Cross-alteration of murine skin and tick microbiome concomitant with pathogen transmission after Ixodes ricinus bite

Abstract

Background: Ticks are major vectors of diseases affecting humans such as Lyme disease or domestic animals such as anaplasmosis. Cross-alteration of the vertebrate host skin microbiome and the tick microbiome may be essential during the process of tick feeding and for the mechanism of pathogen transmission. However, it has been poorly investigated.

Methods: We used mice bitten by field-collected ticks (nymphs and adult ticks) in different experimental conditions to investigate, by 16S rRNA gene metabarcoding, the impact of blood feeding on both the mouse skin microbiome and the tick microbiome. We also investigated by PCR and 16S rRNA gene metabarcoding, the diversity of microorganisms transmitted to the host during the process of tick bite at the skin interface and the dissemination of the pathogen in host tissues (blood, heart, and spleen).

Results: Most of the commensal bacteria present in the skin of control mice were replaced during the blood-feeding process by bacteria originating from the ticks. The microbiome of the ticks was also impacted by the blood feeding. Several pathogens including tick-borne pathogens (Borrelia/Borreliella, Anaplasma, Neoehrlichia, Rickettsia) and opportunistic bacteria (Williamsia) were transmitted to the skin microbiome and some of them disseminated to the blood or spleen of the mice. In the different experiments of this study, skin microbiome alteration and Borrelia/Borreliella transmission were different depending on the tick stages (nymphs or adult female ticks).

Conclusions: Host skin microbiome at the bite site was deeply impacted by the tick bite, to an extent which suggests a role in the tick feeding, in the pathogen transmission, and a potentially important impact on the skin physiopathology. The diversified taxonomic profiles of the tick microbiome were also modified by the blood feeding. Video Abstract.

Keywords: 16S targeted sequencing; Anaplasma; Borrelia; Borreliella; Lyme disease; Metagenome; Metagenomic; Microbiota.

Fig. 1

Description of the three different protocols used in different experiments

Fig. 2

Skin microbiomes from mice bitten by nymphs or adult female ticks are deeply and differently modified. A, B Beta diversity ordination (PCoA) using Bray Curtis (A) and Jaccard (B) distances of the skin microbiome of control mice (red dots), mice bitten by female adult ticks (yellow dots), and mice bitten by nymphs (blue dot) for 1 to 4 days. C Alpha diversity (cluster levels observed, Shannon, Simpson, and inverse Simpson indexes) of the same skin samples. D–G LEfSe (linear discriminant analysis effect size) analysis summarizing the taxa significantly modified (p<0.05 with Mann-Whitney nonparametric test) between the skin of control mice and bitten mice (by nymphs or female ticks) (D), control mice and mice bitten by female ticks (E), control mice and mice bitten by nymphs (F), and mice bitten by female ticks and mice bitten by nymphs (G). H–J Barplots of relative proportions in the skin biopsies of the phyla (H) and top 20 genera (I and J), grouped by duration of feeding (H and I) or by tick stages (J)

Fig. 3

Impacts on the mouse skin microbiome persist 10 days after the removal of the ticks. A Alpha diversity (cluster levels observed, Shannon, Simpson, and inverse Simpson indexes) of the skin microbiome of control mice without a cup (red dots), control mice with a cup (purple dots), mice bitten 3 days by female ticks (yellow dots), and mice bitten 5 days by nymphs (blue dot). Bitten mice were kept 10 days after the removal of the ticks, before being euthanized. B, C Barplots of relative proportions in the skin biopsies of the phyla (B) and top 20 genera (C). D–G LEfSe (linear discriminant analysis effect size) analysis summarizing the taxa significantly modified (p<0.05 with Mann-Whitney nonparametric test) between skin of control mice without cup and control mice with a cup (D), control mice with a cup and mice bitten by female ticks (E), control mice with a cup and mice bitten by nymphs (F), and mice bitten by female ticks and mice bitten by nymphs (G)

Fig. 4

Long-term skin microbiome modifications are different between mice bitten by female ticks or by nymphs. A Alpha diversity (cluster levels observed, Shannon, Simpson, and inverse Simpson indexes) of the skin microbiome of mice 10 days after a blood feeding by tick (female ticks or nymphs) of 1 day (green dots), 2 days (blue dots), or 3 days (red dot). B Barplots of relative proportions in the skin biopsies of the top 20 genera in the samples separated by duration of the feeding. C Alpha diversity (cluster levels observed, Shannon, Simpson, and inverse Simpson indexes) of the same samples separated between mice bitten by female ticks (yellow dots) or by nymphs (blue dots). D Barplots of relative proportions in the skin biopsies of the top 20 genera in the samples separated by tick stages. E, F Beta diversity ordination (PCoA) using Bray Curtis (E) and Jaccard (F) distances of the skin microbiome of mice bitten by female ticks (yellow dots) or by nymphs (blue dots)

Fig. 5

The skin microbiome composition of the mice bitten by ticks gets closer to the microbiome of ticks. A Upset plot representation of the presence and co-occurrence of 15 taxa of interest in the ticks. The presence of the taxa is determined using a fixed threshold of total reads % in the sample mentioned in the figure. Th.: fixed threshold used for the corresponding taxa. Max: % of the taxa in the tick which has the highest proportion. Black horizontal barplots represent the number of ticks with % of the corresponding taxa above the fixed threshold. Colored vertical barplots represent the number of ticks with the corresponding combination of taxa above the fixed threshold, separated by fed nymphs (red), unfed nymphs (green), fed female ticks (blue), and unfed adult ticks (yellow). ¹Candidatus Neoehrlichia; ²B. afzelii or B. burgdorferi; ³Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium; ⁴Candidatus Allocryptoplasma; ⁵Multi-affiliated genera of Alcaligenaceae family (Achromobacter or Bordetella); ⁶Candidatus Midichloria. B Beta diversity ordination (PCoA) using Jaccard distances of the tick microbiome (all ticks, blue dots) and the skin microbiome of mice bitten by ticks (green dots) and of control mice (pink dots). C Beta diversity ordination (PCoA) using Jaccard distances of the tick microbiome separated by maturity and feeding state (blue, yellow, red, and green dots), the skin microbiome of mice bitten by female ticks (dark blue dots) or by nymphs (light blue dots), and the skin microbiome of control mice (pink dots). D Venn diagram of the common clusters present in microbiomes of adult ticks (green), nymph ticks (blue), skin of mice bitten by female ticks (pink), and skin of mice bitten by nymphs (yellow). In the Venn diagram, taxa identified as structural zeros by the ANCOM-II (analysis of compositions of microbiome II) preprocessing method were removed prior to plotting the diagram