A polymeric approach toward resistance-resistant antimicrobial agent with dual-selective mechanisms of action

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Science Advances  27 Jan 2021:
Vol. 7, no. 5, eabc9917
DOI: 10.1126/sciadv.abc9917
  • Fig. 1 Design, synthesis, and characterization of the antimicrobial oligoamidine.

    (A) Structural features of traditional antimicrobial polymers. (B) Structural illustration of groove binders, including pentamidine, the U.S. Food and Drug Administration–approved antiprotozoal agent and an antibiotic sensitizer; furamidine, an antitrypanosomal agent in clinical trials; distamycin, a polyamide-based antibiotic; 4′,6-diamidino-2-phenylindole (DAPI) and Hoechst 33342, commonly used DNA-staining dyes; as well as the two recently reported DNA-targeting antimicrobials, BPH-1358 and MBX-1066. The proposed mechanisms of the designed oligoamidine antimicrobial agent from the polymeric approach are also shown. (C) Synthetic scheme of the oligoamidine studied in this work. (D) MALDI-TOF (matrix-assisted laser desorption/ionization–time-of-flight) spectrum of oligomer 3. (E) Gel permeation chromatogram of oligomer 3 with marked molecular weight in dalton, calculated from a poly(ethylene glycol)–based calibration curve. The negative peak is due to the solvent. m/z, mass/charge ratio.

  • Fig. 2 Evidences for oligoamidine 3’s two distinct antimicrobial mechanisms of action, membrane disruption, and DNA binding.

    (A) 3-treated E. coli (K12) (top) and MDR A. b -1 (bottom) cells showing clear evidence of membrane damage, exhibiting fragments and wrinkled surfaces, similar to previous reports on surfactant-based antimicrobial polymers. Scale bars, 4 μm. (B) Results of membrane permeability assay using E. coli, 3T3 cells and propidium iodide (PI), showing bacterial cell membrane disruption by the addition of 3. PI is a fluorescent dye that can only penetrate compromised cell membranes, so its uptake into cells indicated membrane disruption. Compound treatment time was 4 hours for E. coli. The membrane of the NIH/3T3 cells were not affected by 3 even over an extended time period (24 hours). Fl., fluorescence. (C) Exploration of 3’s cellular internalization mechanism. Chlorpromazine, wortmannin, mβCD/genistein, and 4°C condition, respectively, inhibit clathrin-mediated endocytosis, macropinocytosis, caveolae-mediated endocytosis, and energy-dependent endocytosis in general. (D) Inhibition of 3’s activity by DNA. Externally added DNA substantially inhibited oligomer 3’s antimicrobial effect against MDR A. b -1, whereas colistin’s efficacy was minimally affected by added DNA. (E) Fluorophotometric studies showing that 2 μg/ml of 3 was able to displace >80% of the minor groove binding dye, Hoechst 33342 (4 μg/ml), bound to double-stranded DNA (dsDNA), while kanamycin was not able to incur any change in Hoechst’s fluorescence. a.u., arbitrary units.

  • Fig. 3 Evidences for oligoamidine 3’s selectivity for bacteria over eukaryotic cells on both of its mechanisms of action.

    (A) Overlays of confocal microscopic images of bacteria and mammalian cells stained by DAPI, FM4-64, and 3-FITC. N/A, not applicable. (B) Representative two-dimensional fluorescence intensity histograms of stained bacteria and mammalian cells. (C) Pearson’s correlation coefficient, R, for membrane and DNA staining versus staining with 3-FITC. The statistics covered >20 randomly picked cells in each category. (D) Selective activation of housekeeping gene in bacteria cells by 3. (a) 3 activated recA transcription in A. baumannii at its sub-MIC concentration. (b) The transcription of two housekeeping genes in NIH/3T3 cells, actin and tubulin, was not affected at an even higher concentration of 3. (E) Selective inhibition of protein expression in bacteria cells by 3. (a) Oligomer 3 reduced the expression of protein (RFP) in its sub-MIC concentration. (b) Protein expression of Tubulin and Actin in NIH/3T3 cells was not significantly affected. (F) Heatmap of differential expression analysis showing gene regulation changes in A. baumannii treated with 3. Control and treatment groups each had three replicates. (G) Volcano plot of the transcriptome results using 3-treated A. baumannii. -lgP, -log10 (P of the corresponding protein). P is the octanol-water partition coefficient.

  • Fig. 4 The resistance-resistant nature of 3 and its performance at the presence of eukaryotic cells.

    (A) Resistance generation rate comparison between rifampin, colistin, and 3 in M. smegmatis. (B) Resistance generation rate comparison between kanamycin and 3 in E. coli. (C) Rescue of MDR bacteria-infected RBCs by 3. Oligomer 3 significantly outperformed meropenem by killing all the MDR A. b -1 in the presence of RBCs without causing hemolysis. (D) Rescue of MDR bacteria-infected eukaryotic (NIH/3T3) cells by 3. Scale bars, 100 μm. (E) Cartoon illustration of the intracellular bacteria model study. (F) Confocal microscopic images of M. smegmatis–infected RAW 264.7 cells with or without treatment of rifampin (30 μg/ml) or 3 (10 μg/ml). Only cells treated by 3 survived, with bacteria eliminated. Scale bars, 10 μm. (G) Bacteria burden determination of M. smegmatis–infected RAW 264.7 cells after 1 hour using different treatments. (H) The transcription of actin and tubulin in RAW 264.7 cells (10 μg/ml, 1 hour) was not affected at the condition of the intracellular antimicrobial study.

  • Fig. 5 In vivo efficacy and biocompatibility evaluation of oligoamidine 3.

    (A) Complete eradication of bacteria by oligomer 3 in infected C. elegans models, outperforming several commercial antibiotics. C. elegans were infected with different bacteria as labeled above. (B) Increased survival rate for mice treated with 3 in the mice cutaneous abscess infection model study. (C) Survival analysis result showing that 3 reduced mouse mortality rate from ~55 to <10% in the mice excision wound model study. (D) Significant reduction of the number of bacteria on the infected areas by oligomer 3 in the mice excision wound model study. (E) Comparison of organ bacterial burden in the infected mouse. The results suggest that oligomer 3 should have substantially suppressed the dissemination of bacteria into various organs in infected mice. (F) Tissue immunohistology section images. The subcutaneous treatment of 3 did not damage mice organs at its effective concentration (40 mg/kg).

  • Table 1 MIC and therapeutic indices of 3 and various antibiotics.

    Different Gram-positive, Gram-negative bacteria, and mycobacteria strains, including laboratory strains and clinical isolates with multidrug resistance, were covered in the evaluation. B. s, Bacillus subtilis; S. a, Staphylococcus aureus; E. c, Escherichia coli; E. fa, Enterococcus faecalis; E. fi, Enterococcus faecium; K. p, Klebsiella pneumoniae; A. b, Acinetobacter baumannii; P. a, Pseudomonas aeruginosa; M. s, Mycobacterium smegmatis; M. tb, Mycobacterium tuberculosis; and E. cl, Enterobacter cloacae. USA300 and USA400 are methicillin-resistant S. aureus strains. ND, not determined.

    Bacteria*MIC of 3
    MIC of antibiotics (μg/ml)Therapeutic
    index of 3
    Gram positiveS. a0.5<1<1<1<1>128>128>10,000
    S. a USA3002ND>2500
    S. a USA4002>2500
    S. a -14>12832>12816>128>128>1250
    S. a -24>1283212816>128>128>1250
    B. s4ND>1250
    E. fa4<18<1<1>128>128>1250
    E. fi -12>128ND>12832>128>128>2500
    Gram negativeK. p4>128<164<132<1>1250
    K. p -12>128>12832<1>128<1>2500
    K. p -22>128<164<1<1<1>2500
    K. p -32>32>32>32<1>3232>2500
    K. p -44>32>32>3232>32>32>1250
    K. p -54>32>32>3216>3232>1250
    K. p -62>32>32>3232>3232>2500
    A. b2>1283216<116<1>2500
    A. b -12>128>128>12832128<1>2500
    A. b -22>128<116<116<1>2500
    P. a4128<1128<1>128<1>1250
    P. a -14>1284>128<1>128<1>1250
    P. a -24>128432<1>128<1>1250
    E. cl -12>128<1128<18<1>2500
    E. cl -24>128<1128<164>128>1250
    E. cl -34>32>32>322>3216>1250
    E. cl -42>32>32>32>32>328>2500
    E. cl -54>324>32>32>3216>1250
    E. c (K12)216<132<1>128>128>2500
    E. c -12>128<1644<1<1>2500
    E. c -24>128646416>128<1>1250
    MycobacteriumM. s2.562ND150.5NDND>2000
    M. tb H37Rv2516041282.5>12816>200
    M. tb Erdman25ND>200

    *Strains having their names ending with -x are clinical isolates with multidrug resistance, and ESKAPE strains are shown in bold.

    †Minimal inhibitory concentration.

    ‡The HC50 measured for 3 was >5000 μg/ml.

    Supplementary Materials

    • Supplementary Materials

      A polymeric approach toward resistance-resistant antimicrobial agent with dual-selective mechanisms of action

      Silei Bai, Jianxue Wang, Kailing Yang, Cailing Zhou, Yangfan Xu, Junfeng Song, Yuanxin Gu, Zheng Chen, Min Wang, Carolyn Shoen, Brenda Andrade, Michael Cynamon, Kai Zhou, Hui Wang, Qingyun Cai, Eric Oldfield, Steven C. Zimmerman, Yugang Bai, Xinxin Feng

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