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Insights into the skin microbiome of sickle cell disease leg ulcers

Abstract Leg ulcers are estimated to occur in 1%–10% of North American patients with sickle cell disease (SCD). Their pathophysiology remains poorly defined, but as with other chronic wounds, it is hypothesised that the microbial milieu, or microbiome, contributes to their healing and clinical outcomes. This study utilises 16S ribosomal RNA (rRNA) gene sequencing to describe, for the first time, the microbiome of the SCD leg ulcer and its association with clinical factors. In a cross‐sectional analysis of 42 ulcers, we recovered microbial profiles similar to other chronic wounds in the predominance of anaerobic bacteria and opportunistic pathogens including Staphylococcus, Corynebacterium, and Finegoldia. Ulcers separated into two clusters: one defined by predominance of Staphylococcus and smaller surface area, and the other displaying a greater diversity of taxa and larger surface area. We also find that the relative abundance of Porphyromonas is negatively associated with haemoglobin levels, a key clinical severity indicator for SCD, and that Finegoldia relative abundance is negatively associated with CD19+ B cell count. Finally, ratios of Corynebacterium:Lactobacillus and Staphylococcus:Lactobacillus are elevated in the intact skin of individuals with a history of SCD leg ulcers, while the ratio of Lactobacillus:Bacillus is elevated in that of individuals without a history of ulcers. Investigations of the skin microbiome in relation to SCD ulcer pathophysiology can inform clinical guidelines for this poorly understood chronic wound, as well as enhance broader understanding about the role of the skin microbiome in delayed wound healing.

ulcer pathophysiology can inform clinical guidelines for this poorly understood chronic wound, as well as enhance broader understanding about the role of the skin microbiome in delayed wound healing.

K E Y W O R D S
16S rRNA, chronic wound, leg ulcer, sickle cell disease, skin microbiome

| INTRODUCTION
In the United States, chronic wounds impact millions of patients annually and have been recognised as a major public health concern since the early 2000s. 1 Over the past two decades, a growing body of literature on their pathophysiology has built an evidence base that informs debridement, antimicrobial, and dressing strategies for a variety of wound types, including diabetic foot ulcers, venous ulcers, pressure injuries, and arterial ulcers. 2 While there is a continuing need to improve and refine treatment guidelines for these most common chronic wounds, the phenotype and pathophysiology of one type of ulcer, the sickle cell disease (SCD) leg ulcer, lacks significant investigation and is poorly understood 3 ( Figure 1). SCD is a group of genetic blood disorders in which a mutation in the beta-globin gene causes haemoglobin to polymerise in its deoxygenated state, leading red blood cells to lyse or block blood flow in vessels. 4 Leg ulcers are estimated to occur in 1%-10% of North American SCD patients, with prevalence in other regions of the world ranging from 11% in Ghana to as high as 43% in Brazil. [5][6][7] These wounds are excruciatingly painful, often recalcitrant and recurring, and may result in perceived social stigma. 5,8 Proposed causes include: venous insufficiency 9 ; increased arteriovenous shunting associated with haemolytic anemia 10 ; impaired endothelial function due to the consumption of nitric oxide by cell-free hemoglobin 11 ; and vessel damage from sickled cells, which allows proteins and inflammatory cells to leak into extravascular space. 12 However, these hypotheses remain speculative. Without sufficient knowledge of SCD leg ulcer pathophysiology, treatment strategies vary widely between clinicians and are guided largely by anecdotal experience. 6,13 Treatment of SCD ulcers will benefit from research leveraging a variety of approaches, but investigations of the colonising microbiota may prove especially promising. Wound microbes have been shown to play a role in healing processes by colonising wound beds and overstimulating leukocyte activity, including proteolytic functions and reactive oxygen species production, thereby impairing healing. 14 In recent years, research in this area has become even more fruitful through the advent of 16S rRNA gene sequencing, which has allowed researchers to more effectively identify and study specific communities of cutaneous microbes. [15][16][17] With this information, microbial therapies could be refined to promote or prevent colonisation by beneficial and deleterious organisms, respectively. The wound microbiome may also prove a useful diagnostic tool by serving as an accessible source of information about the inflammatory state of a wound. 18 Despite their potential to inform treatment, microbial studies of SCD ulcers are scant and dated. Some studies have demonstrated that topical antibacterial therapy can reduce SCD ulcer size. 19,20 In others, SCD ulcers were most often colonised by Staphylococcus aureus, 20,21 a known wound pathogen. Each of these studies has been culturebased, limiting their ability to identify taxa which are difficult to culture in laboratory environments. Culture-independent studies utilising 16S rRNA gene sequencing techniques are non-existent.
This paper contributes to the limited collection of studies on SCD leg ulcers, as well as a growing body of literature on chronic wound microbiomes, by describing the SCD leg ulcer microbiome and its association with clinical outcomes. We use 16S rRNA gene sequencing to characterise the bacterial taxa that colonise the F I G U R E 1 Sickle cell disease leg ulcer. Photograph of a sickle cell disease leg ulcer wound bed and surrounding skin, and calculate taxonomic associations with wound severity and other clinical measures salient to SCD. In addition to informing further research on the pathophysiology of SCD ulcers, we establish the SCD leg ulcer as an additional topic of investigation, among diabetic ulcers, venous ulcers, pressure injuries, and other more common chronic wounds, that can provide broader insights into the relationship between the skin microbiome and wound healing.

| Data collection
Medical history and clinical and physical exams were completed and microbiome samples were collected for each participant at the time of visit. Participants were asked to refrain from showering, using soap, or applying lotion to the lower extremities for 24 hours prior to the clinic visit. Microbiome samples were collected from both non-ulcerated and ulcerated skin tissue. If the participant presented with a nonaffected leg(s), non-ulcerated tissue samples were obtained from the medial or lateral malleolus. The swab was pre-moistened and rotated over a 1 cm 2 area of skin. For participants with ulcers, non-ulcerated tissue samples were also obtained from the periwound area, defined as non-ulcerated skin 1 cm from the wound edge. For ulcerated tissue, the wound bed was cleaned with sterile 0.9% sodium chloride, the swab was pre-moistened, and the sample was obtained using the Levine technique. Levine's technique is different than other swab techniques in that it samples fluid from the deep tissue layers, similar to aspiration of wound fluid, and has high concordance with tissue biopsies for recovering microbial load and diversity. 22

| 16S rRNA marker gene sequencing and bioinformatics analysis
DNA was extracted from swab specimens as previously described. 15 Amplicons were prepared using Invitrogen AccuPrime High Fidelity Sequencing data were processed and analysed using the Quantitative Insights Into Microbial Ecology 2 (QIIME2) pipeline. 24 Divisive Amplicon Denoising Algorithm 2 (DADA2), 25 implemented as a QIIME2 plug-in, was used for sequence quality filtering, and taxonomic analysis was done using a Naïve Bayes classifier trained on the Greengenes 13_8 99% operational taxonomic units. The annotation for oxygen tolerance of bacterial taxa was obtained from previous research. 26 Alpha diversity was quantified using three metrics: Faith's phylogenetic diversity (Faith's PD, the representation of species present on the phylogenetic tree), richness (the number of species present), and Shannon's diversity index (Shannon's H, the distribution of relative abundance). A rooted phylogenetic tree was generated for calculation of diversity metrics including Faith's PD and UniFrac distances: first a multiple sequence alignment was performed using Multiple Alignment using Fast Fourier Transform 27 and high variable positions were masked to reduce noise in a resulting phylogenetic tree. A mid-point rooted tree was then generated using FastTree. 28 The Shannon index (H) and richness were calculated using the vegan package version 2.5-6 (https://CRAN.R-project.org/package=vegan) in R (version 3.6.0).

| Statistical analysis
Linear mixed models with patient ID as random effect were used to test the associations between alpha diversity, relative abundances and ratios of taxa, and study groups. Relative abundances and ratios of taxa were log-transformed for these analyses. control for the false discovery rate using the Benjamini-Hochberg procedure. All statistical analyses were conducted in R (version 3.6.0).

| Abundance of aerobes is inversely proportional to proximity to wound skin
We sought to determine if bacterial community diversity was associated with proximity to the wound in participants with active ulcers.
These findings suggest that the ulcer microbiota is distinct from intact skin, with bacterial community diversity lowest in the ulcer.
We next assessed whether the drastic reduction in alpha diversity was associated with a change in the fundamental makeup of the bacterial community, in terms of oxygen tolerance. We annotated the bacterial taxa identified in our samples as aerobes, facultative anaerobes, or obligate anaerobes and tested for associations between the relative abundance of these taxa categories and sample site. The relative abundance of aerobic bacteria decreased as the sampling site shifted from intact to periwound, or periwound to wound (β = À2.79, p adj < 0.001; Figure 2B). The relative abundance of facultative and obligately anaerobic bacteria did not have a statistically significant association with wound proximity (β = À0.01, p adj = 0.92 and β = À0.90, p adj = 0.23, respectively, Figure 2B). Thus, the decrease in bacterial diversity observed in the ulcer was accompanied by a decrease in the relative abundance of aerobic bacteria.

| SCD leg ulcer microbiota are heterogeneous, but most often dominated by Staphylococcus
Consistent with our finding that the relative abundance of aerobes decreased with proximity to the wound skin, wound samples were dominated by the presence of facultative and obligate anaerobes ( Figure 3A). Of the taxa present, Staphylococcus had the highest mean relative abundance at 52%, of which 80% was identified as S. aureus ( Figure 3B; Table S1). This was followed by other facultatively anaerobic bacteria such as Corynebacterium (10%), Streptococcus (4%), Bacillus (2%), and Streptococcaceae, as well as obligately anaerobic bacteria such as Finegoldia (7%), Propionibacterium (3%), Porphyromonas (2%), and Anaerococcus (2%). Of the aerobic bacteria remaining in ulcer samples, Alcaligenes (5%) was the most abundant (Table S1).
However, the mean relative abundance values were not representative of ulcer samples as a whole, which were highly heterogeneous in the composition, relative abundances, and categorical membership of taxa ( Figure 3B). Other than Staphylococcus, which was present in 38 (90%) of ulcer samples, only Corynebacterium and Finegoldia were present in over half the samples (  (Figures 3 and 4A). The two clusters were separated along the first principal coordinate axis, which accounted for 48% of total variation among samples, primarily by relative abundance of Staphylococcus, which was positively correlated with PCoA Axis 1 (ρ = 0.82, p < 0.001; Figure 4B).
We used mixed effects models to test for differences in the relative abundance of bacterial genera between clusters (Table S2)

| Relative abundances are associated with clinical measures
We tested for associations between wound severity measures (surface area, depth, % slough, amount of exudate, colour of exudate) and alpha diversity, as well as relative abundances of the ten taxa with the highest mean relative abundances across all samples. Consistent with our cluster analysis, surface area was negatively associated at the genus level with the relative abundance of Staphylococcus (β = À0.65, p adj < 0.001) but not when considered at the species level (Table S3) (Table S3). Alpha diversity measures were not significantly associated with any wound severity measures.
We also tested for associations between relative abundances of the top ten taxa, alpha diversity measures, and the following clinical variables, which were selected based on their relevance to SCD and previously Alpha diversity measures were not significantly associated with the clinical variables. We also conducted analyses of associations between reports of ulcer-related pain and the top ten taxa and alpha diversity measures. While we did not find significant associations, these findings were limited by the small number of samples with reported pain scores (only 24/42 ulcer samples had associated pain data).

| Intact skin microbiome differs by ulcer history
To investigate the role that intact skin may play in the formation of leg ulcers, we examined ratios of genera between the intact skin Linear mixed models were used to test for associations between ulcer history and log-transformed ratios of bacterial genera. The ratios of Staphylococcus:Lactobacillus, as well as Corynebacterium:Lactobacillus, were elevated in the intact skin of individuals with ulcer histories (β = 2.132, p adj = 0.03 and β = 2.23, p adj = 0.03, respectively). Conversely, the mean ratio of Lactobacillus:Bacillus was higher in individuals without a prior history of ulcers, though our results did not indicate a statistically significant difference from a ratio of 1:1 (β = 2.08, p adj = 0.06). There were no significant ratio differences between individuals with and without active ulcers.

| DISCUSSION
In the past decade, novel sequencing technologies have increasingly been used to examine the interplay between skin wounds and microbial colonisation. 18 To our knowledge, this is the first study to use 16S rRNA marker gene sequencing to describe the SCD leg ulcer microbiome and its association with measures of wound healing and other clinical factors. We find that the presence of anaerobes and common opportunistic pathogens is a comparable finding between the SCD ulcer microbiome and other chronic wounds. We also report several findings that are unique to this wound type and warrant replication and further exploration.
The SCD ulcer microbiome, as characterised by this study, bears many similarities with that of other chronic wounds. The lower alpha diversity and relative abundance of aerobes in the wound bed relative to intact skin aligns with reports of similarly reduced diversity in diabetic foot ulcer skin, 29  Finally, in our analyses of intact skin, we found differences in the ratios of various microbes between individuals with and without histories of ulcers. Specifically, ratios of Corynebacterium:Lactobacillus and Staphylococcus:Lactobacillus were elevated in individuals with a history of SCD leg ulcers, while the ratio of Lactobacillus:Bacillus was elevated in individuals without a history of SCD leg ulcers. Lactobacillus has been implicated in a range of protective mechanisms against cutaneous infection or delayed healing, including immune modulation, promotion of re-epithelialisation, and pathogen clearance. [40][41][42] Conversely, Staphylococcus and Corynebacterium can become opportunistic wound pathogens. Differences between individuals with and without ulcer histories suggest either that the presence of an ulcer induces long-term changes in nearby skin, or that over-or underrepresentation of these taxa can predispose SCD patients to ulcer development. In the latter case, microbial profiles may be useful predictors of future ulcer risk and may inform the development of prophylactic therapies that attenuate, or even, reverse these changes.
This study had some limitations. As a cross-sectional design, this study was not able to assess changes in the skin microbiome over time, although temporal changes have been shown to be important to chronic wound healing. 43 Potentially important clinical factors related to the patient were not measured in this preliminary study, including vaso-occlusion and a rigorous assessment of pain. We did not measure important host-level factors related to the ulcer, including the milieu of growth factors, antimicrobial peptides, and cytokines/ chemokines that interact with the wound microbiota. We were also unable to control patients' topical dressings prior to their study visit.
Moreover, we were limited in our ability to attain species and strainlevel resolution of taxa, despite the fact that species and strain-level variation has previously been associated with wound outcomes. 39 Nonetheless, the findings of our investigation of the SCD ulcer microbiome may spur subsequent research that advances our understanding of the pathophysiology of this understudied chronic wound. More broadly, this research contributes to a growing body of literature on the shifting dynamics of the skin microbiome in relation to wound healing and clinical biomarkers.