 3.5 78.4 18.1 8 32 < 0.015 > 128 32.7 0 67.3 16 >16 2 >16 -non-susceptible (MEM-NS), ceftazidime-non-susceptible (CAZ-NS), and MDR PA isolates: Cefepime 84.0 0 16.0 4 16 < 0.12 > 16 Ceftazidime 0 0 100 32 64 16 > 128  The large number of P. aeruginosa strains non-susceptible to Ceftazidime 84.4 0 15.6 2 32 0.06 >128 20.9 32.7 46.4 4 > 4 0.12 > 4 ceftazidime and meropenem (110 and 158, respectively) should be of All P. aeruginosa MEM-NS PA CAZ-NS PA MDR PA Doripenem 71.2 15.8 13.0 0.5 >4 0.03 >4 41.8 8.2 50.0 >8 >8 1 >8 (n=707) (n=158) (n=110) (n=152) Imipenem 76.8 4.8 18.4 2 >8 0.12 >8 Levofloxacin 28.2 8.2 63.6 > 4 > 4 < 0.03 > 4 concern to practitioners, whose patient population may contain many of Meropenem 34.6 36.4 29.1 8 >8 0.25 >8 Drug MIC90 %Sus MIC90 %Sus MIC90 %Sus MIC90 %Sus Levofloxacin 66.3 6.8 26.9 0.5 > 4 < 0.03 > 4 - 10.0 0 90.0 128 > 128 4 > 128 these strains. Ceftazidime-Avibactam 8 na 8 na 16 na 8 na Meropenem 77.6 12.0 10.3 0.5 >8 < 0.06 > 8 * Interpretive criteria have not been established for ceftazidime-avibactam  MDRPA isolates demonstrate a markedly higher proportion inhibited Ceftazidime 32 84.4 64 54.4 64 0 64 36.8 Piperacillin-tazobactam 78.9 0 21.1 8 128 < 0.25 > 128 * Interpretive criteria have not been established for ceftazidime-avibactam Amikacin 16 88.5 >32 64.6 >32 60.9 >32 57.9 Table 4. Cumulative frequency distribution of ceftazidime-avibactam MICs for all P. aeruginosa by ceftazidime-avibactam at ≤ 8 mg/L than by ceftazidime alone Aztreonam 32 3.5 64 0 128 0 64 0.1 and strains non-susceptible to ceftazidime and meropenem. (90.8% vs 36.8%, respectively). Cefepime 16 84 >16 52.5 >16 32.7 >16 32.2 Doripenem >4 71.2 >4 1.3 >4 20.9 >4 14.5 mg/L  Ceftazidime-avibactam inhibited 98% of all P. aeruginosa at an MIC of Imipenem >8 76.8 >8 10.8 >8 41.8 >8 32.2 N 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 > 64 Table 3. S/I/R, MIC s, and MIC s of agents when tested against 158 meropenem non-susceptible <8 mg/L. Similar results were seen for ceftazidime-avibactam against Levofloxacin >4 66.3 >4 25.3 >4 28.2 >4 14.5 50 90 All P. aeruginosa 707 P. aeruginosa clinical isolates collected from Europe in 2012. Meropenem >8 77.7 >8 0 >8 34.6 >8 25.7 Ceftazidime-Avibactam N 1 2 1 17 124 365 115 68 5 4 3 2 strains non-susceptible to ceftazidime (87.3% inhibited at MIC of <8 Piperacillin-Tazobactam 128 78.9 >128 37.3 >128 10.0 >128 15.1 Antimicrobial Susceptible Intermediate Resistant MIC50 MIC90 Minimum Maximum Pct 0.1 0.4 0.6 3 20.5 72.1 88.4 98 98.7 99.3 99.7 100 mg/L) and against strains non-susceptible to meropenem (91.8% Ceftazidime-avibactam na* na na 4 8 1 > 128 Cetazidime NS 110 Conclusions: Ceftazidime-avibactam was the most active in vitro antimicrobial agent against the combined inhibited at MIC of <8 mg/L). These results are consistent with other Amikacin 64.6 8.9 26.6 8 > 32 0.5 > 32 Ceftazidime-Avibactam N 3 24 31 38 5 4 3 2 P. aeruginosa collected in Europe, including non-susceptible phenotypes. Avibactam restored the in vitro Aztreonam 0 49.4 50.6 32 64 4 > 128 activity of ceftazidime, lowering the MIC values 4- to 8-fold against P. aeruginosa, including meropenem- published studies [5]. 90 Cefepime 52.5 0 47.5 8 >16 0.5 >16 Pct 2.7 24.5 52.7 87.3 91.8 95.5 98.2 100 and ceftazidime-non-susceptible, and multidrug-resistant isolates. Ceftazidime 54.4 0 45.6 8 64 1 > 128 Meropenem NS 158  Of equal concern is that 21.5% of P. aeruginosa isolates in this study Doripenem 1.3 41.8 57.0 >4 >4 0.5 >4 Ceftazidime-Avibactam N 9 36 52 48 5 3 3 2 exhibited multi-drug resistant properties and each antimicrobial Introduction Imipenem 10.8 14.6 74.7 >8 >8 0.12 >8 Pct 5.7 28.5 61.4 91.8 94.9 96.8 98.7 100 Levofloxacin 25.3 11.4 63.3 > 4 > 4 < 0.03 > 4 evaluated showed a significant reduction in susceptibility, with the Pseudomonas aeruginosa is the most common pathogen isolated from hospitalized patients who experience extended stays (>1 week) and is Meropenem 0 53.8 46.2 8 >8 4 >8 Figure 1. MIC frequency distribution comparison of ceftazidime-avibactam and other anti- exception of ceftazidime-avibactam, the MIC of which remained at 8 frequently associated with nosocomial infections. P. aeruginosa infections have been associated with a variety of organs and organ systems Piperacillin-tazobactam 37.3 0 62.7 32 > 128 4 > 128 90 and are especially life-threating to the patient with compromised host defense mechanisms[1]. Treating P. aeruginosa infections has pseudomonal antimicrobials for 152 MDRPA* clinical isolates collected from Europe. * Interpretive criteria have not been established for ceftazidime-avibactam generally been problematic over the years, since there are few agents that are effective against this organism. In recent years, the mg/L. antimicrobial treatment options for practitioners have begun to diminish even further with the increased frequency of multi-drug-resistant P. aeruginosa (MDRPA).  In summary, the new agent ceftazidime-avibactam exhibited in vitro

The AstraZeneca Global Surveillance Study is designed to evaluate current resistance rates of commonly used antimicrobials against a activity against even the most problematic P. aeruginosa phenotypes, variety of pathogens from diverse geographical populations, including diverse demographic venues. Of significant importance in this study is the evaluation of a new non-β-lactam β-lactamase inhibitor – avibactam. Avibactam, combined with the anti-pseudomonal activity of including MDR populations. These findings support the rationale for ceftazidime, was compared to other antimicrobials against P. aeruginosa including MDRPA strains. In this report we compared the susceptibility and resistance levels of recent (2012) P. aeruginosa strains from infections found in hospitalized patients in European countries. performing clinical studies of ceftazidime-avibactam in patients Table 5. S/I/R, MIC50s, and MIC90s of agents when tested against 152 MDRPA** clinical isolates collected from Europe in 2012. infected by P. aeruginosa.

Materials and Methods Antimicrobial Susceptible Intermediate Resistant MIC50 MIC90 Minimum Maximum Ceftazidime-avibactam na* na na 4 8 1 > 128 References • 1,558 Pseudomonas aeruginosa non-duplicate isolates were collected from 132 sites in a 2012 global surveillance program. 707 isolates Amikacin 57.9 9.9 32.2 8 > 32 0.5 > 32 of P. aeruginosa came from European sites. Each site collected and identified consecutive fresh clinical isolates from documented intra- Aztreonam 0.10 30.9 69.1 32 64 4 > 128 1. Obritsch, Marilee D., Fish, Douglas N., et al. Nosocomial Infections Due to Multidrug Pseudomonas aeruginosa: Epidemiology and Treatment Options. abdominal infections (IAI), urinary tract infections (UTI), skin and skin structure infections (SSSI), and lower respiratory tract infections Cefepime 32.2 0 67.8 16 >16 2 >16 Pharmacotherapy 2005; 25(10):1353-1364. (LRTI). Only one isolate of the same species per patient was included in the surveillance study. Each participating site was provided a 2. Clinical Laboratory Standards Institute. 2012. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standards target list of numbers of isolates from selected species. Ceftazidime 36.8 0 63.2 16 64 1 > 128 -- Ninth Edition. CLSI document M07-A9. Clinical and Laboratory Standards Institute (CLSI), Wayne, PA 19087-1898 USA. • Isolates were identified to the species level and sent to a central lab, International Health Management Associates, Inc., Schaumburg, IL, Doripenem 14.5 34.2 51.3 >4 >4 0.12 >4 3. The European Committee on Antimicrobial Susceptibility Testing – EUCAST Clinical Breakpoints, January 2013; http://www.eucast.org/clinical_breakpoints/ USA (IHMA) for susceptibility testing and confirmation of identification using matrix-assisted laser desorption ionization-time of flight Imipenem 32.2 9.2 58.6 >8 >8 1 >8 4. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. CLSI (MALDI-TOF) mass spectrometry (Bruker Daltronics, Bremen, Germany). Levofloxacin 14.5 9.2 76.3 > 4 > 4 < 0.03 > 4 Document M100-S24 (ISBN 1-56238-898-3). Clinical Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 • Minimum inhibitory concentrations (MICs) were determined by the Clinical and Laboratory Standards Institute (CLSI) recommended broth USA, 2014. Meropenem 25.7 32.9 41.5 8 >8 < 0.06 > 8 microdilution testing method using panels prepared and frozen at IHMA [2]. Avibactam was tested at a set concentration of 4 mg/L. MIC 5. Levasseur P, Girard AM, Claudon M, Goossens H, Black MT, Coleman K, Miossec C. In vitro activity of the ceftazidime-avibactam combination against interpretive criteria followed EUCAST 2013 guidelines [3]. No breakpoints have been established for ceftazidime-avibactam. Piperacillin-tazobactam 15.1 0 84.9 64 > 128 < 0.25 > 128 Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother. 2012;56:1606–1609. • MDR P. aeruginosa is defined as resistance to three or more antimicrobial classes. * Interpretive criteria have not been established for ceftazidime-avibactam * MDR - multi-drug resistant to 3 or more antimicrobial classes 6. Flamm RK, Stone GG, Sader HS, Jones RN, Nichols WW. Avibactam reverts the ceftazidime MIC90 of European Gram-negative bacterial clinical isolates to • Quality controls (QC) were performed on each day of testing using appropriate ATCC control strains, following CLSI guidelines [4]. ** MDR - resistant to 3 or more antimicrobial classes CAZ-AVI, ceftazidime-avibactam; Pip-Tazo, piperacillin-tazobactam. the epidemiological cut-off value. J. Chemother. 2014; Published on-line Oct-11 2013.

Presented at the 24th European Congress of Clinical Microbiology and Infectious Diseases, Barcelona, Spain, 10–13 May 2014. This study at IHMA was supported by AstraZeneca Pharmaceuticals LP, which also included compensation fees for services in relation to preparing the abstract/poster. W. W. Nichols is an employee of AstraZeneca. We would like to thank those investigators from Europe who contributed to this global study.