Staphylococcus aureus as dominant bacterial species in the cutaneous microbiome of prurigo nodularis

Introduction

Prurigo nodularis (PN) is a chronic dermatosis of a not fully understood pathogenesis, characterized by the presence of multiple nodular lesions accompanied by intense itch [1]. Data on the role of microorganisms in the development of PN is limited [2, 3]. So far, PN has been most frequently associated with human immunodeficiency virus (HIV) infection [4, 5]. The potential link between PN and hepatitis C has also been suggested in several reports [6], but no large-scale studies have been conducted so far. Additionally, in the study by Mattila et al. [2], acid fast bacilli were identified in 28% of PN lesions using classical Ziehl-Neelsen staining. In the same study, non-tuberculous mycobacteria were cultured from 14% of the lesions. Nevertheless, to the best of knowledge, this is the only research highlighting the direct presence of mycobacteria in PN nodules.
A novel approach to the role of infectious factors in disease pathogenesis is based on the evaluation of the diversity and composition of a pool of microorganisms in a given niche, referred to as “microbiome” [7, 8]. This is possible thanks to the rapid development of next-generation sequencing (NGS)-based techniques. It should be taken into consideration that culture-dependent techniques do not reflect the true composition of microbiome as some microbiota cannot be cultured in standard conditions or inhibit the growth of other slow-growing microorganisms [9]. Microbiome evaluation, using 16S ribosomal RNA (rRNA) analysis, had been predominantly performed in atopic dermatitis (AD) so far [10]. The results of these studies point at significant domination of Staphylococcus aureus in AD microbiome.

Objective

The aim of this preliminary study was to explore the cutaneous microbiome in PN lesions using amplicon-based next-generation sequencing.

Material and methods

Recruitment process

Consecutive adults with PN who presented to the Department of Dermatology in March 2020 were invited to participate in the study. In total, 24 patients with PN (mean age ± standard deviation (SD) 60.5 ±13.5 years) were included.
In all cases, the diagnosis was made by a specialist dermatologist on the basis of the medical history and clinical presentation (presence of chronic pruritus ≥ 6 weeks, history and/or signs of repeated scratching and localized or generalized presence of multiple pruriginous lesions of nodular type) [1]. The diagnosis was confirmed by histopathology in 4 (16.7%) cases. Patients with concomitant diseases that might affect the microbiome analysis or who had been treated with topical or systemic antibiotics within the preceding 4 weeks, were excluded from the study. Similarly, patients who had no active nodules at the time of referral or had been treated for PN with topical or systemic corticosteroids/immunosuppressants/phototherapy within the preceding 3 months were not included. In each patient with PN, physical examination with disease activity assessment was performed.
The control group consisted of 9 healthy volunteers (HV) referred to the Outpatient Clinic for a nevi check-up – mean age 56.9 ±23.0 years. All participants in the control group did not have any concomitant conditions and had not been treated with antibiotics within the preceding 4 weeks.

Skin swab collection

Skin swabs from patients with PN and the control group were collected from areas that were untreated for at least 1 month and were not washed or moisturized with emollients for 1 day prior to collection. No cleaning or disinfection was performed before the procedures.
One to three samples, each from a separate active nodule on the lower extremities, were collected from each patient with PN. Similarly, one to three samples from corresponding skin areas were collected from each HV. Skin swabs were collected with a sterile rod and the material was then dissolved in 0.5 ml sterilized phosphate-buffered saline (PBS).
In total, the microbiome profile of 60 active nodules from 24 patients with PN was analyzed. Control samples (n = 14) were collected from 9 HV.

16S rRNA sequencing

Qiagen Pathogen Lysis Tubes (Cat. No. 19092) and QIAamp UCP Pathogen Mini Kit (Cat. No. 50214) were used for DNA extraction according to the instructions provided. Total amounts of 7.7–20 ng/µl of DNA were obtained. The following primers were used for amplification of the V3-V4 region of 16S rRNA:
16S-Amplicon-PCR-Forward-Primer
5’TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG
and 16S-Amplicon-PCR-Reverse-Primer
5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC.
0.5 µl Primer 16S Amplicon PCR Forward, 0.5 µl Primer 16S Amplicon PCR Reverse, 12.5 µl Polymerase and 11.5 µl DNA were used for PCR reaction. The products were then verified on an agarose gel and samples showing a band of 550 bp were used for further processing. Reamplification was performed according to the protocol using the Nexter XT Index Kit indices (Illumina, San Diego, California, USA) on the C1000 Touch Thermal Cycler (Bio-Rad, Hercules, California, USA). In the next step, DNA libraries were purified by means of AMPure XP magnetic beads (Beckman Coulter, Brea, California, USA).
Sequencing was performed on the Illumina platform using the MiSeq instrument.

Data analysis

Qiime2 v 2021.2 platform was used for analysis. Amplicon sequence variants (ASVs) were selected using DADA2 (parameters: --p-trim-left-f 20 --p-trunc-len-f 240 --p-trim-left-r 20 --p-trunc-len-r 240). Operational taxonomic units (OTUs) were identified. The identification of taxa was performed using Silva v. 138 database.
Data has been uploaded to the BioProject database (ID PRJNA1098942).

Statistical analysis

Demographic data were presented as numbers with percentages or means ± standard deviation (SD). Data were subjected for analysis of variance (Statistica 10 package, StatSoft Inc., USA) followed by Tukey’s post-hoc multiple range test. A p-value < 0.05 was considered statistically significant.

Results

Participant characteristics

Sixty samples from patients with PN (n = 24; 10 men and 14 women) and 14 control samples from HV (n = 9; 5 men and 4 women) were analyzed. There were no statistically significant differences between these groups in terms of age and gender. Nine (37.5%) patients with PN had a positive personal history of atopy, which included atopic asthma in 3 (12.5%) cases and allergic rhinitis in 6 (25.0%) cases. In the control group, none of the patients had atopic background. However, the difference did not reach statistical significance (p = 0.09). The number of active skin lesions varied from several to over 100, however, the majority of the patients (n = 15, 62.5%) had between 20 and 100 lesions. Nodules were the predominant type of lesions in 15 (62.5%) participants. In the remaining patients, although nodules were also observed, papules (n = 4; 16.7%) or plaques (n = 5; 20.8%) predominated. Erosions, resulting from scratching, were observed in 16 (66.7%) patients with PN.
Detailed patient characteristics were presented in table 1.

Alpha diversity

The alpha diversity (intra-sample diversity), reflecting the evenness and richness of microbiota, was measured using Shannon index and Simpson index. Both indices showed a statistically significant difference between the PN and healthy microbiomes (p = 0.02 and p < 0.01, respectively).

Beta diversity

The beta diversity (inter-sample diversity), reflecting the between-subjects differences over time and by location, was evaluated using three indices: Bray-Curtis distance, Jaccard distance and weighted UniFrac. A statistically significant difference was observed between the group subjects in all indices (p < 0.05 in all cases).

Relative abundance

Taxa differentiating the microbiomes of patients with PN and healthy controls were selected using ANCOM analysis. In PN, Firmicutes prevailed over Proteobacteria at the phylum level, while the opposite was observed in HV (i.e. Proteobacteria dominated over Firmicutes).
At the genus level, Staphylococcus spp. dominated in PN microbiome, followed by Pseudomonas and Methylobacterium-Methylorubrum.
At the species level, S. aureus was the most abundant in PN patients, followed by Pseudomonas stutzeri, Staphylococcus uncultured bacterium and S. haemolyticus.
The analysis showed that samples from the PN group contained significantly more S. aureus that the samples from HV. The difference was maintained across all taxonomic levels. It was also shown that P. stutzeri had a greater share in the control samples than in the PN group. The difference was held across all taxonomic levels.
The visualization of the results was displayed in figure 1.

Discussion

The majority of the metagenomic research in dermatology has been focused on atopic dermatitis (AD) so far [11]. The results of these studies unquestionably indicate the predominance of S. aureus in cutaneous microbiome in this entity. In addition, the detection rate of S. aureus was found to be higher in lesional than in non-lesional skin, and to increase with the severity of AD [11–13]. It has also been hypothesized that the increase in the S. aureus abundance in the microbiome may precede and/or trigger disease exacerbations [14]. An issue that is worth considering is the potential association between AD and PN. PN may develop secondary to some of the pruritic skin conditions, AD in particular. In the current study, patients with nodular lesions in the course of AD were not recruited. We also assessed in detail the atopic background of the study participants. None of the patients reported any history of skin lesions consistent with AD. Nine (37.5%) patients with PN had a positive personal history of atopy, which included atopic asthma (12.5%) and allergic rhinitis (25.0%).
The pathogenesis of PN remains not fully elucidated, which prevents the use of targeted therapies. Although many authors suggest potential linkages, both direct and indirect, with infectious factors, these hypotheses have not been reliably proven yet. In the last few years, the concept of “skin-brain axis” has been gaining particular importance. Recent evidence suggests that the cutaneous microbiota may affect the itch-scratch cycle e.g. by producing proteolytic enzymes targeting the protease-activated receptors on sensory nerves [15, 16].
In addition, S. aureus produces ceramidase, which destroys the skin barrier. It is also a source of a-toxins which might impede wound healing [17]. The epidermal barrier disruption may be the first step in the itch-scratch cycle. It is also worth mentioning that the skin microbiome is co-responsible for the immune homeostasis.
In the current study, we found relative abundance of S. aureus in the cutaneous microbiome in PN. As PN is commonly associated with atopy, and similarly to AD, it is characterized by intense pruritus, the predominance of S. aureus in both diseases may not be coincidental. S. aureus is known to produce virulence factors, including superantigens and proteases, which promote inflammation and skin barrier dysfunction [18]. S. aureus is also involved in dynamic interactions with coagulase-negative Staphylococci. Recently, commensal S. hominis A9 has been found to produce potent molecules, inhibiting the growth of S. aureus [19]. In addition, application of commensal staphylococci, such as S. epidermidis, S. capitis, S. hominis, onto the lesional AD skin colonized by S. aureus was demonstrated to be beneficial for the disease course [20]. Hence, interventions aimed at decreasing the
S. aureus colonization and preventing dysbiosis might constitute a promising strategy for the treatment of PN as well.
In AD, the biofilm-forming S. aureus has been particularly suggested to play a role in the disease flares. Such strains may secrete toxins inhibiting the growth of other Staphylococcus species, and, at the same time, escape immune responses more efficiently and are more resistant to antimicrobial therapies [21]. The propensity of S. aureus to form a biofilm was not assessed in the present study, but it certainly represents an interesting direction for research.
In the current study, we also observed decreased abundance of P. stutzeri in PN microbiome, when compared to that of healthy controls. P. stutzeri is a Gram-negative bacillus that may be found in many different ecological niches, mainly because of its properties to grow organotrophically or anaerobically [22]. In clinical settings, it has been considered as an opportunistic pathogen, implicated in several cases of endocarditis, peritonitis, pneumonia, arthritis or meningitis [22–24]. Although P. stutzeri has much lower virulence than other Pseudomonas species (P. auruginosa, in particular), its major threat is linked to a wide range of antibiotic resistance mechanisms [22].
In a very recent study, Kim et al. [16] analyzed the microbiome diversity and composition in lesional skin in two conditions associated with debilitating itch – PN and lichen simplex chronicus (LSC). Their findings, although related to American and Korean populations, were largely consistent with our observations – Staphylococcus spp. dominated in the bacterial microbiome in chronic scratch lesions.
In summary, our results highlight several alterations in the diversity and composition of cutaneous microbiome in PN. However, further research on the role of microorganisms in this debilitating condition is necessary.
The major limitation of the study is a small sample size. Therefore, the correlation between the microbiome composition and the clinical parameters such as disease severity or itch intensity could not be reliably evaluated. It should also be noted that full-length 16S rRNA sequencing is the most reliable method to perform species-level analysis, although V3-V4 region sequencing has been frequently utilized for the evaluation of the cutaneous microbiome as well.

Conclusions

The composition and diversity of bacterial microbiome in PN is altered. The role of S. aureus in PN microbiome and disease pathogenesis needs further elucidation.

Funding

This work was funded by the Mini-Grant of Polish Dermatological Society 2020 (Mini-Grant PTD) awarded to Magdalena Żychowska, MD, PhD.

Ethical approval

The study was conducted according to the Declaration of Helsinki, and was approved by the Ethics Committee of the University of Rzeszow (decision no. 7/2020). All participants signed written informed consent prior to any procedure.

Conflict of interest

The authors declare no conflict of interest.

References