26. in Vitro selection Of enterobacter Cloacae with Cefepime, Meropenem, and Ceftazidime-avibactam Generate Diverse Resistance Mechanism
Authors: Castanheira Mariana , Lindley Jill , Roth Brieanna , Doyle Timothy B , Davis Andrew P , Sader Helio S 
Published: January 2021

Abstract

Background
Acquired β-lactamases are not a common β-lactam resistance mechanism in E. cloacae. In this species, porin mutations decreasing β-lactams permeability are associated with overexpression of the constitutive AmpC with or without increased efflux contributing to β-lactam resistance. We subjected 6 E. cloacae isolates to 10-day serial passage with cefepime (FEP), meropenem (MER), and ceftazidime-avibactam (CAZ-AVI) to evaluate resistance level and mechanism in the mutant strains.

Methods
Serial passaging was performed in broth microdilution (BMD). Isolated colonies growing in the highest antimicrobial concentrations were submitted to short-read whole genome sequencing (WGS) and analyzed for β-lactam resistance mechanisms. Final mutants displaying &gt; 2-fold changes from the baseline and baseline isolates were sequenced using long-read WGS for single nucleotide polymorphism (SNP) analysis.

Results
After 10-day passaging, 3 (50.0%) isolates displayed FEP resistance, but only 1 (15.0%) displayed resistance to MER or CAZ-AVI (Table). MIC changes were 4 to 64-fold for FEP, 16 to 64-fold for MER and 2 to 32-fold for CAZ-AVI. SNP analysis demonstrated that final resistant mutants had diverse mechanisms. MER and CAZ-AVI selected for AmpC mutants E61V, G213D, and V303E. OmpC and AcrA regulator alterations were noted in MER and CAZ-AVI mutants for 1 isolate. Mutations in the ABC transporter UrtB and the cell division inhibitor MioC were observed when 1 isolate was exposed to any of the 3 agents. Mutants for 1 isolate had deletion of OmpC (PCR confirmed). Mutations in the upstream region of various genes and gene regulators were observed. The highest CAZ-AVI MIC values were noted in an isolate displaying a mutation in an RND transported permease.
Table

Conclusion
FEP displayed more resistant mutants when compared to MER and CAZ-AVI. Despite the low MIC values of the mutant isolates for MER, the fold changes for this agent were higher than in FEP and CAZ-AVI. Resistance mechanisms in the mutants were complex. Only a few isolates had alterations in the genes usually associated with β-lactam resistance in E. cloacae. Further studies should be performed to evaluate if the mutations observed in vitro are present in clinical isolates resistant to these agents.

Disclosures
Mariana Castanheira, PhD, 1928 Diagnostics (Research Grant or Support)A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Amplyx Pharmaceuticals (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Fox Chase Chemical Diversity Center (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Paratek Pharma, LLC (Research Grant or Support)Pfizer (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Jill Lindley, Allergan (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Brieanna Roth, n/a, Allergan (Research Grant or Support) Timothy B. Doyle, Allergan (Research Grant or Support)Allergan (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Helio S. Sader, MD, PhD, A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Melinta (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Paratek Pharma, LLC (Research Grant or Support)Pfizer (Research Grant or Support)