The study began with a methodological choice that sets it apart from much of the antibiotic adjuvant literature. Rather than screening libraries of chemicals and then struggling to work out how the hits function, the researchers used CRISPR interference, or CRISPRi, to build a genome-wide gene repression library in Escherichia coli and asked which genes, when silenced, make bacteria more vulnerable to the fluoroquinolone ciprofloxacin. Because CRISPRi represses genes reversibly and without killing the cell, the team could distinguish genes whose loss specifically sensitizes bacteria to antibiotic stress from genes that are simply essential for growth. They exposed the library to ciprofloxacin concentrations ranging from one-eighth to sixteen times the minimum inhibitory concentration, then used high-throughput amplicon sequencing of surviving clones to quantify which guide RNAs had been depleted.

The results pointed unambiguously toward DNA maintenance. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed strong enrichment of sensitivity-related genes in homologous recombination, mismatch repair, and nucleotide excision repair, while Gene Ontology analysis highlighted DNA repair and recombination, the SOS response, membrane organization, and efflux pump regulation. Within the homologous recombination pathway, repression of dnaE, dnaQ, holD, polA, recA, recB, recC, ruvA, and ruvB left bacterial growth essentially untouched in the absence of antibiotics but sharply reduced survival as ciprofloxacin concentrations rose, a pattern the authors describe as synthetic lethality between DNA repair deficiency and antibiotic-induced genotoxic stress. Among these, recA, polA, dnaE, and holD showed the most consistent potentiating effect, not only against quinolones but also against beta-lactams, aminoglycosides, and nitrofurantoin.

Homologous recombination is an attractive target precisely because it is so conserved. Core components such as RecA and RuvABC maintain genome stability, repair DNA damage, and mediate horizontal gene transfer in virtually all bacteria, and their catalytic domains and DNA-binding motifs are nearly identical in organisms as different as E. coli and Bacillus subtilis. To validate the screen, the team deleted several nonessential genes, including recA, polA, holD, ruvB, and dnaQ. Although minimum inhibitory concentrations were unchanged, all five deletion strains showed significantly reduced survival after exposure to one or more bactericidal antibiotics, and recA deletion reduced survival under every bactericidal drug tested while leaving susceptibility to bacteriostatic agents such as tetracycline and tigecycline intact.

The mechanistic story that emerged was broader than simple loss of DNA repair. Compared with the wild-type parent strain, the recA-deficient bacteria displayed significant membrane damage, with increased permeability and fluidity and a reduced membrane potential, all of which promote antibiotic influx. Ethidium bromide accumulation assays revealed impaired efflux pump function, and enzyme-linked immunosorbent measurements confirmed heightened intracellular accumulation of ciprofloxacin. The mutants also produced elevated levels of reactive oxygen species, showed attenuated antioxidant capacity, and suffered pronounced declines in ATP production, respiratory rate, and the transmembrane proton gradient, with swimming motility compromised as well. The authors argue that these phenotypes stem not only from the loss of RecA’s recombinase activity but also from secondary disruption of the SOS response and broader transcriptional dysregulation, effectively turning RecA into a global regulator of bacterial stress physiology.

Perhaps the most striking discovery was that RecA also facilitates the horizontal spread of resistance. Because ATP production, respiration, and motility all regulate plasmid conjugation, the team tested whether recA deficiency affects plasmid transfer. Transconjugation experiments with the RP4-7 plasmid showed that recA-deficient strains, whether serving as donors or recipients, transferred the plasmid at significantly reduced frequency, with the defect most severe when both partners lacked RecA. A target that simultaneously sensitizes bacteria to antibiotics and blocks the dissemination of resistance genes is, in principle, a doubly valuable asset for anti-resistance strategies.

With the target validated genetically, the researchers tested three ways of inhibiting it. First, they delivered a CRISPRi system targeting recA by bacterial conjugation; transcriptional knockdown was successful and sensitized cells to ciprofloxacin and meropenem, but the effect was modest, reflecting the practical limits of plasmid-based delivery. Second, they exploited RecX, a natural RecA inhibitor that suppresses RecA-mediated strand exchange even at substoichiometric levels. Overexpression of recX reduced survival under quinolone treatment in a dose-dependent manner, and a synthetic twenty-amino-acid alpha-helical peptide derived from the RecX-RecA structure, fused to a cell-penetrating motif, potentiated quinolone killing, though its activity did not extend to beta-lactams, aminoglycosides, or colistin.

The third strategy proved the most powerful. Computational docking of a library of 135 candidate compounds against RecA identified cisplatin, the well-known platinum-based anticancer drug, as the strongest potentiator. Cisplatin significantly enhanced ciprofloxacin’s bactericidal effect against wild-type E. coli but showed no synergy in the recA deletion strain, demonstrating that the interaction is RecA-dependent. Surface plasmon resonance confirmed direct, concentration-dependent binding of cisplatin to RecA between 25 and 400 micromolar, with a dissociation constant of 40.3 micromolar, and comet assays revealed DNA fragmentation in cisplatin-treated cells resembling that of recA-deficient mutants. Time-kill assays showed a three-log reduction in viability within twenty-four hours at 8 micrograms per milliliter of cisplatin, with survival falling below the detection limit at 16 micrograms per milliliter after forty-eight hours. The synergy held against twenty multidrug-resistant clinical E. coli isolates and extended to quinolones, beta-lactams, aminoglycosides, polymyxins, and rifampicin in E. coli, Klebsiella pneumoniae, Proteus mirabilis, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecalis. Other platinum drugs, including lobaplatin, nedaplatin, carboplatin, and oxaliplatin, also potentiated antibiotics, though more weakly. Critically, serial passaging showed that cisplatin substantially delayed the emergence of ciprofloxacin resistance.

Cisplatin also suppressed conjugative transfer of clinically critical resistance plasmids, including those carrying tet(X4), bla NDM-5, and mcr-1, across a wide range of temperatures, pH values, plasmid incompatibility types, and donor-recipient combinations spanning E. coli, Salmonella Typhimurium, and K. pneumoniae. In animal models, the combination therapy achieved a seventy-five percent survival rate in Galleria mellonella larvae infected with a multidrug-resistant isolate, significantly outperforming either monotherapy, and reduced bacterial loads in the colon, liver, spleen, lung, and kidney in mouse intestinal and peritonitis-sepsis models while lowering pro-inflammatory cytokines and raising anti-inflammatory ones. In vivo transconjugation assays showed that cisplatin curtailed plasmid transfer in the mouse intestine within forty-eight hours without disturbing bacterial colonization. Safety testing was encouraging: hemolysis remained below five percent at 160 micrograms per milliliter, twenty times the synergistic dose, HEK293T cell viability exceeded ninety-five percent at the same concentration, and histopathology of five major organs in treated mice appeared normal, though the authors caution that cisplatin’s known toxicity and off-target effects will require structural optimization and drug-drug interaction studies before clinical translation.

The work frames what the authors call a defense-targeting adjuvant concept: rather than attacking essential bacterial structures directly, adjuvants disable the resilience machinery that pathogens rely on to survive antibiotic stress. By identifying homologous recombination, and RecA in particular, as a conserved, broadly applicable vulnerability, the study offers a target-driven blueprint for discovering next-generation potentiators and a feasible path toward restoring the efficacy of the antibiotic arsenal already in hand.

Subject of Research: Targeting the bacterial homologous recombination pathway and RecA protein to develop broad-spectrum antibiotic adjuvants against multidrug-resistant pathogens

Article Title: A genome-wide CRISPRi screen identifies homologous recombination pathway as potential target for broad-spectrum antibiotic adjuvants

Article References: Zhang, H., Chen, B., Gu, L., Wang, C., Xu, L., Ji, X., Wang, J., Wang, Z., Xiao, X., & Liu, Y. (2026). A genome-wide CRISPRi screen identifies homologous recombination pathway as potential target for broad-spectrum antibiotic adjuvants. Journal of Advanced Research, 87, 947-962. https://doi.org/10.1016/j.jare.2025.12.015

Image Credits: AI Generated

DOI: 10.1016/j.jare.2025.12.015

Keywords: antibiotic resistance, antibiotic adjuvants, CRISPRi screen, homologous recombination, RecA, cisplatin, horizontal gene transfer, multidrug-resistant bacteria, DNA repair, SOS response, conjugation, antimicrobial potentiators