• Detection of species-specific DNA sequences of Borrelia burgdorferi in infected humans, animal reservoirs, and ixodid tick vectors

      Malloy, Diane Catherine; Nauman, Robert K. (1992)
      Segments of the ospA gene that encode hydrophobic regions of the outer membrane protein, OspA, of Borrelia burgdorferi strain B31 were synthesized for use as oligonucleotide primers in the polymerase chain reaction (PCR). These oligonucleotide primers flank a 309-base-pair segment within the ospA gene. Optimal amplification conditions were achieved in a reaction mixture containing 0.2 uM of each oligonucleotide primer and 2 mMMgCl{dollar}\sb2.{dollar} Dimethyl sulfoxide at a concentration of 10% or higher was found to inhibit amplification and gelatin had no effect at concentrations below 100 ug/ml, and slight inhibition was seen at concentrations higher than 100 ug/ml. After 30 cycles of amplification under optimal conditions, the target fragment could be detected by agarose gel electrophoresis or dot hybridization with a {dollar}\sp{lcub}32{rcub}{dollar}P- or digoxigenin-labeled probe. This segment was amplified in all strains of B. burgdorferi, but it was not detected in other bacterial species. The sensitivity of PCR for the detection of B. burgdorferi in clinical samples was evaluated by seeding blood and urine specimens with B. burgdorferi and subjecting them to amplification. Ten organisms per ml of blood or urine could be detected using PCR with dot hybridization detection. In a blinded study of Lyme disease patients, the OspA PCR was positive in 31% of patients who were early in disease and who had not received oral antibiotic therapy. No patient who had received antibiotics was positive in the PCR. Blood and urine specimens were obtained from canines with clinical and serologic evidence of Lyme disease and subjected to PCR analysis. Of 17 clinical specimens from 15 canines, one blood specimen showed reactivity in the PCR. Two of 32 cerebrospinal fluid specimens from suspected neuroborreliosis patients showed reactivity in the PCR. B. burgdorferi could be detected optimally in tissue only after DNA extraction. Nine of ten mice from a highly endemic Lyme disease area in Wisconsin showed reactivity in the PCR when DNA extracted from heart, kidney, or bladder was used as the target. Two of five punch biopsy tissue samples from skin lesions from suspected Lyme disease patients showed reactivity in the PCR. Of all tissues studied, one yielded a positive spirochete stain and all were negative by immunoperoxidase staining with a polyclonal antibody to B. burgdorferi. The conclusion of this study is that PCR can detect and identify B. burgdorferi in clinical samples from Lyme disease with greater sensitivity than any other currently available method and that this tool can be used to detect the spirochete in tick and animal reservoirs.
    • Structural and molecular interactions of quaternary ammonium compounds with bacteria

      Kim, Sang-Nyun; Nauman, Robert K. (1995)
      The molecular interaction of quaternary ammonium compounds (QAC) with bacterial cells was investigated using various techniques to obtain the knowledge on what kinds of damage are done to the cell that results in cell death. Electron microscopy and flow cytometry were used to evaluate direct effects of didecyldimethyl ammonium chloride (DDAC) to Pseudomonas aeruginosa cells. The characteristic effects of DDAC observed on the bacterial structure were the formation and pinching off of blebs from the outer membrane and the formation of multilamellar structures within the nucleoid area. This was followed by coagulation of the entire nucleoid region and cytoplasm. The morphological changes observed in the intact gram-negative bacterium probably accounted for the bactericidal action of DDAC. An extensive theoretical discussion was provided to explain the observed effects of DDAC on the bacterial envelopes and cytoplasm. A flow cytometric approach was evaluated to determine its use for assessing antibacterial activity of a variety of QAC on P. aeruginosa ATCC 15442. LIVE/DEAD BacLight{dollar}\rm \sp{lcub}TM{rcub}{dollar} viability kit and the SYTOX{dollar}\rm \sp{lcub}TM{rcub}{dollar} kit were used as vital fluorescent stains. The DDAC had the greatest activity by converting the most number of bacteria in the population from green to red, followed by tetradecyl benzalkonium chlorides (BKC), hexadecyl BKC and dodecyl BKC. The flow cytometer provided an excellent means to assess the antibacterial activity of individual QAC. In order to decipher the interaction of DDAC with E. coli lipids, a steady-state and time-resolved emission spectra of 2-dimethylamino-6-lauroyinaphthalene (Laurdan), and steady-state anisotropy decay of 1,6-diphenyl-1,3,5,-hexatriene (DPH) were performed. Finally, {dollar}\sp{lcub}31{rcub}{dollar}P NMR spectroscopy allowed for convenient and qualitative measurements of the effects of DDAC on lipid polymorphism which seems relevant in the DDAC effects on membrane perturbation and possibly bactericidal activity. A schematic model for DDAC-membrane interactions that takes into account Laurdan spectral data, and turbidimetric data in conjunction with {dollar}\sp{lcub}31{rcub}{dollar}P NMR results was presented, which attempts to explain the different modes of action of DDAC on lipid supramolecular structures with respect to molar % of DDAC to lipids.