The First Report of Diversity Analyses of Skin Microbiome In Indonesian Leprosy Patient

Background Skin microbiome is quiet diverse. There are several factors inuencing the skin microbiome, such as skin diseases. However, the effects of leprosy on the skin microbiome remain unclear and there are only a few studies about skin microbiome on leprosy. The aim of this study was to investigate the alpha diversity of skin microbiome on lesional site of multibacillary (MB) leprosy patients who visited the top referral hospital in West Java Indonesia. Here in this study we characterize the skin microbiome in leprosy patient in compared to healthy individual by using next generation 16S rRNA sequencing. A total 18 skin swab samples were collected from 18 samples (14 leprosy patients, 4 healthy individuals). Taxonomic analysis of leprous skin lesions revealed main ve phyla: Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Cyanobacteria. Proteobacteria and Firmicutes were overrepresented in leprosy patients, while Actinobacteria, Bacteroidetes, and Cyanobacteria were diminished in leprosy patients compared to healthy individuals. The main ve genera in leprous skin lesions were Staphylococcus, Acinetobacter, Corynebacterium, Micrococcus, and Propionibacterium. Staphylococcus, Acinetobacter, and Micrococcus were enriched in leprosy patients, while Corynebacterium and Propionibacterium which have a protective role in normal skin, were diminished in leprosy patients when compared with healthy individuals. Twenty-ve species were found in leprous skin lesions that were not typical in human skin and considered as potentially pathogenic. The alpha diversity analysis showed that leprous skin lesions is less diverse than that of the healthy skin microbiome. As a conclusion, the skin microbiome on lesional site of leprosy patient show alteration and less diverse compare to healthy individuals. This suggest that leprosy can affects skin microbiome prole or otherwise.


Introduction
Leprosy is a chronic granulomatous infectious disease caused by Mycobacterium leprae, 1,2 it primarily affects peripheral nerve and skin. 2 Leprosy still became a signi cant health problem in several countries, 3 including Indonesia, which is the third country with the highest cases of leprosy in the world after India and Brazil. 4,5 In 1982, World Health Organization (WHO) divided leprosy into two classi cations, namely paucibacillary (PB) and multibacillary (MB) leprosy. 4 Disability due to leprosy is more common in MB leprosy compared to PB. 6 It is known that leprosy is thought to affect the skin microbiome. To date, there are only two publications from Brazil 7,8 and one publication from India 9 regarding skin microbiome in leprosy, but there is no publication from Indonesia.
Microbiome is all the microorganisms, their genomes, and the surrounding environmental conditions present in a particular ecosystem. 10 It is present in various locations in the body, for example the eyes, nose, mouth, ears, lungs, and skin. The microbiome found in the skin is referred to as the skin microbiome, 11 which play a role in human health. 8 Various factors can affect the skin microbiome, for example diseases 8,12−14 Several cutaneous diseases such as atopic dermatitis, acne, psoriasis, and leprosy can affect microbiome although the effects on pathophysiology of these diseases is unclear. 7,8,15 The interaction of microbiota inter and intraspecies can modulate the innate immune response of the host. 16 The microbiome plays a role in the function and polarization of macrophages towards proin ammatory (M1) as well as anti-in ammatory (M2). 17 Macrophage is one of the keys in the pathogenesis of leprosy. M1 type of macrophages were dominated in PB leprosy, while an M2 type of macrophages were dominated in MB type leprosy. 18 This is the rst report of the diversity and composition of the skin microbiome in Indonesian leprosy patients as compared to healthy individuals by using next generation 16S rRNA sequencing. We report data from 18 study participants, including 3 leprosy patients before treatment, 4 leprosy patients during treatment, 3 leprosy patients during treatment with reversal reaction, and 4 leprosy patients who have been released from treatment, and 4 healthy individuals.

Study design
This research was a descriptive study using a cross-sectional method conducted in Dr.

Specimen and DNA extraction
Person collecting samples wore a fresh pair of gloves and facemask for every participant to avoid contamination. Swabs of lesional skin from leprosy patients and healthy skin from healthy individuals on back area were collected using Puritan® DNA/RNA Shield™ collection tube with swab device. The sampling procedure was undertaken as follows: skin areas were selected and stretched with one hand and the other hand was holding a swab which was already soaked in wetting buffer solution on Puritan® DNA/RNA Shield™ collection tube. Swabbing was performed in one direction for fty times with a rm pressure and make sure that each side of the swab were in contact with the skin. The collected swab samples were stored in -4 °C until DNA extraction. Total genome DNA from the samples was extracted using ZymoBIOMICS™ DNA Miniprep Kit No D4300 protocol. DNA concentration and purity was monitored on 1% agarose gels. According to the concentration, DNA was diluted to 1 ng/µL using sterile water.
The primers 515F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT) was used to amplify V3-V4 hypervariable regions. All polymerase chain reaction (PCR) reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs). Mix same volume of 1x loading buffer (contained SYB green) with PCR products and operate electrophoresis on 2% agarose gel for detection. Samples with bright main strip between 400 bp − 450 bp were chosen for further experiments. PCR products was mixed at equal density ratios. The mixed PCR products were puri ed with Qiagen Gel Extraction Kit (Qiagen, Germany). The libraries generated with NEBNext® Ultra™ DNA Library Prep Kit for Illumina and quanti ed via Qubit and Q-PCR, would be analysed by Illumina platform Bioinformatics analysis Paired-end reads was assigned to samples based on their unique barcodes and truncated by cutting off the barcode and primer sequences. Paired-end reads were merged using FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/). Quality ltering on the raw tags were performed under speci c ltering conditions to obtain the high-quality clean tags according to the Qiime (V1.7.0, http://qiime.org/scripts/split_libraries_fastq.html) quality controlled process. The tags were compared with the reference database (Gold database,http://drive5.com/uchime/uchime_download.html) using UCHIME algorithm (UCHIME Algorithm,http://www.drive5.com/usearch/manual/uchime_algo.html) to detect chimera sequences (https://drive5.com/usearch/manual/chimeras.html). And then the chimera sequences were removed. Then the Effective Tags nally obtained. Sequences analysis were performed by Uparse software (Uparse v7.0.1001 http://drive5.com/uparse/) using all the effective tags. Sequences with ≥ 97% similarity were assigned to the same Operational Taxonomic Units (OTUs). Representative sequence for each OTU was screened for further annotation. For each representative sequence, Mothur software was performed against the SSUrRNA database of SILVA Database (http://www.arb-silva.de/) for species annotation at each taxonomic rank (Threshold:0.8 ~ 1) (kingdom, phylum, class, order, family, genus, species). To obtain the phylogenetic relationship of all OTUs representative sequences, the MUSCLE (Version 3.8.31,http://www.drive5.com/muscle/) can compare multiple sequences rapidly. OTUs abundance information were normalized using a standard of sequence number corresponding to the sample with the least sequences. Subsequent analysis of alpha diversity was performed basing on this output normalized data. The alpha diversity was assessed by Shannon diversity index.

Results
The bacterial composition of leprous skin lesions and healthy individuals normal skin was investigated using NGS. This is the rst study on the diversity and composition of leprous skin lesions and normal skin healthy individuals through the deep sequencing of 16S rRNA genes in Indonesia. A total of 2,033,254 effective tags of 18 samples (3 leprosy patients before treatment (KB), 4 leprosy patients during treatment (KP), 3 leprosy patients during treatment with reversal reaction (KR), and 4 leprosy patients who have been release from treatment (RFT), and 4 healthy individuals (Ko)) was observed.
Skin microbiome composition in leprous skin lesions compared to healthy individuals In this study, it was known that 51 phyla were identi ed, although there were some microbiota whose phylum had not been identi ed. The main ve phyla in all of the samples were Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Cyanobacteria. In leprous skin lesions, the most abundant phyla were Proteobacteria (41.8%) while in healthy individuals were Actinobacteria (41.5%). Proteobacteria and Firmicutes were enriched in leprosy patients, while Actinobacteria, Bacteroidetes, and Cyanobacteria were diminished in leprosy patients compared to healthy individuals. Interestingly, Cyanobacteria phylum were slightly overrepresented in KB group compared to other group and it was slightly more abundance than Bacteroidetes phylum in KB group. A phylum level taxonomic pro le of the healthy skin and leprous skin lesion microbiome is shown in Figs. 1 and 2.
In this study, it was known that 872 genera were identi ed, although there were some microbiota whose genera had not been identi ed. The top ten genera in all of the samples were Staphylococcus (Firmicutes), Acinetobacter, Pseudomonas, Ensifer (Proteobacteria), Corynebacterium, Micrococcus, Propionibacterium, Dietzia (Actinobacteria), Sphingobacterium, Chryseobacterium (Bacteroidetes). In leprous skin lesions, the most abundance genera were Staphylococcus (20.8%) while in healthy individuals were Corynebacterium (15.6%). Staphylococcus, Acinetobacter, and Micrococcus were enriched in leprosy patients, while Corynebacterium and Propionibacterium were diminished in leprosy patients when compared with healthy individuals. Leprous skin lesions and normal skin in healthy individuals were present many similar genera with varied frequency. However, in this study, we also found 22 genera which were only found in leprous skin lesion and was not found in the skin of the healthy individuals, namely Methyloparacoccus, Micropruina, Prevotellaceae UCG-003, Cnuella, Vagococcus, Gluconobacter, hgcl clade, Salinicola, Johnsonella, Solibacillus, Butyrivibrio, Oceanobacillus, Odoribacter, unidenti ed Methylobacteriaceae, Pontibacter, Pleurocapsa, Candidatus Soleaferrea, Selenomonas 3, Saccharomonospora, Nevskia, Myroides, and Fluviicoccus. A genera level taxonomic pro le of the healthy skin and leprous skin lesion microbiome is shown in Figs. 3 and 4.
In this study participants, it was known that 479 species from leprous skin lesions and 454 species from healthy skin individuals were identi ed, although there were some microbiota whose species had not been identi ed. The top ve species in all of the samples were Acinetobacter johnsonii, Micrococcus luteus, Moraxella atlantae, Pseudomonas stutzeri, and Ensifer adhaerens. Acinetobacter johnsonii, Micrococcus luteus, Moraxella atlantae, and Pseudomonas stutzeri were enriched in leprosy patients, while Ensifer adhaerens was diminished in leprosy patients when compared with healthy individuals. The relative frequency at the species level of the leprous skin lesion and healthy individuals is shown in Table 1.

Alpha diversity analysis
The alpha diversity was assessed by Shannon diversity index. In this study, it was known that the alpha diversity of leprous skin lesion (5.09) was higher than healthy individual group (5.25). From each group, it was known that the highest alpha diversity was a KB group (5.47), followed by RFT (5.18), KP 5.10, Ko (5.09), and KR (4.63). The p values were calculated by Wilcoxon rank sum. However, there were no signi cant differences between both group (p > 0.05) (Fig. 5.) In summary, the alpha diversity analysis showed that the healthy skin microbiome is more diverse than that of leprous skin patient.

Discussion
Recent investigations have highlighted the dysbiosis of the skin microbiome in leprosy patients. All of those studies are conducted in Brazil and India. From a 16S rRNA gene dataset, we provide the rst description of Indonesian leprous skin lesions both before treatment, during treatment, during treatment with reversal reaction, and release from treatment compared to healthy individuals.
Leprous skin lesions revealed ve dominant phyla represented by Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Cyanobacteria. The same rst four phyla were consistent with the previous studies on the skin microbiome of leprosy patients in Brazil and India. 7,8,9 However, the fth phyla (Cyanobacteria) has not been mentioned in the previous studies of skin microbiome in leprous skin lesions. Cyanobacteria (blue-green algae) are common inhabitants of water (fresh, brackish, and marine) and terrestrial environments throughout the world. This phylum produces a broad spectrum of secondary metabolites-biologically active products, which could be toxic (cyanotoxins). The cutaneous adverse effects of cyanobacteria and their cyanotoxins are often underdiagnosed. Cyanobacteria has been reported can cause an irritant and allergic contact dermatitis and also generalized urticarial rash. 19 However, until recently the relationship between Cyanobacteria and leprosy is still unknown.
In this study, the result of Proteobacteria which was slightly overrepresented in leprosy patients and Actinobacteria which was underrepresented in leprosy patients compared to healthy individuals were consistent with the previous studies by Silva et al. in 2015. In this study, Firmicutes was overrepresented in leprosy patients and Bacteroidetes was underrepresented in leprosy patients compared to healthy individuals. These ndings were different with previous studies by Silva et al. in 2015 and, and also Bayal in 2019. The differences in the results of these studies are thought to be due to differences in the anatomical locations of the study samples. In this study, all samples were taken from the back, because all leprosy patients had skin lesions on the back, whereas in the study conducted by Silva et al. 7,8 samples were taken from the volar forearm. In addition, other factors that can affect the microbiome such as gender, age, diet, ethnicity, climate, and geographic location can also cause differences in the results of this study. 8,12−14 Staphylococcus and Acinetobacter were the highest abundance genera in leprous skin lesions. These results were different from a previous study conducted by Silva et al. 7 in 2015 in Brazil, which was Bacillus genera was the highest genera found in the leprous skin lesions. From the results of a study conducted by Bayal et al. 9 it was found that the group of leprosy patients during treatment in Hyderabad and Miraj areas of India had a signi cantly different skin microbiome. Based on a study conducted by Blaser et al. 20 in the United States and Venezuela in 2012 which compared the skin microbiome on the forearm between a population of healthy individuals in the United States and Venezuela. It was found that there were differences in the skin microbiome of the two populations. The difference in results between this study and that of Silva et al. and Bayal et al. presumably because the skin microbiome can be in uenced by ethnicity, lifestyle, and/or geographic location. 8,12−14 Propionibacterium and Corynebacterium genera were diminished in leprous skin lesions compared to healthy individuals. Similar results were shown in previous study conducted by Silva et al. 7 Propionibacterium and Corynebacterium were the dominant genera in healthy skin. 21,22 Both of these genera have a protective function in the skin of healthy individuals. 7 One of the species of the genus Propionibacterium, namely Propionibacterium acnes can inhibit the growth of pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pyogenes through the formation of free fatty acids and propionic acid, as well as the secretion of bacteriocins, such as thiopeptide. 13 Therefore, the decreased number of Propionibacterium and Corynebacterium genera in the leprous skin lesions are thought to be due to interference with the skin's protective function. 7 Leprosy reactions can occur due to changes in the immune system in leprosy patients. 23 In reversal reaction, the role of T helper (Th)1 is more dominant than Th2. The existence of dysregulation of the immune system such as increased Th1 activity can cause changes in the composition of the microbiota and otherwise. 24 Based on the results of this study, there were differences in the order of the microbiota phylum (Firmicutes and Actinobacteria) and genera (Micrococcus and Propionibacterium) composition in leprosy patients during treatment with and without reversal reaction. This result indicate the possibility of reversal reaction in uence on microbiome composition. To the best of our knowledge, this was the rst report of the skin microbiome in leprosy with reversal reaction..
The top ve species in all of groups in this study were Acinetobacter johnsonii, Micrococcus luteus, Moraxella atlantae, Pseudomonas stutzeri, and Ensifer adhaerens, with the rst four species were enriched in leprosy patients, while Ensifer adhaerens was diminished in leprosy patients when compared with healthy individuals. Acinetobacter johnsonii, Micrococcus luteus, Moraxella atlantae, and Pseudomonas stutzeri have been reported previously as a pathogen bacteria that can cause some diseases, such as meningitis (Acinetobacter johnsonii, Micrococcus luteus, Pseudomonas stutzeri), bacteriemia in adenocarcinoma patient (Moraxella atlantae), and ecthyma gangrenosum (Pseudomonas stutzeri). [25][26][27][28] Factors that cause the four species mentioned above are found to be more in the leprous skin lesions than healthy individuals is not known, but it is possibly due to autonomic nerve damage in leprous skin lesions which causes dry skin and impaired protective role of the skin. 1 In contrast to the four previous species, Ensifer adhaerens was higher in healthy individuals compared to leprous skin lesions. Ensifer adhaerens is a Gram-negative bacteria from soil and has lysis properties for other Gram-positive and negative bacteria. 29 The presence of this species on human skin has not been previously reported.
Mycobacterium leprae was not found in this study, which was same as the previous study. 7,8 It is possibly because of the skin swab as a sampling procedure, since this bacteria is an obligate intracellular pathogen of macrophages. 1,2,8 Based on this study, there were 22 genera and 25 species of microbiota, which were only found in the leprous skin lesions. These genera and species are not commonly found on human skin. According to those circumstances, these thought to be a potential pathogen, and its existence is probably caused by an impaired skin barrier of protective function in leprosy patients.
The alpha diversity in the leprous skin lesions in this study was lower than that of healthy individuals.
These results were similar to previous study conducted by Silva et al. 8 in 2018 in Brazil and Bayal et al. 9 in 2019 in India. Studies on alpha diversity in skin microbiome have been conducted in several other skin diseases, such as atopic dermatitis and psoriasis. Therefore, it was known that the diversity of the microbiome was found to be lower in these two diseases than in healthy individuals. 30,31 In atopic dermatitis, the decrease in the diversity of the microbiome is related to the degree of disease severity and increased colonization of pathogenic bacteria, such as Staphylococcus aureus. 32 This proves that some diseases can reduce the diversity of the skin microbiome including in leprosy.

Conclussion
The differences of the skin microbiome in leprous skin lesion between Indonesia and previous studies were observed. These ndings indicate the importance of studying skin microbiome in leprosy patients from distinct geographical and cultural settings. There are a taxonomic changes and different abundance levels of the skin microbiome in leprous skin lesions compared to healthy individuals. The alpha diversity in the leprous skin lesions in this study was lower than that of healthy individuals and leprosy with reversal reactions was the lowest diversity of the microbiome compared to the other groups. Altogether, these results contribute to our knowledge of the global human skin microbiome and provide new insights into M. leprae and reversal reaction effects on the microbiome. However, it should be kept in mind that this study represents a minor portion of the Indonesian population. Future studies on a larger number of patients will be needed to strengthen and expand our conclusions.