Susceptibility to hydrophobic molecules and phospholipid composition in Pasteurella multocida and Actinobacillus lignieresii

Abstract

Despite its typically gram-negative cell envelope ultrastructure, Pasteurella multocida is susceptible to the hydrophobic antibiotic novobiocin and is unable to initiate growth on MacConkey agar, a parameter often used to effect its differentiation from other members of the family Pasteurellaceae such as Actinobacillus lignieresii. However, growth on basal medium supplemented with individual selective factors and an agar diffusion assay revealed the bile salts contained in MacConkey agar to be toxic to both organisms. Four P. multocida surface hydrophobicity variants exhibited consistent in vitro susceptibility to the hydrophobic antibiotics novobiocin, rifamycin SV, and actinomycin D as determined by broth dilution. Readily extractable lipid fractions were obtained by chloroform-methanol extraction of freeze-dried whole cells from exponential-phase cultures. No major differences in total cellular readily extractable lipid content were observed among the P. multocida and A. lignieresii stra,ins examined, although hydrophobic P. multocida strains appeared to contain slightly more than did hydrophilic strains. Analytical thin-layer chromatography and quantitation of resolved readily extractable lipid components revealed the major cell envelope phospholipids of both organisms to be phosphatidylethanolamine and phosphatidylglycerol in a molar ratio of approximately 4:1 regardless of cell surface hydrophobicity properties. Similar results were obtained for Pseudomonas aeruginosa, which is notably refractory to hydrophobic molecules. These data support the conclusion that the permeability of the P. multocida cell envelope to structurally unrelated, hydrophobic molecules is not dependent on cell surface hydrophobicity and cannot be explained on the basis of anomalous polar lipid composition.

Carbohydrate patterns, cellular lipoquinones, fatty acids and phospholipids of the genus Pasteurella sensu stricto

“…This agrees with former investigations [13]. It is remarkable that there are no differences between the examined species.…”

“…So far only limited experience has been accumulated as to the taxonomic significance of phospholipid composition [13,33].…”

Kinetics and sequence specificity of processing of prepilin by PilD, the type IV leader peptidase of Pseudomonas aeruginosa

“…However, the kcat values were consistently 10-fold higher for substrate within membranes than for purified substrate when purified PilD was used, while the k5t value To determine whether phospholipids from bacterial membranes could provide a better medium for the cleavage reaction, the relative rates of cleavage of prepilin were determined in the presence of such phospholipids. Total phospholipids were extracted by methanol-chloroform extraction from Escherichia coli DH5ot and the pilA pilD double mutant P. aeruginosa B30-NP by established procedures (17) to determine their ability to substitute for crude membranes in the PilD in vitro assay. These results are shown in Table 3.…”

Influence of methylation on the antibacterial properties of triclosan in Pasteurella multocida and Pseudomonas aeruginosa variant strains

“…Therefore, the effect of triclosan and methyl triclosan on the ability to initiate growth was determined using a modification of a standardized disk agar diffusion bioassay (Hart and Champlin, 1988 …”

“…Enhanced uptake of the hydrophobic probe 1-N-phenylnaphthylamine relative to the refractory control organisms P. aeruginosa and Escherichia coli suggested the presence of regions of surface-exposed phospholipid bilayer in the P. multocida outer membrane. It was concluded that the extreme susceptibility of P. multocida to triclosan (Champlin et al, 2005) as well as other nonpolar antimicrobial agents (Hart and Champlin, 1988) is due to the general inability of the outer membrane to exclude hydrophobic molecules. Low triclosan concentrations which inhibited growth of untreated P. multocida cultures were only effective for P. aeruginosa when rendered susceptible using outer membrane-permeabilizing concentrations of polymyxin Bnonapeptide, compound 48/80, or ethylenediaminetetraacetate (Champlin et al, 2005).…”