Mechanistic Insights to Combating Enterobacterales with Moderate- to High- Risk of AmpC Beta-lactamase Production in Deep Seated Infections

Abstract Background: Limited data suggest that serious infections caused by Enterobacterales with a moderate to high risk of clinically significant AmpC production (AmpC-PE) (e.g., Enterobacter cloacae, K/ebsiella aerogenes, Citrobacter freundii) can be successfully treated with cefepime (FEP) if the FEP minimum inhibitory concentration (MIC) is :::;2 μg/ml. However, isolates with a cefepime-susceptible dose-dependent (SDD) MIC of 4-8 μg/ml should receive a carbapenem due to target attainment and extended-spectrum ~-lactamase (ESBL) concerns. Notably, recent retrospective in a large patient cohort data failed to identify a difference in 30-day mortality between patients infected with FEP-SDD isolates receiving dose-optimized FEP or a carbapenem; however, in multivariable weighted Cox regression models, ceftriaxone (CRO)resistant AmpC and deep-seated infections were identified as independent risk factors of mortality while prolonged infusion beta-lactam was protective. However, pharmacokinetic/pharmacodynamic (PK/PD) data evaluating human-simulated, dose-optimized regimens of FEP versus a carbapenem to treat deep-seated FEP-SDD AmpC-PE is lacking to validate available clinical data. Thus, the purpose of this study is to evaluate and rationally optimize FEP and carbapenem regimens against well-described clinical blood isolates of Enterobacter cloacae by use of well-established and validated high inoculum PK/PD simulated endocardial vegetation (SEV) ex vivo models. Methods: A total of six well-characterized E. cloacae isolates will be selected for testing in 96 h PK/PD ex vivo SEV models. Isolates phenotypic and genotypic characteristics will be representative of that routinely seen clinically (e.g., CRO-susceptible vs. resistant, FEP-SDD, carbapenem-resistant vs. susceptible, and common beta-lactamase genes [CTX-M, SHV-like, KPC]). Human-simulated dose optimized regimens of FEP (2 g q8h via 3 h infusion), meropenem (2 g q8h via 3 h infusion), and ertapenem (1 g q24h via 30 min infusion) will be infused in parallel experiments against each isolate for a total of 18 models. Additionally, we will carry-out transcriptome analysis of beta-lactamase gene expression for each model. An exposureresponse relationship (Emax model) will be used to evaluate ex vivo efficacy assessed as the change in bacterial burden at 96 h compared to O h controls. The association between antibiotic regimen, reduction in bacterial burden (changes in colony-forming units [CFU] per ml), and beta-lactamase expression for each isolate will be evaluated over the course of the SEV model duration (0, 4, 8, 24, 32, 48, 72, 96 h) using Pearson correlation coefficient and ANOVA with Tu key''s post hoc test (P<0.05). Discussion: The experimental methodologies herein are well-established and primed to generate valuable preclinical data to inform future in vivo and clinical trial design for intervention to amplify therapy and improved health outcomes of patients with deep-seated AmpC-PE infections.