UV-B exposure did not alter the fluorescence emission spectra of porphyrins.

<p><i>P. acnes</i> (2×10⁸ CFU) were incubated with 1 mM ALA for 4 h at 42°C and then exposed to UV-B (50 mJ/cm²) (UV-B). Bacteria without UV-B exposure served as a control (C). The porphyrin derivatives were detected in a spectrum between 550 and 700 nm. A spectrum of CpIII (10 µM) (Sigma, St. Louis, MO) exposed to UV-B [0 (C) or 50 mJ/cm² (UV-B)] was presented in an inserted panel. Spectra are representative of three independent experiments.</p

The Response of Human Skin Commensal Bacteria as a Reflection of UV Radiation: UV-B Decreases Porphyrin Production

<div><p>Recent global radiation fears reflect the urgent need for a new modality that can simply determine if people are in a radiation risk of developing cancer and other illnesses. Ultraviolet (UV) radiation has been thought to be the major risk factor for most skin cancers. Although various biomarkers derived from the responses of human cells have been revealed, detection of these biomarkers is cumbersome, probably requires taking live human tissues, and varies significantly depending on human immune status. Here we hypothesize that the reaction of <em>Propionibacterium acnes</em> (<em>P. acnes</em>), a human resident skin commensal, to UV radiation can serve as early surrogate markers for radiation risk because the bacteria are immediately responsive to radiation. In addition, the bacteria can be readily accessible and exposed to the same field of radiation as human body. To test our hypothesis, <em>P. acnes</em> was exposed to UV-B radiation. The production of porphyrins in <em>P. acnes</em> was significantly reduced with increasing doses of UV-B. The porphyrin reduction can be detected in both <em>P. acnes</em> and human skin bacterial isolates. Exposure of UV-B to <em>P. acnes</em>- inoculated mice led to a significant decrease in porphyrin production in a single colony of <em>P. acnes</em> and simultaneously induced the formation of cyclobutane pyrimidine dimers (CPD) in the epidermal layers of mouse skin. Mass spectrometric analysis via a linear trap quadrupole (LTQ)-Orbitrap XL showed that five peptides including an internal peptide (THLPTGIVVSCQNER) of a peptide chain release factor 2 (RF2) were oxidized by UV-B. Seven peptides including three internal peptides of 60 kDa chaperonin 1 were de-oxidized by UV-B. When compared to UV-B, gamma radiation also decreased the porphyrin production of <em>P. acnes</em> in a dose-dependent manner, but induced a different signature of protein oxidation/de-oxidation. We highlight that uncovering response of skin microbiome to radiation will facilitate the development of pre-symptomatic diagnosis of radiation risk in a battlefield exposure, nuclear accidents, terrorist attacks, or cancer imaging/therapy.</p> </div

UV-B exposure decreased the production of porphyrins in bacteria isolated from human faces.

<p>Tape trips were used to isolate facial bacteria from the nose surfaces of five volunteers (▴, •, ▾, ♦, and ▪). Bacteria were exposed to UV-B ranging from 0 to 20 mJ/cm² and incubated with ALA (1 mM) for 4 h at 42°C. The production of porphyrins in individual bacteria was calculated as described in Materials and Methods. The mean values [fluorescence intensity/bacterium (10⁸ CFU)] of porphyrin production in individual bacteria at a single dose of UV-B exposure were denoted ―.</p

The MS/MS spectrum of an internal peptide (THLPTGIVVSCQNER) of <i>P. acnes</i> RF2.

<p>After exposure of <i>P. acnes</i> in PBS with and without UV-B at a dose of 50 mJ/cm², bacterial pellets were digested with trypsin and subsequently subjected to LTQ-Orbitrap XL mass spectrometry analysis. A sequenced peptide (THLPTGIVVSCQNER) is presented and assigned as an internal peptide of <i>P. acnes</i> RF2 (Q6A808). The m/z value of each “y” and “b” ion in collision-induced dissociation (CID) spectra was indicated. Three independent experiments were performed. The oxidized THLPTGIVVSCQNER at H and C is reproducibly and exclusively present in the UV-B irradiated-<i>P. acnes</i>.</p

Fermentation of <em>Propionibacterium acnes,</em> a Commensal Bacterium in the Human Skin Microbiome, as Skin Probiotics against Methicillin-Resistant <em>Staphylococcus aureus</em>

<div><p>Bacterial interference creates an ecological competition between commensal and pathogenic bacteria. Through fermentation of milk with gut-friendly bacteria, yogurt is an excellent aid to balance the bacteriological ecosystem in the human intestine. Here, we demonstrate that fermentation of glycerol with Propionibacterium acnes (P. acnes), a skin commensal bacterium, can function as a skin probiotic for in vitro and in vivo growth suppression of USA300, the most prevalent community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). We also promote the notion that inappropriate use of antibiotics may eliminate the skin commensals, making it more difficult to fight pathogen infection. This study warrants further investigation to better understand the role of fermentation of skin commensals in infectious disease and the importance of the human skin microbiome in skin health.</p> </div

Simultaneous detection of <i>in vivo</i> responses of both <i>P. acnes</i> and skin cells to UV-B exposure.

<p>Mouse ears harboring <i>P. acnes</i> were irradiated with (50 mJ/cm²) and without UV-B. Ears were excised and homogenized immediately (a–c; g–k) and 24 h (d–f; l–p) after radiation. <i>P. acnes</i> in mouse homogenates was incubated with 1 mM ALA and grown on a Brucella broth agar plate. Compared with unexposed control (C) mouse ear (a, d), the intensity of porphyrins in a single colony of <i>P. acnes</i> significantly decreased after UV-B exposure (UV-B) (b, e). The decrease in the porphyrin intensity is still detectable 24 h after UV-B exposure. The intensity of porphyrin fluorescence was presented as % of that in control <i>P. acnes</i> colonies (c, f). No CPDs were detected in an epidermal layer of UV-B-unexposed-ear skin (g, i, l, n). Immunohistochemical staining showed that the CPD formation (red; arrows) detected by a monoclonal antibody to TDM-2 was considerably increased by UV-B (h, j). Merged images showed DAPI (blue) (h) and CPD (purple) (j) staining. However, UV-B-induced CPDs faded away 24 h after UV-B exposure. The percentages of CPD-positive cells in epidermal layers were displayed (k, p). Dash lines indicate the surface of ear skin. Bar (a, b, d, e) = 200 µm; (g–j; l–o) = 50 µm. **<i>P</i><0.01 or ***<i>P</i><0.001 was evaluated using <i>t</i>-tests. Data are the mean ± SD of three separate experiments.</p

Mass spectrometric analysis of oxidized and de-oxidized peptides in UV-B irradiated <i>P. acnes</i>.

<p>Mass spectrometric analysis of oxidized and de-oxidized peptides in UV-B irradiated <i>P. acnes</i>.</p

The production of porphyrins in <i>P. acnes</i> as a function of the doses of UV-B.

<p>After radiation with or without UV-B, <i>P. acnes</i> was then incubated with ALA (1 mM) for 4 h at 42°C under dark conditions. The ALA induced porphyrins were monitored using the fluorescence emission spectra via a Perkin Elmer LS50B fluorescence spectrometer. The number of bacteria was determined by reading the values of OD₆₀₀ as described in Materials and Methods. The production of porphyrins in individual bacteria was calculated by dividing fluorescent intensities of porphyrins by the number of bacteria. ***<i>P</i><0.001 was evaluated using two-tailed <i>t</i>-tests. Data are the mean ± SD of three separate experiments.</p

Glycerol fermentation of <i>P. acnes</i> in skin wounds diminishes the colonization of USA300.

<p><i>P. acnes</i> (10⁷ CFU in 5 µl PBS), P. acnes and glycerol (0.2 mg), PBS (5 µl) alone or glycerol alone were applied onto the skin wounds for 3 days before administration of USA300 (10⁷ CFU in 5 µl PBS) onto the wounded areas. (A, B) Skin lesions pictured 3 days after USA300 application were illustrated. Bar = 0.5 cm. (C) The USA300 numbers in the skin wounds were enumerated 3 days after USA300 application and presented as % of those in skin applied with <i>P. acnes</i> (C) or PBS (D). *<i>P</i><0.05. <i>P</i>-values were evaluated using two-tailed <i>t</i>-tests. Data are the mean ± SD of lesions from five mice per group. NS: Non-significant.</p

NMR validation of fermentation of <i>P. acnes</i> in mouse skin.

<p>(B) The ear of ICR mice was intradermally injected with ¹³C₃-glycerol (0.2 mg) and P. acnes (ATCC6919; 10⁷ CFU in 10 µl PBS) for 3 days. (A) The other ear of the same mouse received ¹³C₃-glycerol (0.2 mg) and PBS (10 µl) as a control. Supernatants of ear homogenates were mixed with 10% D₂O and analyzed by a NMR (400 MHz JEOL JNM-ECS) spectrometer. Data from 1,024 scans were accumulated. The NMR signals (17.1 and 58.4 ppm) of ¹³C-ethanol (Et) metabolized from ¹³C₃-glycerol (Gly) were detected exclusively in the mice injected with ¹³C₃-glycerol and <i>P. acnes</i>. The un-metabolized ¹³C₃-glycerol appears between 60 and 80 ppm in the ¹³C-NMR spectrum. (C) A 2-D ¹H-¹³C HSQC NMR spectrum (600 MHz) was displayed. In addition to glycerol (Gly), ethanol (Et), four SCFAs [butyric acid (B), 3-hydroxy-butyric acid (3HB), lactic acid (L), and propionic acid (P)] were detected in the ear injected with glycerol and <i>P. acnes</i>.</p