3 years ago

Detection of microbial genes in a single leukocyte by polymerase chain reaction following laser capture microdissection

Akiteru Yamada, Kazumi Umeki, Yuji Saeki, Yuuki Hashikura, Hajime Nomura, Ikuo Yamamoto, Kunihiko Umekita, Ichiro Takajo, Chihiro Koshimoto, Akihiko Okayama

Publication date: Available online 10 November 2018

Source: Journal of Microbiological Methods

Author(s): Akiteru Yamada, Kazumi Umeki, Yuji Saeki, Yuuki Hashikura, Hajime Nomura, Ikuo Yamamoto, Kunihiko Umekita, Ichiro Takajo, Chihiro Koshimoto, Akihiko Okayama

Abstract

Although isolation and identification of bacteria in a clinical specimen constitute essential steps for the diagnosis of bacterial infection, positive results of the bacterial culture are not always attained, despite observing the bacteria by Gram staining. As bacteria phagocytosed by the leukocytes are considered as the causative agents of infectious diseases, this study aims to introduce a new approach for the collection of only bacteria phagocytosed by the neutrophils in an animal model using laser capture microdissection (LCM) followed by the DNA identification using polymerase chain reaction (PCR). We inoculated representative bacteria (Escherichia coli and Staphylococcus aureus) into the abdominal cavities of specific pathogen-free C57BL/6 J mice. After 6 h inoculation, we collected the fluid samples from the peritoneal cavities of mice and demonstrated peritonitis by the increase of neutrophils. Then, we smeared the neutrophils on the membrane slides and collected single-cell phagocytosing bacteria by LCM. The supernatant of the cell lysate was supplied for the PCR reaction to amplify the 16S rRNA gene, and we validated the DNA sequences specific for the inoculated bacteria. In addition, PCR using specific primers for E. coli and S. aureus identified each species of bacteria. Hence, this study suggests that the combination of LCM and PCR could be a novel approach to determine bacteria in infectious diseases. Nevertheless, further investigation is warranted to test various additional bacterial taxa to demonstrate the general applicability of this method to clinical samples.

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