Research ArticleANTIMICROBIALS

The antimicrobial peptide SAAP-148 combats drug-resistant bacteria and biofilms

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Science Translational Medicine  10 Jan 2018:
Vol. 10, Issue 423, eaan4044
DOI: 10.1126/scitranslmed.aan4044
  • Fig. 1 SAAP-148 kills MDR ESKAPE pathogens and colistin-resistant E. coli without resistance selection.

    (A) Susceptibility of multidrug-resistant (MDR) ESKAPE (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter species) pathogens and colistin-resistant E. coli to antibiotics and SAAP-148. Bacteria susceptible to all (green boxes) or intermediate/resistant to at least one (red boxes) of the antibiotics per class. Gray boxes are shown if the susceptibility to agents in that class is not assessed. Bactericidal activity of SAAP-148 in PBS and in PBS with 50% human plasma (or 50% urine in case of E. coli). Results are expressed as the LC99.9, the lowest peptide concentration in micromolar that resulted in ≥99.9% killing. Results are medians (and ranges) of three independent experiments. If no range is indicated, then the LC99.9 was identical in all experiments. (B and C) Resistance development of S. aureus JAR060131 (B) and A. baumannii RUH875 (C) to SAAP-148 and the antibiotics rifampicin and ciprofloxacin, respectively. Values are fold changes (in log2) in minimal inhibitory concentration (MIC) relative to the MIC of the first passage.

  • Fig. 2 SAAP-148 prevents biofilm formation, eliminates established biofilms, and kills persisters.

    (A and B) Prevention of biofilm formation by SAAP-148. Biofilm formation by S. aureus JAR060131 (gray lines) and A. baumannii RUH875 (black lines) after 24 hours incubation in uncoated wells (for S. aureus; A) and plasma-coated wells (B) in biofilm medium (BM) 2 medium containing 0 to 12.8 μM of SAAP-148. Results are expressed as the biofilm mass, measured using crystal violet staining, in arbitrary units (au). Values are medians of 14 to 18 replicates from three independent experiments. (C) Bactericidal activity of SAAP-148 against established biofilms of S. aureus JAR060131 (gray lines) and A. baumannii RUH875 (black lines). Results are expressed as the number of viable bacteria [in log10 colony-forming units (CFU)] after 2-hour exposure of 24-hour-old biofilms to SAAP-148. Values are medians of four to six replicates from three independent experiments. (D) Prevention of biofilm formation by SAAP-148. Biofilm formation by S. epidermidis O47 wild type (WT) (black lines) and its ica-deletion mutant Δica (gray dashed lines) after 24 hours in BM2 medium containing 0 to12.8 μM SAAP-148. Results are expressed as the biofilm mass, measured using crystal violet staining, in arbitrary units. Values are medians of 18 replicates from three independent experiments. (E) Bactericidal activity of SAAP-148 against established biofilms of S. epidermidis O47 WT (black lines) and its ica-deletion mutant Δica (gray dashed lines). Results are expressed as the number of viable bacteria (in log10 CFU) after 2-hour exposure of 24-hour-old biofilms to SAAP-148. Values are medians of five to six replicates from three independent experiments. (F) Bactericidal activity of SAAP-148 against persister cells derived from biofilms. Biofilms of S. aureus JAR060131 were treated for 24 hours with 100× MIC of rifampicin. Antibiotic was removed (arrow), and persisters were exposed to PBS containing 1.6 or 3.2 μM SAAP-148 or no peptide for an additional 2 or 4 hours. Results are expressed as the number of viable bacteria in log10 CFU per milliliter. Values are medians of six replicates from three independent experiments. *, significantly different (*P < 0.05, **P < 0.01, and ***P < 0.001) as compared to control (0 μM), as calculated using the Mann-Whitney rank sum test.

  • Fig. 3 SAAP-148 inserts into the bacterial membrane, causing rapid permeabilization and killing of S. aureus and A. baumannii.

    (A) Effect of SAAP-148 on phase transition of DPPG liposomes upon cooling. Results of one representative experiment out of three experiments. (B) Leakage of POPG liposomes in the presence of SAAP-148. Results are expressed as percentage leakage of the fluorescent dye relative to the total amount of the fluorescent dye. Values are medians of four independent experiments. (C) Schematic model of the interaction of SAAP-148 with the bacterial membrane. Peptide structure prediction based on PEP-FOLD. Helical wheel projection indicating the hydrophilic and hydrophobic region along the helical axes. Charged amino acids are in purple, apolar amino acids are in yellow, and C, P, and Q are in red. Net charge of the peptide at neutral pH and hydrophobicity expressed as transfer free energy of peptides from water to n-octanol (ΔGWOCT in kilocalories per mole) calculated from the whole-residue hydrophobicity scale, taking into account the contribution of the C-terminal amidation and N-terminal acetylation, is shown in the middle of the wheel. (D) Membrane permeabilization in S. aureus JAR060131 and A. baumannii RUH875 by SAAP-148, as measured by propidium iodide (PI) influx. After 20 s, SAAP-148(1.6 μM) was added to bacteria (indicated by arrow), and PI fluorescence was measured for 5 min. (E) The percentage of PI-positive S. aureus and A. baumannii cells was calculated at different time intervals after addition of 0.4 to 3.2 μM SAAP-148. Results are medians of three independent experiments. (F) Cryo-transmission electron micrographs of S. aureus LUH14616 exposed to PBS or subinhibitory concentrations of SAAP-148. Arrows indicate membrane disruption and perturbation. Lower figures are magnifications of the area depicted in red in the upper figures. Scale bars, 200 nm. (G) Killing of S. aureus JAR060131 and A. baumannii RUH875 after 0.5- to 120-min exposure to 1.6 to 3.2 μM SAAP-148. Results are expressed as the number of viable bacteria in log10 CFU per milliliter. Values are medians of three independent experiments.

  • Fig. 4 Topical application of SAAP-148 ointment eradicates acute and established infections of MRSA and A. baumannii from the skin.

    (A to F) Ex vivo wounded human skin (A to C) and in vivo abraded murine skin (D to F) were inoculated with MRSA LUH14616 (gray circles) or A. baumannii RUH875 (black circles). Ten minutes after inoculation (D) or 1 hour (A), 24 hours (C and E), or 48 hours (F) after inoculation, the skin was treated with ointments containing no peptide (vehicle) or 0.125, 0.5, or 2% (w/w) SAAP-148 ointment. Results are expressed as the numbers of viable bacteria (in log10 CFU) per skin model of three to six donors and of 16 skin samples for each group of mice (eight mice per group and two skin samples per mouse). Each circle represents one skin sample, and bars indicate medians. *, significantly different (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) as compared to the vehicle, as calculated using the Mann-Whitney rank sum test. (B) Light micrographs of hematoxylin and eosin–stained skin biopsies 24 hours after inoculation with MRSA and A. baumannii and subsequent vehicle treatment. Arrows indicate biofilm formation, which is shown at a higher magnification in the inset.

  • Table 1 Screening of LL-37–derived peptides reveals peptides with improved bactericidal activity against S. aureus.

    Bactericidal activity in phosphate-buffered saline (PBS) and in PBS with 50% human plasma. Results are expressed as the 99.9% lethal concentration (LC99.9), that is, the lowest peptide concentration that resulted in ≥99.9% killing of S. aureus JAR060131. Results are medians (and ranges) of three to four independent experiments. If no range is indicated, then the LC99.9 was identical in all experiments. The most effective synthetic antimicrobial and antibiofilm peptides (SAAPs) are depicted in bold.

    PeptideSequenceLC99.9 (μM)
    PBS50% plasma
    LL-37LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES1.6(1.6–6.4)>204.8
    P139LKKLWKRVFRIWKRIFRYLKRPVR1.6(0.8–1.6)51.2
    P140LRRLWKRLVRIIKRIYRQLKRPVR1.638.4(25.6–51.2)
    P141LRRLYKRVFRLLKRWWRYLKRPVR1.6(0.8–1.6)38.4(25.6–51.2)
    P142LRRLWKRLVKILKRWFRYLRRPVR1.6(0.8–1.6)51.2(51.2–102.4)
    P143LRRLYKRVVKLWKRLFRQLRRPVR1.6(1.6–3.2)51.2(51.2–102.4)
    P144LKKLYKRVAKIWKRWIRYLKKPVR1.638.4(25.6–51.2)
    P145(SAAP-145)LKRLYKRLAKLIKRLYRYLKKPVR1.6(0.8–1.6)12.8(12.8–25.6)
    P146LKKLYKRLFKILKRILRYLRKPVR1.2(0.8–1.6)51.2(25.6–51.2)
    P147LKKLWKRLARLLKRFIRQLRRPVR1.651.2(25.6–51.2)
    P148(SAAP-148)LKRVWKRVFKLLKRYWRQLKKPVR1.612.8(12.8–25.6)
    P149LKKVYKRLARLLKRYIRYLRRPVR1.625.6(25.6–51.2)
    P150LKKVWKRVARLIKRWFRYLRRPVR1.625.6(25.6–51.2)
    P151LKKLYKRLFKLWKRLYRYLKKPVR1.625.6(25.6–51.2)
    P152LRRVYKRLARLIKRYLRQLKKPVR1.6(1.6–3.2)25.6
    P153LRKLWKRVVKIWKRYLRQLRRPVR1.619.2(12.8–25.6)
    P154LRKLWKRLAKIIKRLYRYLRRPVR1.6(0.8–1.6)25.6(12.8–25.6)
    P155LKKVYKRVARLIKRLFRYLKRPVR1.625.6(12.8–25.6)
    P156LRRLWKRLVKLWKRFFRYLKKPVR1.651.2(51.2–102.4)
    P157LKKVWKRVFRILKRFLRYLKRPVR1.6(0.8–1.6)51.2(25.6–51.2)
    P158LRRVYKRLFRLWKRIIRQLRRPVR1.625.6(12.8–25.6)
    P159(SAAP-159)LKRLYKRVFRLLKRYYRQLRRPVR1.6(1.6–3.2)12.8
    P160LKKLWKRLARLWKRIIRQLKKPVR1.6(1.6–3.2)51.2(25.6–51.2)
    P161LRRVWKRVARIIKRLYRYLKRPVR1.6(1.6–3.2)19.2(12.8–25.6)
    P162LKRLWKRLFKILKRYYRYLRRPVR1.625.6(25.6–51.2)
    P163LRRLWKRVFKIIKRLFRQLKKPVR1.6(0.8–1.6)19.2(12.8–25.6)
    P276(SAAP-276)LKRVWKAVFKLLKRYWRQLKKPVR0.86.4(6.4–12.8)
  • Table 2 LL-37–derived peptides are more effective against S. aureus and P. aeruginosa than (pre-)clinical phase peptides.

    Bactericidal activity (LC99.9) in PBS and in PBS with 50% human plasma against S. aureus JAR060131 and P. aeruginosa PAO1. Results are medians (and ranges) of three to four independent experiments. If no range is indicated, then LC99.9 values were identical in all experiments.

    PeptideDevelopment phaseLC99.9 (μM)
    S. aureusP. aeruginosa
    PBS50% plasmaPBS50% plasma
    Ci MAM-A24Preclinical6.451.2(25.6–51.2)4.8(1.6–6.4)51.2
    CZEN-002IIb>102.4>102.4>102.4>102.4
    DPK060II>102.4>102.4>102.4(102.4–>102.4)>102.4
    GhrelinII>102.4>102.4>102.4>102.4
    HB1345Preclinical51.2(25.6–102.4)>102.451.2(25.6–102.4)>102.4
    Hepcidin-25Preclinical>102.4>102.4>102.4>102.4
    hLF1-11I/II102.4>102.4>102.4(51.2–>102.4)>102.4
    IsegananIII3.2(3.2–6.4)12.8(6.4–12.8)2.4(0.8–6.4)51.2(51.2–102.4)
    KABT-AMPPreclinical1.625.6(12.8–25.6)1.625.6(12.8–25.6)
    OmigananIII51.2>102.46.4(6.4–12.8)>102.4
    OP-145I/II1.6(1.6–3.2)>102.43.2(0.8–3.2)>102.4
    PexigananIII12.8(6.4–12.8)25.61.6(0.8–1.6)6.4(6.4–25.6)
    PlectasinPreclinical>102.4>102.4>102.4>102.4
    POL7001Preclinical>102.4>102.451.2(51.2–>102.4)>102.4
    S-ThanatinPreclinical>102.4(102.4–>102.4)>102.4102.4>102.4
    XOMA-629II>102.4>102.4>102.4>102.4
    LL-371.6(1.6–6.4)>102.43.2(1.6–3.2)>102.4
    SAAP-1451.6(0.8–1.6)12.8(12.8–25.6)1.6(0.8–1.6)25.6(12.8–25.6)
    SAAP-1481.612.8(12.8–25.6)1.612.8(6.4–12.8)
    SAAP-1591.6(1.6–3.2)12.81.6(1.6–3.2)12.8(6.4–12.8)
    SAAP-2760.86.4(6.4–12.8)0.8(0.8–1.6)51.2
  • Table 3 Safety of topical application of SAAP-148 ointment in rabbits.

    Single-dose dermal tolerance test in rabbits. Animals with intact or abraded skin were treated for 4 hours with 500 mg of vehicle or 1% (w/w) SAAP-148 ointment. As a control, untreated rabbits were included. Results are expressed as the number of animals out of the total number of animals within the group (n = 3) that showed signs of skin irritation or pathology within 72 hours after treatment.

    Number of rabbits
    IntactAbraded
    UntreatedVehicleSAAP-148UntreatedVehicleSAAP-148
    Skin irritation
      Erythema formation0/30/30/30/30/30/3
      Edema formation0/30/30/30/30/30/3
    Gross pathology findings0/30/30/30/30/30/3
    Histopathological findings
      Lymphohistiocytic infiltration in the dermis0/30/30/33/3*0/32/3*
      Fibrosis in the dermis0/30/30/30/32/3*0/3

    *Minimal change (score 1).

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/423/eaan4044/DC1

      Materials and Methods

      Fig. S1. Antimicrobial activity of SAAP-145/SAAP-148/SAAP-159 alanine-scanning peptides against S. aureus JAR060131 in PBS and in PBS with 50% human plasma.

      Fig. S2. Effect of plasma proteins on antimicrobial activity of SAAPs against S. aureus JAR060131.

      Fig. S3. Release of SAAP-148 from hypromellose gel.

      Fig. S4. Effect of topical application of mupirocin and chlorohexidine ointment on acute and established human ex vivo skin infections of MRSA and A. baumannii.

      Fig. S5. Effect of topical application of SAAP-148 ointment on ex vivo human skin.

      Table S1. Bactericidal activity of peptides in preclinical and clinical development against S. aureus and P. aeruginosa in low-salt buffer.

      Table S2. Antibiogram of ESKAPE pathogens used in this study.

      Table S3. Resistance development of S. aureus and E. coli.

      Table S4. Sequences of peptides in preclinical and clinical development.

      Table S5. Primary data.

      References (4966)

    • Supplementary Material for:

      The antimicrobial peptide SAAP-148 combats drug-resistant bacteria and biofilms

      Anna de Breij, Martijn Riool, Robert A. Cordfunke, Nermina Malanovic, Leonie de Boer, Roman I. Koning, Elisabeth Ravensbergen, Marnix Franken, Tobias van der Heijde, Bouke K. Boekema, Paulus H. S. Kwakman, Niels Kamp, Abdelouahab El Ghalbzouri, Karl Lohner, Sebastian A. J. Zaat, Jan W. Drijfhout, Peter H. Nibbering*

      *Corresponding author. Email: p.h.nibbering{at}lumc.nl

      Published 10 January 2018, Sci. Transl. Med. 10, eaan4044 (2018)
      DOI: 10.1126/scitranslmed.aan4044

      This PDF file includes:

      • Materials and Methods
      • Fig. S1. Antimicrobial activity of SAAP-145/SAAP-148/SAAP-159 alanine-scanning peptides against S. aureus JAR060131 in PBS and in PBS with 50% human plasma.
      • Fig. S2. Effect of plasma proteins on antimicrobial activity of SAAPs against S. aureus JAR060131.
      • Fig. S3. Release of SAAP-148 from hypromellose gel.
      • Fig. S4. Effect of topical application of mupirocin and chlorohexidine ointment on acute and established human ex vivo skin infections of MRSA and A. baumannii.
      • Fig. S5. Effect of topical application of SAAP-148 ointment on ex vivo human skin.
      • Table S1. Bactericidal activity of peptides in preclinical and clinical development against S. aureus and P. aeruginosa in low-salt buffer.
      • Table S2. Antibiogram of ESKAPE pathogens used in this study.
      • Table S3. Resistance development of S. aureus and E. coli.
      • Table S4. Sequences of peptides in preclinical and clinical development.
      • References (5168)

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S5 (Microsoft Excel format). Primary data.

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