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A broad-spectrum antibiotic adjuvant reverses multidrug-resistant Gram-negative pathogens

Nature Microbiology volume 5, pages 1040–1050 (2020)Cite this article

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Abstract

The rapid emergence and dissemination of multidrug-resistant (MDR) bacterial pathogens pose a serious threat to global healthcare. One particular concern is the carbapenem-resistant Enterobacteriaceae (CRE), a group of Gram-negative bacteria that have evolved resistance to all or nearly all available antibiotics. Coupled with the fact of barren antibiotic development pipeline nowadays, a critical approach is to revitalize existing antibiotics using antibiotic adjuvants. We found a short linear antibacterial peptide (SLAP)-S25 carrying four non-natural amino acids of 2,4-diaminobutanoic acid (Dab), which solely showed weak antibacterial activity but boosted the efficacy of antibiotics covering all major classes, including cefepime, colistin, ofloxacin, rifampicin, tetracycline and vancomycin, against MDR Gram-negative pathogens. Mechanistic studies showed that SLAP-S25 triggers membrane damage by binding to both lipopolysaccharide (LPS) in the outer membrane and phosphatidylglycerol (PG) in bacterial cytoplasmic membrane, to potentiate antibiotic efficacy through collaborative strategies. Lastly, SLAP-S25 effectively enhanced the activity of colistin against MDR Escherichia coli-associated infections in three animal models. Our findings provide a potential therapeutic option using existing antibiotics in combination with broad-spectrum antibiotic adjuvants, to address the prevalent infections caused by MDR Gram-negative pathogens worldwide.

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Fig. 1: SLAP-S25 potentiates the efficacy of antibiotics against Gram-negative bacteria.
Fig. 2: SLAP-S25 exerts bactericidal activity by interacting with LPSs and PG.
Fig. 3: Mechanism of SLAP-S25 in combination with antibiotics.
Fig. 4: Metabolic perturbation of E. coli treated with SLAP-S25.
Fig. 5: SLAP-S25 rescues the activity of colistin in three animal infection models.

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Data availability

Source data supporting the findings of the present study are included in the article. RNA-seq data have been deposited in the National Center for Biotechnology Information’s Sequence Read Archive with accession number PRJNA610702. Source data for Figs. 1–3 and 5 and Extended Data Figs. 3, 4, 6, 7 and 10 are provided with the paper.

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Acknowledgements

We thank R. Zhang from the Second Affiliated Hospital of Zhejiang University for kindly sharing clinical isolates, and S. Wang from China Agricultural University for transcriptome analysis. The present study is supported by the National Key Research and Development Programme of China (grant no. 2018YFD0500506), National Natural Science Foundation of China (grant nos. 31922083 and 21861142006) and the Fund of Beijing Dairy Industry Innovation Team.

Author information

Author notes

  1. Yuan Liu

    Present address: Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China

  2. These authors contributed equally: Meirong Song, Yuan Liu.

Authors and Affiliations

  1. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, China

    Meirong Song, Yuan Liu, Xiaoyong Huang, Yang Wang, Jianzhong Shen & Kui Zhu

  2. National Center for Veterinary Drug Safety Evaluation, College of Veterinary Medicine, China Agricultural University, Beijing, China

    Shuangyang Ding, Jianzhong Shen & Kui Zhu

  3. Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety and Beijing Laboratory for Food Quality and Safety, China Agricultural University, Beijing, China

    Shuangyang Ding & Jianzhong Shen

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  1. Meirong Song
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Contributions

J.S. and K.Z. conceived and supervised the project. M.S., Y.L. and X.H. performed all the experiments. M.S. and Y.L. conducted the antibacterial tests, ITC and CD assays, outer-membrane permeability, membrane depolarization, ATP and ROS assays, and animal infection models. X.H. and Y.L. conducted the safety assessment. Y.L. performed the LC–MS/MS assays. M.S. performed the transcriptome assay. K.Z. analysed the data obtained. M.S., Y.L., S.D., Y.W. and K.Z. did the data analysis. M.S., Y.L. and K.Z. wrote the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Jianzhong Shen or Kui Zhu.

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Extended data

Extended Data Fig. 1 Synergistic activity of SLAP-S25 in combination with various antibiotics against MDR E. coli.

Synergy of SLAP-S25 and six antibiotics (cefepime, colistin, ofloxacin, rifampicin, tetracycline, vancomycin and colistin) against E. coli B2 by chequerboard broth microdilution assay. The lowest concentration of antibiotics caused no visible growth of bacteria under sub-MIC of SLAP-S25 was indicated by arrows. Chequerboard data are representative of two biological replicates.

Extended Data Fig. 2 SLAP-S25 dramatically enhances the activity of rifampicin and colistin against different E. coli isolates.

a, Synergy of SLAP-S25 and rifampicin against rifampicin-sensitive and rifampicin-resistant E. coli by chequerboard broth microdilution assay. The lowest concentration of rifampicin caused no visible growth of bacteria under sub-MIC (0.5 μg/mL) of SLAP-S25 was indicated by arrows. b, The MIC values of colistin against MCR-1 positive E. coli isolates were measured under the treatment of SLAP-S25 (0 to 8 μg/mL) by checkerboard assays. The lowest concentration of colistin caused no visible growth of bacteria under sub-MIC (4 μg/mL) of SLAP-S25 was indicated by arrows. c, Growth curves of E. coli ATCC 25922 under the treatments of colistin (0.25 μg/mL), SLAP-S25 (4 μg/mL) and colistin plus SLAP-S25 (0.25 μg/mL + 4 μg/mL). The mean of two biological replicates is shown and error bars represent the standard deviation (SD) (n = 2). d, SLAP-S25 enhances the activity of colistin to three colistin-susceptible E. coli isolates. The lowest concentration of colistin caused no visible growth of bacteria under sub-MIC of SLAP-S25 was indicated by arrows. Data in a, b and d are representative of two biological replicates.

Extended Data Fig. 3 Safety evaluation of SLAP-S25.

a, Hemolytic activity of SLAP-S25 and colistin to the red blood cells of sheep. Different concentrations of SLAP-S25 had no effect on hemolytic toxicity of colistin (2 μg/mL). b, Cytotoxicity of Vero, HEp-2, A549 and H9c2 cells treated with SLAP-S25 was tested based on WST-1 assay. c, Cytotoxicity of Vero, HEp-2, A549 and H9c2 cells treated with colistin was tested based on WST-1 assay. d, Zebrafish embryos were either treated with embryo medium as negative control, SLAP-S25 (32 µg/mL), colistin (32 µg/mL) or 200 µg/mL sodium dehydroacetate as positive control. The represented phenotypes of cardiac hemorrhage were marked by dashed circle. All the data shown in a, b and c are the mean of two biological replicates and error bars represent the standard deviation (SD) (n = 2). The results in d are representative of ten biological replicates.

Source data

Extended Data Fig. 4 SLAP-S25 disrupts the out-membrane integrity through interacting with LPS.

a, Purified E. coli LPS (128 μg/mL) abolished the synergy between SLAP-S25 and non-LPS targeting antibiotic vancomycin in E. coli B2. b, Effects of different cationic ions on the antibacterial activity of SLAP-S25 against E. coli B2. c, Mg2+ inhibits the antibacterial activity of SLAP-S25 in a dose-dependent manner against E. coli B2. d, Exogenous addition of Mg2+ (480 μg/mL) abolishes the potentiation of SLAP-S25 and non-LPS targeting antibiotic vancomycin in E. coli B2. e, Dose-dependent activation of the mgtA/rsmC belongs to PhoPQ two-component system by SLAP-S25. Transcript ratios of the PhoPQ-dependent gene mgtA were quantified by normalizing mgtA using qRT-PCR. The mean of three biological replicates is shown and error bars represent the standard deviation (SD) (n = 3). P values were determined by non-parametric one-way ANOVA. f, Additional Mg2+ inhibited the activation of the PhoPQ two-component system by SLAP-S25 in E. coli B2. Transcript ratios of the PhoPQ-dependent gene mgtA were quantified by normalizing mgtA using qRT-PCR. The mean of three biological replicates is shown and error bars represent the standard deviation (SD) (n = 3). P values were determined by non-parametric one-way ANOVA. g, SLAP-S25 increased the permeability of outer membrane in E. coli probed with 1-N-phenylnaphtylamine (NPN). E. coli was treated with SLAP-S25 for 30 min. The fluorescence intensity of NPN was recorded at the excitation wavelength of 535 nm and emission wavelength of 615 nm. Colistin (4 μg/mL) was used as the positive control. The mean of three biological replicates is shown and error bars represent the standard deviation (SD) (n = 3). P values were determined by non-parametric one-way ANOVA. Data in a-d are representative of three biological replicates.

Source data

Extended Data Fig. 5 SLAP-S25 binds to LPS.

a, The affinity between SLAP-S25 and LPS extracted from mcr-1 negative E. coli DH5α (pUC19) was measured by isothermal titration calorimetry (ITC) in water at 25 °C. Calculated thermodynamic parameters were obtained including equilibrium dissociation constant (KD = 3.7 × 10−6 mol/L), number of binding sites (n = 0.924), molar binding enthalpy (ΔH = 19.12 kJ/mol) and molar binding entropy (ΔS = 167.9 J/mol·K). b, The affinity between SLAP-S25 and LPS extracted from mcr-1 positive E. coli DH5α (pUC190-mcr-1) was measured by ITC in water at 25 °C. KD = 3.2 × 10−6 mol/L, n = 0.971, ΔH = 21.16 kJ/mol and ΔS = 175.89 J/mol·K. c, The affinity between colistin and LPS extracted from mcr-1 positive E. coli DH5α (pUC190-mcr-1) was measured by ITC in water at 25 °C. KD = 5.4 × 10−6 mol/L, n = 2.19, ΔH = 15.25 kJ/mol and ΔS = 152.91 J/mol·K. Data in a-c are representative of two biological replicates.

Extended Data Fig. 6 PG abolishes the antibacterial activity of SLAP-S25 against E. coli B2.

a, Inhibition zones of the mixtures of SLAP-S25 (40 μg) and different kinds of lipids (100 μg). The mean of three biological replicates is shown and error bars represent the standard deviation (SD) (n = 3). P values were determined by non-parametric one-way ANOVA. b, Isothermal titration by 2 mM PG to HEPES buffer without SLAP-S25 at 25 °C. c, Turbidity of the mixtures of SLAP-S25 (2 mg/mL, 50 μL) with different lipids (5 mg/mL, 50 μL) at 600 nm. The mean of three biological replicates is shown and error bars represent the standard deviation (SD) (n = 3). P values were determined by non-parametric one-way ANOVA. d, The main target of SLA-S25 on PG should be the terminal group of glycerol. Length and saturation of fatty acids of PG has no effect on the activity of SLAP-S25. Different kinds of PG were added to the broth and the antibacterial activity of SLAP-S25 against E. coli B2 was performed using the chequerboard microdilution assay. Chequerboard data are representative of two biological replicates. (e and f) No effect on the release of bacterial contents including small molecules such as ATP (MW = 507 Da) (e) and macromolecules such as β-galactosidase (MW = 130 kDa) (f), in the presence of sub-MIC levels of SLAP-S25 (4–8 μg/mL) for 1 h. The mean of three biological replicates is shown and error bars represent the standard deviation (SD) (n = 3). P values were determined by non-parametric one-way ANOVA.

Source data

Extended Data Fig. 7 Accumulation of antibiotics in E. coli B2.

a, Effect of SLAP-S25 on the accumulation of antibiotics. Data were presented as means ± SD (n = 3 biological independent replicates). b, SLAP-S25 had higher accumulation than colistin. Spiked levels of SLAP-S25 and colistin were 4 μg/mL. Data were presented as means ± SD (n = 3 biological independent replicates). c, Dose-dependent accumulation of rifampicin in the presence of SLAP-S25. Data were presented as means ± SD (n = 3 biological independent replicates). P values in a and b were assessed by Student’s t test, that in c by one-way ANOVA.

Source data

Extended Data Fig. 8 Heat map of transcriptome analysis of the genes of E. coli B2.

a, Heat map of transcriptome analysis of E. coli B2 under treatment of SLAP-S25 (0–8 μg/mL) for 8 h; b, Heat map of transcriptome analysis of E. coli B2 after incubation at different time points (2 h, 4 h or 8 h) in the presence of 8 μg/mL SLAP-S25 compared with in the absence of 8 μg/mL SLAP-S25. Total RNA was extracted, sequenced and analyzed as described in Methods. Genes that were identified as significantly different between treatment and control groups (CCs) had Fragments Per Kilobase of transcript Per Million Reads (FPKM) values of at least 1 and a greater than log2-fold increase or decrease in expression level. The heatmap color corresponds to the fold change of each gene in all three comparisons within the same treatment group. Genes were selected from the original list of 4467 genes if the gene were co-upregulated or downregulated in all three comparisons. Based on these criteria, all the differential genes were shown. All data were presented as means (n = 4 biological replicates).

Extended Data Fig. 9 Expression profile of the antimicrobial resistance genes (ARGs).

a, The mRNA expression levels of ARGs in E. coli B2 under the treatment of SLAP-S25 (0–8 μg/mL) for 8 h; The FPKM values of the ARGs were extracted and calculated the log2 fold change individually. b,c, mRNA expression levels (FPKM values) of ARGs in E. coli B2 after incubation at different time points (2 h, 4 h or 8 h) in the absence of SLAP-S25 (b), and in the presence of 8 μg/mL SLAP-S25 (c). All data were presented as means (n = 4 biological replicates). mcr, mobile colistin resistance gene; sul2, sulfonamides resistance gene; floR, florfenicol resistance gene; blaCTX-M-14: β-lactams resistance gene of CTX-M group; fosA3: fosfomycin resistance genes; ahp (4)-Ia: the atypical aminoglycoside antibiotic hygromycin B resistant gene; aac(3)-IV/ aac(6’)-Ib3: aminoglycosides resistant gene; dfrA14/A17, trimethoprim resistance gene; oqxA/oqxB, quinolones resistance gene; aadA1/A5, aminoglycosides resistant gene; arr-2, rifampin resistance gene; blaNDM-5, gene of New Delhi metallo-β-lactamase 5; blaTEM-1B, β-lactams resistance gene of TEM group; blaCTX-M-14: β-lactams resistance gene of CTX-M group; cmlA1: chloramphenicol resistant gene; tetA, tetracyclines resistant gene; tetA*: another copy of tetA in plasmid 4; mdfA, multi-drug resistance gene responsible for resistant to a diverse group of cationic or zwitterionic lipophilic compounds such as ethidium bromide, tetraphenylphosphonium, rhodamine, benzalkonium, rifampin, tetracycline and puromycin. Non-ARGs of mobA (mobilization protein A) and repC (replication protein C) in plasmid 5 were shown as controls.

Extended Data Fig. 10 Combination of colistin plus SLAP-S25 was efficacious in the mouse peritonitis-sepsis model.

a, Different organs in died mice with un-treatment and the treatment of SLAP-S25 or colistin alone were collected for CFU determination in two days. At 48 h post-infection, the survived mice in combination treatment of SLAP-S25 with colistin at levels of 4 mg per kg + 0.5 mg per kg, 8 mg per kg + 1 mg per kg and 8 mg per kg + 8 mg per kg were euthanized by cervical dislocation. Bacterial loads (Log10 CFU of E. coli B2) in the heart, liver, spleen, lung and kidney were counted. All data were presented as means ± SD (n = 6). b, At 168 h post-infection, the survived mice (n = 2 to 10) in combination treatment of SLAP-S25 plus colistin at levels of 4 mg per kg + 0.5 mg per kg, 8 mg per kg + 1 mg per kg and 8 mg per kg + 8 mg per kg or treatment of colistin (8 mg per kg) alone were euthanized by cervical dislocation. Bacterial loads (Log10 CFU of E. coli B2) in the kidney were counted. All data were presented as means ± SD. P values were determined by two side Mann-Whitney U test.

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Song, M., Liu, Y., Huang, X. et al. A broad-spectrum antibiotic adjuvant reverses multidrug-resistant Gram-negative pathogens. Nat Microbiol 5, 1040–1050 (2020). https://doi.org/10.1038/s41564-020-0723-z

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