Phylotypes IA1 Research Articles

Pro-inflammatory activity of Cutibacterium acnes phylotype IA1 and extracellular vesicles: An invitro study.

Acne is a chronic inflammatory skin condition that involves Cutibacterium acnes (C. acnes), which is classified into six main phylotypes (IA1, IA2, IB, IC, II and III). Acne development is associated with loss of C. acnes phylotype diversity, characterised by overgrowth of phylotype IA1 relative to other phylotypes. It was also shown that purified extracellular vesicles (EVs) secreted by C. acnes can induce an acne-like inflammatory response in skin models. We aimed to determine if the inflammatory profile of EVs secreted by C. acnes phylotype IA1 from an inflammatory acne lesion was different from C. acnes phylotype IA1 from normal skin, thus playing a direct role in the severity of inflammation. EVs were produced invitro after culture of two clinical strains of C. acnes phylotype IA1, T5 from normal human skin and A47 from an inflammatory acne lesion, and then incubated with either human immortalised keratinocytes, HaCaT cells, or skin explants obtained from abdominoplasty. Subsequently, quantitative PCR (qPCR) was performed for human β-defensin 2 (hBD2), cathelicidin (LL-37), interleukin (IL)-1β, IL-6, IL-8, IL-17α and IL-36γ, and ELISA for IL-6, IL-8 and IL-17α. We found that EVs produced invitro by C. acnes derived from inflammatory acne lesions significantly increased the pro-inflammatory cytokines and anti-microbial peptides at both transcriptional and protein levels compared with EVs derived from normal human skin. We show for the first time that C. acnes EVs from inflammatory acne play a crucial role in acne-associated inflammation invitro and that C. acnes phylotype IA1 collected from inflammatory acne lesion and normal skin produce different EVs and inflammatory profiles invitro.

Biofilm of Cutibacterium acnes: a target of different active substances.

Acne vulgaris is a chronic inflammatory dermatosis. Cutibacterium acnes plays a crucial role in the acne pathophysiology. Recent works present evidence of C.acnes growing as a biofilm in cutaneous follicles. This development is currently considered one of the leading causes of C. acnes in vivo persistence and resistance to antimicrobials used to treat acne. Our objective was to evaluate the effects of various active compounds (clindamycin, erythromycin, doxycycline, and myrtle extract) on eight distinct, well-characterized strains of C. acnes following their growth in biofilm mode. Cutibacterium acnes isolates from phylotypes IA1 and IA2 produce more biofilm than other phylotypes. No antibiotic effect was observed either during the curative test or preventive test. Myrtle extract at 0.01% (w/v) showed significant efficacy on the biofilm for C. acnes strains (curative assays). Furthermore, it appear that myrtle extract and doxycycline together reduce the overall biomass of the biofilm. A significant dose-dependent effect was observed during the preventive test, greater than the one observed under curative conditions, with an important loss of activity of the myrtle extract observed from 0.001% (w/v) concentration onwards. Transmission electron microscopy showed that bacteria treated withmyrtle extract grew biofilms much less frequently than untreated bacteria. Additionally, when the quantity of myrtle extract grew, the overall number of bacteria dropped, indicating an additional antibacterial action. These findings support the hypothesis that the different C. acnes phylotypes have various aptitudes in forming biofilms. They also suggest that myrtle extract is a promising alternative as an anti-biofilm and antibacterial agent in fighting diseases caused by planktonic and biofilm C. acnes.

Comparative Genomic Analysis of Cutibacterium spp. Isolates in Implant-Associated Infections.

Bacteria of the genus Cutibacterium are Gram-positive commensals and opportunistic pathogens that represent a major challenge in the diagnosis and treatment of implant-associated infections (IAIs). This study provides insight into the distribution of different sequence types (STs) of C. acnes, and the presence of virulence factors (VFs) in 64 Cutibacterium spp. isolates from suspected or confirmed IAIs obtained during routine microbiological diagnostics. Fifty-three C. acnes, six C. avidum, four C. granulosum, and one C. namnetense isolate, collected from different anatomical sites, were included in our study. Using whole-genome sequencing and a single-locus sequencing typing scheme, we successfully characterized all C. acnes strains and revealed the substantial diversity of STs, with the discovery of six previously unidentified STs. Phylotype IA1, previously associated with both healthy skin microbiome and infections, was the most prevalent, with ST A1 being the most common. Some minor differences in STs' distribution were observed in correlation with anatomical location and association with infection. A genomic analysis of 40 investigated VFs among 64 selected strains showed no significant differences between different STs, anatomical sites, or infection-related and infection undetermined/unlikely groups of strains. Most differences in VF distribution were found between strains of different Cutibacterium spp., subspecies, and phylotypes, with CAMP factors, biofilm-related VFs, lipases, and heat shock proteins identified in all analyzed Cutibacterium spp.

Genetic and Functional Analyses of Cutibacterium Acnes Isolates Reveal the Association of a Linear Plasmid with Skin Inflammation

Cutibacterium acnes is a commensal bacterium on the skin that is generally well-tolerated, but different strain types have been hypothesized to contribute to the disease acne vulgaris. To understand how some strain types might contribute to skin inflammation, we generated a repository of C.acnes isolates from skin swabs of healthy subjects and subjects with acne and assessed their strain-level identity and capacity to stimulate cytokine release. Phylotype II K-type strains were more frequent on healthy and nonlesional skin of subjects with acne than those isolated from lesions. Phylotype IA-1 C-type strains were increased on lesional skin compared with those on healthy skin. The capacity to induce cytokines from cultured monocyte-derived dendritic cells was opposite to this action on sebocytes and keratinocytes and did not correlate with the strain types associated with the disease. Whole-genome sequencing revealed a linear plasmid in high-inflammatory isolates within similar strain types that had different proinflammatory responses. Single-cell RNA sequencing of mouse skin after intradermal injection showed that strains containing this plasmid induced a higher inflammatory response in dermal fibroblasts. These findings revealed that C.acnes strain type is insufficient to predict inflammation and that carriage of a plasmid could contribute to disease.

Myrtus communis and Celastrol enriched plant cell culture extracts control together the pivotal role of Cutibacterium acnes and inflammatory pathways in acne.

Acne is a multifactorial inflammatory disease of the pilosebaceous unit in which Cutibacterium acnes is one of the main triggers. A strong predominance of C.acnes phylotype IA1 is present in acne skin with higher biofilm organization and virulence, promoting local immuno-inflammation, especially the Th17 pathway. We evaluated the single and combined pharmacological properties of the plant extracts, Myrtus communis (Myrtacine®) and Celastrol enriched plant cell culture (CEE) extracts on the C.acnes/Th17 pathway. The effect of Myrtacine® on the virulence of C.acnes phylotype IA1 was quantified according to the expression of several related genes. The activity of Myrtacine® and CEE on the inflammatory cascade was assessed using monocytes-derived dendritic cells (Mo-DC) stimulated with membranes or biofilms of the C.acnes phylotype IA1. Finally, the effect of CEE on the Th17 pathway was studied using C.acnes stimulated sebocyte 2D cultures and 3D skin tissue models containing preactivated Th17 cells. Myrtacine® had an anti-virulence effect, evident as a significant and strong inhibition of the expression of several virulence factor genes by 60%-95% compared to untreated controls. Myrtacine® and CEE significantly inhibited proinflammatory cytokine (IL-6, IL-8, IL-12p40 and TNF-α) production by Mo-DC in response to C.acnes phylotype IA1. Interestingly, these two ingredients resulted in synergistic inhibition of most cytokines when used in combination. Finally, we demonstrated an inhibitory effect of CEE, in solution or formulated at 0.3%, specifically on IL-17 release by Th17 lymphocytes in a C.acnes-stimulated sebocyte 2D cultures and by Th17-lymphocytes integrated in a 3D skin models. 2D and 3D models were developed to represent relevant and specific pathways involved in acne. Myrtacine® and CEE were shown to alter one or more of these pathways, indicating their potential beneficial effects on this disease.

Recent advances in understanding inflammatory acne: Deciphering the relationship between Cutibacterium acnes and Th17 inflammatory pathway.

Acne vulgaris is a common chronic inflammatory skin disease of the pilosebaceous units. Four factors contribute to acne: hyperseborrhea and dysseborrhea, follicular hyperkeratinisation, skin microbiome dysbiosis and local immuno-inflammation. Recent key studies have highlighted a better understanding of the important role of Cutibacterium acnes (C.acnes) in the development of acne. Three major findings in the last decade include: (1) the ability of C.acnes to self-organize in a biofilm associated with a more virulent activity, (2) the loss of the C.acnes phylotype diversity and (3) the central role of the Th17 pathway in acne inflammation. Indeed, there is a close link between C.acnes and the activation of the Th17 immuno-inflammatory pathway at the initiation of acne development. These mechanisms are directly linked to the loss of C.acnes phylotype diversity during acne, with a predominance of the pro-pathogenic phylotype IA1. This specifically contributes to the induction of the Th17-mediated immuno-inflammatory response involving skin cells, such as keratinocytes, monocytes and sebocytes. These advancements have led to new insights into the underlying mechanisms which can be harnessed to develop novel treatments and diagnostic biomarkers. A major disadvantage of traditional treatment with topical antibiotics is that they induce cutaneous dysbiosis and antimicrobial resistance. Thus, future treatments would no longer aim to 'kill' C.acnes, but to maintain the skin microbiota balance allowing for tissue homeostasis, specifically, the restoration of C.acnes phylotype diversity. Here, we provide an overview of some of the key processes involved in the pathogenesis of acne, with a focus on the prominent role of C.acnes and the Th17-inflammatory pathways involved.

Skin dysbiosis and Cutibacterium acnes biofilm in inflammatory acne lesions of adolescents

Acne vulgaris is a common inflammatory disorder affecting more than 80% of young adolescents. Cutibacterium acnes plays a role in the pathogenesis of acne lesions, although the mechanisms are poorly understood. The study aimed to explore the microbiome at different skin sites in adolescent acne and the role of biofilm production in promoting the growth and persistence of C. acnes isolates. Microbiota analysis showed a significantly lower alpha diversity in inflammatory lesions (LA) than in non-inflammatory (NI) lesions of acne patients and healthy subjects (HS). Differences at the species level were driven by the overabundance of C. acnes on LA than NI and HS. The phylotype IA1 was more represented in the skin of acne patients than in HS. Genes involved in lipids transport and metabolism, as well as potential virulence factors associated with host-tissue colonization, were detected in all IA1 strains independently from the site of isolation. Additionally, the IA1 isolates were more efficient in early adhesion and biomass production than other phylotypes showing a significant increase in antibiotic tolerance. Overall, our data indicate that the site-specific dysbiosis in LA and colonization by virulent and highly tolerant C. acnes phylotypes may contribute to acne development in a part of the population, despite the universal carriage of the microorganism. Moreover, new antimicrobial agents, specifically targeting biofilm-forming C. acnes, may represent potential treatments to modulate the skin microbiota in acne.

226 Biotic complex to improve acne-prone skin

Cutibacterium acnes (CA) virulent strains can uncontrollably proliferate leading to, redness and pimples in acneic skin. But Staphylococcus epidermidis (SE) can selectively use fermentation initiators such as sucrose to produce short chain fatty acids such as succinic acid reducing CA growth. Based on this knowledge a complex of ingredients for acne care, bacterial and microbiotic reconstructed epidermis (mRHE) study models were developed. The aerobic growth of SE (ATCC12228) or anaerobic growth of virulent CA strain phylotype IA1 (ATCC6919 from facial acne lesions) were evaluated by turbidimetry after 3 days with/without 2% Biotic Complex (BC=biotic blend (sucrose, succinic and lactic acid) loaded in a complex made of pullulan, alginate, hyaluronate, glycerin and glycols).

349 Functional screening of Cutibacterium acnes isolates reveal determinants of skin inflammation.

Cutibacterium acnes (C. acnes) is a ubiquitous commensal bacterium that is generally well tolerated by the human skin immune system. Different strain-types of C. acnes have been reported to be enriched on patients with acne. To understand if these strain-type differences contribute to the develoment of inflammation we generated a library of over 400 C. acnes isolates from skin swabs of healthy and acne subjects and assessed their strain-level identity and inflammatory potential. A higher frequency of phylotype IA-1 C-type strains was observed from acne lesional sites compared to non-lesional sites.

Amplicon-Based Next-Generation Sequencing as a Diagnostic Tool for the Detection of Phylotypes of Cutibacterium acnes in Orthopedic Implant-Associated Infections.

The diagnosis of orthopedic implant-associated infections (OIAIs) caused by the slow-growing anaerobic bacterium Cutibacterium acnes is challenging. The mild clinical presentations of this low-virulent bacterium along with its ubiquitous presence on human skin and human-dominated environments often make it difficult to differentiate true infection from contamination. Previous studies have applied C. acnes phylotyping as a potential avenue to distinguish contamination from infection; several studies reported a prevalence of phylotypes IB [corresponding to type H in the single-locus sequence typing (SLST) scheme] and II (SLST type K) in OIAIs, while a few others found phylotype IA1 (more specifically SLST type A) to be abundant. However, phylotype determination has mainly been done in a culture-dependent manner on randomly selected C. acnes isolates. Here, we used a culture-independent amplicon-based next-generation sequencing (aNGS) approach to determine the presence and relative abundances of C. acnes phylotypes in clinical OIAI specimens. As amplicon, the SLST target was used, a genomic fragment that is present in all C. acnes strains known to date. The aNGS approach was applied to 30 sonication fluid (SF) samples obtained from implants removed during revision surgeries, including 17 C. acnes culture-positive and 13 culture-negative SF specimens. In 53% of the culture-positive samples, SLST types were identified: relative abundances were highest for K-type C. acnes, followed by H- and D-type C. acnes. Other types, including A- and C-type C. acnes that are more prevalent on human skin, had low relative abundances. The aNGS results were compared with, and confirmed by a culture-dependent approach, which included the isolation, whole genome sequencing (WGS) and phylotyping of 36 strains of C. acnes obtained from these SF samples. Besides serving as a powerful adjunct to identify C. acnes phylotypes, the aNGS approach could also distinguish mono- from heterotypic infections, i.e., infections caused by more than one phylotype of C. acnes: in eight out of nine culture-positive SF samples multiple C. acnes types were detected. We propose that the aNGS approach, along with the patient’s clinical information, tissue and SF cultures and WGS, could help differentiate C. acnes contamination from true infection.

Polylysine dendrigraft is able to differentially impact Cutibacterium acnes strains preventing acneic skin.

With a view to reducing the impact of Cutibacteriumacnes (C.acnes) on acne vulgaris, it now appears interesting to modify the balance between acneic and non-acneic strains of C.acnes using moderate approach. In the present study, we identified that a G2 dendrigraft of lysine dendrimer (G2 dendrimer) was able to modify membrane fluidity and biofilm formation of a C.acnes acneic strain (RT5), whereas it appeared no or less active on a C.acnes non-acneic strain (RT6). Moreover, skin ex vivo data indicated that the G2 is able to decrease inflammation (IL1α and TLR-2) and improve skin desquamation after of C.acnes acneic strains colonization. Then, in vivo data confirmed, after C.acnes quantification by metagenomic analysis that the G2 cream after 28days of treatment was able to increase the diversity of C.acnes strains versus placebo cream. The data also showed a modification of the balance expression between C.acnes phylotype IA1 and phylotype II abundances. Taken together, the results confirm the interest of using soft compounds in cosmetic product for modifying phylotype abundances and diversity of C.acnes strains could be a new strategy for prevent acne vulgaris outbreak.

Prepubertal acne: A retrospective study.

BackgroundAcne vulgaris is a common skin disorder, but studies on the epidemiologic features of prepubertal acne are limited.ObjectivesThe aim of this study was to determine the prevalence and severity of prepubertal acne and to identify factors influencing acne severity and poor response to treatment.MethodsA retrospective study was conducted on 683 patients with acne from our database who visited the dermatology department of Nantes University hospital between October 2014 and May 2018. Patients of prepubertal acne (7-12 years) were included in this study.ResultsOf the 683 patients with acne, 24 (3.5%) had prepubertal acne. Prepubertal acne was more common in female patients (75%). Acne severity assessment showed that severe acne (Groupe Expert Acné global acne severity scale 4) was the most common form (33%), and mild and moderate forms (Global Evaluation Acne Group, global acne severity scales 2 and 3) accounted for 25% each. There was a high predominance of phylotype IA1 of Cutibacterium acnes (belonging to CC18 subgroup). The analysis of patients’ lifestyle and acne features identified three factors associated with an increased risk of poor response or resistance to acne treatment. Initially severe acne grading (grade 4) was the most strongly associated parameter (p < .028), followed by regular milk consumption and taking other medications in addition to acne treatment (p < .049 for each).ConclusionThis study reported on prepubertal acne features and identified three factors associated with a high risk of treatment failure or relapse. Adequate and prompt treatment is needed in this subgroup of patients to minimize disease burden and prevent subsequent disease worsening.

Low prevalence of Cutibacterium acnes in prostatic tissue biopsies in a French hospital

We evaluated the Cutibacterium acnes prevalence in prostatic biopsies and characterized the strains at a molecular level. 18 out of 36 biopsies (50%) were sterile after seven days in culture. C. acnes was observed in only two biopsies. Its prevalence was low (5.6%). Finally, the molecular characterization revealed diverse clusters including phylotypes IA1, IB and II.

The Skin Microbiome: A New Actor in Inflammatory Acne.

Our understanding of the role of Cutibacterium acnes in the pathophysiology of acne has recently undergone a paradigm shift: rather than C. acnes hyperproliferation, it is the loss of balance between the different C. acnes phylotypes, together with a dysbiosis of the skin microbiome, which results in acne development. The loss of diversity of C. acnes phylotypes acts as a trigger for innate immune system activation, leading to cutaneous inflammation. A predominance of C. acnes phylotype IA1 has been observed, with a more virulent profile in acne than in normal skin. Other bacteria, mainly Staphylococcus epidermis, are also implicated in acne. S. epidermidis and C. acnes interact and are critical for the regulation of skin homeostasis. Recent studies also showed that the gut microbiome is involved in acne, through interactions with the skin microbiome. As commonly used topical and systemic antibiotics induce cutaneous dysbiosis, our new understanding of acne pathophysiology has prompted a change in direction for acne treatment. In the future, the development of individualized acne therapies will allow targeting of the pathogenic strains, leaving the commensal strains intact. Such alternative treatments, involving modifications of the microbiome, will form the next generation of ‘ecobiological’ anti-inflammatory treatments.

Rhodomyrtus tomentosa Fruit Extract and Skin Microbiota: A Focus on C. acnes Phylotypes in Acne Subjects

Knowing that Rhodomyrtus tomentosa is known to have antibacterial effects, this study investigated the skin microbiota with a focus on Cutibacterium acnes (C. acnes) phylotypes in subjects with acne, and determined microbiota changes after 28 days of treatment with berries Rhodomyrtus tomentosa as an active ingredient (RT). Skin swabs from seventeen acne subjects were collected and the skin microbiome was analyzed using 16S rRNA gene sequencing. A culture-independent next-generation sequencing (NGS)-based SLST (single-locus sequence typing) approach was aimed at evaluating RT extract effects on C. acnes phylotype repartition. Clinical evaluations (lesion counts) were performed at baseline (D0) and after 28 days (D28) of twice-daily application of the RT active ingredient. We determined: (1) the skin microbiota at D0 was dominated by Actinobacteria followed by Firmicutes and Proteobacteria; (2) at the genus level, Cutibacterium was the most abundant genus followed by Staphylococcus and Corynebacterium; (3) C. acnes was the major species in terms of mean abundance, followed by Staphylococcus epidermidis (S. epidermidis) and Staphylococcus hominis (S. hominis); and (4) phylotype IA1 was most represented, with a predominance of SLST type A1, followed by phylotypes II, IB, IA2, IC, and III. After 28 days of RT extract treatment, phylotype repartition were modified with a decrease in abundance (approximately 4%) of phylotype IA1 and an increase in phylotype II and III. Cutibacterium granulosum (C. granulosum) abundance also decreased. Reduction of retentional and inflammatory lesions was also noted only after RT treatment; thus, RT extract acts as a microbiota-regulating agent.

Cutibacterium acnes is an intracellular and intra-articular commensal of the human shoulder joint

Cutibacterium acnes (C acnes) is a mysterious member of the shoulder microbiome and is associated with chronic postoperative complications and low-grade infections. Nevertheless, it is unclear whether it represents a contaminant or whether it accounts for true infections. Because it can persist intracellularly in macrophages at several body sites, it might in fact be an intra-articular commensal of the shoulder joint. In 23 consecutive, otherwise healthy patients (17 male, 6 female; 58 years) who had no previous injections, multiple specimens were taken from the intra-articular tissue during first-time arthroscopic and open shoulder surgery. The samples were investigated by cultivation, genetic phylotyping, and immunohistochemistry using C acnes-specific antibodies and confocal laser scanning microscopy. In 10 patients (43.5%), cultures were Cacnes-positive. Phylotype IA1 dominated the subcutaneous samples (71%), whereas type II dominated the deep tissue samples (57%). Sixteen of 23 patients (69.6%) were C acnes-positive by immunohistochemistry; in total, 25 of 40 samples were positive (62.5%). Overall, 56.3% of glenohumeral immunohistochemical samples, 62.5% of subacromial samples, and 75% of acromioclavicular (AC) joint samples were positive. In 62.5% of the tested patients, Cacnes was detected immunohistochemically to reside intracellularly within stromal cells and macrophages. These data indicate that C acnes is a commensal of the human shoulder joint, where it persists within macrophages and stromal cells. Compared with culture-based methods, immunohistochemical staining can increase C acnes detection. Phylotype II seems to be most prevalent in the deep shoulder tissue. The high detection rate of C acnes in osteoarthritic AC joints might link its intra-articular presence to the initiation of osteoarthritis.

Antibiotic Susceptibility of Cutibacterium acnes Strains Isolated from Israeli Acne Patients.

Antibiotic-resistant Cutibacterium acnes has been reported worldwide, but data from Israeli patients with acne is currently lacking. This study evaluated the antibiotic susceptibility of C. acnes, isolated from 50 Israeli patients with acne to commonly prescribed antibiotics, using the Epsilometer test (E-test). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis, 16S rRNA sequencing and single locus sequence typing (SLST) molecular typing were used to identify and characterize C. acnes. Among 36 strains isolated, phylotype IA1 was most common. Resistance to at least one antibiotic was found in 30.6% of tested strains. Resistance rates were highest for erythromycin (25.0%), followed by doxycycline (19.4%), clindamycin (16.7%), minocycline (11.1%) and tetracycline (8.3%). Significant correlation was found between resistance to multiple antibiotics, with 5.6% of isolates resistant to all antibiotics tested. When reviewing resistances rate worldwide antibiotic resistance was found to be prevalent in Israel. Measures to limit the emergence of antibiotic-resistant strains of Cutibacterium acnes should be taken and alternative treatments should be sought.

Cutibacterium acnes phylotypes diversity loss: a trigger for skin inflammatory process

Acne has long been understood as a multifactorial chronic inflammatory disease of the pilosebaceous follicle, where Cutibacterium acnes (subdivided into six main phylotypes) is a crucial factor. In parallel, the loss of microbial diversity among the skin commensal communities has recently been shown as often accompanied by inflammatory skin disorders. This study investigated the association of C.acnes phylotype diversity loss and the impact on Innate Immune System (IIS) activation. The IIS response of skin after incubation with phylotypes IA1, II or III individually and with the combination of IA1+II+III phylotypes, was studied in an in vitro skin explant system. The inflammatory response was monitored by immunohistochemistry and ELISA assays, targeting a selection of Innate Immune Markers (IIMs) (IL-6, IL-8, IL-10, IL-17, TGF-β). IIMs were significantly upregulated in skin when being incubated with phylotype IA1 alone compared with the combination IA1+II+III. In parallel, ELISA assays confirmed these results in supernatants for IL-17, IL-8 and IL-10. We identify the loss of C.acnes phylotype diversity as a trigger for IIS activation, leading to cutaneous inflammation. These innovative data underline the possibility to set up new approaches to treat acne. Indeed, maintaining the balance between the different phylotypes of C.acnes may be an interesting target for the development of drugs.

472 Differential inflammatory responses induced by Cutibacterium acnes are not dependent on strain-types but are associated with the presence of a linear plasmid

Cutibacterium acnes (C. acnes, formerly Propionibacterium acnes), is normally well tolerated by human skin. Different strain-types of C. acnes are enriched in acne, and it has been hypothesized that strain-types may promote disease. To study the host immune response against strain-types and the genetic basis for such differences, we generated a library of >1000 C. acnes isolates from swabs of lesional and non-lesional sites of acne patients and healthy volunteers. C. acnes strain-level identity was determined by high resolution sequence typing and the inflammatory potential of each isolate was assessed by high-throughput screening of IL-8 secretion from SEB-1 sebocytes and normal human keratinocytes (NHEK) exposed to sterile bacterial supernatants. Consistent with prior findings, a higher frequency of C. acnes phylotype IA1 strains were recovered from acne lesional sites (specifically C1 and C2 sequence types (ST)). However, non-lesional sites from acne patients were found to have K1 and K2 STs, similar to healthy skin. Furthermore, and contrary to predictions, many of the K1/K2 STs from healthy skin and non-lesional acne promoted greater IL-8 production compared to lesional C1/C2 STs (p=0.012). Moreover, clonal isolates from the same ST promoted different cytokine responses, with high and low cytokine-inducing strains identified within K1, C1, C2 and H1 STs. The capacity of identical STs to promote different inflammatory responses was validated by intradermal injection of sterile supernatants into mice and subsequent qRT-PCR for IL-8, CXCL1 and IL-1β. The functional differences within these strains could not be attributed to protease or lipase activity, short chain fatty acid or porphyrin production. Instead, whole genome sequencing revealed the presence of a linear plasmid only in the pro-inflammatory strains. Several putative virulence genes are present in this plasmid. We conclude that a virulence plasmid is most highly associated with the proinflammatory activity of C. acnes.

Comparative analyses of biofilm formation among different Cutibacterium acnes isolates

The Gram-positive anaerobic bacterium Cutibacterium acnes is a commensal of the human skin, but also an opportunistic pathogen that contributes to the pathophysiology of the skin disease acne vulgaris. Moreover, C. acnes, in addition to other skin-colonizing bacteria such as S. epidermidis and S. aureus, is an emerging pathogen of implant-associated infections. Notably, C. acnes isolates exhibit marked heterogeneity and can be divided into at least 6 phylotypes by multilocus sequence typing. It is becoming increasingly evident that biofilm formation is a relevant factor for C. acnes virulence, but information on biofilm formation by diverse C. acnes isolates is limited. In this study we performed a first comparative analysis of 58 diverse skin- or implant-isolates covering all six C. acnes phylotypes to investigate biofilm formation dynamics, biofilm morphology and attachment properties to abiotic surfaces. The results presented herein suggest that biofilm formation correlates with the phylotype, rather than the anatomical isolation site. IA1 isolates, particularly SLST sub-types A1 and A2, showed highest biofilm amounts in the microtiter plate assays, followed by isolates of the IC, IA2 and II phylotypes. Microscopic evaluation revealed well-structured three-dimensional biofilms and relatively high adhesive properties to abiotic surfaces for phylotypes IA1, IA2 and IC. Representatives of phylotype III formed biofilms with comparable biomass, but with less defined structures, whereas IB as well as II isolates showed the least complex three-dimensional morphology. Proteinase K- and DNase I-treatment reduced attachment rates of all phylotypes, therefore, indicating that extracellular DNA and proteins are critical for adhesion to abiotic surfaces. Moreover, proteins seem to be pivotal structural biofilm components as mature biofilms of all phylotypes were proteinase K-sensitive, whereas the sensitivity to DNase I-treatment varied depending on the phylotype.