scholarly journals Comparison of three amplicon sequencing approaches to determine staphylococcal populations on human skin

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Charlotte Marie Ahle ◽  
Kristian Stødkilde-Jørgensen ◽  
Anja Poehlein ◽  
Wolfgang R. Streit ◽  
Jennifer Hüpeden ◽  
...  
Keyword(s):  
Human Skin ◽  
Skin Diseases ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Skin Microbiome ◽  
Staphylococcal Species ◽  
Culture Independent ◽  
Skin Samples ◽  
Mock Communities

Abstract Background Staphylococci are important members of the human skin microbiome. Many staphylococcal species and strains are commensals of the healthy skin microbiota, while few play essential roles in skin diseases such as atopic dermatitis. To study the involvement of staphylococci in health and disease, it is essential to determine staphylococcal populations in skin samples beyond the genus and species level. Culture-independent approaches such as amplicon next-generation sequencing (NGS) are time- and cost-effective options. However, their suitability depends on the power of resolution. Results Here we compare three amplicon NGS schemes that rely on different targets within the genes tuf and rpsK, designated tuf1, tuf2 and rpsK schemes. The schemes were tested on mock communities and on human skin samples. To obtain skin samples and build mock communities, skin swab samples of healthy volunteers were taken. In total, 254 staphylococcal strains were isolated and identified to the species level by MALDI-TOF mass spectrometry. A subset of ten strains belonging to different staphylococcal species were genome-sequenced. Two mock communities with nine and eighteen strains, respectively, as well as eight randomly selected skin samples were analysed with the three amplicon NGS methods. Our results imply that all three methods are suitable for species-level determination of staphylococcal populations. However, the novel tuf2-NGS scheme was superior in resolution power. It unambiguously allowed identification of Staphylococcus saccharolyticus and distinguish phylogenetically distinct clusters of Staphylococcus epidermidis. Conclusions Powerful amplicon NGS approaches for the detection and relative quantification of staphylococci in human samples exist that can resolve populations to the species and, to some extent, to the subspecies level. Our study highlights strengths, weaknesses and pitfalls of three currently available amplicon NGS approaches to determine staphylococcal populations. Applied to the analysis of healthy and diseased skin, these approaches can be useful to attribute host-beneficial and -detrimental roles to skin-resident staphylococcal species and subspecies.

scholarly journals Staphylococcus saccharolyticus: An Overlooked Human Skin Colonizer

2020 ◽  
Vol 8 (8) ◽  
pp. 1105
Author(s):  
Charlotte M. Ahle ◽  
Kristian Stødkilde ◽  
Mastaneh Afshar ◽  
Anja Poehlein ◽  
Lesley A. Ogilvie ◽  
...  
Keyword(s):  
Human Skin ◽  
Staphylococcus Epidermidis ◽  
Amplicon Sequencing ◽  
Growth Conditions ◽  
Skin Microbiota ◽  
Healthy Human ◽  
Staphylococcal Species ◽  
Staphylococcus Capitis ◽  
Culture Independent ◽  
Slow Growing

Coagulase-negative staphylococcal species constitute an important part of the human skin microbiota. In particular, facultative anaerobic species such as Staphylococcus epidermidis and Staphylococcus capitis can be found on the skin of virtually every human being. Here, we applied a culture-independent amplicon sequencing approach to identify staphylococcal species on the skin of healthy human individuals. While S. epidermidis and S. capitis were found as primary residents of back skin, surprisingly, the third most abundant member was Staphylococcus saccharolyticus, a relatively unstudied species. A search of skin metagenomic datasets detected sequences identical to the genome of S. saccharolyticus in diverse skin sites, including the back, forehead, and elbow pit. Although described as a slow-growing anaerobic species, a re-evaluation of its growth behavior showed that S. saccharolyticus can grow under oxic conditions, and, in particular, in a CO2-rich atmosphere. We argue here that S. saccharolyticus was largely overlooked in previous culture-dependent and -independent studies, due to its requirement for fastidious growth conditions and the lack of reference genome sequences, respectively. Future studies are needed to unravel the microbiology and host-interacting properties of S. saccharolyticus and its role as a prevalent skin colonizer.

scholarly journals Finally, Bulk Typing of Bacterial Species down to Strain Level using ON-rep-seq

2018 ◽  
Author(s):  
Łukasz Krych ◽  
Josué L. Castro-Mejía ◽  
Daniel N. Moesby ◽  
Morten B. Mikkelsen ◽  
Morten A. Rasmussen ◽  
...  
Keyword(s):  
High Speed ◽  
Bacterial Species ◽  
Cost Effective ◽  
Species Level ◽  
Strain Level ◽  
Read Length ◽  
High Quality ◽  
Sequencing Platform ◽  
Culture Independent ◽  
Single Flow

AbstractDespite the massive developments within culture-independent methods for detection and quantification of microorganisms during the last decade, culture-based methods remain a cornerstone in microbiology. We have developed a new method for bacterial DNA enrichment and tagmentation allowing fast (< 24h) and cost-effective species level identification and strain level differentiation using the MinION portable sequencing platform (ON-rep-seq). DNA library preparation takes less than 5h and ensures highly reproducible distribution of reads that can be used to generate strain level specific read length counts profiles (LCp). We have developed a pipeline that by correcting the random error of reads within peaks of LCp generates a set (∼10 contigs per sample; 300bp - 3Kb) of high quality (>99%) consensus reads. Whereas, the information from high quality reads is used to retrieve species level taxonomy, comparison of LCp allows for strain level differentiation. With benchmarked 288 isolates identified on a single flow cell and a theoretical throughput to evaluate over 1000 isolates, our method allows for detailed bacterial identification for less than 2$ per sample at very high speed.

scholarly journals Microbiome of human skin - its immunohomeostatic role and the role in pathogenesis of skin diseases

2018 ◽  
Vol 15 (1) ◽  
pp. 63-81
Author(s):  
D D Petrunin
Keyword(s):  
Human Skin ◽  
Skin Diseases ◽  
Secondary Infection ◽  
Treatment Options ◽  
Infectious Complications ◽  
Special Importance ◽  
Skin Microbiome ◽  
Inflammatory Dermatoses ◽  
Pathogenic Microbes

In the last decade new methods of metagenomic analysis allowed to obtain important data regarding the microbiome of human skin. The problem of colonization and secondary infection by pathogenic microbes is of special importance for allergic dermatoses that require topical immunosuppressive therapy. One of treatment options in this case could be topical multicomponent drugs that allow successful treatment of infectious complications of inflammatory dermatoses. But there are still a lot of blanks regarding both fundamental questions regarding human skin microbiome and practice aspects of treatment of skin diseases where it plays a pathogenetic role. This literature review systematizes and structures the accumulated data regarding the composition and the role of human skin microbiome in normal conditions and in various skin diseases as well as summarizes clinical data of use of combinational topical glucocorticosteroid drugs. Furthermore, some algorithms concerning the choice and optimization of topical treatment of secondary infected dermatoses are outlined.

scholarly journals Evaluation of CRISPR Diversity in the Human Skin Microbiome for Personal Identification

mSystems ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Kochi Toyomane ◽  
Ryo Yokota ◽  
Ken Watanabe ◽  
Tomoko Akutsu ◽  
Ai Asahi ◽  
...  
Keyword(s):  
16S Rrna ◽  
Human Skin ◽  
Amplicon Sequencing ◽  
Personal Identification ◽  
16S Rrna Sequencing ◽  
Metagenomic Data ◽  
Skin Microbiome ◽  
Data Set ◽  
Microbiome Diversity ◽  
Rrna Sequencing

ABSTRACT The highly personalized human skin microbiome may serve as a viable marker in personal identification. Amplicon sequencing resolution using 16S rRNA cannot identify bacterial communities sufficiently to discriminate between individuals. Thus, novel higher-resolution genetic markers are required for forensic purposes. The clustered regularly interspaced short palindromic repeats (CRISPRs) are prokaryotic genetic elements that can provide a history of infections encountered by the bacteria. The sequencing of CRISPR spacers may provide phylogenetic information with higher resolution than other markers. However, using spacer sequencing for discrimination of personal skin microbiome is difficult due to limited information on CRISPRs in human skin microbiomes. It remains unclear whether personal microbiome discrimination can be achieved using spacer diversity or which CRISPRs will be forensically relevant. We identified common CRISPRs in the human skin microbiome via metagenomic reconstruction and used amplicon sequencing for deep sequencing of spacers. We successfully reconstructed 24 putative CRISPR arrays using metagenomic data sets. A total of 1,223,462 reads from three CRISPR arrays revealed that spacers in the skin microbiome were highly personalized, and conserved repeats were commonly shared between individuals. These individual specificities observed using CRISPR typing were confirmed by comparing the CRISPR diversity to microbiome diversity assessed using 16S rRNA amplicon sequencing. CRISPR typing achieved 95.2% accuracy in personal classification, whereas 16S rRNA sequencing only achieved 52.6%. These results suggest that sequencing CRISPRs in the skin microbiome may be a more powerful approach for personal identification and ecological studies compared to conventional 16S rRNA sequencing. IMPORTANCE Microbial community diversity analysis can be utilized to characterize the personal microbiome that varies between individuals. CRISPR sequences, which reflect virome structure, in the human skin environment may be highly personalized similar to the structures of individual viromes. In this study, we identified 24 putative CRISPR arrays using a shotgun metagenome data set of the human skin microbiome. The findings of this study expand our understanding of the nature of CRISPRs by identifying novel CRISPR candidates. We developed a method to efficiently determine the diversity of three CRISPR arrays. Our analysis revealed that the CRISPR spacer diversity in the human skin microbiome is highly personalized compared with the microbiome diversity assessed by 16S rRNA sequencing, providing a new perspective on the study of the skin microbiome.

scholarly journals Spermidine-induced recovery of human dermal structure and barrier function by skin microbiome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Gihyeon Kim ◽  
Misun Kim ◽  
Minji Kim ◽  
Changho Park ◽  
Youngmin Yoon ◽  
...  
Keyword(s):  
Human Skin ◽  
Barrier Function ◽  
Skin Diseases ◽  
Lipid Synthesis ◽  
Skin Barrier ◽  
Streptococcus Thermophilus ◽  
Skin Barrier Function ◽  
Skin Microbiome ◽  
Skin Structure

AbstractAn unbalanced microbial ecosystem on the human skin is closely related to skin diseases and has been associated with inflammation and immune responses. However, little is known about the role of the skin microbiome on skin aging. Here, we report that the Streptococcus species improved the skin structure and barrier function, thereby contributing to anti-aging. Metagenomic analyses showed the abundance of Streptococcus in younger individuals or those having more elastic skin. Particularly, we isolated Streptococcus pneumoniae, Streptococcus infantis, and Streptococcus thermophilus from face of young individuals. Treatment with secretions of S. pneumoniae and S. infantis induced the expression of genes associated with the formation of skin structure and the skin barrier function in human skin cells. The application of culture supernatant including Streptococcal secretions on human skin showed marked improvements on skin phenotypes such as elasticity, hydration, and desquamation. Gene Ontology analysis revealed overlaps in spermidine biosynthetic and glycogen biosynthetic processes. Streptococcus-secreted spermidine contributed to the recovery of skin structure and barrier function through the upregulation of collagen and lipid synthesis in aged cells. Overall, our data suggest the role of skin microbiome into anti-aging and clinical applications.

scholarly journals Detection of Staphylococcal Species Diversity within Human Skin Microbiome Using a Simple PCR-SSCP Technique

2018 ◽  
Vol 26 (8) ◽  
pp. 1-6
Author(s):  
Lubab Aqeel ◽  
Rabab Omran
Keyword(s):  
Species Diversity ◽  
Human Skin ◽  
Genomic Dna ◽  
Skin Microbiome ◽  
Healthy Individuals ◽  
Bacterial Strains ◽  
Healthy Human ◽  
Single Strand Conformational Polymorphism ◽  
Staphylococcal Species ◽  
Pcr Products

Objective. The aim of this study was to detect the diversity of Staphylococcus species of a healthy human skin using a simple techniquevPCR-SSCP. Methods: Blood samples, saliva, and skin swaps samples were collected from 50 persons from Hilla City  - Iraq. The genomic DNA was extracted from these samples using the Bacteria Genomic DNA Kit. The concentrations and purity of DNA extract estimated by NanoDrop spectrophotometer. Polymerase chain reaction – single-strand conformational polymorphism (PCR–SSCP) technique was performed to detect the diversity between Staphylococcal species in the human skin microbiome using a specific primer of the 16SrRNA gene. Results: The PCR results, indicated that the Staphylococcal species were found within the ski community, but it's not infected blood and mouth of test healthy individuals. SSCP-heteroduplex patterns of PCR products appeared the presence Staphylococcal species diversity within skin microbiome of test healthy individuals. Conclusion: In spite of the PCR-SSCP, heteroduplex method was simple and cheap and appeared the diversity betweenvStaphylococcalspeciesvin the human skin microbiome, but it's not diagnosed the bacterial strains. So these results required to confirm by DNAvsequencingvtechnique.

scholarly journals High-resolution ISR amplicon sequencing reveals personalized oral microbiome

2018 ◽  
Author(s):  
Chiranjit Mukherjee ◽  
Clifford J. Beall ◽  
Ann L. Griffen ◽  
Eugene J. Leys
Keyword(s):  
High Resolution ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Rrna Gene ◽  
Oral Microbiota ◽  
Community Fingerprinting

AbstractBackground:Sequencing of the 16S rRNA gene has been the standard for studying the composition of microbial communities. While it allows identification of bacteria at the level of species, it does not usually provide sufficient information to resolve at the sub-species level. Species-level resolution is not adequate for studies of transmission or stability, or for exploring subspecies variation in disease association. Current approaches using whole metagenome shotgun sequencing require very high coverage that can be cost-prohibitive and computationally challenging for diverse communities. Thus there is a need for high-resolution, yet cost-effective, high-throughput methods for characterizing microbial communities.Results:Significant improvement in resolution for amplicon-based bacterial community analysis was achieved by combining amplicon sequencing of a high-diversity marker gene, the ribosomal operon ISR, with a probabilistic error modeling algorithm, DADA2. The resolving power of this new approach was compared to that of both standard and high-resolution 16S-based approaches using a set of longitudinal subgingival plaque samples. The ISR strategy achieved a 5.2-fold increase in community richness compared to reference-based 16S rRNA gene analysis, and showed 100% accuracy in predicting the correct source of a clinical sample. Individuals’ microbial communities were highly personalized, and although they exhibited some drift in membership and levels over time, that difference was always smaller than the differences between any two subjects, even after one year. The construction of an ISR database from publicly available genomic sequences allowed us to explore genomic variationwithinspecies, resulting in the identification of multiple variants of the ISR for most species.Conclusions:The ISR approach resulted in significantly improved resolution of communities, and revealed a highly personalized, stable human oral microbiota. Multiple ISR types were observed for all species examined, demonstrating a high level of subspecies variation in the oral microbiota. The approach is high-throughput, high-resolution yet cost-effective, allowing subspecies-level community fingerprinting at a cost comparable to that of 16S rRNA gene amplicon sequencing. It will be useful for a range of applications that require high-resolution identification of organisms, including microbial tracking, community fingerprinting, and potentially for identification of virulence-associated strains.

scholarly journals The Cutaneous Microbiome and Wounds: New Molecular Targets to Promote Wound Healing

2018 ◽  
Vol 19 (9) ◽  
pp. 2699 ◽  
Author(s):  
Taylor Johnson ◽  
Belinda Gómez ◽  
Matthew McIntyre ◽  
Michael Dubick ◽  
Robert Christy ◽  
...  
Keyword(s):  
Wound Healing ◽  
Human Skin ◽  
Host Immune System ◽  
Skin Microbiome ◽  
Epithelial Barrier Function ◽  
Healing Tissue ◽  
Culture Independent ◽  
Skin Commensals ◽  
Chronic Skin

The ecological community of microorganisms in/on humans, termed the microbiome, is vital for sustaining homeostasis. While culture-independent techniques have revealed the role of the gut microbiome in human health and disease, the role of the cutaneous microbiome in wound healing is less defined. Skin commensals are essential in the maintenance of the epithelial barrier function, regulation of the host immune system, and protection from invading pathogenic microorganisms. In this review, we summarize the literature derived from pre-clinical and clinical studies on how changes in the microbiome of various acute and chronic skin wounds impact wound healing tissue regeneration. Furthermore, we review the mechanistic insights garnered from model wound healing systems. Finally, in the face of growing concern about antibiotic-resistance, we will discuss alternative strategies for the treatment of infected wounds to improve wound healing and outcomes. Taken together, it has become apparent that commensals, symbionts, and pathogens on human skin have an intimate role in the inflammatory response that highlights several potential strategies to treat infected, non-healing wounds. Despite these promising results, there are some contradictory and controversial findings from existing studies and more research is needed to define the role of the human skin microbiome in acute and chronic wound healing.

scholarly journals Species-Level Resolution of Female Bladder Microbiota from 16S rRNA Amplicon Sequencing

mSystems ◽  
2021 ◽  
Author(s):  
Carter Hoffman ◽  
Nazema Y. Siddiqui ◽  
Ian Fields ◽  
W. Thomas Gregory ◽  
Holly M. Simon ◽  
...  
Keyword(s):  
16S Rrna ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Classification Schemes ◽  
Common Culture ◽  
16S Rrna Amplicon Sequencing ◽  
Female Bladder ◽  
Culture Independent ◽  
Level Identification

Accurate species-level identification from culture-independent techniques is of importance for microbial niches that are less well characterized, such as that of the bladder. 16S rRNA amplicon sequencing, a common culture-independent way to identify bacteria, is often critiqued for lacking species-level resolution. Here, we extensively evaluate classification schemes for species-level bacterial annotation of 16S amplicon data from bladder bacteria.

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