Volume 3 • Number 4 • 1998 HELICOBACTER © Blackwell Science, Inc. 1083-4389/98/$10.50/269 269–277 269 A Novel Method of Extracting Plasmid DNA from Helicobacter Species Maria Corazon A. De Ungria,* Daniel Tillett,* Brett A. Neilan,* Peter T. Cox, † and Adrian Lee* ABSTRACT *School of Microbiology and Immunology, University of New South Wales, and † Australian Water Technologies, Sydney, Australia Background. Plasmids are extra-chromosomal DNA that may encode products that aid in virulence, pathogene- sis, and the spread of antibiotic resistance among a wide spectrum of bacteria. Plasmids have been detected in Helicobacter pylori, H. felis, H. fennelliae, and H. ci- naedi. However, no function has been attributed to the Helicobacter plasmids studied to date. Moreover, the characterization of plasmids in other Helicobacter spe- cies is an as yet unexplored area of research. Several laboratories have reported difficulties in the extraction and isolation of plasmid DNA from H. pylori and H. felis isolates due to the presence of large amounts of DNase, necessitating cumbersome and time-consuming purification steps. The development of a method for ex- tracting plasmid DNA from Helicobacter species would be useful for future systematic studies of plasmids in this important group of microorganisms. Materials and Methods. Eight H. pylori isolates, includ- ing the Sydney Strain SS1, three H. felis isolates, and one isolate each of H. hepaticus, H. bilis, H. mustelae, and H. rodentium, were screened for plasmid DNA using a novel method that includes a potassium xanthogenate- sodium dodecyl sulfate-phenol (XSP) buffer. A specific PCR targeting a highly conserved plasmid replication protein gene, repA, was used to confirm the presence of plasmids in the H. pylori isolates examined. The PCR primers used were designed based on the sequence of the H. pylori plasmid pHPM180. To demonstrate the effec- tiveness of this method, plasmid DNA extracted from SS1 using XSP buffer was digested using three restriction enzymes (DdeI, SpeI and MaeIII). The relative amount of DNA obtained using the protocol was also compared to the yield derived from four commercial kits commonly used in many laboratories. Results. High and low molecular weight plasmids were extracted from H. pylori (n = 8) and H. felis (n = 3) iso- lates. The size range of these plasmids was from 3 kb to 16 kb. Attempts to isolate plasmids from H. hepaticus ATCC 51488, H. bilis ATCC 51630, H. rodentium MIT-95–2060, and H. mustelae NCTC 11574 were not successful, which was most likely due to the absence of endogenous plasmids from the strains examined. The rel- ative amount of DNA obtained using the XSP buffer protocol was comparable to that obtained from commer- cial kits as assessed by direct examination of plasmid profiles on agarose gels. Plasmid DNA extracted from H. pylori SS1 using XSP buffer was successfully digested with restriction enzymes. Conclusion. This study reports the development of an ef- ficient, inexpensive, and rapid method for extracting high and low molecular weight plasmids from Helico- bacter species. Application of this novel method for the isolation and future characterization of plasmids from different Helicobacter species could promote a better un- derstanding of the role of plasmids in the basic microbial physiology and ecology of this group of microorganisms. P lasmids are extra-chromosomal DNA found in many bacteria that may encode product(s) that often modify the host cell’s phenotype. Examples of these products include the virulence factors en- coded in the Vir plasmids of Yersinia [1], Salmo- nella [2], and Shigella flexneri [3]. Other plasmid- borne factors include antibiotic resistance genes, i.e., penicillin resistance in Neisseriae gonorrheae and Staphylococcus aureus and chloramphenicol resistance in Clostridium perfingens [4], as well as toxin genes encoded on the Ent plasmids of en- terotoxigenic E. coli [5]. In contrast to these well- characterized systems, there is a dearth of infor- mation with regard to the presence and function of plasmids in different Helicobacter species. Stud- ies to investigate the presence of plasmids in H. pylori [6–15], H. felis [16] and H. cinaedi and H. fennelliae [17] have been reported. The sizes of these plasmids vary over a wide range, from 1.5 kb to 148 kb for H. pylori [6–9], 2 kb to 16 kb for H. felis (K. Jalava, manuscript in prepara- tion) and 1.8 kb to 48 kb for H. cinaedi and H. fennelliae [17]. Of all Helicobacter plasmids de- Reprint requests to: Professor Adrian Lee, School of Micro- biology and Immunology, University of New South Wales, Sydney, NSW 2052 Australia. E-mail: A.Lee@unsw.edu.au