title: The exclusive use of flow cytometry to evaluate the antibiotic-susceptibility.
abstract: BACKGROUND
  Live and injured bacteria cannot be successfully discriminated using flow cytometric methods (FCM) with commercial live/dead staining agents because injured cells have intact cell membranes and are counted as live cells. We previously reported that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro (Microbiol. Immunol. 51:763-775, 2007).
  
  
  METHODS
  We report that EMA cleaves the chromosomal DNA of antibiotic-injured, but not live, Listeria monocytogenes. The combination of FCM and EMA treatment was evaluated as a rapid method to discriminate between live and antibiotic-injured L. monocytogenes. Additionally, we evaluated our methodology using blood from pediatric patients infected with other gram-negative and gram-positive bacteria.
  
  
  RESULTS
  For antibiotic-injured, but not live, L. monocytogenes in blood, photoactivated EMA suppressed SYTO9 staining, as the SYTO9 staining of the antibiotic-injured L. monocytogenes was weak compared with that of live cells. Similarly, the rapid and clear discrimination between live and injured bacteria (gram-negative and gram-positive) was performed using the blood of pediatric patients administered antibiotics.
  
  
  CONCLUSIONS
  The combination of FCM with EMA treatment is a rapid method for evaluating the susceptibility of live pathogens in infants with bacteremia without the need for bacterial culture.
  
  
  CONCLUSIONS
  This assay is more rapid than other currently available techniques due to the elimination of the time-consuming culture step and could be used in clinical settings to rapidly determine the success of antibiotic treatment in pediatric bacteremia through the discrimination of injured (i.e., susceptible to the administered antibiotics) and live pathogens.
authors.affiliation: Department of Bacteriology, Kyushu University, Higashi-ku, Fukuoka, Japan. t_soezim@morinagamilk.co.jp
authors.forename: Takashi
authors.initials: T
authors.lastname: Soejima
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Minami
authors.forename: Keiji
authors.initials: K
authors.lastname: Iwatsuki
mesh_terms: D000345:Affinity Labels / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001386:Azides / Q000494:pharmacology; D016470:Bacteremia / Q000188:drug therapy* / Q000378:metabolism / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology; D005434:Flow Cytometry / Q000379:methods*; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology; D006801:Humans; D007223:Infant; D008027:Light; D008089:Listeria monocytogenes / Q000187:drug effects* / Q000254:growth & development; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology; D050296:Microbial Viability / Q000187:drug effects*; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology; D019920:Photoaffinity Labels
chemical_list: D000345:Affinity Labels; D000900:Anti-Bacterial Agents; D001386:Azides; D004269:DNA, Bacterial; D019920:Photoaffinity Labels; C014535:8-azidoethidium