OXA-23 β-Lactamase Overexpression in Acinetobacter baumannii Drives Physiological Changes Resulting in New Genetic Vulnerabilities

β-Lactamase expression is the major mechanism of resistance to penicillins, cephalosporins, and carbapenems in the multidrug-resistant (MDR) bacterium Acinetobacter baumannii. In fact, stable high-level expression of at least one β-lactamase has been rapidly increasing and reported to occur in up to 98.5% of modern A. baumannii isolates recovered in the clinic. Moreover, the OXA-51 β-lactamase is universally present in the A. baumannii chromosome, suggesting it may have a cellular function beyond antibiotic resistance. However, the consequences associated with OXA β-lactamase overexpression on A. baumannii physiology are not well understood. Using peptidoglycan composition analysis, we show that overexpressing the OXA-23 β-lactamase in A. baumannii drives significant collateral changes with alterations consistent with increased amidase activity. Consequently, we predicted that these changes create new cellular vulnerabilities. As proof of principle, a small screen of random transposon insertions revealed three genes, where mutations resulted in a greater than 19-fold loss of viability when OXA-23 was overexpressed. The identified genes remained conditionally essential even when a catalytically inactive OXA-23 β-lactamase was overexpressed. In addition, we demonstrated a synergistic lethal relationship between OXA-23 overexpression and a CRISPR interference (CRISPRi) knockdown of the essential peptidoglycan synthesis enzyme MurA. Last, OXA-23 overexpression sensitized cells to two inhibitors of peptidoglycan synthesis, d-cycloserine and fosfomycin. Our results highlight the impact of OXA-23 hyperexpression on peptidoglycan integrity and reveal new genetic vulnerabilities, which may represent novel targets for antimicrobial agents specific to MDR A. baumannii and other OXA β-lactamase-overexpressing , while having no impact on the normal flora. Acinetobacter baumannii has become a serious pathogen in both hospital and community settings. The β-lactam class of antibiotics is a primary treatment option for A. baumannii infections, and expression of β-lactamases is the most frequent mechanism of resistance in this bacterium. New approaches to treating multidrug-resistant A. baumannii strains are needed. In this study, we demonstrate that overexpressing the OXA-23 β-lactamase leads to significant collateral changes, where peptidoglycan structure is altered. We have identified genes that become selectively essential in OXA-23-expressing strains and confirmed the