title: Evaluation of Propidium Monoazide and Quantitative PCR To Quantify Viable Campylobacter jejuni Biofilm and Planktonic Cells in Log Phase and in a Viable but Nonculturable State.
abstract: Despite being considered fragile and fastidious, Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in the developed world. C. jejuni survives stresses by forming biofilms or entering a viable but nonculturable (VBNC) state. To investigate the number of viable cells in samples exposed to low nutrient and temperature stress, a novel method, propidium monoazide quantitative PCR (PMAqPCR), was compared with Bac Light biovolume analysis and conventional plate counting for the enumeration of C. jejuni-removed biofilm cells and separately grown planktonic cells in late log phase (24 h). There were no significant differences between viable cell counts obtained using PMAqPCR and those from plate counts or Bac Light biovolume analyses for each sample, confirming that this method provides results consistent with those from accepted enumeration methods (P > 0.05). To induce a VBNC state, C. jejuni planktonic cells and dislodged and washed biofilm cells were separately incubated in phosphate-buffered saline at 4°C for up to 60 days. Even when cells exposed to stress were provided with enrichment in Bolton broth before plating, treated biofilm cells lost culturability by day 10, whereas their planktonic counterparts remained culturable to day 60. The nonculturable biofilm cells remained viable in high numbers to day 60, and viable cell counts from the PMAqPCR (6.15 log cells per ml) were not significantly different from those obtained using the Bac Light assay (6.98 log cells per ml) (P > 0.05), confirming that this novel method is also reliable for cells exposed to stress for extended periods. PMAqPCR shows promise for analysis where C. jejuni exists in biofilms or in the VBNC state. Adopting PMAqPCR in routine monitoring, in conjunction with improved biofilm cell collection methods, will allow for more accurate enumeration of viable and potentially virulent cells, leading to improved sanitation and reduced incidence of infection.
authors.affiliation: Department of Biology, Faculty of Science and Environmental Studies, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, Canada P7B 5E1.
authors.forename: Brenda
authors.initials: B
authors.lastname: Magajna
authors.affiliation: Department of Biology, Faculty of Science and Environmental Studies, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, Canada P7B 5E1. heidi.schraft@lakeheadu.ca.
authors.forename: Heidi
authors.initials: H
authors.lastname: Schraft
mesh_terms: D001386:Azides; D018441:Biofilms / Q000254:growth & development*; D002169:Campylobacter Infections / Q000517:prevention & control; D016123:Campylobacter jejuni / Q000166:cytology* / Q000254:growth & development / Q000302:isolation & purification*; D015169:Colony Count, Microbial; D011419:Propidium / Q000031:analogs & derivatives*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
chemical_list: D001386:Azides; C533957:propidium monoazide; D011419:Propidium