Abstract
Pseudomonas aeruginosa infections are difficult to treat due to rapid development of antibiotic drug resistance. The synergistic combination of already-in-use drugs is an alternative to developing new antibiotics to combat antibiotic-resistant bacteria. Here we demonstrate that bismuth-based drugs (bismuth subsalicylate, colloidal bismuth subcitrate) in combination with different classes of antibiotics (tetracyclines, macrolides, quinolones, rifamycins and so on) can eliminate multidrug-resistant P. aeruginosa and do not induce development of antibiotic resistance. Bismuth disrupts iron homeostasis by binding to P. aeruginosa siderophores. Inside cells, bismuth inhibits the electron transport chain, dissipates the proton motive force and impairs efflux pump activity by disrupting iron–sulfur cluster-containing enzymes, including respiration complexes. As a result, bismuth facilitates antibiotic accumulation inside bacteria, enhancing their efficacy. The combination therapy shows potent antibacterial efficacy and low toxicity in an ex vivo bacteraemia model and increases the survival rate of mice in in vivo mouse lung-infection models. Our findings highlight the potential of bismuth-based drugs to be repurposed to combat P. aeruginosa infections in combination with clinically used antibiotics.
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Data availability
The transcriptome (RNA sequencing) data that support the findings of this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) with the accession code GSE223542. Genome sequencing data for the antibiotics resistant isolates are available in the National Center for Biotechnology Information Sequence Read Archive under the accession number PRJNA1089519. Source data are provided with this paper.
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Acknowledgements
We thank the Research Grants Council (R7070-18, 17308921, 17304323, SRFS2122-7S04) and the University of Hong Kong (University Research Committee and Norman and Celia Yip Foundation) for financial support. This work was also supported by the program of China Scholarships Council (number 201906200035 to Y.X.) and the National Natural Science Foundation of China (number 32300155 to Y.X.). R.C. and O.P.K. were partially supported by a grant from the Netherlands Organisation for Scientific Research (number 16433 to R.C. and O.P.K.) on developing novel antimicrobials, and RC was also supported by the Instituto de Salud Carlos III (Miguel Servet program, Spain, number CP21/00113 to R.C.). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.
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O.P.K., H.S. and R.C. conceived and designed the project; Y.X., X.W., P.G., C.W. and R.C. conducted the experiments and data analyses. A.d.J. performed the transcriptome analysis. J.H.K.C. performed the collections of clinical P. aeruginosa isolates. M.J.R.-S., A.R.-N., P.D.-E, J.G. and F.G. performed the toxicity test in C57Bl/6 mice. Y.X., X.W. and P.G. performed the in vivo test in BALB/c mice. W.W., R.Y.-T.K. and H.L. provided the suggestions. Y.X., H.L., R.C., H.S. and O.P.K. prepared the paper with contributions from all other authors. All the authors approved the final paper.
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O.P.K., Y.X. and R.C. are coinventors on a patent application associated with this work entitled ‘synergistic composition against Pseudomonas aeruginosa’, with reference number N2029436 (2021). O.P.K. is also cofounder and board member of the company Omnicin Therapeutics that is developing novel therapies against P. aeruginosa. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Bismuth compounds show strong synergistic effects with multiple antibiotics against PAO1.
The heat map of the FIC indices showing the synergistic effects of 55 antimicrobial agents with five different bismuth drugs against PAO1, while such synergistic effects were not observable for their sodium counterpart salts and other five metal-based compounds.
Extended Data Fig. 2 Bismuth enhances the antimicrobial activity of multiple antibiotics against P. aeruginosa at a low concentration.
Representative heat plots of microdilution checkerboard assay for the combination of antibiotics (azithromycin (a), clarithromycin (b), erythromycin (c), telithromycin (d), ofloxacin (e), levofloxacin (f), ciprofloxacin (g), gatifloxacin (h), moxifloxacin (i), tetracycline (j), doxycycline (k), minocycline (l), tigecycline (m), eravacycline (n), omadacycline (o), chloramphenicol (p), rifampicin (q), cefepime (r), tobramycin (s) and aztreonam (t)) with BSS against PAO1.
Extended Data Fig. 3 Time-killing curves showed the synergistic effects of BSS and antibiotics against PAO1.
Time-killing curves for antibiotics (azithromycin (AZM, a), clarithromycin (CLR, b), telithromycin (TEL, c), chloramphenicol (CHL, d), doxycycline (DOX, e), minocycline (MIN, f), tigecycline (TGC, g), eravacycline (ERV, h), omadacycline (OMC, i), rifampicin (RIF, j), ofloxacin (OFX, k), ciprofloxacin (CIP, l), moxifloxacin (MXF, m), aztreonam (ATM, n), tobramycin (TOB, o), cefepime (FEP, p)) and BSS monotherapy and their combination therapy against PAO1 during 24 h incubation at the indicated concentration (μM). Error bars represent mean ± SD for three biological replicates.
Extended Data Fig. 4 Bismuth disrupts the iron homeostasis of P. aeruginosa.
a, The RT-qPCR confirmed the downregulation of iron uptake-related genes under the treatment of BSS. b, The heat map shows the lack of synergy of bismuth with eravacycline in the presence of 100 μM FeCl3. c, Time killing curves for ERV (0.5 μM) and BSS (16 μM) monotherapy or combination therapy against PAO1 in the absence or presence of 50 μM FeCl3. d, The mass spectra of PVD and bismuth-bound PVD (PVD-Bi). The peak at m/z of 667.8014 and 681.7980, assignable as PVD1 (Cald.667.8022) and PVD2 (Cald. 681.7997), and two different side chains of PVD are noted. The appearance of two new peaks after incubation of PVD with Bi(III) (as Bi(NO3)3) at m/z of 770.7782 and 784.7714 assignable to PVD1-Bi (Cald. 770.7779) and PVD2-Bi (Cald. 784.7753) indicated binding of Bi to PVD at a 1:1 ratio. e, Different UV-vis spectra of PCH upon addition of 0.125–2 molar equivalents of Bi(NTA). The inset shows the changes in absorbance at 360 nm. f, The intracellular bismuth concentrations of PAO1 and pvdA/pchD deletion strain under the treatment of 16 µM BSS. For Fig. a, error bars represent mean ± SEM for three biological replicates. ****. P < 0.0001; by two-sided unpaired t-test, 95% confidence interval. fpvA: P = 0.000048, fpvB: P = 0.000146, fptA: P = 0.000033, piuA: P = 0.000109, chtA: P = 0.000542, fecA: P = 0.000202, pvdA: P = 0.000008, pvdF: P = 0.000245, pchD: P = 0.000003, pchE: P = 0.000118, pvdS: P = 0.000043, tonB1:P = 0.000052. For Fig. c and f, error bars represent mean ± SEM for three biological replicates. For Fig. f, P values were determined using two-sided unpaired t-test, 95% confidence interval.
Extended Data Fig. 5 Bismuth impairs the activity of the electron transport chain (ETC) and promotes intracellular antibiotic accumulation in P. aeruginosa.
a, Time-dependent oxygen consumption rate was quantified by a fluorescence probe in the presence of indicated concentrations of BSS. The decrease in the oxygen consumption rate by BSS suggests the inhabitation of ETC activities. b, BSS inhibits the activity of NADH dehydrogenase in vivo. The bacteria were treated with different concentrations of BSS (μM) for 2 hours, then the inner membrane was collected and membrane-bound NADH-quinone oxidoreductase activity was measured. c–f, BSS dose-dependent inhibition on the activity of NADH dehydrogenase from different gene mutant strains. The bacterial inner membrane was collected and treated with different concentrations of BSS (μM) for 2 hours, then the membrane-bound NADH-quinone oxidoreductase activity was measured. g, The fluorescence of calcein inside of P. aeruginosa under the treatment of indicated concentrations of BSS. h, The fluorescence of calcein inside of P. aeruginosa under the treatment of BSS in M9 medium without iron. The calcein-AM is not able to be quenched by bismuth or the change of metabolism and pH i, The fluorescence of calcein inside of P. aeruginosa under the treatment of different combinations of BSS (10 μM), iron(10 μM) or PVD (10 μM) in M9 medium. When cells were treated with iron and PVD, the fluorescence of calcein-AM was quenched, indicating that the iron was transported inside the cell and the calcein-AM was able be quenched by iron inside this bacterium. While the bacteria were treated with a combination of bismuth, iron, and PVD, iron could not be rapidly transported within the cells, suggesting that bismuth might bind to PVD to inhibit iron transport. For Fig. a–i, the data are presented as mean ± SEM of three biological replicates. For Fig. g and i, P values were determined using two-sided unpaired t-test, 95% confidence interval.
Extended Data Fig. 6 Bismuth disrupts membrane potential and promotes intracellular antibiotic accumulation in P. aeruginosa.
a, Detection of membrane potential in P. aeruginosa. Red/green ratios were calculated using population mean fluorescence intensities for P. aeruginosa incubated with 15 μM DIOC2(3) for 30 min in the presence of BSS or 20 μM CCCP. b, The time-based intracellular bismuth level of PAO1 under the treatment of 32 μM BSS. c–e, Intracellular BSS dose-dependent accumulation of azithromycin, chloramphenicol and ofloxacin. PAβN was used as a positive control. For Fig. a–e, the data are presented as mean ± SEM of three biological replicates. P values were determined using two-sided unpaired t-test for Fig. a, c–e.
Extended Data Fig. 7 The deletion of efflux pump genes oprM and mexCD antagonizes the synergistic effects of BSS and antibiotics.
The heat map showing antagonized synergy of bismuth with eravacycline in the bacterial strain with efflux pump genes oprM and mexCD deleted.
Extended Data Fig. 8 Bismuth enhances the anti-biofilm abilities of antibiotics.
The established biofilms were treated with antibiotics alone or combined with 32 μM of BSS for 24 hours. The biofilms were washed three times with sterile PBS and dispersed by ultrasound, the number of bacteria was determined by plating. The data are presented as mean ± SEM of three biological replicates. P values were determined using two-sided unpaired t-test.
Extended Data Fig. 9 Bismuth enhances the activities of antibiotics ex vivo.
a, Ex vivo bacteremia model for antibiotics, bismuth drugs, and their combined antimicrobial activity. 10 μL of serial decimal dilutions of each one of the combinations of antibiotics with/without bismuth drugs (32 μM) tested in blood were dropped. All the tests were performed in triplicate. b, The appearance of the blood infected with PAO1 after treatment with the indicated concentration of antibiotics alone or the combinations with bismuth for 24 h. Black color of the blood indicates hemolysis and therefore bacterial growth. Red color of the blood indicates the therapy controlled the infection. All the tests were performed in triplicate.
Extended Data Fig. 10 Toxicity studies showing that bismuth drugs are not cytotoxic.
a, b, Bismuth drugs show no toxicity in cells. The cell viability was measured by XTT assay under the treatment of different concentrations of bismuth drugs for 48 hours. The bismuth drugs showed negligible toxicity to human lung epithelial cells and embryonic kidney cells even at very high concentrations. c, Bismuth drugs show negligible hemolytic activity. Percent of hemolysis was calculated concerning the positive control (Triton X-100). (a–c) All the tests were performed in triplicate and all the data are presented as mean ± SD. d, Hematoxylin and eosin (H&E) staining of the mice lungs under treatment of single dose (-1d) or three doses (-3d) of 100 mg/kg CBS. e, Survival curves of mice treated with PBS, 100 mg/kg, and 200 mg/kg of CBS (n = 4) by the intranasal administration. f, The biodistribution of bismuth in mice after treatment by the intranasal administration of 100 mg/kg CBS. g, Proposed the mechanism of action for the synergy of bismuth with antibiotics. Bismuth restores the susceptibility of P. aeruginosa to antibiotics through iron deprivation by interacting with siderophores and Fur. Consequently, the activity of iron-dependent cellular respiration complexes (for example, NADH dehydrogenase) is inhibited, resulting in dissipating the proton motive force (PMF), which further leads to repressed ATP synthesis and impaired multi-drug efflux pump. Ultimately, more antibiotics are rapidly accumulated inside bacterial cells, resulting in the death of cells. For Fig. a–c error bars represent mean ± SEM for three biological replicates. For Fig. d, four mice were included in each group, and similar results were obtained. For Fig. f error bars represent mean ± SEM for four mice samples.
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Xia, Y., Wei, X., Gao, P. et al. Bismuth-based drugs sensitize Pseudomonas aeruginosa to multiple antibiotics by disrupting iron homeostasis. Nat Microbiol 9, 2600–2613 (2024). https://doi.org/10.1038/s41564-024-01807-6
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DOI: https://doi.org/10.1038/s41564-024-01807-6
