title: Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real-time quantitative PCR.
abstract: OBJECTIVE
  To optimize ethidium monoazide (EMA) coupled with real-time quantitative PCR (qPCR) and to evaluate its environmental applicability on quantifying viable legionellae in water and biofilm of cooling towers and hot water systems.
  
  
  RESULTS
  EMA (0.9-45.5 microg ml(-1)) and propidium monoazide (PMA, 0.9 and 2.3 microg ml(-1)) combined with qPCR (i.e. EMA-qPCR and PMA-qPCR, respectively) were applied to unheated and heated (70 degrees C for 30 min) Legionella pneumophila to quantify viable cells, which was also simultaneously determined by BacLight Bacterial Viability kit with epifluorogenic microscopic enumeration (BacLight-EM). The effects of nontarget microflora and sample matrix on the performance of EMA-qPCR were also evaluated. In comparison with BacLight-EM results, qPCR with EMA at 2.3 microg ml(-1) was determined as the optimal EMA-qPCR assay, which performed equally well as PMA-qPCR for unheated Leg. pneumophila but better than PMA-qPCR for heated Leg. pneumophila (P < 0.05). Moreover, qPCR with EMA at 2.3 microg ml(-1) accurately quantified viable Leg. pneumophila, Legionella anisa and Legionella-like amoebal pathogens 6 (LLAP 6) without interferences by heated legionellae, unheated nonlegionellae cells and cooling tower water matrix (P > 0.05). As for water and biofilm samples collected from cooling towers and hot water systems, the viable legionellae counts determined by EMA-qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR.
  
  
  CONCLUSIONS
  The qPCR with EMA at 2.3 microg ml(-1) may accurately quantify viable legionellae (including fastidious LLAP 6) and Leg. pneumophila pretreated with superheating and is applicable for water and biofilm samples obtained from cooling towers and hot water systems.
  
  
  CONCLUSIONS
  The EMA-qPCR assay may be useful in environmental surveillance for viable legionellae and in evaluation of superheating efficacy against legionellae.
authors.affiliation: Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei, Taiwan.
authors.forename: N-T
authors.initials: NT
authors.lastname: Chen
authors.affiliation: Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei, Taiwan.|Center for Research on Environmental and Occupational Health, National Taiwan University, Taipei, Taiwan.|Research Center for Genes, Environment and Human Health, National Taiwan University, Taipei, Taiwan.
authors.forename: C-W
authors.initials: CW
authors.lastname: Chang
mesh_terms: D001386:Azides / Q000737:chemistry*; D018441:Biofilms; D007875:Legionella / Q000235:genetics / Q000302:isolation & purification*; D016952:Legionella pneumophila / Q000302:isolation & purification; D050296:Microbial Viability; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives / Q000737:chemistry; D014871:Water Microbiology
chemical_list: D001386:Azides; C533957:propidium monoazide; D011419:Propidium; C014535:8-azidoethidium