title: The effects of hyperosmosis or high pH on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa: an in vitro study.
abstract: OBJECTIVE
  To investigate the effect of hyperosmotic hyperosmosis or alkaline stress on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa.
  
  
  METHODS
  Biofilms were grown on glass cover slips suspended in bacterial inoculate for 96 h, after which the cover slips with attached biofilms were immersed in brain heart infusion broth (BHI-broth) with 6 mol L(-1) sodium chloride (NaCl) representing the hyperosmotic group or Ca(OH)(2), pH 12.1, representing the alkaline group. Two per cent sodium hypochlorite and BHI- broth served as positive and negative controls, respectively. After treatment, the biofilms were washed, harvested and plated on blood-agar plates after serial dilution. The bactericidal effect was assessed by determining the colony-forming units (CFU). The effect on the biofilm mass was imaged with confocal laser scanning microscopy (CLSM).
  
  
  RESULTS
  Hyperosmosis reduced the CFU of both species significantly after 72 h (P < 0.0001). After 168 h, P. aeruginosa was eradicated and the E. faecalis reduction was more than 99%. High pH could not induce a significant bacterial reduction. CLSM revealed dense flocculation of the biofilms incubated in alkaline broth.
  
  
  CONCLUSIONS
  Hyperosmosis effectively reduced a dual-species biofilm of E. faecalis and P. aeruginosa, whilst high pH had limited bactericidal effect in this model.
authors.affiliation: Department of Cariology, Endodontology, Pedodontology and Oral Microbiology, Academic Centre for Dentistry Amsterdam, Amsterdam, The Netherlands. s.vd.waal@acta.nl
authors.forename: S V
authors.initials: SV
authors.lastname: van der Waal
authors.forename: L W M
authors.initials: LW
authors.lastname: van der Sluis
authors.forename: A R
authors.initials: AR
authors.lastname: Özok
authors.forename: R A M
authors.initials: RA
authors.lastname: Exterkate
authors.forename: J
authors.initials: J
authors.lastname: van Marle
authors.forename: P R
authors.initials: PR
authors.lastname: Wesselink
authors.forename: J J
authors.initials: JJ
authors.lastname: de Soet
mesh_terms: D000468:Alkalies / Q000494:pharmacology; D058491:Bacterial Load / Q000187:drug effects; D001431:Bacteriological Techniques; D018441:Biofilms / Q000187:drug effects*; D018533:Biomass; D002126:Calcium Hydroxide / Q000494:pharmacology*; D018920:Coculture Techniques; D003470:Culture Media; D013293:Enterococcus faecalis / Q000187:drug effects*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D050296:Microbial Viability / Q000187:drug effects; D018613:Microscopy, Confocal; D009995:Osmosis / Q000502:physiology*; D011550:Pseudomonas aeruginosa / Q000187:drug effects*; D012965:Sodium Chloride / Q000494:pharmacology*; D012973:Sodium Hypochlorite / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
chemical_list: D000468:Alkalies; D003470:Culture Media; D012965:Sodium Chloride; D012973:Sodium Hypochlorite; D002126:Calcium Hydroxide