OXA β-lactamases from <i>Acinetobacter</i> spp. are membrane bound and secreted into outer membrane vesicles

β-lactamases from Gram-negative bacteria are generally regarded as soluble, periplasmic enzymes. NDMs have been exceptionally characterized as lipoproteins anchored to the outer membrane. A bioinformatics study on all sequenced β-lactamases was performed that revealed a predominance of putative lipidated enzymes in the Class D OXAs. Namely, 60% of the OXA Class D enzymes contain a lipobox sequence in their signal peptide, that is expected to trigger lipidation and membrane anchoring. This contrasts with β-lactamases from other classes, which are predicted to be mostly soluble proteins. Almost all (>99%) putative lipidated OXAs are present in <i>Acinetobacter</i> spp. Importantly, we further demonstrate that OXA-23 and OXA-24/40 are lipidated, membrane-bound proteins in <i>Acinetobacter baumannii</i>. In contrast, OXA-48 (commonly produced by Enterobacterales) lacks a lipobox and is a soluble protein. Outer membrane vesicles (OMVs) from <i>A. baumannii</i> cells expressing OXA-23 and OXA-24/40 contain these enzymes in their active form. Moreover, OXA-loaded OMVs were able to protect <i>A. baumannii</i>, <i>Escherichia coli</i>, and <i>Pseudomonas aeruginosa</i> cells susceptible to piperacillin and imipenem. These results permit us to conclude that membrane binding is a bacterial host-specific phenomenon in OXA enzymes. These findings reveal that membrane-bound β-lactamases are more common than expected and support the hypothesis that OMVs loaded with lipidated β-lactamases are vehicles for antimicrobial resistance and its dissemination. This advantage could be crucial in polymicrobial infections, in which <i>Acinetobacter</i> spp. are usually involved, and underscore the relevance of identifying the cellular localization of lactamases to better understand their physiology and target them.IMPORTANCEβ-lactamases represent the main mechanism of antimicrobial resistance in Gram-negative pathogens. Their catalytic function (cleaving β-lactam antibiotics) occurs in the bacterial periplasm, where they are commonly reported as soluble proteins. A bioinformatic analysis reveals a significant number of putative lipidated β-lactamases, expected to be attached to the outer bacterial membrane. Notably, 60% of Class D OXA β-lactamases (all from <i>Acinetobacter</i> spp.) are predicted as membrane-anchored proteins. We demonstrate that two clinically relevant carbapenemases, OXA-23 and OXA-24/40, are membrane-bound proteins in <i>A. baumannii</i>. This cellular localization favors the secretion of these enzymes into outer membrane vesicles that transport them outside the boundaries of the cell. β-lactamase-loaded vesicles can protect populations of antibiotic-susceptible bacteria, enabling them to thrive in the presence of β-lactam antibiotics. The ubiquity of this phenomenon suggests that it may have influenced the dissemination of resistance mediated by <i>Acinetobacter</i> spp., particularly in polymicrobial infections, being a potent evolutionary advantage.