Identification of microbial taxa present in Ctenocephalides felis (cat flea) reveals widespread co-infection and associations with vector phylogeny

<p><strong>Background </strong></p> <p><em>Ctenocephalides</em> <em>felis</em>, the cat flea, is the most common ectoparasite of cats and dogs worldwide. As a cause of flea allergy dermatitis and a vector for two genera of zoonotic pathogens (<em>Bartonella</em> and <em>Rickettsia</em> spp.), the effect of the <em>C</em>. felis <em>microbiome</em> on pathogen transmission and vector survival is of substantial medical importance to both human and veterinary medicine. The aim of this study was to assay the pathogenic and commensal eubacterial microbial communities of individual <em>C</em>. <em>felis</em> from multiple geographic locations and analyze these findings by location, qPCR pathogen prevalence, and flea genetic diversity.</p> <p><strong>Methods </strong></p> <p>16S Next Generation Sequencing (NGS) was utilized to sequence the microbiome of fleas collected from free-roaming cats, and the <em>cox1</em> gene was used for flea phylogenetic analysis. NGS data were analyzed for 168 individual fleas from seven locations within the US and UK. Given inconsistency in the genera historically reported to constitute the <em>C</em>. <em>felis</em> microbiome, we utilized the decontam prevalence method followed by literature review to separate contaminants from true microbiome members.</p> <p><strong> Results </strong></p> <p>NGS identified a single dominant and cosmopolitan amplicon sequence variant (ASV) from <em>Rickettsia</em> and <em>Wolbachia</em> while identifying one dominant <em>Bartonella</em> <em>clarridgeiae</em> and one dominant <em>Bartonella henselae/Bartonella</em> <em>koehlerae</em> ASV. Multiple less common ASVs from these genera were detected within restricted geographical ranges. Co-detection of two or more genera (<em>Bartonella</em>, <em>Rickettsia</em>, and/or <em>Wolbachia</em>) or multiple ASVs from a single genus in a single flea was common. <em>Achromobacter</em>, <em>Peptoniphilus</em>, and <em>Rhodococcus</em> were identified as additional candidate members of the <em>C</em>. <em>felis</em> microbiome on the basis of decontam analysis and literature review. <em>Ctenocephalides</em> <em>felis</em> phylogenetic diversity as assessed by the <em>cox1</em> gene fell within currently characterized clades while identifying seven novel haplotypes. NGS sensitivity and specificity for <em>Bartonella</em> and <em>Rickettsia</em> spp. DNA detection was compared to targeted qPCR.</p> <p><strong>Conclusions </strong></p> <p>Our findings confirm the widespread coinfection of fleas with multiple bacterial genera and strains, proposing three additional microbiome members. The presence of minor <em>Bartonella</em>, <em>Rickettsia</em>, and <em>Wolbachia</em> ASVs was found to vary by location and flea haplotype. These findings have important implications for flea-borne pathogen transmission and control. </p>