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Objective  The aim of this study was to characterize the bacterial microbiome of hard ticks with affinity to bite humans in La Rioja (North of Spain).    Methods  A total of 88 adult ticks (22  Rhipicephalus sanguineus  sensu lato, 27  Haemaphysalis punctata , 30  Dermacentor marginatus  and 9  Ixodes ricinus ) and 120  I .  ricinus  nymphs (CRETAV collection, La Rioja, Spain), representing the main anthropophilic species in our environment, were subjected to a metagenomic analysis of the V3-V4 region of the 16S rRNA gene using an Illumina MiSeq platform. Data obtained with Greengenes database were refined with BLAST. Four groups of samples were defined, according to the four tick species.    Results  Proteobacteria was the predominant phylum observed in all groups. Gammaproteobacteria was the most abundant class, followed by Alphaproteobacteria for  R .  sanguineus ,  H .  punctata  and  D .  marginatus  but the relative abundance of reads for these classes was reversed for  I .  ricinus . This tick species showed more than 46% reads corresponding to ‘not assigned’ OTUs (Greengenes), and >97% of them corresponded to ‘ Candidatus  Midichloriaceae’ using BLAST. Within Rickettsiales, ‘ Candidatus  Midichloria’,  Rickettsia ,  Ehrlichia , ‘ Candidatus  Neoehrlichia’ and  Wolbachia  were detected.  I .  ricinus  was the most alpha-diverse species. Regarding beta-diversity,  I .  ricinus  and  H .  punctata  samples grouped according to their tick species but microbial communities of some  R .  sanguineus  and  D .  marginatus  specimens clustered together.    Conclusions  The metagenomics approach seems useful to discover the spectrum of tick-related bacteria. More studies are needed to identify and differentiate bacterial species, and to improve the knowledge of tick-borne diseases in Spain.

Exploring the bacteriome in anthropophilic ticks: To investigate the vectors for diagnosis