Cultivation of Enterobacter Hormaechei from Human Atherosclerotic Tissue

Abstract

To determine whether culturable bacterial strains are present in human atheromatous tissue and to investigate their properties using culture, quantitative PCR, metagenomic screening, genomic and biochemical methods. We analyzed femoral atherosclerotic plaque and five pairs of diseased and healthy arterial tissue for the presence of culturable bacteria using cell cultures and genomic analysis. Gram negative aerobic bacilli were cultivated from the plaque tissue. Ribosomal 16S DNA amplification and sequencing identified the isolates as Enterobacter hormaechei. The isolate was resistant to ampicillin, cefazolin, and erythromycin. A circular 10 kb plasmid was isolated from the strain. Antibiotic protection assays of the isolate demonstrated invasive ability in a human monocytic cell line. To extend the study, five matched pairs of diseased and healthy aortic tissue were analyzed via quantitative PCR. Eubacterial 16S rDNA was detected in all specimens, however, E. hormaechei DNA was detected in surprisingly high numbers in two of the diseased tissues only. While it is well documented that inflammation is an important risk factor for vascular pathophysiology, the association of bacteria with atherosclerosis has not been clearly established, in large part due to the inability to isolate live bacteria from atheromatous tissue. This is the first study providing direct evidence of Enterobacter spp. associated with atheromatous tissues. The data suggest that chronic infection with bacteria may be an under-reported etiologic factor in vascular pathogenesis. Importantly, characterization of the clinical isolate supports a model of atherogenesis where systemic dissemination of bacteria to atherosclerotic sites may occur via internalization in phagocytic cells.