Benchmarking surface decontamination methods for reliable Ixodes ricinus microbiome profiling

Ticks harbor complex microbial communities that influence their physiology and vector competence. Accurate characterization of tick-associated bacterial communities is challenged by microorganisms associated with or adhering to the tick exoskeleton, which may contribute bacterial signals to sequencing-based analyses and influence the resulting microbiome profiles. This study establishes a quantitative benchmarking framework for the objective comparison of tick surface decontamination protocols for microbiome analyses. Adult Ixodes ricinus ticks and nymphs were experimentally contaminated with a defined bacterial mock community and subjected to five decontamination treatments: 70% ethanol, 1% bleach, DNA/RNA Zap, lysozyme followed by bleach, and lysozyme followed by DNA/RNA Zap. Microbial communities were characterized using Oxford Nanopore full-length 16S rRNA gene sequencing. The mock-community approach provided a direct benchmark of the relative sequence representation of experimentally introduced surface bacteria, revealing substantial differences among treatments. Ethanol consistently showed the weakest effect, whereas bleach- and DNA/RNA Zap-based protocols, with or without lysozyme pre-treatment, produced near-complete depletion of mock-community reads. Comparative analysis of tick legs and leg-free bodies was consistent with Brevibacterium being predominantly surface-associated, whereas the endosymbiont' Candidatus Midichloria' remained readily detectable in leg-free bodies following decontamination. Community-level diversity analyses suggested treatment-associated differences in recovered bacterial profiles, although these patterns were exploratory. Importantly, Borrelia -associated sequences remained readily detectable in experimentally infected nymphs following surface treatment. Under the experimental conditions tested here, bleach and DNA/RNA Zap produced the strongest reduction in the relative sequence abundance of experimentally introduced surface bacteria, while dominant tick-associated bacterial taxa and pathogen-associated sequences remained detectable. Lysozyme pre-treatment did not confer measurable advantages. The choice of surface-decontamination strategy should also consider the biological compartment and microbial fraction of interest. The controlled mock-community approach provides an initial quantitative framework for benchmarking surface-decontamination procedures, although validation using field-collected ticks, additional tick species, more diverse surface-associated microbial communities, and independent laboratory settings will be required before broader methodological standardization can be proposed.