Detection of Bartonella henselae and Bartonella quintana by a simple and rapid procedure using broad-range PCR amplification and direct single-strand sequencing of part of the 16S rRNA gene.

Abstract

OBJECTIVE: To detect directly Bartonella henselae and Bartonella quintana using culture-independent, molecular techniques, and to evaluate a simple and rapid procedure that allows uncultivable bacteria to be detected in usually sterile clinical specimens in a diagnostic laboratory. METHODS: From four clinical specimens proven to contain B. henselae (n=3) or B. quintana (n=1) DNA, part of the 16S rRNA gene was amplified using the polymerase chain reaction (PCR) and broad-range bacterial primers followed by reamplification and direct, single-strand sequencing. The partial 16S rRNA sequences were compared to reference sequences in databases. RESULTS: Similarities between sequences derived from clinical samples and those of B. henselae and B. quintana, respectively, were in the range 98.7--100%, indicating a strong association to the genus Bartonella. Intraspecies sequence variations within the B. henselae sequences were observed. CONCLUSIONS: The method described is a rapid, sensitive and reliable tool to generate partial 16S rRNA sequences of B. henselae and B. quintana directly from normally sterile clinical specimens. It is compatible with adequate prevention of contamination as is needed for diagnostic purposes, and it possesses the potential to detect other pathogens, including those as yet unknown.