Outer membrane permeability for nonpolar antimicrobial agents underlies extreme susceptibility of Pasteurella multocida to the hydrophobic biocide triclosan

Abstract

Pasteurella multocida exhibits nonspecific susceptibility to nonpolar antimicrobial agents such as triclosan, despite possessing an ultrastructurally typical gram-negative cell envelope. Capsulated and noncapsulated cell surface variants were examined to investigate the role outer membrane permeability plays in triclosan susceptibility. Test strains were unable to initiate growth in the presence of bile salts and were susceptible to triclosan with minimal inhibitory concentrations (MICs) ranging from 0.06 to 0.25 μg/ml. Disk agar diffusion bioassays revealed triclosan susceptibility to be dose dependent and all strains were susceptible to the hydrophobic antibiotics novobiocin, rifamycin SV, and chloramphenicol. Triclosan minimal bactericidal concentrations were greater than MICs, thereby suggesting that dose dependency reflected both bacteriostatic and bactericidal effects. Total and viable cell density growth kinetic determinations revealed a triclosan concentration of 2.0 μg/ml resulted in loss of batch culture viability within 4–24 h. Concentrations of 0.02 and 0.2 μg/ml exerted either a bacteriostatic or bactericidal effect depending on the strain. Uptake of the hydrophobic probe 1- N-phenylnaphthylamine was greater in P. multocida strains than refractory control organisms Pseudomonas aeruginosa and Escherichia coli thereby suggesting the presence of phospholipid bilayer regions in the outer membrane. Because triclosan inhibits a conserved enoyl-ACP reductase necessary for bacterial fatty acid biosynthesis, these data support the notion that extreme susceptibility in P. multocida is due to the general inability of the outer membrane to exclude nonpolar compounds. Moreover, susceptibility is independent of the presence of capsular material and the biocide is bactericidal in a concentration dependent manner.