| Summary: | Next-generation sequencing (NGS) studies show that mosquito and tick microbiomes influence the transmission of pathogens, opening new avenues for vector-borne pathogen control. Recent microbiological studies of Australian ticks highlight fundamental knowledge gaps of tick-borne agents. This investigation explored the composition, diversity and prevalence of bacteria in Australian ticks (n = 655) from companion animals (dogs, cats and horses). Bacterial 16S NGS was used to identify most bacterial taxa and a Rickettsia-specific NGS assay was developed to identify Rickettsia species that were indistinguishable at the V1-2 regions of 16S. Sanger sequencing of near full-length 16S was used to confirm whether species detected by 16S NGS were novel. The haemotropic bacterial pathogens Anaplasma platys, Bartonella clarridgeiae, “Candidatus Mycoplasma haematoparvum” and Coxiella burnetii were identified in Rhipicephalus sanguineus (s.l.) from Queensland (QLD), Western Australia, the Northern Territory (NT), and South Australia, Ixodes holocyclus from QLD, Rh. sanguineus (s.l.) from the NT, and I. holocyclus from QLD, respectively. Analysis of the control data showed that cross-talk compromises the detection of rare species as filtering thresholds for less abundant sequences had to be applied to mitigate false positives. A comparison of the taxonomic assignments made with 16S sequence databases revealed inconsistencies. The Rickettsia-specific citrate synthase gene NGS assay enabled the identification of Rickettsia co-infections with potentially novel species and genotypes most similar (97.9–99.1%) to Rickettsia raoultii and Rickettsia gravesii. “Candidatus Rickettsia jingxinensis” was identified for the first time in Australia. Phylogenetic analysis of near full-length 16S sequences confirmed a novel Coxiellaceae genus and species, two novel Francisella species, and two novel Francisella genotypes. Cross-talk raises concerns for the MiSeq platform as a diagnostic tool for clinical samples. This study provides recommendations for adjustments to Illuminaʼs 16S metagenomic sequencing protocol that help track and reduce cross-talk from cross-contamination during library preparation. The inconsistencies in taxonomic assignment emphasise the need for curated and quality-checked sequence databases.
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