Acne microbiome: From phyla to phylotypes.

Acne vulgaris is a chronic inflammatory skin disease with a complex pathogenesis. Traditionally, the primary pathophysiologic factors in acne have been thought to be: (1) altered sebum production, (2) inflammation, (3) excess keratinization and (4) colonization with the commensal Cutibacterium acnes. However, the role of C. acnes has been unclear, since virtually all adults have C. acnes on their skin yet not all develop acne. In recent years, understanding of the role of C. acnes has expanded. It is still acknowledged to have an important place in acne pathogenesis, but evidence suggests that an imbalance of individual C. acnes phylotypes and an alteration of the skin microbiome trigger acne. In addition, it is now believed that Staphylococcus epidermidis is also an actor in acne development. Together, C. acnes and S. epidermidis maintain and regulate homeostasis of the skin microbiota. Antibiotics, which have long been a staple of acne therapy, induce cutaneous dysbiosis. This finding, together with the long-standing public health edict to spare antibiotic use when possible, highlights the need for a change in acne management strategies. One fertile direction of study for new approaches involves dermocosmetic products that can support epidermal barrier function and have a positive effect on the skin microbiome.

Unravelling the eco-specificity and pathophysiological properties of Cutibacterium species in the light of recent taxonomic changes.

In 2016, a new species name Cutibacterium acnes was coined for the well-documented species, Propionibacterium acnes, one of the most successful and clinically important skin commensals. The nomenclatural changes were brought about through creation of the genus Cutibacterium, when a group of propionibacteria isolates from the skin were transferred from the genus Propionibacterium and placed in the phylum Actinobacteria. Almost simultaneously, the discovery of two novel species of Cutibacterium occurred and the proposal of three subspecies of C. acnes were reported. These dramatic changes that occurred in a long-established taxon made it challenging for the non-specialist to correlate the huge volume of hitherto published work with current findings. In this review, we aim to correlate the eco-specificity and pathophysiological properties of these newly circumscribed taxa. We envisage that this information will shed light on the pathogenic potential of new isolates and enable better assessment of their clinical importance in the foreseeable future. Currently, five species are recognized within the genus: Cutibacterium acnes, Cutibacterium avidum, Cutibacterium granulosum, Cutibacterium modestum (previously, "Propionibacterium humerusii"), and Cutibacterium namnetense. These reside in different niches reflecting their uniqueness in their genetic makeup. Their pathogenicity includes acne inflammation, sarcoidosis, progressive macular hypomelanosis, prostate cancer, and infections (bone, lumbar disc, and heart). This is also the case for the three newly described subspecies of C. acnes, which are C. acnes subspecies acnes (C. acnes type I), subspecies defendens (C. acnes type II), and subspecies elongatum (C. acnes type III). C. acnes subspecies acnes is related to inflamed acne and sarcoidosis, while subspecies defendens to prostate cancer and subspecies elongatum to progressive macular hypomelanosis. Because the current nomenclature is based upon polyphasic analyses of the biochemical and pathogenic characteristics and comparative genomics, it provides a sound basis studying the pathophysiological roles of these species.

Thermaurantimonas aggregans gen. nov., sp. nov., a moderately thermophilic heterotrophic aggregating bacterium isolated from microbial mats at a terrestrial hot spring.

A moderately thermophilic and heterotrophic bacterial strain, LAT, was isolated from microbial mats sampled at a hot spring in Nakabusa, Nagano, Japan. The cells of strain LAT were aerobic, Gram-stain-negative, non-sporulating, non-motile and rod-shaped (2.0-4.1 µm long). The formation of dense cell aggregates in liquid medium was a unique characteristic of the strain. Strain LAT grew optimally at 50°C and at pH 7.5. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the nearest neighbour of strain LAT was Schleiferia thermophila JCM 30197T with 94.1 % sequence similarity. The draft genome sequence of strain LAT (2 671 880 bp) showed 18.0 % digital DNA-DNA hybridization, 70.9 % average nucleotide identity and 72.1 % average amino acid identity (AAI) values in comparison with the genome sequence of S. thermophila JCM 30197T (2 606 763 bp); the 16S rRNA gene sequence similarity and AAI values are lower than the cutoffs used for assignment to a separate genus. On the basis of phenotypic features, major cellular fatty acid composition, genome sequencing and phylogenetic position, a novel genus and species are proposed for strain LAT, to be named Thermaurantimonas aggregans (= JCM 31826T=DSM 106522T).

Cutibacterium modestum sp. nov., isolated from meibum of human meibomian glands, and emended descriptions of Cutibacterium granulosum and Cutibacterium namnetense.

An anaerobic and aerotolerant bacterium, strain M12T, was isolated from the meibum of inflamed human meibomian glands. Cells of the strain was Gram-stain-positive, non-spore-forming and non-motile rods. Growth on trypticase soy agar plates supplemented with 5 % sheep blood was fastest at 30-37 °C under anaerobic conditions. The 16S rRNA gene sequence of the strain revealed that it belongs to the genus Cutibacterium with a 98.0 % similarity value to the closest species, Cutibacterium acnes. Genome analysis of the strain with type strains of the other Cutibacterium species resulted in digital DNA-DNA hybridization values of 32.3-22.3% and average nucleotide identity (OrthoANI) values of 86.7-73.6 %. Biochemical and physiological analyses using API rapid ID 32A and API Coryne kits revealed relatively low reactivity of the strain compared with C. acnes and Cutibacterium namnetense. The most abundant major cellular fatty acid was iso-C15 : 0. Fermentation end-products from glucose were propionate, lactate, succinate and acetate. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. Major menaquinones were MK-9(H4), MK-9(H2) and MK-9. The major peaks of the MALDI-TOF mass spectrometry spectrum were at 3493, 3712, 6986 and 7424 Da. The DNA G+C content was 59.9 mol%. Based on these findings, we propose a novel species, Cutibacterium modestum. The type strain of C. modestum is M12T (=JCM 33380T=DSM 109769T). On the basis of further genomic analysis, we also provide emended descriptions of Cutibacterium granulosum (Prévot 1938) Scholz and Kilian 2016 and Cutibacterium namnetense (Aubin et al. 2016) Nouioui et al. 2018.

Proposal of new combination, Cutibacterium acnes subsp. elongatum comb. nov., and emended descriptions of the genus Cutibacterium, Cutibacterium acnes subsp. acnes and Cutibacterium acnes subsp. defendens.

In 2016, division of the genus Propionibacterium into four distinct genera was proposed. As a consequence, the species Propionibacterium acnes was transferred to Cutibacterium gen. nov. as Cutibacterium acnes comb. nov. The three recently proposed subspecies of P. acnes were not, however, accommodated in this proposal. Following a very recent validation of a new combination for C. acnessubsp.defendens and an automatically created C. acnessubsp.acnes, we now propose the new combination, C. acnessubsp. elongatum comb. nov. The type strain of Cutibacterium acnes subsp. elongatum is JCM 18919T (=NCTC 13655T). On the basis of further genomic and phenotypic (haemolysis and MALDI-TOF mass spectrometry) analyses of these subspecies, we also provide emended descriptions of the genus Cutibacterium Scholz and Kilian 2016, C. acnessubsp.acnes (Gilchrist 1900) Nouioui et al. 2018, and C. acnessubsp.defendens (McDowell et al. 2016) Nouioui et al. 2018.

Dissecting the taxonomic heterogeneity within Propionibacterium acnes: proposal for Propionibacterium acnes subsp. acnes subsp. nov. and Propionibacterium acnes subsp. elongatum subsp. nov.

Propionibacterium acnes subsp. acnes subsp. nov. and Propionibacterium acnes subsp. elongatum subsp. nov. are described. These emanate from the three known phylotypes of P. acnes, designated types I, II and III. Electron microscopy confirmed the filamentous cell shape of type III, showing a striking difference from types I/II, which were short rods. Biochemical tests indicated that, in types I/II, either the pyruvate, l-pyrrolidonyl arylamidase or d-ribose 2 test was positive, whereas all of these were negative among type III strains. Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra, which profile mainly their ribosomal proteins, were different between these two groups. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) spectra of all phylotypes revealed a specific protein biomarker that was overexpressed in type III strains compared with types I/II only when grown aerobically. Reference strains had high whole-genome similarity between types I (>91 %) and II (>75 %), but a considerably lower level of 72 % similarity with type III. recA and gyrB sequence dendrograms confirmed the distant relatedness of type III, indicating the presence of two distinct centres of variation within the species P. acnes. On the other hand, cellular fatty acid profiles and 16S rRNA gene sequence relatedness (>99.3 %) circumscribed the species. Thus, we propose two subspecies, Propionibacterium acnes subsp. acnes subsp. nov. for types I/II and Propionibacterium acnes subsp. elongatum subsp. nov. for type III. The type strain of Propionibacterium acnes subsp. acnes is NCTC 737T ( = ATCC 6919T = JCM 6425T = DSM 1897T = CCUG 1794T), while the type strain of Propionibacterium acnes subsp. elongatum is K124T ( = NCTC 13655T = JCM 18919T).

Interleukin-8 content in the stratum corneum as an indicator of the severity of inflammation in the lesions of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is an inflammatory skin disease characterized by both acute and chronic eczema. Various markers are used to clinically evaluate the severity of AD. In order to identify a marker of local severity of AD, we measured IL-8, IL-18, vascular endothelial growth factor (VEGF), and transforming growth factor-α (TGF-α) levels in the stratum corneum (scIL-8, scIL-18, scVEGF and scTGF-α) and evaluated the correlation between the levels of these cytokines and the clinical severity scores of localized skin lesions. METHODS: Stratum corneum samples were collected from the skin lesions of 50 patients with AD using the tape-stripping technique, and the scIL-8, scIL-18, scVEGF and scTGF-α levels were evaluated using the ELISA method. The trans-epidermal water loss and skin water content of the lesions were also measured prior to tape stripping. RESULTS: The levels of scIL-8, scIL-18, scVEGF and scTGF-α were significantly higher in patients with AD than in healthy controls. Additionally, the levels of scIL-8, scIL-18 and scVEGF significantly correlated with the severity of AD. CONCLUSIONS: Among these cytokines, scIL-8 showed the highest correlation with the severity scores of lesions in AD as well as other parameters. Our results also suggest that measuring cytokines in the stratum corneum by using ELISA combined with tape stripping is a convenient method to evaluate the severity of skin lesions in AD.

Higher allergenicity of high molecular weight hydrolysed wheat protein in cosmetics for percutaneous sensitization.

BACKGROUND: Wheat protein derivatives are used in a variety of products worldwide. Gluten is commercially used 'as is' or with modifications such as hydrolysis, which is carried out to overcome its insolubility. Several cases of contact urticaria following exposure to hydrolysed wheat protein (HWP) in cosmetics or of anaphylaxis caused by deamidated gluten in food or non-food products have been described. OBJECTIVES: To evaluate the types of HWP that have higher allergenicity for percutaneous sensitization. METHODS: We enrolled 7 patients with wheat-dependent exercise-induced anaphylaxis who had been sensitized to HWP primarily through the percutaneous and/or the rhinoconjunctival route by using facial soap containing HWP. Reaction to wheat proteins was confirmed by IgE immunoblotting and basophil CD203c expression with six HWP variants. RESULTS: The IgE of all the patients reacted to HWPs composed of large polypeptide aggregates. High molecular weight (MW) HWPs were also found to induce significant enhancement of basophil CD203c expression. CONCLUSIONS: HWPs composed of large polypeptide aggregates possibly induce sensitization to a greater degree than lower-MW HWPs. Basophil surface CD203c expression is useful for evaluating the allergenicity of HWPs.

Content of vascular endothelial growth factor in stratum corneum well correlates to local severity of acute inflammation in patients with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is an inflammatory skin disease, characterized by existence of both acute and chronic eczema. Various markers are used to clinically evaluate the severity of AD as a whole. However, little is known regarding markers that can efficiently indicate the severity of a localized lesion. Vascular endothelial growth factor (VEGF), a potent activator of vascular permeability, is known to be increased in AD lesions. In order to establish whether the VEGF content in the stratum corneum (scVEGF) can be used as a marker to evaluate severity of AD lesions, we evaluated the association between scVEGF and symptom scores of localized lesions. METHODS: Fifty patients with AD and 12 healthy subjects were enrolled. Skin lesions were evaluated and transepidermal water loss and skin water content of the lesions were measured. Stratum corneum samples were collected from the skin of back, neck and arm by the tape stripping technique. The scVEGF were evaluated using a VEGF-specific ELISA method after extracting protein from the scales. RESULTS: The scVEGF levels were significantly higher in patients with AD than in healthy controls. Moreover, the scVEGF levels highly correlated with the manifestation scores of erythema and edema/papulation, and weakly correlated with the scores of excoriation, xerosis and itch. They also correlated significantly with transepidermal water loss and skin water content. CONCLUSIONS: The scVEGF levels correlated well with the severity of clinical conditions, especially erythema and edema/papulation. scVEGF level is considered to be a useful marker to evaluate acute inflammatory conditions in individual AD lesions.

What Do We See in Spectra?: Assignment of High-Intensity Peaks of Cutibacterium and Staphylococcus Spectra of MALDI-TOF Mass Spectrometry by Interspecies Comparative Proteogenomics.

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is a widely used and reliable technology to identify microbial species and subspecies. The current methodology is based on spectral fingerprinting, analyzing protein peaks, most of which are yet to be characterized. In order to deepen the understanding of these peaks and to develop a more reasonable identification workflow, we applied proteogenomic approaches to assign the high-intensity peaks of MALDI-TOF spectra of two bacterial genera. First, the 3-22 kD proteomes of 5 Cutibacterium strains were profiled by UPLC-MS/MS, and the amino acid sequences were refined by referring to their genome in the public database. Then, the sequences were converted to m/z (x-axis) values based on their molecular masses. When the interspecies comparison of calculated m/z values was well-fitted to the observed peaks, the peak assignments for the five Cutibacterium species were confirmed. Second, the peak assignments for six Staphylococcus species were performed by using the above result for Cutibacterium and referring to ribosomal subunit proteins coded on the S10-spc-alpha operon (the S10-GERMS method), a previous proteomics report by Becher et al., and comprehensive genome analysis. We successfully assigned 13 out of 15 peaks for the Cutibacterium species and 11 out of 13 peaks for the Staphylococcus species. DNA-binding protein HU, the CsbD-like protein, and 50S ribosomal protein L7/L12 were observed in common. The commonality suggests they consist of high-intensity peaks in the MALDI spectra of other bacterial species. Our workflow may lead to the development of a more accurate species identification database of MALDI-TOF mass spectrometry based on genome data.

Common Features and Intra-Species Variation of Cutibacterium modestum Strains, and Emended Description of the Species.

Cutibacterium modestum is a new species coined in 2020 as the fifth species of genus Cutibacterium, which includes Cutibacterium acnes. The species is predicted as a minor but common member of skin microbiome and includes a group tentatively named as "Propionibacterium humerusii". The description of the species has been provided only with a single strain. To establish the characteristics of C. modestum and search for possible disease-related subtypes, we investigated the biochemical characteristics of eight live strains and performed in silico comparison of nine genomes. The common features, which included the morphology of Gram-stain positive short rods, the negativity of phenylalanine arylamidase, and several unique MALDI-TOF MS spectral peaks, were considered useful in laboratory identification. Pairwise comparisons of the genomes by in silico DNA-DNA hybridization showed similarity values of 98.1% or larger, which were far higher than the subspecies cutoff of 79-80%. The 16S rRNA gene sequences of thirteen isolates and genomes were identical. Their recA gene sequences were identical except for two strains, HM-510 (HL037PA2) and Marseille-P5998, which showed unique one-nucleotide polymorphisms. The biochemical features using API kits were slightly different among the isolates but far closer than those of the nearest other species, C. acnes and Cutibacterium namnetense. Spectra of MALDI-TOF mass spectrometry showed slight differences in the presence of m/z 10,512 (10 kD chaperonin GroS) and three other peaks, further clustering the eight isolates into three subtypes. These results indicated that these isolates did not separate to form subspecies-level clusters, but subtyping is possible by using recA gene sequences or MALDI-TOF mass spectrometry spectra. Moreover, this work has confirmed that a group "P. humerusii" is included in C. modestum.

Actinic lichen planus in a Japanese man: first case in the East Asian population.

Actinic lichen planus (ALP) is a rare variant of lichen planus in which lichen planus develops on the light-exposed areas of the skin. ALP is reported to occur in the African, Middle Eastern,and Indian populations, with very few cases reported in Caucasians. Here, we report a case of ALP in a Japanese man; to the best of our knowledge, this is the first reported occurrence of ALP in the East Asian population. A 52-year-old Japanese man developed recurrent painful annular erythema on the face and hands. Histopathological examination of his skin biopsy revealed lichenoid-type infiltrates of lymphocytes and histiocytes. We established a diagnosis of ALP on the basis of the distribution of eruptions only on the sunlight-exposed areas and histological findings. Oral administration of systemic steroids proved effective in improving his condition. Lichen planus is known to be induced by an irritant (Koebner phenomenon);we believe that our patient is genetically susceptible to sunlight exposure and that sunlight acted as an irritant stimulating the development of ALP.

Sequence analysis of filaggrin gene by novel shotgun method in Japanese atopic dermatitis.

BACKGROUND: Recent reports indicated that nonsense mutations in filaggrin (FLG) found in ichthyosis vulgaris (IV) patients are predisposing factors for atopic dermatitis (AD) with asthma. The exon 3 of FLG contains tandemly repeated, highly homologous, 11-13 sequence units of 972 or 975 bp, each of which corresponds to the coding sequence of the processed filaggrin with slight sequence difference. This unique gene structure has hampered the precise DNA sequence determination. OBJECTIVE: We developed a novel DNA sequencing method "FLG-shotgun" to directly characterize the mutations in Japanese AD patients. METHODS: We examined 24 Japanese AD patients with "FLG-shotgun" method. RESULTS: Multiple units of FLG were amplified by PCR using several sets of common primers for the conserved regions, and DNA sequences of each cloned PCR product were determined. Multiple reads of DNA sequences in both alleles were aligned and re-constructed to cover the entire coding regions. We found three major genotypes (A, B, and C) which represent different numbers (11-13) of homologous sequence units. Furthermore, we found two novel nonsense mutations; one mutation 8666-8667CC>GA on the unit 9 of allele B that causes a nonsense mutation S2899X in two patients and the other mutation 9887C>A on the unit 10 of allele B that causes a nonsense mutation S3296X in two patients. CONCLUSION: We found two novel FLG mutations by directly analyzing Japanese patients with AD. FLG-shotgun will provide a valuable tool to further define the nature of the AD phenotype associated with FLG mutations.

Characterization of skin microbiota in patients with atopic dermatitis and in normal subjects using 16S rRNA gene-based comprehensive analysis.

A previous study using bacterial 16S rRNA gene-based clone libraries revealed that the microbiota in healthy human skin included uncultured micro-organisms, although the micro-organisms in skin exposed to disease conditions remain to be examined. To compare the profiles of skin microbiota in 13 patients with atopic dermatitis (AD) and 10 healthy controls, terminal RFLP analysis of bacterial 16S rRNA genes was applied to 23 swab-scrubbed samples from facial skin. This culture-independent analysis successfully revealed the complex bacterial members of the microbiota as peak patterns following capillary electrophoresis of terminal restriction fragments (T-RFs). Each T-RF peak reflected a micro-organism, and the micro-organism to which each peak was assigned could be identified by computer simulation of T-RF length using the nucleotide sequence data of bacterial species residing in the skin. Among 18 species detected in the study, Stenotrophomonas maltophilia was detected significantly more commonly in AD patients (5/13 for AD patients vs 0/10 for controls), whilst Dietzia maris was detected significantly more commonly in normal controls (8/10 for controls vs 2/13 for AD patients). Moreover, Streptococcus species, which are considered to be uncommon in uninfected skin, were detected in seven patients and eight normal controls. Although further studies should be undertaken to investigate the roles of these micro-organisms in AD, the microbiota were presumed to include hitherto uninvestigated bacterial species in the major population of patients with AD and of healthy controls.

Abnormal keratin expression in circumscribed palmar hypokeratosis.

BACKGROUND: Circumscribed palmar or plantar hypokeratosis (CPH) is a rare skin disorder only recently described. OBJECTIVE: To determine the diagnostic features and to provide insight into the pathogenesis of CPH, with analysis of two new Japanese cases. METHODS: Dermoscopy, immunohistochemistry, electron microscopy, polymerase chain reaction amplification for human papillomavirus (HPV) DNA and 16S microbial rRNA gene profiling were conducted. RESULTS: Dermoscopy showed characteristic features using both dry and jelly immersion observation; step-like desquamation and a homogeneous erythema with regularly distributed whitish spots. Immunohistochemistry revealed strong staining with anti-pankeratin antibody (AE1+AE3) and anti-keratin 16 antibody, and decreased expression of keratin 2e. EM revealed a breakage of the corneocytes within their cytoplasm, but structures for cell attachment were intact. HPV and lesion-specific bacteria were not detected. LIMITATIONS: The number of cases analyzed was two. CONCLUSION: Hyperproliferative epidermal state along with enhanced corneocyte fragility may account for the unique features in CPH.