Naturally competent Acinetobacter baumannii clinical isolate as a convenient model for genetic studies

Abstract

Acinetobacter baumannii A118 was isolated from a patient's blood culture. It is susceptible to several antibiotics, is naturally competent, and supports replication and stable maintenance of four plasmid replicons. A. baumannii A118 took up a fluorophore-labeled oligonucleotide analog. These characteristics make this isolate a convenient model for genetic studies.

FIG. 1.

(A) Amplified ribosomal DNA restriction analysis (ARDRA). Ethidium bromide-stained 1.5% agarose gel electrophoresis of products obtained after treatment of the amplified 16S rRNA gene with CfoI, AluI, or MboI is shown. Molecular size DNA standard values are shown on the right side of the gel. Numbers at the bottom of each lane correspond to the ARDRA pattern (http://users.ugent.be/∼mvaneech/ARDRA/Acinetobacter.html). Ab, A. baumannii. (B) Plasmid DNA extracted from A. baumannii A118. Ethidium bromide-stained 0.7% agarose gel electrophoresis of linearized plasmid DNA isolated from transformed A. baumannii A118 is shown. Molecular size DNA standard values are shown on the right side of the gel. The enzymes used to digest the plasmids were as follows: XhoI (pVK102 and pHCMW1), EcoRV (pAADB and pAADA1KN), and SpeI (pMET1). (C) Internalization of fluorescent PS. Bright field (upper) and fluorescent (lower) microscopy of A. baumannii A118 cells incubated in the presence of Oregon green-labeled PS (green). The membranes were labeled using FM5-95 (red) as described before (14). Cells were visualized with a ×100 objective on a Nikon Eclipse TE2000-U microscope equipped with a Photometrics Cool-SNAP HQ charge-coupled-device camera. The images were analyzed and processed using the Adobe Photoshop software program. Arrows show cells that took up labeled PS. (D) Internalization of fluorescent LNA/DNA. Cells in stationary phase were incubated in saline buffer with Alexa Fluor 488-conjugated LNA/DNA for 30 min at 37°C in the absence of light. Then, the cells were washed twice by centrifugation, resuspended in saline buffer, and labeled with FM5-95. Images were taken by laser scanning confocal microscopy (LSCM) in a Zeiss Pascal confocal microscope. Deconvolution of the overlay picture focused at 1.6 μm was performed at the Oregon Health and Science University Molecular Microbiology and Immunology Imaging Core Facility Microscopy.