Objectives:

Although infective endocarditis due to HACEK group of bacteria (Haemophilus spp., Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens and Kingella spp.) is a rare occurrence, the identification of the organisms is still important diagnostically for the specific therapy. The HACEK bacteria are classified as fastidious Gram-negative coccobacilli, and the biochemical characteristics resemble each other. Thus, the identification of HACEK bacteria has been rather hard and sometimes inconclusive. In this study, we developed a rapid and highly sensitive identification method for HACEK bacteria by means of 16S rRNA gene PCR amplification followed by restriction fragment length polymorphism analysis (PCR-RFLP).

Methods:

H. aphrophilus ATCC 33894, A. actinomycetemcomitans ATCC 33384, C. hominis ATCC 12826, E. corrodens ATCC 23834 and K. kingae ATCC 23330 were used. DNA samples were prepared by a DNA purification kit. After PCR amplification using the primers corresponding to Escherichia coli 16S rRNA gene, the PCR products were digested with 4 U of either f HinfI and f MspI at 37°C for 1.5 h. The samples were then separated on 1.8% agarose gel, and the restriction patterns were recorded.

Results:

The RFLP patterns of five species of HACEK bacteria obtained by combing use of HinfI and MspI digestion were readily distinguished from each other and from other pathogens of infective endocarditis including viridans streptococci. Furthermore, the PCR-RFLP analysis yielded a definitive identification of C. hominis from one of the blood samples of the patients with infective endocarditis in which causative pathogens could not be unidentified by biochemical identification kits. The result was confirmed by a 16S rRNA gene sequence analysis of the isolate.

Conclusion:

The PCR-RFLP analysis developed in this study was a rapid and highly sensitive identification method for HACEK group of bacteria, and could be applicable for a definitive diagnostic detection of HACEK group of bacteria.