High bacterial colonization and lipase activity in microcomedones

Abstract

Background: Although acne vulgaris has a multifactorial aetiology, comedogenesis and bacteria colonization of the pilosebaceous unit are known to play a major role in the onset of inflammatory acne lesions. However, many aspects remain poorly understood such as where and when is the early stage of the Propionibacterium acnes colonization in follicular unit? Our research aimed at providing a precise analysis of microcomedone's structure to better understand the interplay between Propionibacterium acnes and follicular units, and therefore, the role of its interplay in the formation of acne lesions.

Methods: Microcomedones were sampled using cyanoacrylate skin surface stripping (CSSS). Their morphology was investigated with multiphoton imaging and their ultrastructure with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Bacterial lipase activity in the microcomedones was quantified using a dedicated enzymatic test as well as a Fourier Transform Infra-Red (FTIR) analysis. The porphyrin produced by bacteria was analysed with HPTLC and fluorescence spectroscopy.

Results: The imaging analysis showed that microcomedones' structure resembles a pouch, whose interior is mostly composed of lipids with clusters of bacteria and whose outer shell is made up of corneocyte layers. The extensive bacteria colonization is clearly visible using TEM. Even after sampling, clear lipase activity was still seen in the microcomedone. A high correlation, r = .85, was observed between porphyrin content measured with HPTLC and with fluorescence spectroscopy. These observations show that microcomedones, which are generally barely visible clinically, already contain a bacterial colonization.

Keywords: Propionibacterium acnes; Acne vulgaris; Inflammation; electron microscopy; fluorescence; fourier transform infrared; hair follicle; multiphoton microscopy.

Figure 1

Structural analysis of microcomedones from cyanoacrylate skin surface stripping sample. A, Representative microscopic images of cyanoacrylate skin surface stripping sample (CSSS) samples using dark field mode, showing the presence of microcomedones, corneocytes network and hair follicles on skin surface layer. B, Representative images of lipid accumulation around and inside microcomedones on CSSS sample using O Red Oil staining in bright field microscopy. C, High‐resolution 3D images of microcomedones observed on CSSS samples by multiphoton microscopy using autofluorescence mode

Figure 2

Ultrastructural analysis of microcomedones by electron microscopy. A, Representative images of microcomedones analysed by scanning electron microscopy, showing the overall view of a microcomedone. B, Representative images of microcomedones analysed by transmission electron microscopy, showing the overall pouch shape of microcomedone: corneocyte layers at exterior, cluster of Propionibacterium acnes bacteria inside the microcomedone and presence of lipids around the bacteria

Figure 3

Determination of autofluorescence properties of porphyrins and quantification of microcomedone density on cyanoacrylate skin surface stripping samples. A, A1, Autofluorescence of porphyrins standards (dotted line: CPI; full line: CPIII; mixed line: PpIX). A2, Autofluorescence of solubilized microcomedones. In small red autofluorescence quantification (AUC between 560 and 665 nm) for all samples. A3, Correlation between red autofluorescence intensity (AUC between 560 and 665 nm) and the porphyrin amount from solubilized microcomedones determined by HPTLC analysis. B, B1‐B2 Multiphoton images with NDD detectors (B1) and with spectral detector (B2) of label‐free microcomedones on CSSS samples using autofluorescence mode. B3, Autofluorescence profiles of microcomedone computed from three different parts indicated in the image B2. C, Representative image of CSSS samples in epifluorescence microscopy using red autofluorescence mode. Microcomedone contour and density were automatically detected by an homemade software

Figure 4

Endogenous lipase activity. A, Analysis of Propionibacterium Acnes endogenous lipase activity directly measured on CSSS samples using the EnzCheck^® substrate. A1, Image at different times showing the fluorescence increase. A2, Fluorescence quantification during time (dotted line with squares: lipase of porcine pancreas as positive control; dotted line with triangles: blank; full line with triangles: microcomedones samples). In small: variation in fluorescence intensity at 3H for all samples. B, Determination by FTIR of free fatty acids (FFA)/ triglycerides (TG) ratio from microcomedones. FTIR spectra of microcomedones samples (full line) compared to a spectra of normal skin sebum (dotted line). In small: FFA/TG ratio for all samples