Liver microbiota in healthy rats: the hidden inhabitants of hepatocytes

Abstract

The tumor and tissue microbiomes of human beings have recently been investigated. Gut permeability was known as a possible resource for the positive detection of tissue bacteria. Herein, we report that bacteria were detected in high abundance in the hepatocyte nucleus of healthy rats, and that they were shared with gut microbiota to an extent. We assessed male Sprague Dawley (SD) rats for the 16S ribosomal ribonucleic acid (rRNA) gene. After rats were sacrificed by blood drainage from the portal vein, we extracted total deoxyribonucleic acid (DNA) from their ileal and colonic contents and liver tissues. The V3–V4 region of the 16S rRNA gene was amplified by polymerase chain reaction (PCR) and sequenced using an Illumina HiSeq 2500 platform. Sequences were assigned taxonomically by the SILVA database. We also detected bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA) in situ using immunofluorescence (IF) and western blotting and the 16S rRNA gene using fluorescent in situ hybridization (FISH). In the livers of six rats, we detected 54,867.50 ± 6450.03 effective tags of the 16S rRNA gene and clustered them into 1003 kinds of operational taxonomic units (OTUs; 805.67 ± 70.14, 729–893). Individuals showed conservation of bacterial richness, abundance, and evenness. LPS and the 16S rRNA gene were detected in the nuclei of hepatocytes. The main function composition of genomes of the annotated bacteria was correlated with metabolism (79.92 ± 0.24%). Gram negativity was about 1.6 times higher than gram positivity, and the aerobic and anaerobic phenotypes were nearly the same. Liver microbiota also showed high expression of mobile elements, biofilm formation, and stress tolerance, as well as potentially pathogenic phenotypes. Chemoheterotrophy, fermentation, and aerobic chemoheterotrophy were the main heterotrophic functions. Liver bacteria were shared with both the small and large intestines but showed significantly higher richness, evenness, and the β-diversity results showed that the liver microbiota exhibited significantly higher similarity than the small and large intestines (P < 0.05). The liver microbiota were hidden intracellular inhabitants in healthy rat livers and were shared with the gut microbiota.