Strategy for identification & characterization of Bartonella henselae with conventional & molecular methods

Abstract

Background & objectives: Bartonella henselae is a fastidious gram-negative bacterium usually causing self limiting infections in immunocompetent individuals but often causes potentially life threatening infection, such as bacillary angiomatosis in immunocompromised patients. Both diagnosis of infections and research into molecular mechanisms of pathogenesis have been hindered by lack of appropriate and reliable diagnostic techniques. We undertook this study to standardize methods to characterize B. henselae in clinical samples to diagnose Bartonella infection correctly.

Methods: B. henselae ATCC 49882 strain was procured from American type culture collection, USA. This strain was revived and maintained in the laboratory, and identification and characterization of this strain was done by conventional and molecular techniques, which included culture on various media, staining by different methods including electron microscopy, biochemical analysis by conventional methods and API, polymerase chain reaction (PCR) for amplification of citrate synthase gene followed by restriction fragment length polymorphism (RFLP).

Results: This organism was biochemically inert due to slow growth and generated unique identification code with API. The amplification of the citrate-synthase gene with primers yielded a 381 bp product followed by specific RFLP profile for B. henselae.

Interpretation & conclusions: Bartonella is fastidious and fragile organism and should be handled carefully. Extra effort and careful observation are required to isolate and characterize this organism.

Fig. 1

Tryptic soy agar showing of growth B. henselae.

Fig. 2

Gram's staining showing gram negative, pleomorphic bacilli in clumps at 100× magnification.

Fig. 3

Electron microscopic photograph of B. henselae.

Fig. 4

Agarose gel showing bands obtained by electrophoresis of PCR products from B. henselae using primers targeted at conserved bacterial citrate synthase gene sequence. Lane at left extreme is with marker DNA (100bp). Lanes 1 and 2 show B. henselae PCR product (381 bp); lane 3-negative control.

Fig. 5

Agarose gel showing bands obtained by electrophoresis of RFLP products of PCR product of B. henselae using restriction enzymes Taql and Acil. Lanes at right and left extreme are with marker DNA. Lanes 1 and 4 - positive control (B. henselae PCR product), lane 2 Taql and lane 3 Acil restriction profile.