Disparate efficacy of tobramycin on Ca2+-, Mg2+-, and HEPES-treated Pseudomonas aeruginosa biofilms

Abstract

Mucoid exopolysaccharide (MEP) obtained from Pseudomonas aeruginosa 579 was suspended in 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) pH 7.2 containing 0.1-10.0 mM of CaCl2∙2H2O or MgCl2∙4H2O. MEP treated with HEPES or <5.0 mM of the Ca2+ or Mg2+ salts remained soluble and bound tobramycin in an equilibrium dialysis bioassay. MEP treated with 5.0 or 10.0 mM of the Ca2+ or Mg2+ salts did not bind tobramycin. Five and 10 mM Ca2+-treated MEP precipitated but Mg2+-treated MEP did not. Pseudomonas aeruginosa 579 biofilms formed using a defined growth medium having <1 mM Ca2+ or Mg2+ were treated for 1 h with 10 mM HEPES ± 5.0 mM CaCl2∙2H2O or MgCl2∙4H2O, prior to an 8-h exposure to HEPES, or the defined growth medium, ± 125 μg/mL of tobramycin. The tobramycin kill kinetics for the HEPES-, Mg2+-, and Ca2+-treated biofilms were similar and gradual from T = 0–6 h. The viability of the HEPES- and Mg2+-treated populations declined sharply (from 6 to 8 h). Bacteria dispersed from the MEP in control biofilms at 0 and 8 h did not grow in the presence of 7.81 μg/mL of tobramycin. Thus, binding of tobramycin of P. aeruginosa 579 MEP may not be as influential to the impediment of tobramycin diffusion as is the steric hindrance imposed by the Ca2+ condensation of the polymer. Key words: Pseudomonas aeruginosa, mucoid exopolysaccharide, tobramycin, biofilm.