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Bacteroides fragilis is the most significant anaerobic pathogen in humans, which is attributable to a number of virulence factors and to its ability to acquire antibiotic resistance by DNA transfer. Although transfer of antibiotic resistance has been demonstrated in vitro, the mechanisms of DNA transfer are unknown. To date, three types of transfer factors have been identified. They include conjugal plasmids, mobilizable transposons, and tetracycline transfer elements. These factors function independently but also interact with one another to facilitate and enhance transfer. The goal of my laboratory's research has been to determine the mechanisms of transfer of plasmids pBFTM10 and pLV22a and the mobilizable transposon Tn5520 as models for understanding DNA transfer in B. fragilis. We use transposon mutagenesis, subcloning, DNA sequencing, gel retardation assays, DNA footprinting, and complementation studies to determine which proteins are required for plasmid transfer. Thus far, depending on the transfer factor, we have determined that one, two, or three proteins plus a nick site are required for transfer, and we are currently determining protein functions. We are also determining which specific protein-DNA interactions are required for transfer initiation. Remaining questions include: (1) What are the functions of the transfer proteins? (2) What does the host provide in the mating process, and how is this controlled? (3) What controls DNA transfer? Answers to these questions will allow us to then ask what the molecular relationships are between plasmid transfer and other transfer factors, such as the tetracycline transfer element. Selected Publications Hurtuk MG, He LK, Szilagyi A, Gamelli RL, Hecht DW, Kennedy RH, Rhys-Williams W, Love WG, Shankar R, The novel antibacterial drug XF-70 is a potent inhibitor of Staphylococcus aureus infection of the burn wound, J Burn Care Res. 2010 May-Jun;31(3):462-9.PMID: 20453736 [PubMed - in process]. Johnson S, Schriever C, Patel U, Patel T, Hecht DW, Gerding DN. Rifaximin Redux: treatment of recurrent Clostridium difficile infections with rifaximin immediately post-vancomycin treatment. Anaerobe. 15(6):290-1 (2009). O'Connor JR, Galang MA, Sambol SP, Hecht DW, Vedantam G, Gerding DN, Johnson S. 2008. Rifampin and rifaximin resistance in clinical isolates of Clostridium difficile. Antimicrob Agents Chemother. 52(8):2813-7. Snydman DR, Jacobus NV, McDermott LA, Golan Y, Hecht DW, Goldstein EJ, Harrell L, Jenkins S, Newton D, Pierson C, Rihs JD, Yu VL, Venezia R, Finegold SM, Rosenblatt JE, Gorbach SL, Lessons learned from the anaerobe survey: historical perspective and review of the most recent data (2005-2007). Sears CL, Islam S, Saha A, Arjumand M, Haque Alam N, Faruque ASG, Salam MA, Shin J, Hecht DW, Weintraub A, Sack RB, Qadri F. Enterotoxigenic Bacteroides fragilis infection is associated with inflammatory diarrhea. Clin Infect Diseases (2008, in press). Thomas J, Hecht DW. Interaction of Bacteroides fragilis pLV22a relaxase Transfer DNA with E. coli RP4 TraG coupling protein. Molecular Microbiology 66(4):948-60, 2007. Hecht, DW, Kos I, Knopf S, Vedantam G. Characterization of BctA, a mating apparatus protein required for transfer of the Bacteroides fragilis conjugal element BTF-37. Res Microbiol, 158(7):600-7 , 2007. Hecht DW, Galang M, Osmolski JR, Johnson SB, Gerding DN. In Vitro activity of Fifteen Antimicrobial Agents Against 110 Toxigenic Clostridium difficile Clinical Isolates Collected from 1983-2004. Antimicrob Agents Chemother 51(8):2716-9, 2007. Hecht DW. Routine anaerobic blood cultures: back where we started? Clin Infect Dis, 44:901-3, 2007 (editorial commentary). Snydman DR, Jacobus NV, McDermott LA, Ruthazer R, Golan Y, Goldstein EJ, Finegold SM, Harrell LJ, Hecht DW, Jenkins SG, Pierson C, Venezia R, Yu V, Rihs J, Gorbach SL. National Survey on the Susceptibility of Bacteroides fragilis Group: Report and Analysis of Trends in the United States from 1997 to 2004. Antimicrob Agents Chemother.51:1649-55, 2007. Barron WM, Reed RL, Forsythe S, Hecht DW, Glen J, Murphy B, Lach R, Flores S, Tu J, Concklin M. Successful Implementation of Computerized Provider Order Entry Using an Existing Clinical Information System. Joint Commission Journal on Quality and Patient Safety 32:506-516, 2006. Sprandel KA, Drusano GL, Hecht DW, Rotschafer JC, Danziger LH, Rodvold KA. Population pharmacokinetic modeling and Monte Carlo simulation of varying doses of intravenous metronidazole. Diag Micro and Infect Dis 55:303-309, 2006 Rooney AP, Swezey, JL, Friedman R, Hecht DW and Maddox CW. Analyses of Core Housekeeping and Virulence Genes Reveals Cryptic Lineages of Clostridium perfringens Genetics 172(4):2081-92, 2006. Hecht DW. Anaerobes: Antibiotic resistance, Clinical Significance, and the role of susceptibility testing. Anaerobe 12:115-122, 2006. Vedantam G, Knopf S, Hecht DW. (2006).
Bacteroides fragilis mobilizable transposon Tn5520 requires a 71 base pair origin of transfer sequence and a single mobilization protein for relaxosome formation during conjugation.
Molecular Microbiology 59:288-300. |
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