title: Alternative methods to limit extracellular bacterial activity for enumeration of intracellular bacteria.
abstract: The gentamicin survival assay, a method routinely used to estimate bacterial infection of eukaryotic host cells, depends on the presumed limited penetration of gentamicin across the eukaryotic cell membrane. However, some studies have suggested that gentamicin may in fact enter eukaryotic cells and kill intracellular bacteria. In this study we devised alternative methods to enumerate intracellular Salmonellae using a lytic bacteriophage, SP6, and an amino acid auxotroph, Pro- mutant, which replicates selectively within host cells in the presence of its uptake inhibitor, 3,4-dehydro-L-proline. The conventional gentamicin survival assay was systematically compared with the alternative methods for the enumeration of intracellular Salmonellae. We found that gentamicin decreases the survival of intracellular Salmonellae when added to extracellular media at concentrations above 20 microg/ml. The alternative methods do not suffer from this disadvantage, suggesting that they should be used to replace the gentamicin survival assay. In addition, the proline auxotroph method could be applied to detect bacterial release from host cells.
authors.affiliation: Genome Research Center for Enteropathogenic Bacteria and Research Institute of Vibrio Infection, South Korea.
authors.forename: Hyun-Ju
authors.initials: HJ
authors.lastname: Kim
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Kim
authors.forename: Yeongjin
authors.initials: Y
authors.lastname: Hong
authors.forename: Joon Haeng
authors.initials: JH
authors.lastname: Rhee
authors.forename: Hyon E
authors.initials: HE
authors.lastname: Choy
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015169:Colony Count, Microbial / Q000379:methods*; D005110:Extracellular Space / Q000382:microbiology*; D005839:Gentamicins / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006367:HeLa Cells / Q000382:microbiology; D006801:Humans; D042541:Intracellular Space / Q000382:microbiology*; D018613:Microscopy, Confocal; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D012483:Salmonella Phages; D012486:Salmonella typhimurium / Q000187:drug effects / Q000254:growth & development* / Q000821:virology; D016019:Survival Analysis
chemical_list: D000900:Anti-Bacterial Agents; D005839:Gentamicins; D049452:Green Fluorescent Proteins; C002195:3,4-dehydroproline; D011392:Proline