Contribution of RND superfamily multidrug efflux pumps AdeABC, AdeFGH, and AdeIJK to antimicrobial resistance and virulence factors in multidrug-resistant <i>Acinetobacter baumannii</i> AYE

<i>Acinetobacter baumannii</i> is a critical priority gram-negative bacterial species characterized by multidrug resistance. The latter is significantly attributable to the resistance-nodulation-cell division (RND) superfamily of tripartite multidrug efflux systems represented by AdeABC, AdeFGH, and AdeIJK. By constructing isogenic deletion mutants, this investigation assessed the impact of RND efflux pumps on planktonic and biofilm cell antimicrobial susceptibility as well as on biofilm formation and virulence factors in a multidrug-resistant reference strain, <i>A. baumannii</i> AYE. Inactivation of individual genes encoding the aforementioned three RND pumps or regulators (i.e., AYE△<i>adeA</i>, △<i>adeB</i>, △<i>adeC</i>, △<i>adeRS</i>, △<i>adeFGH</i>, and △<i>adeIJK</i> mutants) demonstrated that the three efflux pumps, particularly AdeB, contribute to resistance in both planktonic and biofilm cells to structurally unrelated anti-<i>A</i>. <i>baumannii</i> drugs, including carbapenems, fluoroquinolones, macrolides, polymyxins, and/or tetracyclines/tigecycline. The pump inactivation also altered other functions, changes in bacterial motility and adhesion, reduction of biofilm formation, and decreased expression of the genes related to biofilm formation and virulence factors (<i>abaI</i>, <i>bap, bfmR</i>, <i>csuE</i>, <i>ompA,</i> and <i>pgaA</i>, except for <i>abaR</i> whose expression was increased). The virulence assay measured through the survival rates of <i>A. baumannii</i>-infected <i>Galleria mellonella</i> revealed the relation between RND pumps (particularly AdeB) and pathogenicity. The findings together expand the understanding of specific <i>A. baumannii</i> RND pumps or components for their roles in resistance and virulence/pathogenicity in the presence of high-level multidrug resistance, highlighting the RND pumps as potential therapeutic intervention targets against <i>A. baumannii</i> infection.