Related Experiment Videos
Isolation of a Kawasaki disease-associated bacterial sequence from peripheral blood leukocytes
1Department of Pediatrics, Chukyo Hospital, Nagoya, Japan. shibatam@med.nagoya-u.ac.jp
Background:
The clinical and epidemiologic features of Kawasaki disease (KD) suggest an infectious etiology, but the agent(s) remains unknown. We aimed to isolate the causative bacterial gene from peripheral blood leukocytes of patients with acute KD.
Methods:
Nested polymerase chain reaction (PCR) assay was used to amplify the bacterial 16S ribosomal RNA gene (rDNA). The amplified DNA were cloned into a plasmid vector and sequenced. Phylogenetic analysis was performed with clustal W program and the neighbour-joining method.
Results:
First, the PCR reagents were examined by the PCR assay using conservative primers and we found more than 10 16S rDNA sequences contaminating the reagents. We then examined five KD patients using the PCR assay, excluding the contaminated sequences, and obtained five 16S rDNA sequences as possible KD-associated sequences. The primers specific to each 16S rDNA sequence were synthesized and used for specific PCR assays. Only the PCR assay specific to the 16S rDNA sequence termed 16S71-33 did not show any false positives with the control DNA from non-KD patients. The 16S71-33 sequence was positive in three of 20 patients with acute KD before gamma-globulin therapy, but it became negative after therapy. The phylogenetic analysis showed a new species of the genus Corynebacterium as the origin of the 16S71-33 sequence.
Conclusions:
These data show that an infectious KD agent is traced in peripheral leukocytes and that a new Corynebacterium species may be responsible for KD in some cases. The true frequency and the role of the new Corynebacterium in KD would be clarified by measuring specific antibodies to it.
Insights
Researchers identified a new Corynebacterium species in Kawasaki disease (KD) patients. This bacterial gene, found in leukocytes, may be the infectious agent causing KD in some cases.
Area of Science:
- Microbiology
- Immunology
- Pediatrics
Background:
- Kawasaki disease (KD) clinical and epidemiologic features suggest an infectious cause, but the specific agent remains unidentified.
- Previous research has not definitively isolated the causative agent of KD.
Purpose of the Study:
- To isolate the causative bacterial gene from peripheral blood leukocytes of patients with acute Kawasaki disease (KD).
- To identify potential infectious agents responsible for KD.
Main Methods:
- Nested polymerase chain reaction (PCR) was used to amplify bacterial 16S ribosomal RNA gene (rDNA).
- Amplified DNA was cloned, sequenced, and subjected to phylogenetic analysis using clustal W and neighbor-joining methods.
- Specific PCR assays were developed to validate findings, controlling for reagent contamination and non-KD patient DNA.
Main Results:
- PCR reagent contamination with over 10 16S rDNA sequences was identified and excluded.
- Five potential KD-associated 16S rDNA sequences were obtained from five KD patients.
- A specific sequence, 16S71-33, identified as a new Corynebacterium species, was positive in 3/20 acute KD patients before therapy and negative after therapy, with no false positives in controls.
- Phylogenetic analysis confirmed the 16S71-33 sequence originated from a novel Corynebacterium species.
Conclusions:
- An infectious agent, potentially a new Corynebacterium species, has been traced in peripheral leukocytes of Kawasaki disease patients.
- This novel Corynebacterium species may be responsible for KD in a subset of cases.
- Further research measuring specific antibodies is needed to clarify the frequency and role of this new Corynebacterium in KD.