Bacterial microbiota profiling of human-biting ticks in Yozgat Province, Türkiye

In the present study, the bacterial microbiota of ticks infesting humans in Yozgat Province, Türkiye, was investigated. Ticks were collected from 54 patients who were presented to the Emergency Service of the Yozgat Bozok University Research and Training Hospital between March and November 2019. The following tick species were identified: Hyalomma marginatum, Rhipicephalus turanicus, Rhipicephalus bursa, Haemaphysalis parva, Dermacentor marginatus, and Hyalomma aegyptium. 16S rRNA libraries were prepared from tick DNA pools using a two-step PCR protocol targeting the V3-V4 region. In the second (indexing) PCR, dual Illumina indexes and adapters were added using the Nextera XT Index Kit. The resulting libraries were then sequenced on an Illumina NovaSeq platform. A total of 3782 operational taxonomic units (OTUs) were identified; an average of 127,823 reads were obtained per sample, and an average of 31,561 reads were retained after taxonomic assignment. The OTUs were classified into 20 phyla, 41 classes, 94 orders, 168 families, 344 genera, and 452 putative species-level taxa. At the phylum level, Proteobacteria were predominant (averaging 63%), followed by Actinobacteriota and Bacteroidota (each approximately 10%) and Firmicutes (9%). At the class level, Alphaproteobacteria (average 37%) and Gammaproteobacteria (26%) exhibited the highest relative abundance, while at the order level, Rickettsiales (34%) and Francisellales (17%) were prominent. At the family and genus levels, the dominant taxa were identified as Rickettsiaceae (33%), Francisellaceae (17%), Rickettsia (33%), Francisella (17%), and Coxiella (7%), respectively. The most prevalent species included symbiotic bacteria such as Francisella sp. and Candidatus Midichloria mitochondrii. The Kruskal-Wallis test, conducted from phylum to genus level to evaluate taxonomic composition differences among tick species, revealed no statistically significant differences (p ≥ 0.05 for all comparisons). In alpha diversity analyses, no significant differences were found between tick species in terms of Shannon and Simpson indices (Shannon: H = 7.69; p = 0.26, Simpson: H = 8.07; p = 0.23). Similarly, no significant differences were observed between sex and developmental stages (Shannon: p = 0.86, Simpson: p = 0.73). The Chao1 and observed OTU values indicated higher richness trends, particularly in the Hyalomma marginatum and Hyalomma sp. pools. However, Spearman correlations between sample size and diversity indices were not significant (p > 0.05). These findings provide a baseline overview of the bacterial microbiota associated with human-biting ticks in Yozgat Province and may serve as a foundation for future studies employing higher-resolution methods to assess pathogen presence and public health risk.