Date Published: March 19, 2019
Publisher: Public Library of Science
Author(s): Aránzazu Portillo, Ana M. Palomar, María de Toro, Sonia Santibáñez, Paula Santibáñez, José A. Oteo, Ben J. Mans.
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).
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.
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.
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.
The identification of microorganisms from biological samples has been dominated by the use of traditional culture-dependent methods and conventional molecular biology techniques (mostly polymerase chain reaction, PCR). The isolation of most tick-borne bacteria in synthetic media or in cell culture is difficult to obtain, and a high number of microbes remain uncultured. For the last two decades, the identification of Rickettsia spp. and other tick-associated pathogens has been mainly based on the use of specific PCR assays and sequence analysis [1,2]. Until recently, most studies focused on the detection of pathogens in vectors were able to detect a unique or a few microorganisms in a single assay. Metagenomic approaches, based on the development of the Next Generation Sequencing (NGS) techniques, and primary focused on the 16S rRNA study combined with bioinformatics tools, is revolutionizing the research in the fields of epidemiology and diagnosis of infectious diseases, among others, overcoming the limitation of detecting only one or few microorganisms at a time . Metagenomic analysis can reveal the complexity of the microbiota of a given sample . The number of pathogens associated with ticks has increased over the last years. Currently, there is a worldwide rising incidence of patients with a history of a tick-bite [5,6]. The importance of tick-borne diseases (TBDs) as a growing threat for public health has been recently underlined, and ‘what is not sought, is not found’ . As ticks are able to transmit different microorganisms at one bite, it is necessary to be aware of possible co-infections. To investigate the microbial community composition harbored by ticks can facilitate the knowledge about the interactions among tick-associated microorganisms, the discovery of new uncultured microorganisms and subsequently, their implications as human pathogens.
A total of 19,977,253 read counts (average counts per sample = 201,790) and 227 OTUs were observed. The rarefaction curves reached a plateau, demonstrating that bacterial diversity had been satisfactorily detected for all samples (S1–S4 Figs).
Many TBDs have been recognized for the first time in the last few years, and emerging tick-borne pathogens are being detected [3,22–24]. Not only the clinical observation but also the application of new diagnostic methods (based on culture and molecular biology assays) has contributed to this progress . Nevertheless, TBDs are dangerously expanding and they constitute underestimated causes of human illness worldwide . The implementation of NGS platforms aimed to diagnosis is being developed, although reports about the contribution of this technique to the clinical diagnostic of TBDs are sporadic . Herein, the bacteriome of tick species with affinity to bite humans was analysed using the 16S metagenomic approach to investigate tick-related microorganisms and to improve the diagnosis of TBDs, particularly in cases with unknown etiologic agents.