Investigating Viable Monobactam and 𝜷 - lactamase Inhibitor - based Combinations Versus Pan - resistant K. pneumoniae Utilizing the Disc Diffusion Synergy Test Chloe A. Matecki, John F. Klem , Albert Chen, Brian T. Tsuji, Raymond Cha Center for Antimicrobial Pharmacodynamics, Department of Pharmacy Practice, School of Pharmacy and Pharmaceutical Sciences, U niv ersity at Buffalo • Carbapenem - resistant Enterobacteriales (CRE) are resistant to nearly all antimicrobial agents that are currently available for the treatment of positive cases • Recent estimates put the total of carbapenem - resistant K pneumoniae species at over 7 , 900 1 • A patient in Nevada passed after contracting strain 920 of K pneumoniae when it was found to be resistant to all 26 antimicrobial agents tested for susceptibility 2 • Resistance to K pneumoniae is fostered in large part due to the presence of bla NDM - 1 carbapenemase and extended - spectrum - beta - lactamase (ESBL) blaCTX - M - 15 2 , 3 , 4 • Precedence for using the combination of aztreonam with ceftazidime/avibactam synergistically comes from its success previously described using disc diffusion methods 3 , 4 • Existing literature and studies have not adequately addressed the need to find effective methods for treating superbug infections • This study aims to investigate the possible identification of novel combinations against the pan - resistant Nevada strain utilizing new beta - lactam combinations involving aztreonam and ceftazidime/avibactam to improve upon the already existing synergy of the combination • Effective combinations against pan - resistant K pneumoniae remains a significant public health priority • While some baseline inhibition was noted with the backbone of aztreonam and ceftazidime/avibactam, there was no augmentation of inhibition with any of the tested agents The loss of diameter when using the two antimicrobials thought to give the combination of aztreonam plus ceftazidime/avibactam its synergy is something that also brings into question the mechanistic utility of ceftazidime and double β - lactams • Limitations of this work include the lack of concentration array and modeling of the dynamic concentrations that exist in vivo Both of these offer opportunities to look further into the combinations, using the recent phase 1 trial of aztreonam plus ceftazidime/avibactam • Further investigations with avibactam in novel, expansive combinations in concert with rationalized and pharmacokinetically - optimized designs are warranted, as they will guide the future use of β - lactam and β - lactamase inhibitor combinations Study Design : Disc diffusion synergy testing Literature Search : • Baseline Term : Aztreonam+Ceftazidime - Avibactam • Exclusion criteria : Did not contain use of base triple therapy non - K pneumoniae organism of focus, use in determination of susceptibility in hospitals, use of Polymyxin B Disc Diffusion Synergy Test : • Concentrations : Clinical and Laboratory Standards Institute (CLSI) breakpoints were targeted for each agent 5 Created using 1 to 10 dilutions of agents from laboratory stores of the antimicrobial agents Bacterial Strain : A Pan - resistant (non - susceptible to 26 antibiotics) K pneumoniae strain from the CDC was used harboring bla NDM - 1 and bla CMY - 6 , bla CTX - M - 15 and bla NSHV - 28 • Plates : Cation - adjusted Mueller - Hinton agar plates were made in the laboratory via sterile procedures for the purpose of this experiment • Cultures : Colonies from a fresh overnight cultured plate of the Nevada CRE strain were resuspended and diluted to target an inoculum of 1 x 10 8 CFU/mL utilizing spectrophotometry • Interpretation of Results : Zone of inhibition diameters were measured using vernier calipers once the 16 - hour disc incubation was achieved Synergy was assessed through visual observation of a significant improvement of the zone of inhibition from the baseline of aztreonam plus ceftazidime/avibactam • Author and co - authors have no conflicts of interest to disclose • The MICs for K. pneumoniae were confirmed for all antimicrobials utilized in this experiment via broth microdilution in duplicate according to CLSI guidelines. • Validation was observed for the resistance of individual agents. • Synergy was not observed in and of the Aztreonam+Ceftazidime - Avibactam combinations • +0.2 mm was observed as the highest differential for imipenem. • - 0.2 mm loss of diameter was observed for gentamicin. • The double therapy of aztreonam plus avibactam did not produce the same diameter as aztreonam plus ceftazidime/avibactam • 4.9 mm was observed as the differential between the combinations • As the discs were impregnated with the recommended concentrations only, it is possible that higher concentrations of both the adjunctive agents and backbone aztreonam plus ceftazidime/avibactam using higher serial dilutions could produce meaningful expansions to the diameters. 1. Tan X, Kim HS, Baugh K, Huang Y, Kadiyala N, Wences M, Singh N, Wenzler E, Bulman ZP Therapeutic Options for Metallo - β - Lactamase - Producing Enterobacterales Infect Drug Resist 2021 ; 14 : 125 - 142 doi org/ 10 2147 /IDR S 246174 2. de Man TJB, Lutgring JD, Lonsway DR, et al Genomic Analysis of a Pan - Resistant Isolate of Klebsiella pneumoniae, United States 2016 mBio 2018 ; 9 ( 2 ) : e 00440 - 18 Published 2018 Apr 3 doi : 10 1128 /mBio 00440 - 18 3. Marshall, S , Hujer , A M , Rojas, L J , Papp - Wallace, K M , Humphries, R M , Spellberg , B , Hujer , K M , Marshall, E K , Rudin, S D , Perez, F , Wilson, B M , Wasserman, R B , Chikowski , L , Paterson, D L , Vila, A J , van Duin , D , Kreiswirth , B N , Chambers, H F , Fowler, V G , Jr, Jacobs, M R , ... Bonomo , R A ( 2017 ) Can Ceftazidime - Avibactam and Aztreonam Overcome β - Lactam Resistance Conferred by Metallo - β - Lactamases in Enterobacteriaceae? Antimicrobial agents and chemotherapy, 61 ( 4 ), e 02243 - 16 https : //doi org/ 10 1128 /AAC 02243 - 16 4. Smith NM, Boissonneault KR, Chen L, et al Mechanistic Insights to Combating NDM - and CTX - M - Coproducing Klebsiella pneumoniae by Targeting Cell Wall Synthesis and Outer Membrane Integrity Antimicrob Agents Chemother 2022 ; 66 ( 9 ) : e 0052722 doi : 10 1128 /aac 00527 - 22 5. CLSI Performance Standards for Antimicrobial Susceptibility Testing 32 nd ed CLSI supplement M 100 Wayne, PA : Clinical and Laboratory Standards Institute ; 2022 Figure 1. Literature search refinement process algorithm A literature search was performed to both assess previous studies or case reports that utilized combinations to combat CRE with the backbone of aztreonam plus ceftazidime/avibactam and the breadth of data available for combinations that had not yet been tested in vitro Agent(s) Zone of Inhibition (mm) Aztreonam plus avibactam 15.6 Aztreonam plus ceftazidime/avibactam 20.5 Aztreonam plus ceftazidime/avibactam + mecilinam 20.6 Aztreonam plus ceftazidime/avibactam + gentamicin 20.3 Aztreonam plus ceftazidime/avibactam + imipenem 20.7 Aztreonam plus ceftazidime/avibactam + ertapenem 20.6 Figure 3. Individual agent and double therapy controls Figure 2. Experimental triple and quadruple therapy discs Table 1. Zones of inhibition for experimental combinations and selected controls Contact Chloe Matecki at chloemat@buffalo.edu for any questions