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Elevated expression of Resistance-Nodulation-Cell Division (RND) drug transporters is commonly observed in clinical isolates of , a nosocomial pathogen associated with multidrug-resistant (MDR) infections. We describe here a CRISPRi platform directed toward identifying essential gene hypomorphs that preferentially change resistance to the fluoroquinolone antibiotic ciprofloxacin in RND pump overproducers. An sgRNA library including single and double nucleotide mutations directed against essential genes of was constructed and introduced into multiple strain backgrounds, allowing strain-specific, titratable knockdown efficiencies to be analyzed. Other than NusG depletions, there were few candidates in the absence of drug treatment that showed lowered fitness specifically in strains overexpressing the RND efflux pumps AdeAB, AdeIJK, or AdeFGH. In the presence of ciprofloxacin, the hypomorphs that caused hypersensitivity were predicted to result in outer membrane dysfunction, with the AdeFGH overproducer appearing particularly sensitive to such disruptions. Increased ciprofloxacin susceptibility in two pump hyper-expressers was observed after depletions of a monovalent cation-proton antiporter protein and a nucleoid-associated protein, the latter of which was necessary for pump hyper-activation. On the other hand, depletions of translation-associated proteins as well as components of the proton-pumping ATP synthase resulted in fitness benefits in the presence of the drug for at least two pump-overproducing strains. Therefore, pump overproduction exacerbated stress caused by defective outer membrane integrity, while the efficacy of drug resistance in efflux pump overproducers was enhanced by slowed translation or defects in the proton-pumping ATP synthase.

Acinetobacter baumannii is a nosocomial pathogen often associated with multidrug resistance (MDR) infections. Fluoroquinolone resistance (FQR) due to drug target site mutations and elevated expression of RND drug transporters is common among clinical isolates. We describe here a CRISPRi platform that identifies hypomorphic mutations that preferentially altered drug sensitivity in RND pump overproducers. An sgRNA library against essential genes of A. baumannii was constructed with single and double nucleotide mutations that produced titratable knockdown efficiencies and introduced into multiple strain backgrounds. Other than nusG depletions, there were few candidates in the absence of drug treatment that showed lowered fitness specifically in strains overexpressing clinically relevant RND efflux pumps AdeAB, AdeIJK, or AdeFGH. In the presence of ciprofloxacin, the hypomorphs causing hypersensitivity were predicted to result in outer membrane dysfunction, to which the AdeFGH overproducer appeared particularly sensitive. Depletions of either the outer membrane assembly BAM complex, LOS biogenesis proteins, or Lpt proteins involved in LOS transport to the outer membrane caused drug hypersensitivity in at least two of the three pump overproducers. On the other hand, depletions of translation-associated proteins, as well as components of the proton-pumping ATP synthase pump resulted in fitness benefits for at least two pump-overproducing strains in the presence of the drug. Therefore, pump overproduction exacerbated stress caused by defective outer membrane integrity, while the efficacy of drug resistance in efflux overproducers was enhanced by slowed translation or defects in ATP synthesis linked to the control of proton movement across the bacterial membrane.Competing Interest StatementThe authors have declared no competing interest.

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against and . Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of . To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in . CRISPRi-induced partial depletion of lipooligosaccharide transport genes ( - , ) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on genes in Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains.