Research ArticleMicrobiome

Antimicrobials from human skin commensal bacteria protect against Staphylococcus aureus and are deficient in atopic dermatitis

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Science Translational Medicine  22 Feb 2017:
Vol. 9, Issue 378, eaah4680
DOI: 10.1126/scitranslmed.aah4680
  • Fig. 1. Dysbiosis of the skin microbiome in AD is associated with S. aureus colonization.

    (A) Live S. aureus CFUs on skin of non-AD subjects and on nonlesional and lesional skin of subjects with AD. Bar, median. (B) Shannon diversity index of bacterial community on skin from non-AD and S. aureus culture–positive or S. aureus culture–negative subjects with AD. Data are shown as box and whisker plots. (C) Principal coordinate analysis plot analysis displaying composition of bacterial communities on non-AD and S. aureus culture–positive or S. aureus culture–negative subjects with AD. SA+, S. aureus culture–positive; SA, S. aureus culture–negative; L, lesional; NL, nonlesional. (D) Ratio of Staphylococcus spp. CFU abundance determined by live colony counting compared to rCFU of Staphylococcus determined by qPCR with species-specific primers. Bar, median. P values were calculated by two-tailed paired t test for lesional versus nonlesional samples or two-tailed independent t test for non-AD versus AD groups.

  • Fig. 2. S. aureus colonization correlates with a lack of antimicrobial activity in CoNS.

    (A) Schematic of a high-throughput antimicrobial screening of CoNS against S. aureus. (B) CFU of CoNS without (red) or with antimicrobial activity against S. aureus (green). Each bar represents data from individual subjects. (C) Frequency of CoNS colonies with activity against S. aureus. Data are reported for 29 healthy subjects and 41 nonlesional or 40 lesional sites from AD subjects. Subjects that had less than 25 CoNS colonies per swab were not included. Each point represents the frequency that anti–S. aureus activity was detected in all colonies isolated from each individual. Bar, mean. ****P < 0.0001. P values were calculated by two-tailed independent t test. (D) Correlation between proportion of CoNS strains that have the capacity to inhibit S. aureus growth and abundance of live S. aureus CFU on the skin surface. Each dot represents data from one individual. Quadrants are divided on the basis of the frequency of antimicrobial CoNS (>50 or <50%) and detection of live S. aureus (<1 or >1 CFU/ cm2). The proportion (%) of subjects in each quadrant to total subjects is shown. L, lesional; NL, nonlesional.

  • Fig. 3. Antimicrobial activity is detected in diverse strains of CoNS and not predictable at the species level.

    (A) Relative abundance of CoNS species with antimicrobial activity from five non-AD subjects and seven subjects with AD, who were randomly selected. Up to 48 CoNS isolates were randomly selected from each individual for 16S rRNA sequencing to identify species. Refer to table S2 for details. (B) Relative abundance of CoNS species without anti–S. aureus activity isolated from six subjects with AD. Refer to table S2 for details.

  • Fig. 4. Colonization by an antimicrobial CoNS strain is protective against S. aureus.

    (A) Anti–S. aureus activity secreted from a representative antimicrobial S. hominis strain (A9) isolated from a non-AD subject. A live colony of S. hominis A9 (upper), conditioned medium (10 μl) from an S. hominis A9 overnight culture (middle), or unconditioned medium [tryptic soy broth (TSB)] served as a negative control were applied on TSB agar containing S. aureus. The black area represents zone of growth inhibition of S. aureus. (B and C) Effect of S. hominis on the survival of S. aureus on ex situ pigskin (B) or S. hominis applied to live mouse skin (C). S. aureus was first applied to skin as described in Materials and Methods. The action of S. hominis A9 was compared to controls, including UV-killed and washed A9 or live S. hominis strains that do not produce AMP activity in solution assay (C4, C5, and C6). All CoNS bacteria were applied at 1 × 105CFU/cm2. Data represent means ± SEM of data from five pigskin sheets (B) or six mice (C). (D) Effect of multiple applications of S. hominis A9 on survival of S. aureus on mouse skin. S. hominis A9 strain or vehicle was applied twice a day to mouse back skin colonized by S. aureus over the indicated periods. Skin swabs were collected to count S. aureus survival. Data represent means ± SEM from 10 (day 3) or 5 (day 7) independent mice. *P < 0.05 by two-tailed independent t test.

  • Fig. 5. S. hominis A9 isolated from normal human skin produces unique lantibiotics.

    (A) Reverse-phase high-performance liquid chromatography elution profile of peptides purified from culture supernatant of S. hominis A9 strain. The insert is a radial diffusion activity against S. aureus from the indicated fractions. Molecular mass of fractions 30 and 32 was measured by MALDI-TOF mass spectrometry (MS) (fig. S4). (B) Amino acid sequence and predicted thioester bonds from the two lantibiotics purified from S. hominis A9. Dha, 2,3-didehydroalanine; Dhb, (Z)-2,3-didehydrobutyrine. (C) Organization of the gene cluster encoding Sh-lantibiotic precursors and lantibiotic biosynthetic genes in S. hominis A9. (D) List of lantibiotic-related genes, gene locus, and putative functions. (E) Effect of application of Sh-lantibiotics (0.5 nmol) or conditioned medium from S. hominis A9 (50 μl) on survival of S. aureus on pigskin. Data represent means ± SEM of four independent assays. **P < 0.01 by two-tailed independent t test. (F) Dose-response curves for the antimicrobial activity of Sh-lantibiotic-α and Sh-lantibiotic-β against S. hominis A9 strain. Data represent means ± SEM of triplicate assays. m/z, mass/charge ratio.

  • Fig. 6. Sh-lantibiotics are commonly found on healthy human skin and synergize with a host AMP.

    (A) Frequency of detecting Sh-lantibiotic-α by colony PCR using gene-specific primers in CoNS isolates from human skin. Each point represents analysis of one individual. Bar, mean. P value was calculated by Wilcoxon-Mann-Whitney test. (B) Detection of Sh-lantibiotic-α peptide by Western blotting from extracts of skin swabs taken from two non-AD subjects who were colonized by bacteria having the Sh-lantibiotic-α gene and two AD subjects who were PCR-negative for the Sh-lantibiotic-α gene. S. hominis culture supernatant was loaded as a positive control. A total of 20 μg of protein was loaded in each lane. The uncropped image is shown in fig. S9. The membrane was restained with antibody against cytokeratin-10, a predominant protein in the stratum corneum, as a loading control. (C and D) Dose-response curves for the antimicrobial activity of Sh-lantibiotic-α (C) and Sh-lantibiotic-β (D) against S. aureus and their synergistic antimicrobial activity with human LL-37. Data represent means ± SEM of triplicate assays. Arrow shows minimal bactericidal concentration.

  • Fig. 7. Transplantation of antimicrobial CoNS reduces survival of S. aureus on human skin.

    (A) Characterization of CoNS clones used for autologous microbiome transplant (AMT). Antimicrobial class of each clone was identified by whole-genome sequencing (refer to fig. S11 for more details). (B) Radial diffusion assay for anti–S. aureus activity secreted from each active CoNS strain used for AMT. Radial diffusion assay of bacteria and conditioned medium was conducted as described previously. (C) Effect of transplantation of antimicrobial CoNS or vehicle on the survival of S. aureus on the skin of five subjects with AD. S. aureus survival was measured by colony counting before transplant (pre) and 24 hours after a single application of bacteria (post). Application and analysis were done in a blinded fashion, and samples are from the contralateral arm of subjects treated with the identical vehicle containing antimicrobial bacteria or vehicle only. Data of AMT or placebo treatment (vehicle) are compared with data from four untreated control subjects with AD. P value was calculated by two-tailed paired t test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/378/eaah4680/DC1

    Methods

    Fig. S1. Analysis of the skin bacterial community by 16S rRNA gene sequencing and its relationship to the capacity to culture S. aureus from the skin.

    Fig. S2. Abundance of live Staphylococcus and Staphylococcus-specific DNA on the skin of non-AD and AD subjects.

    Fig. S3. Specificity of Staphylococcus genus–specific primers for real-time qPCR.

    Fig. S4. Anti–S. aureus activity in CoNS is stable over time.

    Fig. S5. Effect of vehicle treatment on the survival of S. aureus on ex situ pigskin or live mouse skin.

    Fig. S6. MALDI-TOF MS analysis for two AMPs purified from S. hominis.

    Fig. S7. Representation of amino acid losses in genome-guided MALDI-TOF/TOF analysis for Sh-lantibiotic-β.

    Fig. S8. Skin isolate strains of CoNS exert selective antimicrobial activity against S. aureus.

    Fig. S9. Detection of Sh-lantibiotic-α peptide by Western blotting from extracts of skin swabs taken from two non-AD subjects and two AD subjects.

    Fig. S10. List of hypothetical antimicrobial genes identified in anti–S. aureus CoNS clones used for AMT.

    Fig. S11. List of previously known lantibiotic genes identified in antimicrobial S. epidermidis clones isolated from non-AD skin.

    Fig. S12. Study design of AMT therapy for patients with AD.

    Fig. S13. Effect of transplantation of antimicrobial CoNS or vehicle on the survival of S. aureus on the skin of five subjects with AD.

    Table S1. Clinical characteristics of AD and non-AD subjects.

    Table S2. Proportion of CoNS species identified with antimicrobial and nonantimicrobial activity.

    Table S3. Clinical characteristics of AD subjects receiving AMT.

    Table S4. Sequences of PCR primers.

    Table S5. Primary data (provided as an Excel file).

    References (4563)

  • Supplementary Material for:

    Antimicrobials from human skin commensal bacteria protect against Staphylococcus aureus and are deficient in atopic dermatitis

    Teruaki Nakatsuji, Tiffany H. Chen, Saisindhu Narala, Kimberly A. Chun, Aimee M. Two, Tong Yun, Faiza Shafiq, Paul F. Kotol, Amina Bouslimani, Alexey V. Melnik, Haythem Latif, Ji-Nu Kim, Alexandre Lockhart, Keli Artis, Gloria David, Patricia Taylor, Joanne Streib, Pieter C. Dorrestein, Alex Grier, Steven R. Gill, Karsten Zengler, Tissa R. Hata, Donald Y. M. Leung, Richard L. Gallo*

    *Corresponding author. Email: rgallo{at}ucsd.edu

    Published 22 February 2017, Sci. Transl. Med. 9, eaah4680 (2017)
    DOI: 10.1126/scitranslmed.aah4680

    This PDF file includes:

    • Methods
    • Fig. S1. Analysis of the skin bacterial community by 16S rRNA gene sequencing and its relationship to the capacity to culture S. aureus from the skin.
    • Fig. S2. Abundance of live Staphylococcus and Staphylococcus-specific DNA on the skin of non-AD and AD subjects.
    • Fig. S3. Specificity of Staphylococcus genus–specific primers for real-time qPCR.
    • Fig. S4. Anti–S. aureus activity in CoNS is stable over time.
    • Fig. S5. Effect of vehicle treatment on the survival of S. aureus on ex situ pigskin or live mouse skin.
    • Fig. S6. MALDI-TOF MS analysis for two AMPs purified from S. hominis.
    • Fig. S7. Representation of amino acid losses in genome-guided MALDI-TOF/ TOF analysis for Sh-lantibiotic-β.
    • Fig. S8. Skin isolate strains of CoNS exert selective antimicrobial activity against S. aureus.
    • Fig. S9. Detection of Sh-lantibiotic-α peptide by Western blotting from extracts of skin swabs taken from two non-AD subjects and two AD subjects.
    • Fig. S10. List of hypothetical antimicrobial genes identified in anti–S. aureus CoNS clones used for AMT.
    • Fig. S11. List of previously known lantibiotic genes identified in antimicrobial S. epidermidis clones isolated from non-AD skin.
    • Fig. S12. Study design of AMT therapy for patients with AD.
    • Fig. S13. Effect of transplantation of antimicrobial CoNS or vehicle on the survival of S. aureus on the skin of five subjects with AD.
    • Table S1. Clinical characteristics of AD and non-AD subjects.
    • Table S2. Proportion of CoNS species identified with antimicrobial and nonantimicrobial activity.
    • Table S3. Clinical characteristics of AD subjects receiving AMT.
    • Table S4. Sequences of PCR primers.
    • References (4563)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S5. Primary data (provided as an Excel file).

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