1230. Evaluation of the Interplay Between β-lactamases and Porin Production on β-Lactam MICs in Klebsiella pneumoniae

Abstract Background K. pneumoniae can emerge resistant to β-lactam antibiotics through the production of β-lactamase enzymes and/or loss of the outer membrane porins, OmpK35, OmpK36, and/or PhoE. While both mechanisms are hypothesized to work synergistically, β-lactamases have been the focus of previous studies. As a result, the contribution of outer membrane porin loss to the β-lactam minimum inhibitory concentration (MIC) is unknown. The objective of this study was to evaluate the contribution of specific β-lactamases and porin production to β-lactam susceptibility. We hypothesize that production of a β-lactamase in a clinical isolate deficient in 3 major porins will result in higher β-lactam MICs but not always a resistant phenotype. Methods The structural gene and promoter of CTX-M-14, CTX-M-15, and CMY-2 were cloned into a low copy number vector and transformed into Kp 23, a wild-type clinical isolate, and KPM 20, a clinical isolate deficient in OmpK35/36 and PhoE. MICs to ceftolozane/tazobactam, cefotaxime, ceftazidime, cefepime, and meropenem were determined by E-test. Kp 23 and KPM 20 were characterized by Western blot and whole genome sequencing. Results Production of CMY-2 alone led to a resistant phenotype for ceftolozane/tazobactam, cefotaxime, and ceftazidime regardless of porin production (Figure 1). CMY-2 production in KPM 20 resulted in non-susceptibility to meropenem. Both clones were susceptible to cefepime. Production of CTX-M-14 and CTX-M-15 in Kp 23 resulted in only cefotaxime resistance. Production of CTX-M-14 and CTX-M-15 in KPM 20 resulted in isolates non-susceptible to all antibiotics tested. Figure 1. MICs of K. pneumoniae clones against panel of β-lactam antibiotics. Conclusion When evaluating clinical isolates, it is impossible to determine the contribution of individual resistance mechanisms in the susceptibility pattern. This study demonstrated that resistance is not solely dependent on the β-lactamase produced and that the impact of porin deficiency varies with the antibiotic being evaluated. These data suggest that antibiotic selection may be more nuanced and that a broader range of therapeutics may be available given the appropriate diagnostic tools. Understanding the contributions of all resistance mechanisms is necessary to inform selection of the most appropriate antibiotic therapy. Disclosures Nancy D. Hanson, PhD, Merck (Grant/Research Support)

into Kp 23, a wild-type clinical isolate, and KPM 20, a clinical isolate deficient in OmpK35/36 and PhoE. MICs to ceftolozane/tazobactam, cefotaxime, ceftazidime, cefepime, and meropenem were determined by E-test. Kp 23 and KPM 20 were characterized by Western blot and whole genome sequencing.
Results. Production of CMY-2 alone led to a resistant phenotype for ceftolozane/tazobactam, cefotaxime, and ceftazidime regardless of porin production ( Figure  1). CMY-2 production in KPM 20 resulted in non-susceptibility to meropenem. Both clones were susceptible to cefepime. Production of CTX-M-14 and CTX-M-15 in Kp 23 resulted in only cefotaxime resistance. Production of CTX-M-14 and CTX-M-15 in KPM 20 resulted in isolates non-susceptible to all antibiotics tested. Conclusion. When evaluating clinical isolates, it is impossible to determine the contribution of individual resistance mechanisms in the susceptibility pattern. This study demonstrated that resistance is not solely dependent on the β-lactamase produced and that the impact of porin deficiency varies with the antibiotic being evaluated. These data suggest that antibiotic selection may be more nuanced and that a broader range of therapeutics may be available given the appropriate diagnostic tools. Understanding the contributions of all resistance mechanisms is necessary to inform selection of the most appropriate antibiotic therapy.

Session: P-72. Resistance Mechanisms
Background. β-lactamase-producing Enterobacterales (Ebact) frequently co-carry resistance to antimicrobials from other classes, limiting treatment options. Avibactam (AVI) inhibits class A, class C, and some class D serine β-lactamases, while aztreonam (ATM) is refractory to hydrolysis by class B metallo-β-lactamases (MBLs). ATM-AVI is being developed for use against drug-resistant isolates of Ebact, especially those co-producing MBLs and serine β-lactamases. This study evaluated the in vitro activity of ATM-AVI and comparators against Ebact collected in 2017-2019 from patients with lower respiratory tract infections (LRTI) as part of the Antimicrobial Testing Leadership and Surveillance (ATLAS) program.
Methods. Non-duplicate clinical isolates were collected in 52 countries in Europe, Latin America, Asia/Pacific (excluding mainland China and India), and Middle East/ Africa. Susceptibility testing was performed by CLSI broth microdilution and interpreted using CLSI 2021 and FDA (tigecycline) breakpoints. ATM-AVI was tested at a fixed concentration of 4 µg/mL AVI. MDR was defined as resistant (R) to ≥3 of 7 sentinel drugs: amikacin, aztreonam, cefepime, colistin, levofloxacin, meropenem, and piperacillin-tazobactam. PCR and sequencing were used to determine the β-lactamase genes present in all isolates with meropenem MIC >1 µg/mL, and Escherichia coli, Klebsiella spp. and Proteus mirabilis with ATM or ceftazidime MIC >1 µg/mL.

Results Table
Conclusion. Based on MIC 90 values, ATM-AVI was the most potent agent tested against drug-resistant and MBL-positive subsets of Ebact collected from LRTI. The promising in vitro activity of ATM-AVI warrants further development of this combination for treatment of LRTI caused by drug-resistant Ebact.
Disclosures  Background. Cefiderocol (CFDC) represents a new addition to the antimicrobial armamentarium with broad activity against Gram-negative bacteria (GNB). CFDC remains stable to hydrolysis in the presence of serine β-lactamases (ESBLs, KPC and OXA-type carbapenemases) and metallo-β-lactamases. The CFDC and comparator activities were analyzed against Enterobacterales (ENT), including molecularly characterized carbapenem-resistant isolates (CRE), as a part of the SENTRY Antimicrobial Surveillance Program in the USA.

In Vitro Activity of Cefiderocol and Comparator Agents against Molecularly characterized Carbapenem-resistant Enterobacterales Clinical Isolates Causing Infection in United States
Methods. 4,053 ENT were collected from 31 sites in 2020. Susceptibility testing was performed by broth microdilution and CFDC testing used iron-depleted media. CLSI/FDA breakpoints were used. Isolates displaying MIC values ≥4 µg/mL for imipenem (excluded for P. mirabilis, P. penneri and indole-positive Proteus) or meropenem (MER) were subjected to genome sequencing and screening of β-lactamase genes.
Conclusion. The CFDC activity was consistent, regardless of phenotypes or genotypes, including against isolates carrying genes other than bla KPC , where approved β-lactam/β-lactamase inhibitor combinations showed limited activity. These data confirm CFDC as an important option for the treatment of infections caused by ENT and resistant subsets.