Uncovering nitroxoline activity spectrum, mode of action and resistance across Gram-negative bacteria

Abstract

Nitroxoline is a bacteriostatic quinoline antibiotic, known to form complexes with metals. Its clinical indications are limited to uncomplicated urinary tract infections, with a susceptibility breakpoint only available for Escherichia coli. Here, we test > 1000 clinical isolates and demonstrate a much broader activity spectrum and species-specific bactericidal activity, including Gram-negative bacteria for which therapeutic options are limited due to multidrug resistance. By combining genetic and proteomic approaches with direct measurement of intracellular metals, we show that nitroxoline acts as a metallophore, inducing copper and zinc intoxication in bacterial cells. The compound displays additional effects on bacterial physiology, including alteration of outer membrane integrity, which underpins nitroxoline's synergies with large-scaffold antibiotics and resensitization of colistin-resistant Enterobacteriaceae in vitro and in vivo. Furthermore, we identify conserved resistance mechanisms across bacterial species, often leading to nitroxoline efflux.

Fig. 1. Nitroxoline is active beyond UTI pathogens, including intracellular bacteria, and exerts bactericidal activity.

a Nitroxoline structure. b Overlap between Gram-negative bacterial genera or species tested with three orthogonal susceptibility testing methods in this study and according to EUCAST. All seven EUCAST-specific entries are genera for which species resolution is missing. The number of overlapping species/genera between EUCAST and our methods is shown as intersection size, whereas the number of genera/species assessed by each approach is shown as set size. c Nitroxoline is active against several Gram-negative bacterial species. MICs were determined against 30 bacterial species in broth microdilution. The total number of strains tested is indicated next to species, ordered by phylogeny according to GTDB (Methods). The clinical breakpoint for E. coli (16 µg/ml) is indicated (black line). MIC₅₀ values are framed in black and listed in the Source Data Fig. 1c together with MIC₉₀ values. d Nitroxoline is active against intracellular S. Typhi. Intracellular bacterial counts were assessed with the gentamicin protection assay in two S. Typhi clinical isolates (Methods, Supplementary Data 2). Cell counts were determined before and after treatment with nitroxoline (5 µg/ml) or solvent control (DMSO) at 7 h p.i. (MOI 100). Mean and standard error are shown across four independent experiments. ns p > 0.05; *p = 0.022; **p = 0.003 (two-sided Welch’s t-test). e Nitroxoline is bactericidal against A. baumannii ATCC 19606^T. Mean and standard deviation across at least three biological replicates are shown for each condition. f Nitroxoline induces lysis in A. baumannii ATCC 19606^T. White arrowheads mark the release of cytoplasmic material and loss of the pericellular halo. Representative images of phase-contrast videos were acquired after 8 µg/ml nitroxoline treatment (4x MIC, Methods, Supplementary Movie 1). The scale bar denotes 5 µm. Source data are provided as a Source Data file.

Fig. 2. Nitroxoline interacts with other antibiotics in E. coli and resensitizes colistin-resistant E. coli and K. pneumoniae.

a Nitroxoline interacts with several antibiotics in E. coli. Nitroxoline combinations were tested in 8 × 8 broth microdilution checkerboards in E. coli BW25113 (Supplementary Fig. 3). Bliss interaction score distributions are shown for each combination (n = 98 scores corresponding to 7 × 7 dose-combinations in two biological replicates). The median (central line), first (lower hinge) and third quartile (upper hinge) are shown for each boxplot. Whiskers correspond to 1.5x IQR from each hinge. The numbers stand for cumulative Bliss scores for each combination (Methods). b Nitroxoline resensitizes colistin-resistant K. pneumoniae and E. coli. Growth (OD_595 nm at 10.75 h, corresponding to the beginning of stationary phase for the untreated control for each strain) was measured in the presence of serial twofold dilutions of colistin, supplemented or not with 0.75 µg/ml nitroxoline and normalised by no-drug controls. Three K. pneumoniae and two E. coli strains (dashed lines) and their isogenic colistin-resistant descendants (solid lines) were tested, including experimentally evolved and clinical isolates (framed in black, Supplementary Data 2). One K. pneumoniae clinical isolate carries the mcr-1 positive natural plasmid pKP2442 and, therefore, lacks a parental strain. Mean and standard error across four biological replicates are shown. c Nitroxoline resensitizes a colistin-resistant K. pneumoniae clinical isolate in vivo. G. mellonella larvae were infected with the indicated isolate and treated with single drugs, their combination or were left untreated (solvent-only control). The mean and standard error are shown across four independent experiments for each condition. p = 0.0255 and p = 0.0098 comparing colistin-nitroxoline with colistin and untreated, respectively (log-rank test). NX nitroxoline, COL colistin. Source data are provided as a Source Data file.

Fig. 3. Nitroxoline directly perturbs the OM in E. coli.

a Nitroxoline decreases the abundance and stability of outer membrane proteins and Lpt machinery. Volcano plots depicting abundance (left) or stability (right) changes upon nitroxoline exposure in whole-cell 2D-TPP. Results are based on n = 5 independent experiments (four drug concentrations and a vehicle control). Effect size and statistical significance as log₂ (F-statistic) (Methods) are represented on the x- and y-axis, respectively. The F-statistic was transformed to 1 when 0 before the log₂ transformation. Proteins are colour-coded according to their Gene Ontology (GO) annotation (Supplementary Fig. 5a). b Nitroxoline effects profiled by chemical genetics on an E. coli whole-genome single-gene deletion mutant library. Effects are expressed as multiplicative changes of mutant fitness compared to the plate median (approximating wild-type). Significance was obtained from an empirical Bayes’ moderated two-sided t-statistics, Benjamini–Hochberg adjusted (two independent clones per mutant, three replicates per condition, Methods, Supplementary Data 4). Genes are colour-coded as in Fig. 3a (GO in Supplementary Fig. 5c). c Nitroxoline directly affects OM permeability. NPN fluorescence upon exposure of E. coli BW25113 to nitroxoline, positive (polymyxin B, EDTA) and negative (chloramphenicol, untreated samples) controls. Data points represent the average for each of the four biological replicates per condition. The horizontal line and error bars indicate mean and standard error. ns p > 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 (two-sided Welch’s t-test using the chloramphenicol control as the reference group). EDTA, p = 0.008; Polymyxin B, p = 0.00004; no-drug control, p = 0.053; nitroxoline 0.45 µg/ml, p = 0.0008; nitroxoline 0.9 µg/ml, p = 0.003; nitroxoline 1.8 µg/ml, p = 0.002. d Nitroxoline is more potent upon chemical perturbation of the OM. EOP assays with tenfold serial dilutions of E. coli BW25113 cells plated onto no-drug control plates, 0.5% SDS + 0.8 mM EDTA, 0.45 µg/ml nitroxoline, or their combination. Four biological replicates were tested for each condition. Source data are provided as a Source Data file.

Fig. 4. Nitroxoline increases intracellular levels of copper and zinc.

a–c Nitroxoline affects metal homoeostasis inducing copper and zinc detoxification responses, as determined by 2D-TPP. Heatmaps show the relative remaining soluble fraction compared to the vehicle control at each temperature to highlight changes in protein abundance and thermal stability profiles of the Cu(I) exporter CopA, the periplasmic copper oxidase CueO (a), the transcriptional regulators Zur and ZntR, zinc importer ZnuA and exporter ZntA (b), and the manganese importer MntH (c), are shown. d Nitroxoline increases intracellular levels of copper, zinc and manganese. Synchrotron-based nano-XRF measurements on E. coli untreated or exposed to nitroxoline (1 µg/ml), expressed as elemental areal density (ng/cm²). The mean and standard error across ≥5 cells are shown (Methods). ns p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 (two-sided Welch’s t-test). Copper, p = 0.01; iron, p = 0.73; manganese, p = 0.0006; zinc, p = 0.0005. Source data are provided as a Source Data file.

Fig. 5. Resistance to nitroxoline is associated with efflux pump upregulation across species.

a Whole-genome sequencing of experimentally evolved nitroxoline-resistant strains (Supplementary Data 2). Nitroxoline MIC values are indicated below each strain. Mutation effects are colour-coded. Strains on which proteomics was performed (Fig. 5b) are indicated in bold. K. pneumoniae strains whose sensitive parental strain lacks oqxR are marked with an asterisk. The in-patient evolved K. pneumoniae clinical isolate 8_R1 is indicated in italics. b Protein abundance changes in nitroxoline-resistant strains. Selected proteins annotated as efflux pumps or porins are shown and clustered according to Pearson’s correlation. Hits are marked with an asterisk (adjusted p value ≤0.05 and at least twofold abundance change (Supplementary Fig. 6a, b and Supplementary Data 6). Species are colour-coded, as in Fig. 5a. c Wild-type emrR and oqxr complementation restores nitroxoline susceptibility. The experimentally evolved E. coli strain 1_R4 with emrR D109V mutation (Fig. 5a and Supplementary Fig. 6c) and K. pneumoniae strain 4_R1 harbouring the oqxR G60-L67 duplication (Fig. 5a, d and Supplementary Fig. 6d) are shown. Nitroxoline MIC was measured by broth microdilution. Mean and standard error across four biological replicates are shown. ns p > 0.05; *p ≤ 0.05 (Wilcoxon test. For E. coli: empty plasmid vs no-plasmid, p = 0.608; empty plasmid vs wild-type efflux pump, p = 0.042; no-plasmid vs wild-type efflux pump, p = 0.042. For K. pneumoniae: empty plasmid vs no-plasmid, p = 0.217; empty plasmid vs wild-type efflux pump, p = 0.042; no-plasmid vs wild-type efflux pump, p = 0.042). d Amino acid changes resulting from oqxR mutations. The domain annotation of OqxR was obtained from its closest annotated structural homologue NsrR (Methods). e Efflux pump inhibitors resensitize nitroxoline-resistant strains. Nitroxoline MIC was measured by broth microdilution. Mean and standard error across four biological replicates are shown. Resistant strains are shown as shaded plots next to their parental-sensitive strains. For results on all strains, see Supplementary Fig. 6g. p values are only shown when significant: *p ≤ 0.05; **p ≤ 0.01 (Wilcoxon test). Source data are provided as a Source Data file.