Is there a relationship between rosacea with gut dysbiosis? Yilmaz K, Altindis M, Dikicier BS, Yüksekal G, Köroglu M

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Is there a relationship between rosacea with gut dysbiosis?  Department of Medical Microbiology, Sakarya University School of Medicine, Sakarya, Turkey  Department of Dermatology, Sakarya University School of Medicine, Sakarya, Turkey Correspondence Address: Dr. Kerem Yilmaz Department of Medical Microbiology, Sakarya University School of Medicine, Sakarya Turkey Source of Support: None, Conflict of Interest: None Background: Rosacea is a common chronic inflammatory dermatosis that affects about 10% of the population. Although various environmental stimulants and endogenous factors have been shown to stimulate the innate immune response and abnormal neurovascular signaling in the etiology, the variety of clinical forms leads to a poor understanding of the pathophysiology of rosacea. Objectives: In this study, we aimed to determine the relationship between rosacea disease and the intestinal microbiome. Methods: For this purpose, 20 patients with clinical diagnosis of rosacea in the Education and Research Hospital of Sakarya University and 10 healthy volunteers with age and sex matched to the control group were included. 16s ribosomal RNA sequence and metagenomic analyses were performed from fecal samples. Results: We determined the relationships between various changes in the rosacea clinic and intestinal microbiome. According to the results of metagenomic DNA analysis in rosacea patients according to healthy volunteers, Lachnospira, Lachnoclostridium, Roseburia, and Roseburia intestinalis were found to be higher and Coriobacteriaceae, Ruminococcaceae, Butyricimonas virosa, and Clostridiales bacteria were found to be higher. Conclusion: These differences were thought to be related to indirect dysbiotic pathways with rosacea clinic. Since only one study examining the relationship between rosacea and intestinal microbiome can be reached in the literature, it is needed to have more and more sampling studies in order to make sense of the microorganisms that stand out. How to cite this article: Yilmaz K, Altindis M, Dikicier BS, Yüksekal G, Köroglu M. Is there a relationship between rosacea with gut dysbiosis?. Dermatol Sin 2020;38:88-93 How to cite this URL: Yilmaz K, Altindis M, Dikicier BS, Yüksekal G, Köroglu M. Is there a relationship between rosacea with gut dysbiosis?. Dermatol Sin [serial online] 2020 [cited 2020 Jun 3];38:88-93. Available from: http://www.dermsinica.org/text.asp?2020/38/2/88/285350 Rosacea is a common chronic inflammatory dermatosis which affects about 10% of the population. Symptoms are generally present in various combinations and levels of severity fluctuating between exacerbation and remission periods. Based on morphologic properties, rosacea is generally divided into four main subtypes: erythematotelangiectatic, papulopustular, rhinophyma, and ocular. Erythematotelangiectatic rosacea is characterized with temporary facial erythema (blushing), combined with a background of persistent centrofacial erythema and also present in most telangiectasia patients. In clinical representation of the disease, patients frequently have the morphologic properties of multiple rosacea subtypes and complaints of burning, stinging, rash, and increased sensitivity. Although it has been demonstrated in its etiology that the increase in various environmental stimuli and endogenous factors stimulates the innate immune response and abnormal neurovascular signal, the variety of clinical forms prevents completely understanding the pathophysiology of rosacea. The intestinal microbiome is relatively stable despite varying between persons; however, studies have demonstrated that antibiotic treatment, international travel, and sickness can alter the normal intestinal microbiome. In addition, the microbiome is affected by various parameters including age, gender, diet, and method of delivery. In the human gastrointestinal system, more than 100 trillions of bacteria of more than a thousand species colonize the intestines. A large part of the organisms present in the intestinal microbiome is from two phylums: Firmicutes and Bacteroidetes. The role of the intestinal microbiome as an important determiner of human health and sickness has recently been discovered as an exciting field of study in medicine. Instability in the intestinal microbiome has been associated with many diseases such as obesity, type 2 diabetes, atopy, and inflammatory intestinal diseases. The balance between the intestinal microbiome and the pathogenesis of dermatological diseases has also started to be studied and discovered lately. The intestinal microbiome is comprised a large bacteria community, their metabolites, and byproducts. In addition, the skin and intestines function as neuroimmunoendocrine organs and take part in the main communications with the nervous system, immune system, and endocrine system. The “gut–brain axis” is comprehensively documented in the literature, and it was first documented in 1930 by Stokes and Pillsbury that depression led to modifications in the intestinal microbiome and inflammatory dermatological diseases. The intestinal microbiome can generate neurotransmitter substances as a reaction to stress and other external stimuli which can modulate the skin function with neural functions. For example, communal organisms in the intestines can produce norepinephrine, serotonin, and acetylcholine or stimulate the emission of neuropeptides from nearby enteroendocrine cells. These neurotransmitters can transgress the intestinal epithelium, mix with blood flow, and lead to systemic effects. In addition to neurotransmitters, the intestinal microbiome excretes short-chain fatty acids (SCFAs) including propionic acid, butyric acid, acetic acid, and lactic acid obtained from dietary polysaccharide fermentation. Most of these SCFAs are produced in the large intestine. The colon is highly efficient in the reabsorption of fatty acids. These metabolites and neurotransmitters are suggested to be produced by the intestinal microbiome, reach clinically significant levels in blood flow, and affect the skin. Moreover, studies have observed intestinal and dermatologic microbiome dysbiosis concurrently with various inflammatory skin diseases such as rosacea, psoriasis, and atopic dermatitis. The purpose of this study is to shed a light on the rosacea pathogenesis and assist new diagnosis and treatment approaches by identifying the relationships between rosacea and the intestinal microbiome. For this purpose, it was studied if there was any difference in the intestinal microbiome of patients diagnosed with rosacea and the intestinal microbiome of healthy volunteers and if so which microorganisms were prominent. Ethics The approval of Sakarya University Faculty of Medicine Non-Pharmacologic Interventional Ethical Board was obtained for this study (SAUTF-KAEK No. 2018/06/27). All participants in the study provided their informed consent. Study design This study was conducted at Sakarya University Training and Research Hospital, Medical Microbiology Laboratory, between July 2018 and December 2018. The study included 20 female patients clinically diagnosed with rosacea upon their application to the dermatology polyclinics of the same hospital and 10 health female volunteers who were matched with rosacea patients in terms of age and gender as the control group. The rosacea patients were selected among the stable erythematous-telangiectatic and moderate-type patient in order to make a homogeneous patient group. All volunteering patients and control group members were applied a mini questionnaire which included questions about their drug use (antibiotics and proton-pump inhibitor), dietary habits, smoking, and use of alcohol to implement the criteria of inclusion and exclusion. The criteria for inclusion in the study group included being diagnosed with rosacea and in the 18–49 age group, whereas the exclusion criteria included other than incompatibility with the diagnosis and age group requirements, having used antibiotics, proton-pump inhibitor and/or probiotics, and prebiotics in the past 4 weeks. Collection of fecal samples In our study, the patient samples were scored using the Bristol stool scale. The stool samples were measured in pH with indicator tapes (Merck, NJ, USA). Approximately 1-g stool samples obtained from the patient and control groups were divided into two equal parts and out in DNA/RNA Shield™ Collection Tube (Zymo Research, CA, ABD) (includes genetic material preservative solution). The samples were kept at −20°C for a couple of days until the study period. The second samples were kept at −80°C. Nucleic acid isolation and 16S ribosomal RNA sequence analysis The genomic DNA was isolated from the stool samples using ZymoBIOMICS DNA Mini Kit (Zymo Research, CA, ABD) according to the manufacturer's instructions. Bacterial 16S ribosomal RNA (rRNA) gene target sequencing was realized with the genetic materials obtained in our study. 341f (CCTACGGGNGGCWGCAG) and 805r (GACTACHVGGGTATCTAATCC) universal bacterial 16S primer pairs which target V3–V4 area of 16S rRNA gene were used. Amplicon libraries were cleaned with (Zymo Research, Select-a-Size DNA Clean & Concentrator™) selecting fragments larger than 200 bp. Then, there were normalized and gathered together. The sequence analysis of final libraries was made with Illumina MiSeq. The ZymoBIOMICS Microbial Community Standards (both cellular standard and DNA standard) were used as positive controls for each run. Negative controls (e.g., blank extraction control and blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process. Bioinformatic analyses Trimmomatic-0.33 program was used to extract adapters from Illumina data in the first place as a result of raw sequence reading. Amplicon sequences and smaller than 320 bp and chimeric amplicon sequences were identified and excluded from the system. Full-amplicon sequences were collected for each sample. Microbial operational taxonomic units (OTUs) were analyzed with Qiime 1.9.1 (QIIME is an open-source bioinformatics pipeline for performing microbiome analysis from raw DNA sequencing data). The GreenGene database was selected as the reference database for OTUs. Samples were collected randomly up to 50.000 sequences to minimize potential bias caused by unequal sampling in every sample. The similarity threshold of OTU was 97% in this study. Alpha- and beta-diversity analyses were made according to the sequence results. Bray–Curtis dissimilarity principal coordinate analysis was used for beta-diversity analysis. The phylogenetic diversity index was used for alpha-diversity analysis. Statistical analysis Taxonomic maps were prepared with the hierarchical clustering of taxons and samples with bioinformatic analyses. LEfSe analysis helps to identify taxa whose distributions are significantly and statistically different among predefined groups. LEfSe uses statistical analysis to identify taxa whose distributions among predefined groups are significantly different. It also utilizes the concept of effect size to allow researchers to focus on the taxa of dramatic differences. By default, LEfSe identifies taxa whose distributions among different groups are statistically different with P < 0.05, and the effect size ([Lineer Discriminant Analysis (LDA) score] higher than 2. LEfSe analysis is only possible if group information is given. It can conveniently help researchers identify biomarkers among/between groups (e.g., control group vs. disease group). All rosacea-diagnosed participants in the study were women. The average age was 38.3 ± 8.9 for the rosacea patients and 35.7 ± 2.9 for the healthy volunteers. The stool samples were rather among Type 5–7 for the rosacea group and Type 1–3 for the healthy group. Comparing stool pH average values of the rosacea and healthy groups, the average stool pH of the rosacea group (average stool pH = 7.5) was higher than the healthy group (average stool pH = 6.7) (P < 0.001, Mann–Whitney U-test). Fourteen of the rosacea patients (70%) were Type 1 (Bacteroides dominant gut microbiota), six (30%) were Type 2 (Prevotella gut microbiota), and seven of the healthy volunteers (70%) were Type 1 and 3 (30%) were Type 2. After 25 million readings in total, chimeric amplicon sequences and amplicon sequences smaller than 320 bp were excluded before the analysis. Negative controls (blank extraction control and blank library preparation control) were used to evaluate the biological load. The genomic DNAs obtained from the stool samples of rosacea patients and healthy volunteers were sequenced with Illumina MiSeq, and analyses were conducted with suitable databases. The increased distance in the coordinate axis shows the differentiated patient and healthy microbiomes. The PC1 maximum variation rate was 11.09%. Comparing the rosacea patients and healthy volunteers, reduced phylogenetic dissimilarity (microbial diversity) was observed in the intestinal microbiome of the rosacea patients [Figure 1]. Figure 1: (a) β-diversity based on Bray–Curtis algorithm. (b) α-diversity comparison between rosacea patients and rosacea-free controls (phylogenetic diversity index) Click here to view Considering the distribution between the groups of rosacea patients and healthy volunteers, statistically different taxons which had P < 0.05 an effect size and LDA score bigger than 2 were identified [Figure 2]. According to the metagenomic DNA analysis results, the rosacea patients had higher rates of Lachnospira, Lachnoclostridium, Roseburia, and Roseburia intestinalis, and the healthy volunteers had statistically higher rates of factors on type, species, and family basis such as Prevotellaceae, Prevotella copri, Coriobacteriaceae, Desulfovibrio, Ruminococcaceae, Ruminococcus, Clostridiales, Clostridiales family 13, Selenomonadales, Succinivibrio, Hungatella, Mollicutes, Butyricimonas virosa, Alisitipes, Oscillospira, and Erysipelotrichaceae [Figure 2]. Figure 2: Prominent microorganisms and LDA scores in healthy volunteers and rosacea patients Click here to view The intestinal microbiome components lower the intestinal pH with the SCFAs they produce with the microbial fermentation of dietary carbohydrate sources which are digestible by the intestinal microorganisms but cannot be digested by the host enzymes. Low intestinal pH shortens the intestinal transit time and triggers sooth muscles which encourage peristalsis. Low intestinal pH supports the reproduction of useful intestinal microbiota members such as Bifidobacterium and Lactobacillus; as these bacteria lower the intestinal pH, the reproduction of pathogen bacteria is inhibited. Increased pH suggests reduced SCFA and unbalanced intestinal microbiome and thus skin dyshomeostasis with gut skin axis. In our study too, supporting intestinal dysbiosis, the average stool pH of the rosacea patients (average stool pH = 7.5) was higher than the average stool pH of the healthy volunteers (average stool pH = 6.7). Bristol stool scoring can offer preliminary information about the intestinal microbiome like stool pH. Lower scores (1 and 2) indicate slow transit and higher scores (5–7) indicate fast transit and increased rectal sensitivity. Blake et al. (2016) obtained much higher scores in irritable bowel syndrome (IBS) patients with a dysbiotic intestinal microbiome than healthy volunteers in their study on healthy volunteers (n = 169) and patients diagnosed with IBS (n = 19) using Bristol stool scoring. The Bristol scores of rosacea patients were much higher than those of the healthy volunteers in our study too. This suggests reduced colonic transit time, intestinal dysbiosis, and thus skin dyshomeostasis with gut skin axis. A publication which compiled numerous studies on the patients diagnosed with atopic dermatitis, one of the common dermatological diseases, emphasized that significant differences were obtained in the intestinal microbiome compared to healthy volunteers and disease could develop with gut skin axis. A study conducted using 16S rRNA metagenomic sequence analyses on the intestinal microbiome of patients with atopic dermatitis (n = 90) identified a strong relationship between Faecalibacterium prausnitzii and atopic dermatitis clinics. The same study demonstrated reduced SCFA levels in atopic dermatitis patients, supporting dysbiotic formation. A study made on patients with chronic urticaria (n = 20) and healthy volunteers (n = 20) obtained a significantly higher frequency of Akkermansia muciniphila, Clostridium leptum, and F. prausnitzii in the stool samples of healthy controls than chronic urticaria patients. A study on the relationship between another chronic dermatological disease, psoriasis, and the intestinal microbiome associated increased Faecalibacterium and decreased Bacteroides with psoriasis. The comprehensive literature scanning only reached a study of Nam et al. which was conducted with a low number of participants (n = 12) on the relationship between rosacea and the intestinal microbiota using new-generation sequencing methods. The same systems, primaries, and analysis methods used in our study were used in this concerned study. The respective study had 12 female patients diagnosed with rosacea whose average age was 42.58, and the average age of the healthy control group (n = 251) was 43.02. The concerned study obtained higher rates of Acidaminococcus and Megasphaera and lower rates of Slackia, Coprobacillus, Citrobacter, and Desulfovibrio in the rosacea patients according to 16S rRNA sequence analysis results from the stool samples from healthy volunteers. Compared to the healthy volunteers, the rosacea patients had statistically higher rates of Acidaminococcus, Megasphaera, and Lactobacillales and were associated with rosacea clinics. In our study, different results were obtained other than the low rates of Desulfovibrio in the patient group. According to 16S rRNA sequence analysis results, in our study, the rosacea patients had higher rates of Lachnospira, Lachnoclostridium, Roseburia, and R. intestinalis (P < 0.05 and LDA >2) [Figure 2]. Roseburia and R. intestinalis bacteria which were obtained at higher rates in the rosacea patients in this study were also obtained at higher rates compared to the healthy volunteers in another study conducted on obese patients. It is believed that these bacteria extract huge amounts of energy from undigested fibers, which affect the balance of energy positively. It was considered that the impact on the energy balance might have an important role in the change in the microbial composition. Geng et al. identified a significant increase in Roseburia species as a result of 16S rRNA sequence analyses in colorectal cancer patients and emphasized that this increase might be associated with dysbiosis and disease development. Some Roseburia species can cause interleukin-8 secretion from the intestines with special proteins they excrete and lead to pro-inflammatory effects. This mechanism is suggested to be effective in the pathogenesis of the diseases it is associated with. However, on the contrary, it is argued that Roseburia species are good for health and they can even be bioindicators which might indicate good health. The mechanism discussed here is explained by Roseburia species colonizing the mucin layers in the intestines and exhibiting inflammatory properties with SCFA production. In our study, 65% of the rosacea patients were on a high carbohydrate diet according to the obtained information. It is suggested to be effective in the higher rates of some bacteria with high fermentative properties such as Roseburia. The presence of high rates of Roseburia is associated with faster colonic transit time. It was thought that it might be one of the reasons for reduced diversity in the patients. Considering Lachnospira, Lachnoclostridium bacteria which we had at higher levels in the rosacea patient group, the colonization of these bacteria caused a significant increase in liver and mesenteric adipose tissue weights as well as fasting blood glucose and drops in plasma insulin levels. The abundance of these bacteria was associated with type 2 diabetes development. These bacteria, measured in high quantities in the patients, are all from the Lachnospiraceae family despite looking like different bacteria. Therefore, in more general terms, there is an increase in the Lachnospiraceae family in the intestines of rosacea patients. The same family members [on rows 22, 29, and 36 in [Figure 2] were only high in two samples in the control group. It is clearly significant for rosacea patients. It is believed to be a component taking part in the pathophysiology of the disease. Furthermore, according to the sequence analysis results, the rosacea patients and healthy volunteers had the same rates of enterotypes. This result limits our interpretation of the potential relationship between the rosacea clinics and enterotyping. It was considered that all these results might be potential factors to play a role in the dysbiosis development and rosacea clinics. It was also considered that prebiotics and probiotics might be tried as alternatives of treatment. The patients diagnosed with rosacea had higher stool pH average and Bristol scores than the control group. This was evaluated as a potential factor for shortened colonic transit time and increased pH dysbiosis. According to 16S rRNA sequence analysis results, the microbiome diversity of which has been reduced and which has differentiated is a risk factor for rosacea development. Lachnospira, Lachnoclostridium, Roseburia, and R. intestinalis were statistically higher and Coriobacteriaceae, Ruminococcaceae, B. virosa, and Clostridiales bacteria were statistically lower in the rosacea patients compared to the healthy volunteers. This difference was suggested to be associated with indirect dysbiotic paths with rosacea clinics. The study suggested a relationship with rosacea and intestinal dysbiosis as a result of increased and decreased bacterial groups. As there was only one study on the rosacea and intestinal microbiome relationship in the literature, more studies with larger samples are needed to interpret the prominent microorganisms. Financial support and sponsorship This work was supported by Sakarya University Scientific Research Projects (SAU: 2018-2-9-177). 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