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The scalp hair shaft microbiota is distinct from that of the scalp skin. The composition of scalp hair microbiota is greatly influenced by the scalp microbiome, but it is also shaped by various intrinsic factors, including gender, as well as extrinsic factors like hair washing and styling. What We Know: Differences in bacterial community structures between the microbiome of hair shafts and scalp include variances in cell density and relative abundances of Firmicutes and Proteobacteria. There is also correlation between Actinobacteria and Firmicutes abundances between an individual's hair and scalp (Watanabe et al., 2020). The primary bacteria found on human scalp hair shafts are native inhabitants originating from the hair roots. Phyla Actinobacteria, Proteobacteria and Firmicutes are the species present in the greatest abundance, with the former two competing for dominance (Watanabe et al., 2021). Scalp hair shafts are known to harbour the hair-specific genus, Pseudomonas, alongside the skin-derived genera Cutibacterium and Staphylococcus, which is distinct from other human skin microbiomes. Cutibacterium, Lawsonella, Moraxella and Staphylococcus were notably elevated in males compared to females. Conversely, the bacterial cell count of Pseudomonas was higher in females than in males (Watanabe et al., 2021). Females using hair wax had reduced cell counts of Cutibacterium, Lawsonella and Moraxella, while hair bleach showed lowered Pseudomonas counts. In males, hair colour application decreased Cutibacterium, Lawsonella and Staphylococcus counts, and using hair dryers reduced Staphylococcus. Additionally, males who washed their hair in the morning had lower Lawsonella counts than those who washed it the night prior (Watanabe et al., 2021). Industry Impact and Potential: Given the shared bacterial species between scalp skin and hair shafts, environmental distinctions on scalp skin could potentially contribute to microbial differences observed on hair shafts (Watanabe et al., 2021). This discovery opens up a promising and largely uncharted frontier of scalp and hair care products. By focusing on nurturing the scalp microbiome, new innovative products can offer targeted support to the hair and understanding the interplay between scalp and hair microbiomes not only sheds light on individualised hair care, but also presents promising opportunities for product development. Our Solution: With a database of 20,000 microbiome samples and 4,000 ingredients, alongside a global network of 10,000 testing participants, Sequential offers tailored solutions to build your custom microbiome studies and product formulation. Our focus and emphasis on creating microbiome safe and friendly products guarantees the preservation of biome integrity, making us the perfect partner for your scalp and hair care product development needs. References: Watanabe, K., Yamada, A., Nishi, Y., Tashiro, Y. & Sakai, K. (2021) Host factors that shape the bacterial community structure on scalp hair shaft. Scientific Reports. 11 (1), 17711. doi:10.1038/s41598-021-96767-w. Watanabe, K., Yamada, A., Nishi, Y., Tashiro, Y. & Sakai, K. (2020) Relationship between the bacterial community structures on human hair and scalp. Bioscience, Biotechnology, and Biochemistry. 84 (12), 2585–2596. doi:10.1080/09168451.2020.1809989.

The interplay between skin wounds and the skin microbiome presents a captivating area of study. While the precise mechanisms remain elusive, ongoing research is unravelling how the microbiome influences wound healing, shedding light on both facilitative and inhibitory roles. What We Know: When the skin barrier is compromised due to injury, it enables the colonisation of microbes not typically present on the skin or the transfer of microbiota components to areas where they aren't normally found. Research indicates that microbiota may play a beneficial role in wound healing by regulating the innate immune response and promoting tissue regeneration (Yang et al., 2024). Burn injuries are an example of acute wounds, which increase the permeability of the skin, thus allowing skin microbes to penetrate deeper tissues, leading to possible infections. Therefore, burns significantly change the skin's microbial balance, favouring heat-loving microbes like Aeribacillus, Caldalkalibacilus and Nesterenkonia, while reducing helpful bacteria like Cutibacteria, Staphylococci and Corynebacteria. These shifts are linked to specific wound healing outcomes; higher levels of Corynebacterium are associated with infections, while Staphylococci and Cutibacteria are linked to lower infection rates post-burn (Yang et al., 2024). Research found that invasive moulds, including those from the Mucorales, Aspergillus and Fusarium species, were found to considerably prolong wound closure compared to non-fungal infected wounds with similar injury patterns. Enterococci was often observed in conjunction with invasive fungal infections, heightening the complexity of traumatic wounds (Warkentien et al., 2015). Industry Impact and Potential: Understanding microbe-host interactions in wounds can inform future therapeutic interventions, paving the way for microbiota-based mechanisms in wound treatment (Yang et al., 2024). Research has demonstrated that commensals play a vital role in initiating innate immune responses. Resident commensals and even generally pathogenic Staphylococcus aureus promote skin regeneration and wound-induced hair follicle growth (Wang et al., 2021). Studies have shown that Lactobacillus spp. enhance keratinocyte proliferation and migration and prevent biofilm formation. Animal models also showed the beneficial effects of these probiotics, as well as Bifidobacteria and Saccharomyces cerevisiae, in reducing pathogen colonisation and biofilm formation, facilitating tissue repair and reducing excessive scarring (Yang et al., 2024). Our Solution: Unlock the potential of microbiome-based wound healing products with Sequential's comprehensive end-to-end Microbiome Product Testing Solution. Whether used independently or alongside our expert-guided product development and formulation services, this holistic approach empowers your business to pioneer innovative solutions for addressing skin conditions. References: Wang, G., Sweren, E., Liu, H., Wier, E., Alphonse, M.P., et al. (2021) Bacteria induce skin regeneration via IL-1β signaling. Cell Host & Microbe. 29 (5), 777-791.e6. doi:10.1016/j.chom.2021.03.003. Warkentien, T.E., Shaikh, F., Weintrob, A.C., Rodriguez, C.J., Murray, C.K., Lloyd, B.A., Ganesan, A., Aggarwal, D., Carson, M.L., Tribble, D.R., & on behalf of the Infectious Disease Clinical Research Program Trauma Infectious Disease Outcomes Study Group (2015) Impact of Mucorales and Other Invasive Molds on Clinical Outcomes of Polymicrobial Traumatic Wound Infections. Journal of Clinical Microbiology. 53 (7), 2262–2270. doi:10.1128/jcm.00835-15. Yang, Y., Huang, J., Zeng, A., Long, X., Yu, N. & Wang, X. (2024) The role of the skin microbiome in wound healing. Burns & Trauma. 12, tkad059. doi:10.1093/burnst/tkad059.

It is a known fact that each and every individual has a unique microbiome, comprising a community of bacteria, fungi, viruses and protozoa (commonly referred to as microbes). Interestingly, the origin of a person's microbiome stems from the moment they were born. Studies show that regardless of birthing route (vaginal or caesarean), 58.5% of a baby’s microbiome is acquired from their mother (Bogaert., et al 2023). In recent years, the mother-to-child microbiome transfer has become a topic of note within the microbiome space. Despite growing literature about the baby microbiome, it is still an area that needs further exploration. This article will aim to highlight current research on the topic, whilst focusing on Atopic Dermatitis as seen within newborns and infants. It is crucial to understand and expand upon this research as it might lead to uncovering new microbial candidates that could impact skin conditions, such as Atopic Dermatitis, in early life and unlock their treatments. A Background on Atopic Dermatitis There are numerous paediatric skin disorders that can affect an infant in early life. Typically, we find that Atopic Dermatitis (AD), is the most prevalent skin disorder amongst infants and is an important condition to consider (Gilaberte et al., 2020). It has been seen that infants that suffer from AD can go on to develop a highly contagious condition known as impetigo (a bacterial infection that typically affects infants and young children)( Mannschreck et al.,2020).  According to a study published in 2015 “the median impetigo prevalence in children was 12.3%, (IQR 4.2–19.3%)” (Bowen et al., 2015).  It is known that the skin microbiome of infants is completely different to that of adults, however we also know that the pathophysiology and the inflammatory cytokines that are triggered are alike. The pathogenesis of AD is incredibly complex, which is why pinpointing its treatment has been difficult. Most of the drugs that are currently under investigation or are showing some positive results in clinical trials targeting skin cells, but we now understand that there are more solutions that intend to target the microbiome component (Baldwin et al., 2020). At present, there are two ways of exploring AD. One way is to look directly at the source and extract samples of the skin microbiome to observe changes in its profile. The alternative is to look at the gut microbiome, pulling from previously published literature that suggests that there is a convincing link between the gut microbiome and skin health. The Role of Staphylococcus aureus in AD. AD sites on the body are known to be dominated by Staphylococcus aureus (S. aureus), which is the most studied and well-described bacteria linked to AD. It is known that S. aureus goes through a stage of growth which leads to AD flare-ups in individuals (Khadka et al., 2021). This means that by identifying the growth of S. aureus early a threshold can be determined that when surpassed could be used to predict when a flare-up could be triggered, which may give rise to the opportunity for prevention. Mother-to-child Microbiome Transfer When exploring early colonisation of the baby microbiome, the transfer from mother-to-child is more commonly talked about. Until recently, it has been agreed that the womb is sterile. However, new research has been carried out on amniotic fluid which suggests that certain microbes might be present there as well (Kaisanlahti et al., 2023). Although the evidence used to support this research in scientific literature is generally regarded as weak, more studies are crucial before this can be proven or disputed. Another theory surrounding the amniotic fluid explores the idea that a baby might in fact ingest the amniotic fluid, resulting in bacterial transfer from the mother. This is another area that is currently lacking in evidence and requires further research. There is a significant transfer of microbes from a mother to her child during vaginal delivery as well as through skin-to-skin contact in a baby’s early moments of life. During breastfeeding, the bacteria surrounding the nipple is also transferred orally. According to recent publications, 58.5% of a baby’s microbiome is a direct result of their mother. The rest is attributed to their environment and other external factors (Bogaert., et al 2023). Whether there is a link between birthing route (vaginal or caesarean) and AD is currently unclear, with mixed study results and a vast majority of scientists agreeing that a clear link cannot consistently be established. Infant Early Life and AD: Skin Microbiome There are several published papers which explore the association of the baby microbiome with AD. For the purposes of this article “The skin microbiome in the first year of life and its association with atopic dermatitis” will be evaluated more closely (Rapin., et al 2023). This study was conducted in Oslo, Norway, in which the skin of babies was sampled at four timepoints (At birth, 3 months, 6 months and 12 months). Various investigational factors, including the composition of the skin microbiome, birthing methods, environmental influences, parental factors, and breastfeeding, were analyzed to assess potential associations with skin immunities. Thee results of this study ultimately showed that these factors were all correlated and instrumental in the development of the baby’s microbiome. The study further showed that each variable held a different influence on the skin microbiome depending on the timepoint the sample was taken at. For example, at birth the mode of delivery was most instrumental. However, as time went on different factors became more influential, such as birth location, breastfeeding, maternal AD, maternal food allergies, and exposure to pets. It is known that the delivery of a baby shapes the microbiome in early life. However, can an association be found between the birthing route and the development of AD? This Oslo study concluded that ultimately there is not a strong association between delivery mode and AD pathogenesis. The reasoning behind this conclusion was that the few differences that were noted at birth levelled out by 12 months and no longer held differentiable significance (Rapin., et al 2023). Infant early life and AD: Gut Microbiome It is interesting to note that the conclusions drawn from the skin microbiome study in Oslo is mirrored by another study that was conducted on the gut microbiome in association with AD. The study in question “The associations of maternal and children’s gut microbiota with the development of atopic dermatitis for children ages 2 years” (Fan et al., 2022) compared the gut microbiome of mothers and babies, and found that mothers of infants and toddlers with AD had higher abundance of Candidatus_Stoquefichus and Pseudomonas in pregnancy. The study also found that infants and toddlers with AD had a higher abundance of Eubacterium_xylanophilum group at birth, Ruminococcus_gauvreauii group at 1 year of age, UCG-002 at 2 years, and lower abundance of Gemella and Veillonella at 2 years of age. It is particularly interesting that the study also demonstrated a lower abundance of Prevotella in mothers of infants and toddlers with AD compared to mothers of the control group. The Skin Microbiome: New Findings A subsequent study performed shotgun metagenomic sequencing on the skin microbiome, it found that a dysbiosis in the microbiome exists prior to the onset of AD (Chaudhary et al., 2023). Firstly, the study affirmed that birth mode and demographics in fact did not associate with subsequent AD development. However, what the study did find was that by measuring the skin of babies, reduced Prevotella abundance could be a predictor of subsequent AD development. The benefits of using shotgun metagenomics meant that more functional analysis could be conducted, ultimately showing that there was a significant reduction in Prevotella abundance in the AD group compared to the control group. Additionally, there are some differences in host and bacterial features in certain genes that are interesting to target based on what is seen in shotgun metagenomic sequencing. When looking at lipid profiling they showed a complete difference between the AD group and control group. Prevotella has been found to be a good candidate as a potential predictor of AD development, as demonstrated by this study. Conclusion There is considerable research being conducted on the baby microbiome as well as AD in adults, however more research needs to be carried out in order to make stronger links between what might be the root cause of AD in babies and infants. The publications highlighted within the article give some good insights into AD and some of the positive and negative correlations between different variables. Notable, the role of Prevotella in AD might be an interesting one to explore further as there is strong evidence to suggest that it could be a good diagnostic target to better understand how AD might develop. At present, the majority of treatments within the personal care and pharmaceutical industry continue to target Staphylococcus aureus, with proven improvements and more clinical data to show that this approach is effective in adults with AD. However, new candidates must be studied so that the industry can adapt the way it approaches AD treatment and intervention in the future, particularly regarding the development of AD in children. Reference Baldwin H, Aguh C, Andriessen A, Benjamin L, Ferberg AS, Hooper D, Jarizzo JL, Lio PA, Tlougan B, Woolery-Lloyd HC, Zeichner J. Atopic Dermatitis and the Role of the Skin Microbiome in Choosing Prevention, Treatment, and Maintenance Options. J Drugs Dermatol. 2020 Oct 1;19(10):935-940. doi: 10.36849/JDD.2020.10.36849/JDD.2020.5393. PMID: 33026777. Bogaert D, van Beveren GJ, de Koff EM, Lusarreta Parga P, Balcazar Lopez CE, Koppensteiner L, Clerc M, Hasrat R, Arp K, Chu MLJN, de Groot PCM, Sanders EAM, van Houten MA, de Steenhuijsen Piters WAA. Mother-to-infant microbiota transmission and infant microbiota development across multiple body sites. Cell Host Microbe. 2023 Mar 8;31(3):447-460.e6. doi: 10.1016/j.chom.2023.01.018. PMID: 36893737. Bowen AC, Mahé A, Hay RJ, Andrews RM, Steer AC, Tong SY, Carapetis JR. The Global Epidemiology of Impetigo: A Systematic Review of the Population Prevalence of Impetigo and Pyoderma. PLoS One. 2015 Aug 28;10(8):e0136789. doi: 10.1371/journal.pone.0136789. PMID: 26317533; PMCID: PMC4552802. Chaudhary PP, Myles IA, Zeldin J, Dabdoub S, Deopujari V, Baveja R, Baker R, Bengtson S, Sutton A, Levy S, Hourigan SK. Shotgun metagenomic sequencing on skin microbiome indicates dysbiosis exists prior to the onset of atopic dermatitis. Allergy. 2023 Oct;78(10):2724-2731. doi: 10.1111/all.15806. Epub 2023 Jul 8. PMID: 37422700; PMCID: PMC10543534. Fan X, Zang T, Dai J, Wu N, Hope C, Bai J, Liu Y. The associations of maternal and children's gut microbiota with the development of atopic dermatitis for children aged 2 years. Front Immunol. 2022 Nov 17;13:1038876. doi: 10.3389/fimmu.2022.1038876. PMID: 36466879; PMCID: PMC9714546. Gilaberte Y, Pérez-Gilaberte JB, Poblador-Plou B, Bliek-Bueno K, Gimeno-Miguel A, Prados-Torres A. Prevalence and Comorbidity of Atopic Dermatitis in Children: A Large-Scale Population Study Based on Real-World Data. J Clin Med. 2020 May 28;9(6):1632. doi: 10.3390/jcm9061632. PMID: 32481591; PMCID: PMC7356227. Kaisanlahti, A., Turunen, J., Byts, N. et al. Maternal microbiota communicates with the fetus through microbiota-derived extracellular vesicles. Microbiome 11, 249 (2023). https://doi.org/10.1186/s40168-023-01694-9 Khadka VD, Key FM, Romo-González C, Martínez-Gayosso A, Campos-Cabrera BL, Gerónimo-Gallegos A, Lynn TC, Durán-McKinster C, Coria-Jiménez R, Lieberman TD, García-Romero MT. The Skin Microbiome of Patients With Atopic Dermatitis Normalizes Gradually During Treatment. Front Cell Infect Microbiol. 2021 Sep 24;11:720674. doi: 10.3389/fcimb.2021.720674. PMID: 34631601; PMCID: PMC8498027. Mannschreck D, Feig J, Selph J, Cohen B. Disseminated bullous impetigo and atopic dermatitis: Case series and literature review. Pediatr Dermatol. 2020 Jan;37(1):103-108. doi: 10.1111/pde.14032. Epub 2019 Nov 22. PMID: 31755570. Rapin A, Rehbinder EM, Macowan M, Pattaroni C, Lødrup Carlsen KC, Harris NL, Jonassen CM, Landrø L, Lossius AH, Nordlund B, Rudi K, Skjerven HO, Cathrine Staff A, Söderhäll C, Ubags N, Vettukattil R, Marsland BJ. The skin microbiome in the first year of life and its association with atopic dermatitis. Allergy. 2023 Jul;78(7):1949-1963. doi: 10.1111/all.15671. Epub 2023 Feb 24. PMID: 36779606.

Introduction: What are biosensors? Biosensors are described as analytical devices used to detect biomarkers, or analytes, which can be anything from toxic chemicals, to small molecules, microbes or even peptides and nucleotides (Bhalla et al., 2014). These devices are made up of key parts. Firstly, biological components which uphold the biosensing capacity, for example antibodies detecting antigens or aptamers binding to their specific targets. Then, physical or chemical information transducers which signal the presence of a target. Finally, the signal is processed and we obtain an output (Figure 1). Traditionally these kinds of biosensors rely on electrochemistry, and we often think of them in the context of diabetes and Continuous Glucose Monitoring Devices, allowing patients with the condition to control their blood sugars. However, biosensors have very diverse applications which can range from disease monitoring to environmental monitoring, and there is a continuing need to make cheap and reliable, as well as simple biosensors that can be used across a wide range of applications. Figure 1: Schematic of biosensors. Image taken from Verma and Gahlaut (2019) Synthetic biology biosensors Synthetic biology mainly relies on exploiting the power of nature to create useful tools, thus this approach could provide a new generation of biosensing systems that present great advantages over traditional devices. The first of these advantages lies in the fact that they are custom-made biological tools which allow for highly specific target identification. Moreover, there are two approaches in designing biosensors. On the one hand, they can be created by genetically engineering live cells to create Whole-Cell Biosensors (WCB) that are able to respond in real-time to their environment and detect specific analytes (Chen et al., 2023). On the other hand, we can generate cell-free biosensors which are essentially solutions consisting of all the required machinery, proteins and genetic circuits that allow for the detection of our targets (Zhang et al., 2020). Ultimately, both approaches produce a distinguishable qualitative or quantitative signal. They allow for quick and accurate sensing of targets, which as discussed is crucial for fulfilling the demand across a range of applications. The rationale behind the workings of such biosensors is quite simple. They have an input, which can be anything from environmental signals to light or even small molecules, this input gets converted into a signal that gets processed by a genetic network which can be designed to perform diverse functions depending on our applications (Wang et al., 2023). In a similar way to electrical circuits, we can add in different ranges of signal processing steps. As a result, our biosensor is able to generate an output, for instance a change in colour by production of pigments visible to the naked eye, emission of fluorescence that we can see and quantify with the right equipment or even liberation of gases that can be monitored using ultrasound (Figure 2). There is a level of adaptability and it is ultimately up to us to decide on the purpose of these biosensors. Figure 2: Schematic of synthetic biology based biosensors. (diagram from Xinyi Wan, University of Edinburgh, PhD Thesis) Bacterial biosensing Biosensors uphold great promises for bacterial biosensing whether it is from water, soil, human or food samples. It all starts with sample collection, treatment of the samples, and then detection can either be done directly on the bacteria by detecting exposed biomarkers or unique DNA and RNA sequences, or by indirect detection of unique toxins, peptides, or volatile organic compounds (VOCs) produced by the bacteria (Mazur et al. 2023). Point-of-care (POC) devices POC devices are medical diagnostic tools designed to provide rapid and convenient testing and analysis of patient samples. These devices aim to bring testing closer to the patients and aim to reduce the need for the sample to be sent into a central laboratory, thus allowing quicker decision making by healthcare providers. POC devices play a crucial role in improving patient outcomes, especially in scenarios where time sensitive results are required. A great example of those would be lateral-flow tests which can give simple informative results in less than 30 minutes without requiring the assistance of trained experts. Key features of POCs include: quick turnaround times of test results, great accessibility and ease of use in various healthcare and nontraditional settings and research areas, and crucially they must be user friendly even to those who are not laboratory staff (Mazur et al., 2023). These devices allow for great diagnostic ranges, for example diagnostic of infections, monitoring chronic conditions, and screening for patients before sourcing a study. Paper-based biosensors The vast majority of us will have been exposed to and acquainted with the use of paper-based biosensors during the COVID-19 pandemic, in the form of lateral flow tests. In simple terms, it corresponds to a sheet of paper where we have an ensemble of biological elements that can react with each other and generate our desired output upon contact with the target. They are cost-effective, provide rapid responses (usually between 5-30 minutes), are a simple portable solution, and can be adapted for remote or resource limited settings. Indeed, many of these advantages have been seen through the use of paper-based biosensors during the COVID-19 pandemic. These paper-based cell-free biosensors, and can be used alongside genetic networks to generate a whole new range of specific functions. The workflow behind paper-based biosensors follows as described in figure 3; firstly we have our platform which is the microchannel paper, it has great microfluidic capacities and is designed to use a special paper which contains microchannels and spots. These channels are created using a technique such as printing or wax patterning, and within those channels we can embed our capture molecules as well as our synthetic gene network and cell-free extracts. The capture molecules are substances that can specifically bind to the bacteria of interest, and they are immobilised within the paper's channels. The liquid sample is inserted into the biosensor, and if the sample contains the bacteria, it will be able to be processed by our synthetic genetic circuit. The liquid flows along the paper channels carrying the bacteria with it, and as it comes into contact with the capture molecules our systems work and a signal is generated (Pardee et al., 2014). Figure 3: Assembling a paper-based synthetic biology biosensor. (diagram taken from Pardee et al., 2014) Why are they relevant to us? There already exists a few golden standard techniques such as ELISA, qPCR, FTIR, etc., however while these techniques are very robust, accurate and sensitive to strain level, they can often also be costly, time consuming, require centralised laboratories, trained personnel,  extensive sample pretreatment and multi-step processing. There is now a need to develop point-of-care devices that are fast, cheap, portable, and do not require any specialist training. This is especially important as low-income regions too often struggle to access adequate diagnostic tools for the detection of pathogens, ultimately leading to higher mortality rates (Pardee et al. 2014). We will focus our attention on the article ‘A low-cost paper-based synthetic biology platform for analysing gut microbiota and host biomarkers’ (Takahashi et al, 2018). This study is of great importance, especially considering that the microbiome make-up is key to understanding health and diseases. This study was focused towards trying to detect the hypervariable regions of the 16S rRNA genes in order to perform taxonomic profiling of the microbiome. The study aimed to develop an approach that is affordable, on demand and allows for simple analysis of the microbiome from stool samples. In addition they aimed to develop a platform that could accurately identify species-specific mRNA from 10 different bacteria as well as the mRNA of 3 key biomarkers involved in inflammation (calprotectin, CXCL5 and IL-8) and one cytokine (oncostatin M) which helps to predict the efficacy of TNF-α therapies in IBD patients. Furthermore, they pushed their research for rapid and inexpensive detection of toxin mRNA in the diagnosis of C. difficile infections. As a result of the study, a platform was successfully developed for analysis of the gut microbiota for clinical research and adaptability and low resource settings.Their device allowed for the orthogonal detection of species-specific mRNA from 10 different bacteria associated with gut health and disease with 3 fM limit of detection (LOD) achieved. In other words, this tool was able to discriminate between different bacteria and accurately report which ones were detected without having any cross-talk or reporting the wrong species. On top of a great accuracy, it is important to consider the LOD, which refers to the lowest amount of a substance (here mRNA) detected by the sensor. The lower the LOD, the more sensitive the biosensor is, making it efficient in detecting extremely small amounts of the bacteria's genetic material. A LOD as low as the one they achieved is reflective of great sensitivity. For comparison, traditional methods like qPCR often have LODs in the range of picomolars (pM) or femtomolars (fM). As a result, this biosensor is great for accurately detecting bacteria even if they are present in very low concentrations! The Takahashi group was able to develop a simple approach to study relative abundances of bacteria in the stool samples via a semi-quantitative determination of the concentrations of each target mRNA. Although this semi-quantitative approach initially only provided them with a rough idea of mRNA amounts rather than exact concentrations, the group compared the samples with known referencing standards and created a standard curve which allowed them to estimate mRNA concentrations in the samples. Results were even validated by a comparison with RT-qPCR which showed very similar performance. Finally, they were also able to identify different toxin mRNA expression levels from pathogenic C.difficile strains that were otherwise indistinguishable by using standard DNA-based qPCR diagnosis. The system they generated is a translation-based biosensor which relies on a technology called “Toehold-switch sensors” that control the translation of genes. In simple terms, toehold switches correspond to a strand of RNA that can form a hairpin structure based on complementary base pairing. This RNA strand has a region specifically complementary to our target sequences and a region that encodes for the protein we want to express for the detection - in this case a green fluorescent protein (GFP). Due to the secondary structure of the RNA, the GFP cannot be expressed as it is made inaccessible to the transcriptional machinery. The only way of expressing the GFP is by exposing the toehold switch to the target mRNA sequence from the bacteria we want to detect. The two strands will be able to bind to each other, causing a conformational change in the toehold switch which allows GFP to be transcribed and expressed (Figure 4). The advantage of this technique is that RNA folding is universal and many computer design softwares can predict the 3D structure which facilitates their design. However, on the other hand RNA folding is highly sensitive to physiological conditions, so unless the right conditions are met, the ability of the biosensor to function might be hindered. Figure 4: Toe-hold switch mechanism. (Diagram taken from Takahashi et al., 2018) Advantages of this approach Some of the main advantages of this approach are that it provides us with a great ease of use with simple results being observed. Moreover, the biosensors can be adapted for low resource settings, as the reactions that are happening do not require highly specialised equipment. For example, the GFP detection can be monitored on affordable and easy to build portable electronic readers that can quantify the changes in absorbance. The sample amplification of the mRNA is enabled through Nucleic acid sequence based amplification (NASBA) by using simple isothermic incubators, so there is no real need for thermocyclers with greater powers such as the ones which are required for qPCR (Kia et al. 2023). Additionally an important aspect of this is the cost, using this technique the mRNA can be quantified in around 3-4 hours for $16 per transcript using commercially available kits, with the potential to decrease this cost to $2 per transcript by using in-house cell-free extracts. Compared to qPCR which costs significantly more, it is clear that this is a more cost effective alternative (Takahashi et al., 2018). All of this could be applied to a broad range of studies, including skin samples. This technology is easily adaptable to target other microorganisms, such as fungi, bacteriophages and human viruses. It could also be adapted for point-of-care use and at home monitoring for patients to respond to one of the greater goals: being able to regularly monitor disease progression in patients. Limitations of the study The first limitation that they encountered upon initial design was the low limit of detection of the toehold switch: with the sensor alone, the limit of detection was in the 10-30 nM range. They therefore decided to pair their approach with NASBA which is a technique done during the sample processing step to amplify the bacterial RNA prior to detection. This technique is very robust, particularly well-suited for RNA, and presents less risk of DNA contamination, therefore minimising the risk of a false positive result. As a result, this step greatly improved the sensitivity of this device to 3 fM. Additionally, as the 16S sensors they first generated presented significant crosstalk in closely related bacteria and were therefore not suitable for discriminating amongst highly related bacterial species, they chose to target different mRNA sequences which are unique to the desired bacteria based on a bioinformatics pipeline they generated and obtained a perfectly orthogonal detection of the different bacterial species. Their sensors were however not yet optimised to identify down to strain level. Moreover, despite requiring easily accessible equipment, their approach still involves some sample processing steps and is still not yet fully adapted for at-home testing, and therefore is not yet at the point-of-care application levels. However, we can still get a lot of important and valuable information in a more accessible way than traditional techniques. Pioneering skin microbiome biosensors There is very little research around biosensors in the field of the skin microbiome. However, translating this methodology and finding a wide array of specific targets for skin microbiome studies is achievable. An interesting avenue in the future would be introducing ribocomputing circuits for bacterial interaction studies to allow us to track the social interactions of bacteria in different dysbiosis conditions. Nowadays, more advanced paper-based biosensors allow for detection of signals directly with smartphones which can save the data into a cloud to monitor changes over time (Kim et al., 2021). The rapid advances in synthetic biology and growing interest in the skin microbiome will certainly open doors to a future where these proof-of-concept studies will serve as a basis to create wearable devices capturing in real time  the changes of our beloved skin microbial communities. References Bhalla, N., Jolly, P., Formisano, N. & Estrela, P. (2016) Introduction to biosensors. Essays in Biochemistry. 60 (1), 1-8. 10.1042/EBC20150001. Chen S, Chen X, Su H, Guo M, Liu H. Advances in Synthetic-Biology-Based Whole-Cell Biosensors: Principles, Genetic Modules, and Applications in Food Safety. Int J Mol Sci. 2023 Apr 28;24(9):7989. doi: 10.3390/ijms24097989. PMID: 37175695; PMCID: PMC10178329. Damour, A., Robin, B., Deroche, L., Broutin, L., Bellin, N., Verdon, J., Lina, G., Leclère, F. M., Garcia, M., Cremniter, J., Lévêque, N. & Bodet, C. (2021) Phenol-soluble modulins α are major virulence factors of Staphylococcus aureus secretome promoting inflammatory response in human epidermis. Virulence. 12 (1), 2474-2492. 10.1080/21505594.2021.1975909. Kia, V., Tafti, A., Paryan, M. & Mohammadi-Yeganeh, S. (2023) Evaluation of real-time NASBA assay for the detection of SARS-CoV-2 compared with real- time PCR. Irish Journal of Medical Science. 192 (2), 723-729. 10.1007/s11845-022-03046-2. Kim, S., Lee, M. H., Wiwasuku, T., Day, A. S., Youngme, S., Hwang, D. S. & Yoon, J. (2021) Human sensor-inspired supervised machine learning of smartphone-based paper microfluidic analysis for bacterial species classification. Biosensors & Bioelectronics. 188 113335. 10.1016/j.bios.2021.113335. Mazur, F., Tjandra, A. D., Zhou, Y., Gao, Y. & Chandrawati, R. (2023) Paper-based sensors for bacteria detection. Nature Reviews Bioengineering. 1 (3), 180-192. 10.1038/s44222-023-00024-w. Nai, Y. H., Doeven, E. H. & Guijt, R. M. (2022) An improved nucleic acid sequence-based amplification method mediated by T4 gene 32 protein. PloS One. 17 (3), e0265391. 10.1371/journal.pone.0265391. Pardee, K., Green, A. A., Ferrante, T., Cameron, D. E., DaleyKeyser, A., Yin, P. & Collins, J. J. (2014) Paper-Based Synthetic Gene Networks. Cell. 159 (4), 940-954. 10.1016/j.cell.2014.10.004. Takahashi, M. K., Tan, X., Dy, A. J., Braff, D., Akana, R. T., Furuta, Y., Donghia, N., Ananthakrishnan, A. & Collins, J. J. (2018) A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers. Nature Communications. 9 (1), 3347-12. 10.1038/s41467-018-05864-4. Wan, X. (2019) Synthetic biology enabled cellular and cell-free biosensors for environmental contaminants. The University of Edinburgh. Wang, C., Zeng, H., Liu, K., Lin, Y., Yang, H., Xie, X., Wei, D. & Ye, J. (2023) Biosensor-based therapy powered by synthetic biology. Smart Materials in Medicine. 4 212-224. 10.1016/j.smaim.2022.10.003. Zhang L, Guo W, Lu Y. Advances in Cell-Free Biosensors: Principle, Mechanism, and Applications. Biotechnol J. 2020 Sep;15(9):e2000187. doi: 10.1002/biot.202000187. Epub 2020 Jul 23. PMID: 32667120.

Introduction In this post we will be summarising multiple different articles looking at the biology of bacteriophages and their potential applications, with a focus on exploring what we can do to harness their potential. We will start by introducing the most common bacteriophages of the microbiome, and moving onto how these interact and manifest within the microbial communities of the skin, and then concluding with a discussion about whether we can harness them to develop technologies that can engineer the skin microbiome as a way to treat dermatological conditions that affect millions of people annually. We will primarily focus on the sebaceous (oily) sites of the skin such as the face, even though there are indeed a variety of other sites which are also important to consider when looking at phageomes and disease. The Virome To understand the role of bacteriophages, it is necessary to first look at defining the virome in relation to host health and microbiome ecology. The virome can be defined as ‘a subset of the core human microbiome consisting only of the viral biomass in a given community’. This applies to the skin also, which is colonised by numerous groups of viruses with their own ecology and interactions. It includes many different classes of virus, the three most common being Eukaryotic DNA viruses, Viral Genetic Elements and Bacteriophages (Phages) (Virgin, 2014). Virome composition is shaped by a variety of forces, both ecological and evolutionary. Some studies have found that the skin microenvironment, including physical properties (e.g. whether the skin is dry, moist or sebaceous) which appear to be a key driving force behind viral community dynamics (Byrd et al., 2018), with viral blooms appearing in specific sites along the skin including sebaceous regions of the face such the cheeks and the forehead (Oh et al., 2016). Other factors which might impact a person’s microbial community include geography such as pollution levels and UV exposure, in addition to lifestyle factors such as diet, health and personal care. However, it is important to note here that no core DNA virome has yet been found to exist, with lots of individual variation in virome composition. This could be due to a number of different factors, including biogeography of the skin, ethnicity, genetics, parental imprinting and transmission between and across populations (Byrd et al., 2018). Individual variation of the types of virus found in humans presents a possible future avenue of study to further explore why and how this is the case, and furthermore whether it has any key implications for individual differences in skin conditions. The Phageome Despite this lack of a cohesive virome in the human population, there does appear to be a subset of this community which possesses a more concerted signature than its larger counterpart. This is where the phageome comes in. The phageome refers to the net biomass of bacteriophages found within the human microbiome (Townsend et al., 2021). It differs from the virome in that it has been found to possess a conserved signature across human populations, despite any interspecific differences which may exist within the microbiome as a whole, therefore indicating the possibility of a shared core phageome existing (Oh et al., 2016). Bacteriophages fall under the umbrella of the phageome, and are described as ‘viruses which are capable of infecting and/or killing bacteria’ (Castillo et al., 2018). In the case of most humans, the bacteria on the skin microbiome are primarily infected by two core groups of phages found across sebaceous sites of the skin; Cutibacterium phages and Staphylococcus phages. These phages can be found across sebaceous areas of the face, usually with a single strain of phages dominating these sites. Like other phages, their populations tend to remain largely stable within their microenvironments compared to the transient eukaryotic DNA viruses, which suggests a level of fixation within their bacterial-host population (Oh et al., 2016). In regards to the types of interactions which manifest between bacteriophages and their respective host, these can vary depending on the evolutionary adaptations to the host species they decide to colonise (Hannigan et al., 2015). Interactions usually range from lysogenic, to pseudolysogenic, or purely lytic. Lysogenic phages integrate into the host genome as a prophage and continue to propagate in this form as the host replicates and divides, meaning that their fitness is ultimately linked to the survival of their bacterial host. On the other hand, pseudolysogenic phages can alternate between the two extremes, existing as prophages until they are exposed to environmental stress at which point they are excised and circularised to enter the lytic stage. Finally, the lytic viruses are a group which infect and kill the host while using them as a medium of replication, these types may go onto wipe out large populations of bacteria within a community causing dysbiosis that affect the human host, or in some cases having the opposite effect of preventing pathogenic bacterial species from propagating in the microbiome. C. acnes and The Skin C. acnes phages is a subset of Cutibacteria-infecting phages which colonise sebaceous areas of the body, such as the face, with most individuals possessing a single strain of this phage within the skin microbiome (Castillo et al., 2018). C. acnes phages, like many other Cutibacteria-infecting species, tend to be lacking in genetic diversity, with between 85 - 100% sequence identity observed between strains (Liu et al., 2015). Its presence is usually found associated with that of its target bacterial species, C. acnes, with which they form antagonistic interactions, going on to have a reductive effect on bacterial abundance and population size (Oh et al., 2016). Such antagonistic dynamics have played a role in modulating the position of the microbiome through the selective removal of certain C. acnes populations. However, the population sizes of some C. acnes have been found to positively correlate with phage prevalence, this indicates a certain amount of variability of the type of interaction which manifests between the two partners (Oh et al., 2016). These phages tend to assume a mostly lytic state, going onto infect and lyse their target host cells and effectively reduce the population size of  C. acnes species within the microbial community. However, some have been observed to have the ability to adopt a pseudolysogenic state. This shows that the type of bacteriophage that manifests is largely constrained by C. acnes lineage (Liu et al., 2015). This level of flexibility in C. acnes phage lifestyle could have evolved in order to enhance phage survival, but no comprehensive explanation has yet been uncovered for this. The overpopulation of certain C. acnes strains is associated with skin conditions such as acne. C. acnes phages are capable of lysing and destroying C. acnes strains from most lineages including pathogenic strains which can have the effect of reducing the relative abundance of C. acnes in the host microbiome by shifting the skin microbiome away from acne-associated dysbiosis (Liu et al., 2015). Additionally, C. acnes phages are more abundant in the facial skin samples of the individuals with healthy, non-acne infected skin, indicating the regulatory function of these phages in preventing proliferation of pathogenic C. acnes strains associated with the onset of acne (Liu et al., 2015). These findings suggest a link between phage abundance and the development of acne, and the role of these bacteriophages in regulating microbiome balance. S. aureus Phages and The Skin Staphylococcus aureus phages form interaction with Staphylococcus aureus strains of bacteria that are also highly abundant within the microbiome of sebaceous skin sites. These bacteria have been associated with the onset of many skin diseases, such as atopic dermatitis and psoriasis (Natarelli et al., 2023). Infection of these S. aureus strains by phages has also recently been linked as another potential risk factor in increasing the virulence of these bacterial strains, with phages helping to facilitate transmission of antibiotic resistant genes and virulence factors to these bacteria from other host reservoirs that harbour these genetic elements such as S. epidermidis (Hannigan et al., 2017). This allows these bacteria to fortify their defences against the human immune system, and cause disorders associated with the skin or in some cases even increase severity of skin-disorders. Many of these lysogenic S. aureus phages carry virulence factors that upon integration into the host genome can confer fitness benefits such as host propagation and survival. They can also carry many genes that might add to this fitness or cause genomic rearrangements that enhance the pathogenicity and virulence of S. aureus strains, going onto improve host fitness and survival and worsening the severity of certain skin conditions as these bacteria grow (Hannigan et al., 2017). The effects of this lysogeny on human skin has to some extent been associated with atopic dermatitis, with greater abundances of S. aureus being detected in patients possessing these lesions (Bjerre et al., 2021). Potential Applications of Phages We will now go on to explore the potential applications of these phages, and discuss technologies that have been devised to harness the power of some bacteriophages in the treatment of skin conditions such as acne or atopic dermatitis. It is important to note that suitable candidates for phage therapy must be lytic, non-lysogenic, and free of virulence factors and antibiotic resistant genes in order to properly target these problem strains without transferring virulence factors or genetic elements that might cause the bacteria being targeted more infectious (Kim et al., 2022). Acne and C. acnes phage therapy In the case of acne it is increased sebum production that can induce the growth of pathogenic C. acnes strains and this overgrowth is what might exacerbate the conditions and drive inflammation across the skin and bring about global effects (Natarelli et al., 2023). The overpopulation of these strains can go on to trigger dysbiosis by further reducing the already low levels of microbiome diversity, including wiping out other C. acnes populations regardless of whether they are commensal or pathogenic. This phenomenon suggests that acne might have less to do with increasing pathogenic C. acnes strain abundance, but rather it is the loss of C. acnes phylotype diversity in the microbiome (Mias et al., 2023). Certain C. acnes strains have been found to be more strongly associated with acne pathogenesis, while others are associated with healthy skin microbiome composition, this indicates that certain C. acnes phages can be used to target C. acnes bacterial population types associated with acne allowing the selective suppression of bacterial growth while still maintaining commensal or beneficial strain population structure (Fitz-Gibbon et al., 2013; Liu et al., 2015). Personalised phage therapies might also be developed to target particular strains of C. acnes present in the skin microbiome depending on individual bacterial community structure, in order to restore dysbiotic skin. Resistant C. acnes strains might require some more engineering of phages to be able to overcome host immune mechanisms and anti-virulence factors. However, preliminary studies attempting to treat pathogenic C. acnes strains with specific bacteriophages have already shown some promising results (Xuan et al., 2023), with near total population reduction upon application of these phages (Kim et al., 2019). Therefore, showing the potential to treat, or at least minimise, the effects of acne. Atopic dermatitis and S. aureus phage therapy In the case of atopic dermatitis, disease can also be brought about by dysbiosis of the skin microbiome. An overabundance of S. aureus populations relative to other species can trigger atopic dermatitis, by causing immune dysregulation and impairing the skin barrier function which leads to inflammation and flaking of the skin which is characteristic of this condition (Tham et al., 2020). The symptoms of AD can be worsened through the acquisition of virulence genes from bacteria that increase S. aureus strains pathogenicity. While some strains of phage support and promote the survival of S. aureus, a variety of other groups have been proven to have powerful anti S. aureus agents with several cases reporting complete eradication of S. aureus and/or patient improvement upon administration of these phage strains (Hatoum-Aslan, 2021). Many of these phages have small genomes which are too compact to support the integration of virulence-associated genes that can be transferred between the microbiome during transduction, thus eliminating the risk of increasing pathogenicity or inducing mutations. These can be engineered to express antibacterial compounds in their genomes that have the effect of destroying the infected cell upon expression (Hatoum-Aslan, 2021). Other studies have also demonstrated the effect of phage derived compounds against AD, showing the potential of specific S. aureus phages in combating these AD associated bacterial strains (Tham et al., 2020). The Potential of Phage Therapy Acne and atopic dermatitis industries in the US alone are worth $2.5 billion (Castillo et al., 2018) and $5 billion (Adamson, 2017) respectively, with treatments often being costly and difficult to develop. These conditions present a fresh, untapped market in which phage therapies can enter. Phage therapies can offer an alternative to traditional methods of treatment of bacterial infections requiring the use of antibiotics. This is especially important when considering the global burden of antibiotic resistance and the rising numbers of resistance annually observed, and demonstrates how antibiotics are not a sustainable method of treating many bacterial infections. These phages can be made to target bacteria that have developed some level of multidrug resistance, which presents the potential of them also reducing antibiotic resistance in a population and making the administration of antibiotics all the more effective (McCutcheon et al., 2020). Unlike traditional medicines, bacteriophage therapy is able to treat diseases in a target specific manner, without harming other components of the microbiome or human host. They are also easier and less expensive to produce en masse (Jończyk-Matysiak et al., 2017), therefore they are practical, economically feasible and able to cover a large area of the skincare market. Most importantly, as a natural part of the human microflora, they are safe and well tolerated with no adverse effects of their administration being reported as of yet (Castillo et al., 2018). Future Perspectives Phage therapy, as with all budding technologies, still has a considerably long way to go before it can become a conventional treatment for dermatological conditions. More research on the interactions between bacteriophages and the skin microbiome must be conducted to investigate any global effects which might be observed upon removal of any particular bacteria subpopulation, particularly as this is something which might have adverse effects that we are currently unaware of (Castillo et al., 2018). It will also help us get a better understanding of the extent to which these phages produce such bactericidal in regulatory effects on the microbiome. This will also help to identify the right phages in targeted treatment. Despite impressive findings surrounding the effectiveness of phages in removing disease-associated micropopulations, none of these involve the use of human models as a basis of study. Indeed, the conduction of more human trials are necessary before this field can expand and move forward towards being released to the market (Jończyk-Matysiak et al., 2017; Castillo et al., 2018). Caution must also be exercised when taking phage species which express a certain level of lysogeny within their strains, such as those strains targeting S. aureus. These lysogenic phages can have the opposite effect of reinforcing pathogenic bacteria, rather than having the desired effect of destroying them (Jończyk-Matysiak et al., 2017). The long term impact of population wide eradication on the microbiome health and ecology is not yet fully understood or characterised. This ultimately means we must proceed with caution moving forward. However, many remain optimistic that this technology will move forward to have transformative effects on the skin and healthcare industry, presenting an exciting new avenue for disease to be targeted and treated more efficiently than ever before. Improvements in technology and recent advances in our understanding of phage biology have certainly made it more likely for these expectations to one day become a reality. References Adamson, A. S. Adv Exp Med Biol 1027, 79–92 (2017) Bjerre, R. D. et al. BMC Microbiology 21, 256 (2021) Byrd, A. L. et al. Nat Rev Microbiol 16, 143–155 (2018) Castillo, D. E. et al. Dermatol Ther (Heidelb) 9, 19–31 (2018) Cheng, L. et al. BMC Microbiology 18, 19 (2018) Fitz-Gibbon, S. et al. J Invest Dermatol 133, 2152–2160 (2013) Hannigan, G. D. et al. PeerJ 5, e2959 (2017) Hatoum-Aslan, A. Trends in Microbiology 29, 1117–1129 (2021) Jończyk-Matysiak, E. et al. Front Microbiol 8, 164 (2017) Kim, S. et al. Antibiotics (Basel) 11, 1041 (2022) Liu, J. et al. ISME J 9, 2078–2093 (2015) McCutcheon, J. G. et al. International Journal of Molecular Sciences 21, 6338 (2020) Mias, C. et al. Journal of the European Academy of Dermatology and Venereology 37, 3–11 (2023) 15 Natarelli, N. et al. International Journal of Molecular Sciences 24, 2695 (2023) Oh, J. et al. Cell 165, 854–866 (2016) Tham, E. H. et al. Biotechnology Journal 15, 1900322 (2020) Townsend, E. M. et al. Frontiers in Cellular and Infection Microbiology 11, (2021) Virgin, H. W. Cell 157, 142–150 (2014) Xuan, G. et al. Microbial Pathogenesis 180, 106111 (2023)

Introduction The microbiome is an organic ecosystem of trillions of bacteria that live and interact with one another on and within our bodies (Berg et al., 2020). In recent years, more research has been carried out suggesting that the microbiome plays a critical role in our overall well-being, including our gut and skin health (Human Microbiome Project Consortium, 2012). It is widely accepted that every individual’s microbiome is unique, influenced by factors such as sex, age, physical health, lifestyle and environmental factors. However, recent research has emerged which begs the question: is the microbiome influenced by one's genetic makeup or rather by environmental factors? To answer this question, we look at a study conducted on twins to explore how their overlapping and diverse microbial communities give clues about what influences shape our microbiome. Due to limited research on twin skin microbiomes, we will rely on studies conducted predominantly on the gut and some on the oral microbiome. Defining Twins To better unearth the relationship between twin microbiomes, it is crucial to understand that not all twins are made alike. Identical twins (monozygotic) share the same DNA sequences, allowing us to better differentiate what elements of the microbiome are linked to genetics and which are influenced by environmental factors. On the other hand, fraternal twins (dizygotic) share about 50% of their DNA. Analysing genetically identical and fraternal twins can allow us to identify the environmental and genetic effects on their microbiome (Martin et al.,1997). Genetic Influence on Microbial Composition in the Gut A study (Goodrich et al., 2016) conducted 16S rRNA analysis on the gut microbiomes of approximately 1,126 twins in the United Kingdom. The results showed that the presence of specific genetic variants in the LCT (Lactase) gene locus was associated with relative abundances of the heritable genus Bifidobacterium. It is understood that Bifidobacterium metabolises lactose resulting in individuals exhibiting higher levels of Bifidobacterium if they are unable to produce enough Lactase (Goodrich et al., 2016). Another study conducted in Missouri, focused on four pairs of female adolescent twins (1 monozygotic and 3 dizygotic pairs). Within each pair, there was significant variability in weights, with one having lower body fat percentages and the other having higher levels. For the purpose of the study, fecal samples were collected from each participant and transplanted into healthy mice. The results of this study showed that the microbiota from the twins with a lower body fat percentage was better at breaking down and fermenting polysaccharides (carbs formed of sugar molecules) than the microbiota of the twins with higher body fat percentages. It was also found that mice who were transplanted with the microbiota of the twins with lower body fat percentage demonstrated protection from obesity phenotype (Ridaura et al, 2013). Genetic Influence on Microbial Composition in the Oral Cavity A study published under the name “Longitudinal Study of Oral Microbiome Variation in Twins” in 2020 evaluated the genetic and environmental factors attributed to oral caries (cavities) in both monozygotic and dizygotic twins. Dental plaque samples were extracted and analysed through 16S rRNA. It was noticed that changes in the oral microbiome were strongly influenced by the environment when compared to participant’s genetics. Other elements driving changes in the oral microbiome of twins was their age, the age at which they started brushing their teeth and their actions after brushing. The study identified the relevance of heritability on the microbiome by way of Capnocytophaga and Actinomyces in monozygotic twins, and Kingella within dizygotic twins. Certain bacteria were more associated with ageing: Veillonella and Corynebacterium. On the other hand, younger subjects were associated with Aggregatibacter. Streptococcus was found to decrease over time and Selenomonas increased with more frequent brushing per day (Freire et al., 2020). It was reported that unearthing the true biological mechanisms behind caries could unlock the potential to understand biomarkers and pathways that could help with prevention in early ears. However, further research needed to be carried out to reduce this knowledge gap. Genetic Influence and the Skin Microbiome Finally, another study conducted in Korea in 2015 evaluated the relationship between genetic and environmental factors on the skin microbiome of twins (Si et al, 2015). Results from Genetic associations and shared environmental effects on the skin microbiome of Korean twins (Si et al, 2015) Participants: The study in question included 45 subjects with 16 monozygotic twins, 8 dizygotic twins between the ages of 26 to 55, as well as their mothers and an additional 5 unrelated subjects. In 32 subjects (mothers and twins), skin traits such as pigmentation and skin humidity were measured. Sample collection: skin swabs were taken from each subject from the upper right arm to reduce variations due to external factors such as different personal care routines and varying cosmetic usage. Extraction: The entirety of the microbial DNA was extracted from the samples and the V2 and V3 regions of 16S rRNA genes and pyro-sequenced. Analysis: The 16S rRNA sequencing results were then analysed through Bioinformatics. Results from Genetic associations and shared environmental effects on the skin microbiome of Korean twins (Si et al, 2015) The results of this study demonstrated that Propionibacterium (now referred to as Cutibacterium), Staphylococcus and Streptococcus were the richest skin microbiota on a genus level, however as expected there was some variability amongst individuals. It was found that skin pigmentation has a significant impact on the skin bacteria with medium-skinned individuals having an increased microbial diversity. The results also showed that the highest similarities in skin microbiota existed between monozygotic twins followed by dizygotic twins and finally between mothers and twins. Furthermore, a negative correlation was found with an abundance of C. jeikeium and the allele T, which is a single polymorphism nucleotide (SNP) localised to a gene that plays a significant role in skin barrier function (filaggrin). From past research, we know that defects in filaggrin can be known to result in allergic skin conditions such as ichthyosis vulgaris and atopic eczema, both linked to excessive dryness of the skin (McAleer et al, 2013). This leads us to the conclusion that there might be a link between filaggrin processing and bacteria leading to pathogenesis (when an infection turns into a disease). To conclude the findings of this study on twin pairs, it was proven that both genetics and environmental factors can shape the skin microbiome (we saw this with pigmentation). A strong correlation between C. jeikeium from the skin microbiota and human genetic factors allele T was also found in relation to skin barrier function (Si et al, 2015). Next Steps Further research is needed in order to build on this study conducted in 2015. The question of whether the microbiome is influenced predominantly by genetics or environmental factors plays a crucial role into how we address the skin microbiome and the diseases that have been found to be linked to its disbalance. A significant next step to the work published above would be to include higher resolution taxonomic profiling, quantifying differences at species and strain level between identical twins would be of much importance. If you are interested in carrying out any research with us in studying the differences between identical twins’ microbiome, or you have questions about our testing platform for your own clinical studies - you can reach us through www.sequential.bio Lexicon Dizygotic: fraternal twins who are simply as alike as any other siblings, occurring when two eggs are released at a single ovulation and are fertilised by two different sperm. Microbiome: The microbiome is a characteristic microbial community occupying a reasonably well-defined habitat which has distinct physio-chemical properties. The microbiome not only refers to the microorganisms involved but also encompasses their theatre of activity, which results in the formation of specific ecological niches. This includes their genetic material, and also structural molecules, like enzymes, membrane lipids or polysaccharides (Definition based on Berg et al., 2020). Monozygotic: identical twins who develop when one egg is fertilised by a single sperm and then during the first two weeks of conception the developing embryo splits into two, causing two genetically identical babies to develop. Polysaccharides: Carbs formed of sugar molecules. Pathogenesis: The process by which an infection deteriorates into a disease. References Freire, M., Moustafa, A., Harkins, D.M. et al. Longitudinal Study of Oral Microbiome Variation in Twins. Sci Rep 10, 7954 (2020). https://doi.org/10.1038/s41598-020-64747-1 Martin, N., Boomsma, D. & Machin, G. A twin-pronged attack on complex traits. Nat Genet 17, 387-392, doi:10.1038/ng1297-387 (1997). Human Microbiome Project Consortium. (2012). Structure, function, and diversity of the healthy human microbiome. Nature, 486(7402), 207-214. Goodrich, J. K. et al. Genetic Determinants of the Gut Microbiome in UK Twins. Cell Host Microbe 19, 731-743, doi:10.1016/j.chom.2016.04.017 (2016). Ridaura, V. K. et al. Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science 341, 1241214, doi:10.1126/science.1241214 (2013). Si, J., Lee, S., Park, J. M., Sung, J. & Ko, G. Genetic associations and shared environmental effects on the skin microbiome of Korean twins. BMC Genomics 16, 992, doi:10.1186/s12864-015-2131-y (2015). McAleer, M. A. & Irvine, A. D. The multifunctional role of filaggrin in allergic skin disease. J Allergy Clin Immunol 131, 280-291, doi:10.1016/j.jaci.2012.12.668 (2013).

Abstract Understanding the oral microbiome in health and disease is essential as it will give further directions to explore the functional and metabolic alterations associated with the diseased states, and to identify molecular signatures for drug development and targeted therapies which will ultimately help in rendering personalized and precision medicine. In this article we explore the development of the oral microbiome, ask what might constitute a ‘healthy’ oral microbiome, and question what still needs to be done in terms of research in order to fully understand the link between the oral microbiome, oral health, and systemic health. The Oral Microbiome The oral microbiome is thought to have the third highest diversity of any microbiome niche, and given its ease of collection has become one of the most well-studied (Dewhirst et al. 2010). Previously, studying the microbiome was limited to the conventional culture-dependent techniques, but the abundant microflora present in the oral cavity could not be cultured. Hence, studying the microbiome was difficult. The emergence of new genomic technologies including next-generation sequencing and bioinformatics has revealed the complexities of the oral microbiome, and has provided a powerful means of studying it. The oral microbiome comprises a complex and diverse community of microorganisms living within the oral cavity. It is understood to be the third most diverse and largest after the gut and skin microbiota with over 700 species of bacteria alone (excluding fungi, protozoa and viruses). The microbiome is crucial to the oral health of humans as it is formed of an abundance of bacteria that live on multiple different surfaces: teeth, soft tissues, and mucosa. It is also the first point of digestion and is therefore thought to be crucial in maintaining oral and systemic health (Deo & Deshmukh, 2019). Development of the Oral Microbiome Despite the womb being considered as sterile, recent studies have found colonization by oral microorganisms within the intrauterine environment in as many as 70% of pregnant women, namely Streptococcus spp. and Fusobacterium nucleatum. The development of the oral microbiota is understood to begin after birth, and grows as an infant is exposed to varying microorganisms from its environment. This happens as soon as a baby’s first weeks of life. At birth, a newborn acquires all the necessary species of microbes to keep it healthy, namely Streptococcus salivarius. Within the first year, an infant will go on to develop a more diverse microbiome with Lactobacillus, Actinomycetes and Neisseria (Deo, Deshmukh, 2019). The increase of microorganisms in the oral cavity is the result of a variety of factors including oral hygiene practices, diet, and environmental factors. As children grow and their teeth begin to emerge, the complexity of the microbiome increases, and the microbial community shifts to reflect changes in the oral environment. What is a ‘Healthy’ Oral Microbiome? According to Berg et al. (2020), the term "microbiome" pertains to the collective microbial population residing in a specific environment with unique characteristics. It encompasses not only the microorganisms co-existing in that environment but also their interactions with each other. It is generally accepted that for the microbiome to be healthy it needs to be balanced. The concept of a "core microbiome" in oral microbial communities refers to the presence of similar bacterial communities in the mouths of healthy individuals who are not related to each other, regardless of their age or ethnicity. This means that there are certain types of bacteria that tend to be present in the oral cavity of healthy people, regardless of their individual differences. A healthy microbiome is a barrier to external pathogens and possible exterior aggression. It must be balanced. The balance between bacteria is different between each individual because it depends on multiple factors. However, the species found in individuals overlap (Zaura, et al, 2009). To maintain this balance, oral hygiene with products targeting the microbiome is recommended, meaning the use of products that do not upset the balance of the microbiome. Pre and probiotics can also be interesting ingredients in oral health products for a balanced microbiome (Nanavati et al, 2021). An unbalanced oral microbiome can allow the overgrowth of pathogenic bacteria and lead to an oral infection locally in the mouth, for example Filifactor alocis, Porphyromonas, Synergistetes, and Peptostreptococcaceae are genera known to be a causal agent for periodontal disease (Lovegrove, 2004). Amazingly, pathogenic bacteria in the mouth might contribute to systemic diseases, for example, an increase in Fusobacterium and other bacterial species has been found in oral cancer patients and diabetics, reinforcing the importance of a balanced oral microbiome (Matsha, et al, 2020). Ectopic colonization of oral bacteria in other tissues or organs such as the stomach, heart, brain, placenta or even tumors could influence these diseases through an inflammatory response. Some recent studies also point to the possible usefulness of the oral microbiome as a treatment for infections of different organs of the body. For example, it has been shown that the use of oral firmicutes spores could help treat C.difficile infections. (Feuerstadt, et al, 2022) Next steps for research The link between the oral microbiome and systemic health is still an evolving area of research, thanks to the emergence of sequencing and bioinformatics we are closer to understanding this link. Further research on the correlation between systemic diseases and the oral microbiome is needed in order to gather such consistent and reliable results required to fully understand the important role of oral cavity bacteria. To date, we have conducted numerous oral microbiome studies. If you are interested in carrying out any research with us and testing your oral care products, you can reach us at team@sequential.bio. Lexicon: Oral Microbiome: All genomes of microorganisms in the oral cavity (Deo, Deshmukh,2019) Systemic disease: Disease that impact the whole body C. difficile infections: Clostridium difficile, also called C. difficile, is a type of bacteria that can cause a bowel infection Fusobacterium: Bacteria normally present in the mouth, but can cause infections when  unbalanced Prebiotics: ingredients that promote the balance of the microbiome Probiotics: Microorganisms that have health benefits References: Deo, P.N. and Deshmukh, R. (2019) “Oral microbiome: Unveiling the fundamentals.,” Journal of Oral and Maxillofacial Pathology, 23(1), pp. 122–128. Available at: https://doi.org/10.4103/jomfp.jomfp_304_18. Feuerstadt, P. et al. (2022) “SER-109, an Oral Microbiome Therapy for Recurrent Clostridioides difficile Infection,” New England Journal of Medicine, 386(3), pp. 220–229. Available at: https://doi.org/10.1056/nejmoa2106516. Lovegrove JM. Dental plaque revisited: bacteria associated with periodontal disease. J N Z Soc Periodontol. 2004;(87):7-21. PMID: 15143484. Marsh, P.D. (2000) “Role of the Oral Microflora in Health,” Microbial Ecology in Health and Disease, 12(3), pp. 130–137. Available at: https://doi.org/10.1080/089106000750051800. Matsha, T.E. et al. (2020) “Oral Microbiome Signatures in Diabetes Mellitus and Periodontal Disease,” Journal of Dental Research, 99(6), pp. 658–665. Available at: https://doi.org/10.1177/0022034520913818. Nanavati, G., Prasanth, T., Kosala, M., Bhandari, S.K. and Banotra, P., 2021. Effect of probiotics and prebiotics on oral health. Dental Journal of Advance Studies, 9(01), pp.01-06. Zaura, E. et al. (2009) “Defining the healthy ‘core microbiome’ of oral microbial communities,” BMC Microbiology, 9(1), p. 259. Available at: https://doi.org/10.1186/1471-2180-9-259.

Abstract When it comes to the personal care industry for intimate female care products, there is still a lot to be explored and infinitely more that we do not know. For this reason, it is crucial to shed light on current and past research so that we can learn from it and make efforts to widen the literature to bring more effective formulations to the industry. The Intimate Care Industry The intimate care industry for female products is projected to reach USD $69,853 million by 2030 (Acumen Research and Consulting, 2022). For years there has been a stigma linked to women’s intimate care and a lack of willingness to discuss such topics. However, we are seeing a shift towards one that favours openness and education. Women’s intimate care products account for approximately 17% of the personal care market, with popularity rising for hygiene products the likes of intimate washes (FMI, 2019). As companies begin to formulate their own products for the needs of this niche of the market, the interesting question arises of what one should look for within these formulations. As we have seen in recent years, the skincare industry is seeing a drastic turn towards science-backed products with an increase in demand for microbiome-friendly formulas. However, what do we truly know about the vaginal and vulvar microbiome? Vaginal Microbiome The microbiome refers to the microbial population that occupies a habitat with distinct properties. It doesn’t just refer to the microorganisms that live together, but how they interact with one another (Berg et al., 2020). The vaginal microbiome consists of about 9% of the total human microbiome. Ordinarily, we find that the microbiome is at its healthiest when the population of bacteria is diverse. This is the case for the skin as well as the gut microbiome. However, research informs that the vaginal and vulvar microbiome act in the opposite way. When there is a high diversity in the vulvar and vaginal microbiome, it draws attention to an underlying problem that needs addressing (Saraf et al., 2021). In females of reproductive age, it is noted that the Lactobacillus species is a key characteristic of a healthy vaginal microbiome. These include: L. iners, L. crispatus, L. gasseri, and L. jensenii (Saraf et al., 2021). We also find that the same can be said about Bifidobacterium, which suggests protective characteristics as well (Freitas and Hill, 2017). If we look on the opposite side of the spectrum, we note that species such as Prevotella, Atopobium, Gardnerella, Megasphaera, and Mobiluncus are associated with an unhealthy or abnormal vaginal microbiota (Ravel et al., 2011). Diving Deeper into Vaginal Lactobacillus species Lactobacillus is characterized as a Gram-positive bacteria with a rod shape. These bacteria are known to produce lactic acid and make the vagina's pH more acidic (<4). According to current research, an acidic environment can restrict the growth of non-indigenous bacteria, which can foster a healthier environment. As a general rule, the lactobacillus species converts sugar into pyruvate which in turn converts it to lactic acid. They can make two types of lactic acid known as D-Lactic acid and L-Lactic acid (Saraf et al., 2021). Characterizing the Vaginal Microbiome In 2011 the concept of Community State Types (CSTs) was introduced with the aim of categorizing the vaginal microbiome communities (Ravel et al., 2011). This categorization was introduced after sampling women of asymptomatic ages through to reproductive women with 16S rRNA Sequencing. As a result, 5 CSTs were found, namely: I (L. crispatus): Type 1 is understood to be the healthiest, with research showing that this type can even prevent infections such as STIs, BV, and UTIs. II (L. gasseri): Type 2 is also linked to a healthy vaginal microbiome, with Lactobacillus gasseri dominating the population of bacteria. III (L. iners): Type 3 is neutral meaning it could be disruptive or protective to the vaginal community and is dominated by a species of Lactobacillus known as Lactobacillus iners. IV (Diversity group): Type 4 is understood to be an unhealthy vaginal environment with a high diversity of bacteria and low population of lactobacilli. V (L. jensenii): Type 5 is another state type acknowledged as healthy with a dominance of Lactobacillus jensenii. It is interesting to note that within this research females of different ethnic backgrounds fell under different community types. It is crucial to acknowledge that ethnicity plays a key role in the vaginal microbiome and further research is necessitated to understand how it varies from female to female (Ravel et al., 2011). This image has been pulled out from “Vaginal microbiome of reproductive-age women” (Ravel et al., 2011) In a paper published in 2020, it is disputed that CSTs needs to go a step further to help classify the vaginal microbiome. That’s where Valencia comes in (or VAginaL community state typE Nearest CentroId clAssifier). It is a centroid-based tool for measuring vaginal microbial communities based on composition (France et al, 2020).  The aim is to classify samples based purely on similarities against a reference that has been defined from 13,160 taxonomic profiles from 1,975 women within the United States. This large dataset allows for a more comprehensive process of identifying, characterizing and defining CSTs. Vaginal Microbiota Through the Female Lifespan Just as with any element of the human body, the vaginal microbiome alters throughout a female’s lifespan, particularly during the menstruating period and after menopause. This is due to the structural and hormonal features of the vaginal tissue (epithelium), which results in a changing environment. It is still not entirely clear what specific changes we can expect to see in each of these stages, as there are many factors at play, including lifestyle, environment and yes, even ethnicity. In order to uncover more about the vaginal and vulvar microbiome, it is crucial to conduct further studies and test products made for feminine intimate care with the correct methodologies and quantitative analysis to understand their true effect on the microbiome. Sequential is a testing company with years of expertise in the field of skin microbiome and genetics. We utilise deep molecular analysis and next-generation sequencing (NGS) technology to understand the impact on an individual’s microbiome from products they use, and the effect from their environment. All of our testing is carried out in-vivo and with the utmost care for unearthing the secrets that lie on the surface of the skin.  If you are interested in carrying out any research with us and testing products, you can reach us at team@sequential.bio. Lexicon Community State Types (CSTs): introduced with the aim of categorizing the vaginal microbiome communities (Ravel et al., 2011). Microbiome: The microbiome is a characteristic microbial community occupying a reasonably well-defined habitat which has distinct physio-chemical properties. The microbiome not only refers to the microorganisms involved but also encompasses their theater of activity, which results in the formation of specific ecological niches. This includes their genetic material, and also structural molecules, like enzymes, membrane lipids or polysaccharides. (Definition based on Berg et al., 2020) Skin microbiome: is present on the whole skin surface, including oral cavity and mucosal surfaces of the external genital organs. The composition of the skin microbiome is dynamic, site-specific but also differs from individual to individual. (Definition based on Byrd et al., 2018) Valencia: is a centroid-based tool for measuring vaginal microbial communities based on composition (France et al, 2020). References Acumen Research and Consulting. (2021). Feminine hygiene products market size worth around $38.5 billion by 2028. Acumen Research and Consulting. https://www.acumenresearchandconsulting.com/feminine-hygiene-products-market#:~:text=The%20Global%20Feminine%20Hygiene%20Products,have%20grown%20over%20the%20years. France MT, Ma B, Gajer P, Brown S, Humphrys MS, Holm JB, Waetjen LE, Brotman RM, Ravel J. VALENCIA: a nearest centroid classification method for vaginal microbial communities based on composition. Microbiome. 2020 Nov 23;8(1):166. doi: 10.1186/s40168-020-00934-6. PMID: 33228810; PMCID: PMC7684964. Freitas, A. C., and Hill, J. E. (2017). Quantification, isolation and characterization of Bifidobacterium from the vaginal microbiomes of reproductive aged women. Anaerobe 47, 145–156. doi: 10.1016/j.anaerobe.2017.05.012 Ravel, J., Gajer, P., Abdo, Z., Schneider, G. M., Koenig, S. S. K., McCulle, S. L., … Forney, L. J. (2010). Vaginal microbiome of reproductive-age women. Proceedings of the National Academy of Sciences, 108(Supplement_1), 4680–4687. doi:10.1073/pnas.1002611107 Future Market Insights. (2019). Women intimate care market: Global industry analysis 2013-2017 and opportunity assessment 2018-2028. Future Market Insights. https://www.futuremarketinsights.com/reports/women-intimate-care-market De Seta, F., Campisciano, G., Zanotta, N., Ricci, G., & Comar, M. (2019). The Vaginal Community State Types Microbiome-Immune Network as Key Factor for Bacterial Vaginosis and Aerobic Vaginitis. Frontiers in Microbiology, 10. doi:10.3389/fmicb.2019.02451 Saraf, V. S., Sheikh, S. A., Ahmad, A., Gillevet, P. M., Bokhari, H., & Javed, S. (2021). Vaginal microbiome: normalcy vs dysbiosis. Archives of Microbiology, 203(7), 3793–3802. doi:10.1007/s00203-021-02414-3

Abstract: Understanding the Baby Microbiome How important is it to understand the baby's microbiome? This post will explore what we know so far about the baby microbiome, and concludes by highlighting that in order to keep the very sensitive skin of infants as healthy as possible finding out more about the composition of the baby microbiome is crucial. What Do We Know About the Baby Microbiome The skin is the human body’s largest organ. The skin microbiome is made up of an organic ecosystem of trillions of bacteria that sit on the surface of the skin. It acts as a barrier against the threats and infections posed by the outside world, and works in a team to keep your skin healthy by fighting infection, supporting the immune system, healing wounds and controlling inflammation. Therefore, the microbiome needs to remain dynamic and responsive to the changing environments throughout the human life cycle. This is also true for the baby microbiome, and an infant’s exposure to environmentally sourced microbes may vary across different settings. It is interesting to consider how differences in childcare practices across sociocultural contexts could play a part in differential microbial exposures, for example, those linked to hygiene practices, attending daycare, and physical contact with siblings (Manus et al, 2020). At Birth Fetal skin is colonized by surrounding microorganisms immediately after birth. There are a variety of factors at birth that can influence the skin microbiome. Firstly is the mode of delivery. In vaginally delivered newborns, the skin’s bacterial signature resembles the mother’s vaginal bacteria. Whereas newborns delivered by cesarean resemble the bacteria relating to the skin. Moreover, newborns delivered by cesarean were found to have reduced skin microbial diversity (Dominguez-Bello et al, 2010). A second factor is whether the newborn is carried to full term. Where a newborn is born prematurely, the overall variety of species of skin bacteria and the relative abundance of the community are likely to be lower than babies born full-term (Pammi et al, 2017). However, it is important to note that the regional differences between preterm and full-term infants disappear after the first month of life. Maturation of the Infant Microbiome Within the first 6 weeks of life, the significant re-organization of the infant microbiota is primarily driven by body site, rather than by the mode of delivery (Chu et al, 2017). More significant differences have been discovered between the infant and maternal stool. Amazingly, it has been found that the baby microbiota becomes very similar to that of the adult by just the fourth week of life (Gaitanis et al, 2019). The Benefits of Understanding the Baby Microbiome Baby’s skin is known to be especially sensitive, highly prone to inflammatory conditions like eczema and dermatitis, and susceptible to infections such as candidiasis. The key benefit of understanding the composition of the baby microbiome is that strategies, for example, specific prebiotics and probiotics targeting the skin, can be developed to prevent the excessive growth of opportunistic pathogens and help us to ensure the baby's skin is kept healthy. Sequential Bio is a testing company with years of expertise in the field of skin microbiome and genetics. We utilise deep molecular analysis and next-generation sequencing (NGS) technology to understand the impact on an individual’s microbiome from products they use, and the effect from their environment. Having previously carried out skin microbiome testing on infants down to as little as 4 months, we have the knowledge and technology to help companies better understand the infant microbiota as they go about formulating their targeted products. All of our testing is carried out in-vivo and with the utmost care for unearthing the secrets that lie on the surface of the skin.  If you are interested in carrying out any research with us and testing products, you can reach us at team@sequential.bio. Lexicon Microbiome: The microbiome is a characteristic microbial community occupying a reasonably well-defined habitat which has distinct physio-chemical properties. The microbiome not only refers to the microorganisms involved but also encompasses their theatre of activity, which results in the formation of specific ecological niches. This includes their genetic material, and also structural molecules, like enzymes, membrane lipids or polysaccharides. (Definition based on Berg et al., 2020) Skin microbiome: is present on the whole skin surface, including the oral cavity and mucosal surfaces of the external genital organs. The composition of the skin microbiome is dynamic, and site-specific but also differs from individual to individual. (Definition based on Byrd et al., 2018) Probiotics: Live microorganisms that are intended to have health benefits when consumed or applied to the body. They can be found in yoghurt and other fermented foods, dietary supplements, and beauty products. Prebiotics: Non-living ingredients that are used to support the balance of both good and bad bacteria on your skin throughout your skin and within your body. References Chu DM, Ma J, Prince AL, Antony KM, Seferovic MD, Aagaard KM. Maturation of the infant microbiome community structure and function across multiple body sites and in relation to mode of delivery. Nat Med. 2017 Mar;23(3):314-326. doi: 10.1038/nm.4272. Epub 2017 Jan 23. PMID: 28112736; PMCID: PMC5345907. Capone KA, Dowd SE, Stamatas GN, Nikolovski J. Diversity of the human skin microbiome early in life. J Invest Dermatol. 2011 Oct;131(10):2026-32. doi: 10.1038/jid.2011.168. Epub 2011 Jun 23. PMID: 21697884; PMCID: PMC3182836. Dominguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, Fierer N, et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci USA. 2010;107(26):11971–11975. doi: 10.1073/pnas.1002601107. Gaitanis G, Tsiouri G, Spyridonos P, Stefos T, Stamatas GN, Velegraki A, et al. Variation of cultured skin microbiota in mothers and their infants during the first year postpartum. Pediatr Dermatol. 2019;36(4):460–465. Manus MB, Kuthyar S, Perroni-Marañón AG, Núñez-de la Mora A, Amato KR. Infant Skin Bacterial Communities Vary by Skin Site and Infant Age across Populations in Mexico and the United States. mSystems. 2020 Nov 3;5(6):e00834-20. doi: 10.1128/mSystems.00834-20. PMID: 33144313; PMCID: PMC7646528. Pammi M, O'Brien JL, Ajami NJ, Wong MC, Versalovic J, Petrosino JF. Development of the cutaneous microbiome in the preterm infant: a prospective longitudinal study. PLoS One. 2017;12(4):e0176669. doi: 10.1371/journal.pone.0176669.

Abstract Topical Steroid Withdrawal (TSW) can affect the quality of life of individuals, and the fact that there is currently no cure means that this is a particularly important area of research to consider. The role of the microbiome in TSW is not yet fully understood. Although some studies have begun looking at different types of therapy as a possible new treatment for TSW, this article explores the condition and where we are we research. What is Topical Steroid Withdrawal? Topical Steroid Withdrawal (TSW) describes the appearance of symptoms occurring after putting an end to long-term topical steroid use. TSW can happen after immediate use of treatment, but can also happen after misuse of treatment, decreasing the strength of treatment, and applying the treatment less frequently or to fewer parts of the body. Side effects are typically placed into two different categories; local and systemic. Local side effects describe symptoms which generally occur with prolonged treatment, and are conditional on the strength of the topical steroid, its vehicle and area of application. Most common examples of local side effects include skin atrophy, rosacea, perioral dermatitis, acne, and purpura. The other category of side effects, systemic adverse effects, are seen when very strong topical steroids are used for prolonged periods on the skin. What is important to highlight about TSW is that there is not currently any medication that provides a cure, patients can only attempt to manage the symptom. Treatments aimed at controlling and suppressing symptoms usually include cool compresses on the skin, antibiotics for inflammation, and over-the-counter antihistamine treatment to reduce redness and itching. Topical Steroid Withdrawal and the Skin Microbiome The skin microbiome is made up of an organic ecosystem of trillions of bacteria that sit on the surface of the skin. It acts as a first-line of defense against the outside world, and works in a team to keep your skin healthy by fighting infection, supporting the immune system, healing wounds and controlling inflammation. In order for the skin microbiome to work together efficiently with the human host, it needs to be balanced with a diversity of bacteria populating the skin. As the skin microbiome performs such a crucial function in keeping the skin healthy, understanding the role of the skin microbiome within the context of TSW may be essential to making progress in controlling the associated symptoms. Next Steps For Research The role of the microbiome in TSW is not fully explained. Considering little to know research exists on the impact of TSW on the skin microbiome, it is clear that further research should be conducted with a sufficient sample size to evaluate if a pre-/pro-/post-biotic treatment could improve the symptoms of TSW. Moreover, in order to find out whether the changes in symptoms are linked to the alterations in the microbiome community and changes in diversity, further work is required. Sequential is a testing company with years of expertise in the field of skin microbiome and genetics. We utilise deep molecular analysis and next-generation sequencing (NGS) technology to understand the impact on an individual’s microbiome from products they use, and the effect from their environment. All of our testing is carried out in-vivo and with the utmost care for unearthing the secrets that lie on the surface of the skin. If you are interested in carrying out any research with us and testing products, you can reach us at team@sequential.bio. Lexicon Microbiome: The microbiome is a characteristic microbial community occupying a reasonably well-defined habitat which has distinct physio-chemical properties. The microbiome not only refers to the microorganisms involved but also encompasses their theatre of activity, which results in the formation of specific ecological niches. This includes their genetic material, and also structural molecules, like enzymes, membrane lipids or polysaccharides. (Definition based on Berg et al., 2020) Skin microbiome: is present on the whole skin surface, including oral cavity and mucosal surfaces of the external genital organs. The composition of the skin microbiome is dynamic, site-specific but also differs from individual to individual. (Definition based on Byrd et al., 2018) Probiotics: Viable (active or dormant) microorganisms added to a cosmetic product with an intended cosmetic benefit to the host at the application site, either directly or via an effect on the host microbiome, when utilized in adequate amounts. Reference List Moreno-Indias, I. et al. Neonatal Androgen Exposure Causes Persistent Gut Microbiota Dysbiosis Related to Metabolic Disease in Adult Female Rats. Endocrinology 157, 4888-4898, doi:10.1210/en.2016-1317 (2016). Yurkovetskiy, L. et al. Gender bias in autoimmunity is influenced by microbiota. Immunity 39, 400-412, doi:10.1016/j.immuni.2013.08.013 (2013)

Abstract Retinoids refer to a variety of topical vitamin A-based products used on the skin, they are used to treat mild acne and as a preventative measure to reduce signs of ageing. Despite widespread usage and prescription by doctors, the full effects on the skin microbiome are unclear. Since the skin microbiome has an essential function in the health of the skin, understanding this further is critical. In this article, we will explore the evidence defining the relationship between retinoids and the skin microbiome. In summary, we find that studies suggest that Vitamin A and its metabolite may aid the self-regulatory process of the skin microbiome. However, we also need to be aware of the limitations of these studies, and consequently the need to conduct further studies in order to explore the questions which are still left unanswered. What are Retinoids? “Retinoids” is an umbrella term referring to a variety of vitamin A-based products used on the skin. Retinoids are available over-the-counter in weaker strengths in the form of creams, gels and serums to be applied directly on the skin, and are generally used to treat mild acne and used as a preventative measure to reduce signs of ageing. Prescription-strength treatments are also available, usually to treat moderate to chronic skin conditions. How are Retinoids Used? A lack of Vitamin A is associated with increased susceptibility to skin infection and inflammation (Chen et al, 2019), particularly as a result of an increase in the population of bacteria such as Staphylococcus aureus which become dominant within the skin (Wiedermann et al, 1996). Therefore, retinoids could have the potential to be beneficial for skin health by ensuring the skin barrier and microbiome are in check. Retinoids work by increasing blood flow and boosting skin cell turnover, therefore accelerating the cell renewal process and preventing dead skin cells from clogging pores, which may ultimately help the skin to repair itself quicker and reduce inflammation. However, retinoids are also known to potentially cause an initial ‘skin purging’ period (Leyden et al, 2017), where irritation may occur within the first few weeks but then later subsides, which can be an alarming and uncomfortable experience. Retinoids are well known for their benefits in reducing the signs of ageing by targeting fine lines, wrinkles, and pigmentation. Retinoids are advised to be used by individuals in their mid-twenties as a preventative measure, aimed at slowing down the natural ageing process by increasing the production of collagen and stimulating the production of new blood vessels in the skin. Yet it is important to highlight that retinoids are not typically considered a “quick fix” solution, as optimal results generally require a certain level of consistency and patience. In general, improvements are expected to begin to become visible within 3-6 months of regular use, while the best results are expected to take closer to 12 months. Skin Microbiome and Retinoids The skin microbiome is made up of an organic ecosystem of trillions of bacteria that sit on the surface of the skin. It acts as the first line of defence against the outside world and works in a team to keep your skin healthy by fighting infection, supporting the immune system, healing wounds and controlling inflammation. In order for the skin microbiome to be at its healthiest, it needs to be balanced with a high diversity of bacteria populating the skin. Retinoids are generally acknowledged to be quite harsh treatments, and so it is necessary that the potential effects their use may have on the skin microbiome are explored to ensure overall skin health is not compromised. Current Studies Linked to Retinoids Studies have suggested that Vitamin A and its metabolite can maintain the homeostasis of the skin microbiome, by regulating the innate immune system in a number of ways (Silvestre, Sato, & Reis, 2018). The key elements of the innate immune system in the skin are Toll-like receptors (TLRs), these TLRs act as a guard by recognising and fighting off pathogenic bacteria. In the skin, Vitamin A deficiency means that certain TLRs (specifically TLR2 and TLR3) are unable to function normally and cannot recognise any potentially bad bacteria growing on the pores or hair follicles. Additionally, sometimes TLRs do not activate until there is a certain number of pathogens reached (Roche & Harris-Tryon, 2021). Studies have suggested this could indicate a potential link between Vitamin A deficiency and skin conditions such as psoriasis and atopic dermatitis (Chen et al,2019). Topical retinoids can activate these TLRs so that they are able to function effectively, and therefore the use of retinoids may have therapeutic benefits in skin and hair regeneration (Kim et al, 2019). Next Steps for Research While we know that retinoids alter the skin microbiome and the potential benefits are clear, current research does not explore the specific strains of microbes that derive benefits from retinoid use and the impact this has on microbial diversity. In order to have a clearer and fuller understanding of the effects of retinoids on the skin microbiome, more focused studies are clearly necessary. Sequential is a testing company with years of expertise in the field of skin microbiome and genetics. We utilise deep molecular analysis and next-generation sequencing (NGS) technology to understand the impact on an individual’s microbiome from products they use, and the effect from their environment. All of our testing is carried out in-vivo and with the utmost care for unearthing the secrets that lie on the surface of the skin. If you are interested in carrying out any research with us and testing products, you can reach us at team@sequential.bio. Lexicon Skin Microbiome: refers to the collection of genomes from all the microorganisms in the environment (on your skin). Retinoids: a catch-all for an array of vitamin A-based products used on the skin, used to treat mild acne and reduce fine lines and wrinkles. Bacteria: bacteria are single-cell organisms that live everywhere on earth, including on the surface of the skin. Homeostasis: the self-regulating process by which biological systems maintain stability while adjusting to changing external conditions. Staphylococcus aureus (S.aureus): S. aureus is a key bacterium, typically considered to be harmful because it can cause skin infection as well as inflammation in the outer skin barrier. Reference List Chen, W., Zhao, S., Zhu, W., Wu, L. & Chen, X. Retinoids as an Immunity-modulator in Dermatology Disorders. Arch Immunol Ther Exp (Warsz) 67, 355-365, doi:10.1007/s00005-019-00562-5 (2019). Eichner, R. Epidermal effects of retinoids: in vitro studies. J Am Acad Dermatol 15, 789-797, doi:10.1016/s0190-9622(86)70235-1 (1986). Harris, T. A. et al. Resistin-like Molecule alpha Provides Vitamin-A-Dependent Antimicrobial Protection in the Skin. Cell Host Microbe 25, 777-788 e778, doi:10.1016/j.chom.2019.04.004 (2019). Idres, N., Marill, J., Flexor, M. A. & Chabot, G. G. Activation of retinoic acid receptor-dependent transcription by all-trans-retinoic acid metabolites and isomers. J Biol Chem 277, 31491-31498, doi:10.1074/jbc.M205016200 (2002). Kim, D. et al. Noncoding dsRNA induces retinoic acid synthesis to stimulate hair follicle regeneration via TLR3. Nat Commun 10, 2811, doi:10.1038/s41467-019-10811-y (2019). Leyden, J., Stein-Gold, L. and Weiss, J. Why topical retinoids are mainstay of therapy for acne. Dermatology and therapy, 7(3). 296-297, doi:https://doi.org/10.1007/s13555-017-0185-2 (2017). Leyden J, Stein-Gold L, Weiss J. Why Topical Retinoids Are Mainstay of Therapy for Acne. Dermatol Ther (Heidelb). 2017 Sep;7(3):293-304. doi: 10.1007/s13555-017-0185-2. Epub 2017 Jun 5. PMID: 28585191; PMCID: PMC5574737. Piipponen, M., Li, D. & Landen, N. X. The Immune Functions of Keratinocytes in Skin Wound Healing. Int J Mol Sci 21, doi:10.3390/ijms21228790 (2020). Rittie, L., Varani, J., Kang, S., Voorhees, J. J. & Fisher, G. J. Retinoid-induced epidermal hyperplasia is mediated by epidermal growth factor receptor activation via specific induction of its ligands heparin-binding EGF and amphiregulin in human skin in vivo. J Invest Dermatol 126, 732-739, doi:10.1038/sj.jid.5700202 (2006). Roche FC, Harris-Tryon TA. Illuminating the Role of Vitamin A in Skin Innate Immunity and the Skin Microbiome: A Narrative Review. Nutrients. 2021 Jan 21;13(2):302. doi: 10.3390/nu13020302. PMID: 33494277; PMCID: PMC7909803. Silvestre, M. C., Sato, M. N. & Reis, V. Innate immunity and effector and regulatory mechanisms involved in allergic contact dermatitis. An Bras Dermatol 93, 242-250, doi:10.1590/abd1806-4841.20186340 (2018). Schroeder, M. & Zouboulis, C. C. All-trans-retinoic acid and 13-cis-retinoic acid: pharmacokinetics and biological activity in different cell culture models of human keratinocytes. Horm Metab Res 39, 136-140, doi:10.1055/s-2007-961813 (2007). Wiedermann, U. et al. Vitamin A deficiency predisposes to Staphylococcus aureus infection. Infect Immun 64, 209-214, doi:10.1128/iai.64.1.209-214.1996 (1996).

Abstract Benzoyl peroxide (BPO) is well known to be used for people that have acne. Although the effect of this drug in reducing acne is clear, the effect on the skin microbiome is less known which may have a role to play in the long-term effects and symptoms of BPO usage. In this post, we will discuss what is known, and how the skin microbiome might be affected. What is Benzoyl Peroxide and How Is It Used? Benzoyl Peroxide (BPO) is a drug that is commonly used in the treatment of mild to moderate acne. It is often formulated with active ingredients, antibiotics, or retinoids and infused into topical skincare products such as gels, face washes, and spot treatments. Since BPO can be quite strong, each formulation will typically contain a maximum of 5% of the drug. BPO products are generally recommended in controlled amounts, therefore are most often sold as spot creams to be used only in targeted areas. Individuals are advised to gradually increase the number of applications up to a maximum of twice in one day. Common Side Effects of Benzoyl Peroxide The reason that BPO should be applied with caution and control is due to common side effects that include dry skin, peeling skin, and skin irritation. These are reported to occur in just over 10% of individuals, which can be alarming, especially for those who have sensitive skin. Excessive use of BPO has been reported to cause the skin to become very dry and flaky. In addition, the prolonged use of BPO products can also cause the skin to become over-dependant, which consequently risks the return or worsening of the acne when the treatment is stopped. What does this all mean in the context of the skin microbiome? Skin Microbiome and Benzoyl Peroxide The skin microbiome is made up of an organic ecosystem of trillions of bacteria that sit on the surface of the skin and acts as a first-line defence against external factors. For the skin microbiome to be at its healthiest, it needs to be balanced with a high diversity of bacteria populating the skin. Now when looking at it through the context of BPO, we are left with quite an interesting situation. BPO claims to fight acne and potentially prevent new breakouts by working as an antiseptic to attack and reduce the bacteria on the surface of the skin. It also removes dead skin in order to unclog and minimize the appearance of pores. As far as attacking and reducing bacteria is concerned, scientists are left with a crucial question to answer. How does BPO affect the skin microbiome and the diversity of bacteria on the skin? Current Studies Linked to Benzoyl Peroxide Research on this subject is mixed, with some academic papers reporting that BPO may have a link to reduced microbial diversity (as well as acne), however, other research fails to show any statistical change. A recent study published (Zhou et al, 2022) compared skin microbiome changes in people who suffer from acne before and after the use of BPO, but found that among the top 20 bacteria species two of them (Staphylococcus, Acinetobacter) increase in abundance whilst one decreased Corynebacterium. Another research study (Ahluwalia et al., 2019) found that although there weren’t statistically significant changes in the diversity of the skin microbiome, the use of BPO resulted in a statistically significant increase in Streptococcus mitis (S.mitis) population. Taking a different look at things, it was found that after the use of BPO S.epidermidis increased, which could potentially inhibit the growth of harmful bacteria such as Cutibacterium acnes (C.acnes), certain strains of which are a known culprit in individuals with acne (Wang et al., 2014). However, the argument arose that even if Staphylococcus epidermidis (S. epidermidis) rose to help stave off C.acnes, could the benefits outweigh the ill effects of using BPO in general? Next Steps for Research To be able to answer questions like this in more depth, there is a strong need to conduct further research in a controlled environment, with the aim of decoding the impacts of BPO on the skin microbiome and the diversity of not just the most common bacteria, but also the obscure ones that one might not think to be significant. Other questions might be, does the skin microbiome significantly change if the use of BPO becomes chronic? And does this have lasting effects and long-term ill effects on the skin? Sequential is a testing company with years of expertise in the field of skin microbiome and genetics. We utilise deep molecular analysis and next-generation sequencing (NGS) technology to understand the impact on an individual’s microbiome from products they use, and the effect from their environment. All of our testing is carried out in-vivo and with the utmost care for unearthing the secrets that lie on the surface of the skin. If you are interested in carrying out any research with us and testing products, you can reach us at team@sequential.bio. —-------------------------------------------------------------------------------------------------------------- Lexicon Skin Microbiome: refers to the collection of genomes from all the microorganisms in the environment (on your skin). Skin Microbiota: refers to microorganisms that are found within a specific environment. Microbiota can refer to all the microorganisms found in an environment, including bacteria, viruses, and fungi. Bacteria: bacteria are single-cell organisms that live everywhere on earth, including on the surface of the skin. Benzoyl Peroxide: Benzoyl Peroxide (BPO) is a drug that is commonly used in the treatment of mild to moderate acne. Streptococcus mitis (S.mitis): S.mitis is a gram-positive bacteria found mainly in the mouth but low in the skin and is usually opportunistic/pathogenic in Adults. In fact, several anti-bacterial lipids have been tested against this bacteria. On the other hand, the pre-adolescent microbiome might have more S.mitis and very little C.acne because sebaceous glands have not developed fully yet. Only after puberty do we start to see C.acne increasing and other bacteria such as S.mitis decreasing. Cutibacterium acnes (C.acnes): is a highly prevalent bacterium that inhabits pores where sebum/natural oils are formed. it is linked to skin conditions such as acne, but only with some strains. Staphylococcus epidermidis (S.epidermidis): is one of more than forty species of bacteria that belongs to the Staphylococcus family and is part of the organisms that normally inhabit humans, specifically the skin. Reference List Ahluwalia, J., Borok, J., Haddock, E. S., Ahluwalia, R. S., Schwartz, E. W., Hosseini, D., Amini, S., & Eichenfield, L. F. (2019, Mar). The microbiome in preadolescent acne: Assessment and prospective analysis of the influence of benzoyl peroxide. Pediatr Dermatol, 36(2), 200-206. https://doi.org/10.1111/pde.13741 Coughlin, C. C., Swink, S. M., Horwinski, J., Sfyroera, G., Bugayev, J., Grice, E. A., & Yan, A. C. (2017, Nov). The preadolescent acne microbiome: A prospective, randomized, pilot study investigating characterization and effects of acne therapy. Pediatr Dermatol, 34(6), 661-664. https://doi.org/10.1111/pde.13261 Karoglan et al, 2019 https://pubmed.ncbi.nlm.nih.gov/31573666/ Oh, J., Conlan, S., Polley, E. C., Segre, J. A., & Kong, H. H. (2012). Shifts in human skin and nares microbiota of healthy children and adults. Genome Med, 4(10), 77. https://doi.org/10.1186/gm378 Wang, Y., Kuo, S., Shu, M., Yu, J., Huang, S., Dai, A., Two, A., Gallo, R. L., & Huang, C. M. (2014, Jan). Staphylococcus epidermidis in the human skin microbiome mediates fermentation to inhibit the growth of Propionibacterium acnes: implications of probiotics in acne vulgaris. Appl Microbiol Biotechnol, 98(1), 411-424. https://doi.org/10.1007/s00253-013-5394-8 Zhou, L., Chen, L., Liu, X., Huang, Y., Xu, Y., Xiong, X., & Deng, Y. (2022, Mar). The influence of benzoyl peroxide on skin microbiota and the epidermal barrier for acne vulgaris. Dermatol Ther, 35(3), e15288. https://doi.org/10.1111/dth.15288