Decontamination of ticks to assess unbiased internal tick-borne microbiome (DNA)

Various microbial pathogens have been found in Ixodes ricinus. However, most studies assessed tick microbiomes without prior decontamination of the tick surface, which may alter the results and mislead conclusions of the tick-borne microbiome. The aim of this study was to test four different decontamination methods namely i.) 70% ethanol, ii.) DNA Away, iii.) 5 % sodium hypochlorite and iv.) Reactive Skin Decontamination Lotion (RSDL), that were previously reported for tick, surface, animal or human skin decontamination. To test efficiency of decontamination, we contaminated each tick with a defined mixture of Escherichia coli, Micrococcus luteus, Pseudomonas fluorescens, dog saliva and human sweat. As negative control no contamination or no decontamination strategy as positive control were carried out. After nucleic acid extraction, the recovery rate of contaminants was determined on RNA and DNA level by quantitative PCR and tick-borne microbiome analyses by bacterial 16S rRNA gene amplicon sequencing. 5% sodium hypochlorite treated ticks revealed the lowest amount of contaminants followed by DNA Away, RSDL and 70% ethanol. Moreover, tick microbiomes after 5% sodium hypochlorite decontamination clustered with negative controls. Decontamination of external microbiome from ticks is essential to retrieve unbiased internal tick-borne microbiome and to unambiguously detect its pathogens.