Optimization of a sampling and analysis process to study the effects of skin care products on the microbial skin flora.

Abstract (Swedish)

Vår hud är hem för en stor grupp av mikroorganismer, hudens mikroflora, som påverkar vår hälsa och vårt välbefinnande. Ändå finns det många aspekter av detta ämne som ännu är outforskade. Detta projekt syftade till att optimera en metod för analys av hudens mikroflora och effekten av hudvårdsprodukter på hudens mikroflora, från provtagning och provberedning till analys med kvantitativ polymeraskedjereaktion (qPCR). Olika testparametrar, såsom provtagningsmetoder, extraktionskitt och PCR mastermixar, utvärderades i en laboratoriemiljö med Staphylococcus aureus. Genom att tillsätta bakteriell lösning till en yta av Vitro-skin, samla bakterierna genom provtagning, extrahera provet och analysera med qPCR formulerades en metodbeskrivning. S. aureus -lösning användes i två olika koncentrationer, 5*106 och 5*108 CFU/ml och 20μL per test, totalt en ympning av antingen 105 eller 107 celler till varje testyta. Några utvalda hudvårdsprodukter undersöktes med avseende på hämmande effekter på qPCR-analysen.
Fem provtagningsmetoder, två extraktionskitt och två master mixar testades totalt. De två extraktionskitten, Qiagen DNA-mikrobiomkitt och ThermoFisher PureLink- mikrobiomkitt, visade sig ha olika styrkor där DNA-mikrobiomkittet var mycket tillförlitlig och användbart för olika provtagningsmetoder och PureLink-mikrobiomkittet som var mer specialiserat producerade både högre och lägre DNA-mängd beroende på vilken provtagningsmetod som användes. Av de två testade mastermixarna, Genesig Oasig master mix och Quantabio Toughmix master mix, visade sig Toughmix vara mer motståndskraftig medan Oasig master mixens prestanda påverkades av tillsatsen av hudvårdsprodukter till analysen. Kvalitetskontroll utfördes i form av extraktion- skontroller som ingick i PCR-analysen och total DNA-koncentrationsmätningar gjorda på prover utöver qPCR. Resultaten bekräftades med statistisk analys, Anova och bonferroni-justerade t-test.
I slutändan bedömdes Copan ESwab 480C topsen, Thermofisher PureLink Microbiome DNA-extraktionskitt och Quantabio Toughmix 2x master mix vara den bästa kombinatio- nen. Vidare fanns hämmande effekter från vissa hudvårdsprodukter på qPCR-analysen, där tillsats av lösnigen från en intimvåtservett resulterade i en ökning på 15 cykler och lösningen från en sminkborttagningsservett en ökning på fyra cykler av Cq-värdet jämfört med prover utan tillsatts. På grund av COVID-19-pandemin var det brist på laborato- riematerial, och det fanns några planerade tester som inte kunde genomföras. Arbetet kommer att fortsätta efter detta specifika projekt, inklusive ytterligare arbete för att säkerställa kvaliteten på den föreslagna metoden.

Abstract

Our skin is home to a vast community of microorganisms, the skin micro flora, which affect our health and well-being. Yet, there are many aspects of this topic that are still unexplored. This project aimed to optimize a method for analysis of the skin micro flora and the effect of skin care products on the skin micro flora, from sampling and sample preparation to analysis with quantitative polymerase chain reaction (qPCR). Different testing parameters, such as sampling methods, extraction kits and master mixes, were evaluated in a lab setting using Staphylococcus aureus. By adding bacterial solution to a surface of Vitro-skin, collecting the bacteria through sampling, extracting the sample, and analyzing the sample using qPCR, a method description was formulated. The S. aureus solution was used in two different concentrations, 5*106 and 5*108 colony forming units (CFU)/ml and 20 μL per test, totalling an inoculation of either 105 or 107 CFU to each test site. Some chosen skin care products were investigated for inhibitory effects on the qPCR analysis.
Five sampling methods, two extraction kits and two master mixes were tested in total. The two extraction kits, Qiagen DNA microbiome kit and ThermoFisher PureLink mi- crobiome kit, proved to have very different strengths with the DNA microbiome kit being very reliable and applicable to many different sampling methods and the PureLink mi- crobiome kit being more specialized and producing both higher and lower DNA yields depending on the sampling method used. Out of the two master mixes tested, the Gene- sig Oasig master mix and Quantabio Toughmix master mix, the Toughmix proved to be more resilient while the Oasig master mix’s performance was affected by the addition of skin care products to the analysis. Quality control work was performed in the form of extraction controls included in the PCR analysis and total DNA concentrations made on samples in addition to the qPCR. Results were confirmed by statistical analysis, Anova and bonferroni-corrected t-tests.
In the end the Copan ESwab 480C flocked swab, ThermoFisher PureLink Microbiome DNA purification kit and Quantabio Toughmix 2x master mix were shown to be the best combination. Furthermore there were inhibitory effects from some skin care products on the qPCR analysis, where the addition of a moist toilet paper solution resulted in a 15-cycle increase and a Demakup solution a four-cycle increase in Cq values compared to samples without any added additives. Because of the COVID-19 pandemic there was a shortage of lab material, some planned tests could not be carried out, and work will be continued beyond this particular project, including further work in verifying the quality of the proposed method.

Popular Abstract

It has been said that the human body consists of more bacterial cells than human. Whether this is true or not it is a fact that we are landlords to millions of tiny ten- ants living on and inside our bodies. Most people have heard of the gut microbiome, the microbial community of the intestines, but it is not the only bacterial community paying rent to us. The skin is also home to a vast selection of bacteria, and it is becoming more and more clear that it might equal the role the gut microbiome plays in our health and well-being. From arming our budding immune systems as babies to acting as security guards against pathogenic bacteria colonizing our skin, the skin microflora fulfills a lot of different functions that help keep us healthy throughout our entire lives.
However, there is still much to learn about the skin micro flora, how is it affected by the hosts’ diet and hygiene routines? Which bacteria are responsible for certain skin condi- tions and which helps keep others away? To answer these questions more research and investigation is needed. This project has focused on developing a method which can be used for that purpose, by testing different sampling and analysis techniques. To assess the different techniques samples were collected and the amount of bacterial cells col- lected with the method counted, quantified, using PCR-technique. The resulting method uses the ESwab 480C from Copan, PureLinkTM Microbiome DNA Purification Kit from Thermofischer and the ToughMix® 2x master mix from Quantabio for sampling, sample processing and qPCR analysis. The method was then tested with samples containing various skin care products to investigate what effects the presence of skin care products would have on the method. It was shown that skin care products can have an adverse ef- fect on the qPCR analysis, but that the Toughmix master mix is very adapt at mitigating these effects, producing accurate results anyway.
The analysis did only involve quantification at this stage, although PCR is a very powerful and versatile tool for microbiological analysis, and could be adapted to produce more results than simple quantification of bacteria present in a sample.The PCR protocols can be adapted to enable detection and se- quencing of bacteria with very high accuracy, meaning that bacteria present
even in very small quantities could be detected By doing this bacterial profiles can be generated for different subjects, and then screened for changes with time, change in hygiene routine, etc. By understand- ing the skin flora we might find treatments for various skin conditions, find ways to prevent infections caused by antimicrobial-resistant bacteria and mechanisms to boost weak immune systems. To do this we need to have a better understanding of the interactions between ourselves and the microorganisms living on our skin. Hopefully this research method can contribute to the improvement of that understanding and help uncover more knowledge in the future. Illustration created by author from sourced image, see ref. list. (Origimm 2021)

• author: Gifvars, Emma
• supervisor: Johannes Hedman; Linda Jansson; Karin Bjerre; Charlotta Löfström; Erik Nygren
• organization: Applied Microbiology
• alternative title: Optimering av en provtagnings- och analysprocess för att studera effekterna av hudvårdsprodukter på den mikrobiella hudfloran hos människor.
• course: KMBM05 20211
• year: 2021
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering; Biology and Life Sciences
• keywords: Method development, Method optimization, Microbial skin flora, Biotechnology, PCR analysis, Quantification of bacteria, Degree project in applied microbiology
• language: English
• id: 9043350
• date added to LUP: 2021-06-14 14:23:38
• date last changed: 2021-06-14 14:23:38