• Role of subtyping in detecting Salmonella cross contamination in the laboratory.

      De Lappe, Niall; Connor, Jean O; Doran, Geraldine; Devane, Genevieve; Cormican, Martin; National Salmonella Reference Laboratory, Department of Medical Microbiology, Galway University Hospitals, Galway, Ireland. niall.delappe@hse.ie (2009)
      BACKGROUND: With the exception of M. tuberculosis, little has been published on the problems of cross-contamination in bacteriology laboratories. We performed a retrospective analysis of subtyping data from the National Salmonella Reference Laboratory (Ireland) from 2000-2007 to identify likely incidents of laboratory cross contamination. METHODS: Serotyping and antimicrobial susceptibility testing was performed on all Salmonella isolates received in the NSRL. Phage typing was performed on all S. Typhimurium and S. Enteritidis isolates while multi-locus variance analysis (MLVA) was performed on selected S. Typhimurium isolates. Pulsed field gel electrophoresis (PFGE) using the PulseNet standard protocol was performed on selected isolates of various serovars. RESULTS: Twenty-three incidents involving fifty-six isolates were identified as likely to represent cross contamination. The probable sources of contamination identified were the laboratory positive control isolate (n = 13), other test isolates (n = 9) or proficiency test samples (n = 1). CONCLUSION: The scale of laboratory cross-contamination in bacteriology is most likely under recognized. Testing laboratories should be aware of the potential for cross-contamination, regularly review protocols to minimize its occurrence and consider it as a possibility when unexpected results are obtained.
    • Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane.

      Knodler, Leigh A; Winfree, Seth; Drecktrah, Dan; Ireland, Robin; Steele-Mortimer, Olivia; Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT 59840, USA. lknodler@niaid.nih.gov (2009-11)
      The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella-containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono-ubiquitinated. Substitution of these six lysine residues with arginine, SopB-K(6)R, almost completely eliminated SopB ubiquitination. We found that ubiquitination does not affect SopB stability or membrane association, or SopB-dependent events in SCV biogenesis. However, two spatially and temporally distinct events are dependent on ubiquitination, downregulation of SopB activity at the plasma membrane and prolonged retention of SopB on the SCV. Activation of the mammalian pro-survival kinase Akt/PKB, a downstream target of SopB, was intensified and prolonged after infection with the SopB-K(6)R mutant. At later times, fewer SCV were decorated with SopB-K(6)R compared with SopB. Instead SopB-K(6)R was present as discrete vesicles spread diffusely throughout the cell. Altogether, our data show that ubiquitination of SopB is not related to its intracellular stability but rather regulates its enzymatic activity at the plasma membrane and intracellular localization.