Reduced Staphylococcus Abundance Characterizes the Lesional Microbiome of Actinic Keratosis Patients after Field-Directed Therapies

ABSTRACT Skin microbiome dysbiosis with a Staphylococcus overabundance is a feature of actinic keratosis (AK) and squamous skin carcinoma (SCC) patients. The impact of lesion-directed treatments for AK lesions such as diclofenac (DIC) and cold atmospheric plasma (CAP) on the lesional microbiome is not established. We studied 321 skin microbiome samples of 59 AK patients treated with DIC 3% gel versus CAP. Microbial DNA from skin swabs taken before start of treatment (week 0), at the end of the treatment period (week 24), and 3 months after end of treatment (week 36) was analyzed after sequencing the V3/V4 region of the 16S rRNA gene. The relative abundance of S. aureus was scrutinized by a tuf gene specific TaqMan PCR assay. The total bacterial load and both, relative and absolute abundance of Staphylococcus genus was reduced upon both therapies at week 24 and 36 compared to week 0. Notably, the lesional microbiome of patients responding to CAP therapy at week 24 was characterized by an increased relative abundance of Corynebacterium genus compared to nonresponders. A higher relative abundance of Staphylococcus aureus at week 36 was a feature of patients classified as nonresponders for both treatments 12 weeks after therapy completion. The reduction of the Staphylococcus abundance after treatment of AK lesions and alterations linked to treatment response encourage further studies for investigation of the role of the skin microbiome for both, the carcinogenesis of epithelial skin cancer and its function as predictive therapeutic biomarker in AK. IMPORTANCE The relevance of the skin microbiome for development of actinic keratosis (AK), its progression into squamous skin cancer, and for field-directed treatment response is unknown. An overabundance of staphylococci characterizes the skin microbiome of AK lesions. In this study, analyses of the lesional microbiome from 321 samples of 59 AK patients treated with diclophenac gel versus cold atmospheric plasma (CAP) revealed a reduced total bacterial load and reduced relative and absolute Staphylococcus genus abundance upon both treatments. A higher relative Corynebacterium abundance was a feature of patients classified as responders at the end of CAP-treatment period (week 24) compared with nonresponders and the Staphylococcus aureus abundance of patients classified as responders 3 months after treatment completion was significantly lower than in nonresponders. The alterations of the skin microbiome upon AK treatment encourage further investigations for establishing its role for carcinogenesis and its function as predictive biomarker in AK.

major line 118 ff "The reduction of the Staphylococcus abundance was mainly attributed to a reduction of CoNS, while the mean relative S. aureus abundance was lower but not significantly different between the different timepoints ( Figure 2D)." Change wording to relative abundance (& throughout the manuscript where appropriate); how does this translate into absolute abundance? of Staphylococcus vs. S. aureus? Important given the pro-tumorigenic role of S. aureus "Section increased Corynebacterium relative abundance at capillitium in responders"; how does this translate to absolute abundance? could the authors give a little bit more information about the species of Corynebacterium? was it just one species or different ones?
The differences between the two treatments (DIC vs CAP) on the skin microbiome could be described in an individual paragraph for clearness minor wording line 117 "Staphylococcus was the only genus taxon" remove taxon since Staphylococcus per se is specified as a genus (use this also throughout the manuscript where appropriate e.g. line 149) line 129ff LDA score {greater than or equal to} 3 .. not shown.... please show this data in the supplemental material Propionibacterium .... I assume Cutibacterium (which is the current correct designation)

Discussion
Authors should elaborate more on the different pathogenic vs physiologic roles of S. aureus vs. CoNS (e.g. S. epidermidis) vs. Corynebacterium and their relation to the described treatment effects; there is a plethora of literature out investigating these taxa in the context of skin diseases, tumors and inflammation. How does this fit to their findings? Is the reduction of certain taxa (or increase) somehow mechanistically beneficial (e.g. by increasing beneficial taxa by the treatments) or is the harsh treatment just killing certain taxa? How acts DIC on skin microbiota directly or via the immune-system? Reviewer #2 (Comments for the Author): In this manuscript, the use of diclofenac (DIC) and cold atmospheric plasma (CAP) on the skin microbiome in the treatment of actinic keratosis was studied. The following comments are made: 1. Line 36. "tuff" is a gene? If so, put it. 2. Lines 86, 245-246. How did the treatments with CAP and DIC? You don't say it. It is a fundamental part of your work, there should be a section to explain the treatments. 3. Line 90. What extremities are you talking about? explain it 4. Line 100. What does "LefSe" mean? put it 5. Line 132-133. "The reduction of the relative abundance of Staphylococcus of 16S rRNA sequences was due largely to CoNSreduction". It is not clear why you say they are CoNS. How did you measure and verify it? explain it. 6. Line 150. In line 142-143, it is well established that it is an R, but in line 150 nR appears and you do not say what it means or what its definition is. Correct it and define what is an nR. 7. Line 163. "NR", should be nR 8. Line 179-183. What interpretation do you give to these results, discuss it. 9. Line 194. Capitalizing Gram is a proper name. 10. You do not discuss the effect of CAP on the microorganisms found and their differences. Discuss the mechanism of CAP on bacteria and why the differences found. 11. Lines 306-307. What control strains of S. aureus and S. epidemidis did you use? 12. Line 387 and Figure 5. Staphylococcus aureus abbreviated as S. aureus Staff Comments:

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Point-to-point Reply
We are grateful for the positive and helpful reviews that helped to clearly improve the manuscript. We have complied with all comments and answer them in the following point-topoint reply. Answer from the authors: Thank you for the summary, your positive feedback and valuable suggestions. We have now included all your suggestions and performed qPCR for determining absolute abundances for individual samples. With the information regarding the total bacterial abundance of each sample and the relative abundances of our 16S rRNA sequencing data, we calculated the absolute abundances of different bacterial genera or species of interest. We have supplemented our revised version with absolute Staphylococcus, S. aureus, CoNS and Corynebacterium abundances as detailed described below and in the revised version of our manuscript. We also describe which Corynebacterium spp. were identified from amplicon sequence variants of 16S rRNA gene sequencing. Both is described in your comments below and in the revised version of the manuscript in detail. major line 118 ff "The reduction of the Staphylococcus abundance was mainly attributed to a reduction of CoNS, while the mean relative S. aureus abundance was lower but not significantly different between the different timepoints ( Figure 2D)." Change wording to relative abundance (& throughout the manuscript where appropriate); how does this translate into absolute abundance? of Staphylococcus vs. S. aureus? Important given the protumorigenic role of S. aureus Answer from the authors: We have changed the wording into relative abundance throughout the manuscript where appropriate. In addition, we have now performed qPCR to determine absolute abundances using the Femto Bacterial DNA quantification kit (Zymo Research, Freiburg, Germany), which allows for calculation of the 16S rRNA gene copy numbers in individual samples (described in the Materials and Methods section, lines 380-386 of the clean version). The total number of 16S rRNA copies is significantly lower at week 24 compared to week 0, before start of treatment ( Figure 6A). Interestingly, the absolute Staphylococcus and CoNS abundances are both significantly lower at week 24 compared with week 0. For week 36, only the absolute Staphylococcus abundance but not CoNS abundance is significantly lower compared with week 0 ( Figure 6B and 6C). However, the S. aureus load decreases also after treatment completion and the abundance is lowest at week 36 and significantly lower at that time point compared with week 0 ( Figure 6D), indicating that not only killing effects of the treatments are responsible for S. aureus reduction observed, but that also other factors, like (modulation of immune and other host cells) might play a role in microbiome alterations, as the absolute S. aureus abundance is even lower 12 weeks after completion of treatments compared to the end of treatment period. S. aureus has been considered as pathogen promoting tumor growth, which is in contrast to the CoNS, which had for long periods been regarded as non-or less pathogenic. However, although considered as less pathogenic, CoNS was linked to disease progression in transplant patients with SCC, that exhibit an overabundance of S. epidermidis (Krueger A et al. 2022. Changes in the skin microbiome associated with squamous cell carcinoma in transplant recipients. ISME Communications 2:13.). We have included these issues in the results and discussions section, lines 204-223 and in the discussion of the revised manuscript.
"Section increased Corynebacterium relative abundance at capillitium in responders"; how does this translate to absolute abundance? could the authors give a little bit more information about the species of Corynebacterium? was it just one species or different ones?

Answer from the authors:
We thank the reviewer for this comment. In contrast to the Staphylococcus absolute abundance, that significantly decreased at treatment completion compared to week 0, the absolute Corynebacterium abundance was not significantly lower at therapy completion (week 24) and 12 weeks after therapy completion (week 36) compared to week 0 before start of treatment, indicating that treatment effects do not affect all bacteria equally ( Figure 6E). A tendency for higher absolute abundances of the Corynebacterium genus was present for patients classified as R for week 24 (Supplemental Figure 3) and week 36 (Figure 7) compared with nR for the majority of time points. However, a significant higher Corynebacterium abundance was observed only for DIC treated patients classified as R at week 36 compared with nR for the timepoint week 24 (Figure 7). We have included these results in the manuscript (lines 223-227 and lines 231-236) Identification of bacteria to the species level using 16S rRNA gene sequencing is not always possible and the databases may not always reliably identify the bacteria to the species level. However, among the bacteria of the Corynebacterium genus, Corynebacterium kroppenstedtii, Corynebacterium bovis and two species of the Corynebacterium genus that were not identified by the Greengenes database were the four most abundant species accounting for about 98.9% of the capillitium reads that were assigned to bacteria of the Corynebacterium genus. Furthermore, reads of the Corynebacterium species C. durum, C. lubricantis, C. mastitidis, C. simulans, C. stationis and C. variabile were identified using the Greengenes database. We have now included these information in the results section, lines 184-189 and discussed the results line 298-305.

The differences between the two treatments (DIC vs CAP) on the skin microbiome could be described in an individual paragraph for clearness:
Answer from the authors: Thank you for this suggestion. We have performed analyses regarding the differences in the skin microbiome between CAP vs DIC treatment for the time points at week 24 and 36. Interestingly the differences in the skin microbiome composition between both treatments were minor and LEfSe did not identify biomarkers linked to the treatment with LDA score >3. We have now supplemented the manuscript with the additional absolute abundance data and as the differences between both treatments were minor, we prefer not to add another paragraph but we have described the differences between both treatments in the existing paragraphs in the results section (lines 149-162 and lines 226-239). Answer from the authors: We thank the reviewer for this comment. LEfSe did not identify a biomarker with LDA score ≥ 3. But we have now added Supplemental Figure 1 showing taxa with an LDA score ≥ 2 and <3 between CAP and DIC treatment for week 24 and 36.

Propionibacterium .... I assume Cutibacterium (which is the current correct designation)
Answer from the authors: We agree with the reviewer that Cutibacterium is the correct nomenclature of the genus previously designated as Propionibacterium. However, the Greengenes database used Propionibacterium for the taxonomy assignment. We have now replaced Propionibacterium by Cutibacterium in the manuscript text and explained the former nomenclature in brackets, see line 123 of the revised version of the manuscript.

Discussion
Authors should elaborate more on the different pathogenic vs physiologic roles of S. aureus vs. CoNS (e.g. S. epidermidis) vs. Corynebacterium and their relation to the described treatment effects; there is a plethora of literature out investigating these taxa in the context of skin diseases, tumors and inflammation. How does this fit to their findings? Is the reduction of certain taxa (or increase) somehow mechanistically beneficial (e.g. by increasing beneficial taxa by the treatments) or is the harsh treatment just killing certain taxa? How acts DIC on skin microbiota directly or via the immune-system? Answer from the authors: We thank the reviewer for this comment. We have now supplemented our discussion by a more detailed inclusion of studies investigating the pathogenic role of S. aureus and the less pathogenic role of CoNS in the context of skin malignancies. The decrease in relative and absolute S. aureus abundance which occurs after treatment completion, with median and mean reduction of S. aureus abundance and its association with treatment response, supports other reports, claiming S. aureus as important bacterial species with pro-tumorigenic potential. However, although most studies focussed on S. aureus as most important species of the Staphylococcus genus, there is also literature describing S. epidermidis, the most common species of CoNS, linked to squamous skin cancer progression in transplant patients (Krueger A, et al. 2022. Changes in the skin microbiome associated with squamous cell carcinoma in transplant recipients. ISME Communications 2:13.) Recent reports suggest that pathology may be context dependent and indicate that pathogens like S. aureus may asymptomatically colonize the skin but mutualists like S. epidermidis or corynebacteria may then promote disease. We have discussed these aspects in lines 307-328 of the revised version of the manuscript.

Reviewer #2 (Comments for the Author):
In this manuscript, the use of diclofenac (DIC) and cold atmospheric plasma (CAP) on the skin microbiome in the treatment of actinic keratosis was studied. The following comments are made: 1. Line 36. "tuff" is a gene? If so, put it.
Answer from the authors: We have now supplemented this statement as suggested.
2. Lines 86, 245-246. How did the treatments with CAP and DIC? You don't say it. It is a fundamental part of your work, there should be a section to explain the treatments.

Answer from the authors:
We have now explained the treatment performed by CAP and DIC in the Materials and methods section, lines 354-360: "The ACTICAP study design included a washout phase of potentially prohibited medication, including photosensitizing agents, immunomodulators, antineoplastic agents and systemic retinoids for 4-12 weeks before treatment with CAP or DIC was started, depending on drug type. Patients were included after a minimum of 6 months after physical procedures (e.g. surgery, laser, PDT). The identified treatment areas were computationally randomized to either CAP that was applied for 180 seconds twice a week or topical DIC (diclofenac 3%) twice a day (8)."