title: Comparative analysis of selected methods for the assessment of antimicrobial and membrane-permeabilizing activity: a case study for lactoferricin derived peptides.
abstract: BACKGROUND
  Growing concerns about bacterial resistance to antibiotics have prompted the development of alternative therapies like those based on cationic antimicrobial peptides (APs). These compounds not only are bactericidal by themselves but also enhance the activity of antibiotics. Studies focused on the systematic characterization of APs are hampered by the lack of standard guidelines for testing these compounds. We investigated whether the information provided by methods commonly used for the biological characterization of APs is comparable, as it is often assumed. For this purpose, we determined the bacteriostatic, bactericidal, and permeability-increasing activity of synthetic peptides (n = 57; 9-13 amino acid residues in length) analogous to the lipopolysaccharide-binding region of human lactoferricin by a number of the most frequently used methods and carried out a comparative analysis.
  
  
  RESULTS
  While the minimum inhibitory concentration determined by an automated turbidimetry-based system (Bioscreen) or by conventional broth microdilution methods did not differ significantly, bactericidal activity measured under static conditions in a low-ionic strength solvent resulted in a vast overestimation of antimicrobial activity. Under these conditions the degree of antagonism between the peptides and the divalent cations differed greatly depending on the bacterial strain tested. In contrast, the bioactivity of peptides was not affected by the type of plasticware (polypropylene vs. polystyrene). Susceptibility testing of APs using cation adjusted Mueller-Hinton was the most stringent screening method, although it may overlook potentially interesting peptides. Permeability assays based on sensitization to hydrophobic antibiotics provided overall information analogous - though not quantitatively comparable- to that of tests based on the uptake of hydrophobic fluorescent probes.
  
  
  CONCLUSIONS
  We demonstrate that subtle changes in methods for testing cationic peptides bring about marked differences in activity. Our results show that careful selection of the test strains for susceptibility testing and for screenings of antibiotic-sensitizing activity is of critical importance. A number of peptides proved to have potent permeability-increasing activity at subinhibitory concentrations and efficiently sensitized Pseudomonas aeruginosa both to hydrophilic and hydrophobic antibiotics.
authors.affiliation: Department of Microbiology and Parasitology, University of Navarra, 31080 Pamplona, Spain. ssanchez@alumni.unav.es
authors.forename: Susana
authors.initials: S
authors.lastname: Sánchez-Gómez
authors.forename: Marta
authors.initials: M
authors.lastname: Lamata
authors.forename: José
authors.initials: J
authors.lastname: Leiva
authors.forename: Sylvie E
authors.initials: SE
authors.lastname: Blondelle
authors.forename: Roman
authors.initials: R
authors.lastname: Jerala
authors.forename: Jörg
authors.initials: J
authors.lastname: Andrä
authors.forename: Klaus
authors.initials: K
authors.lastname: Brandenburg
authors.forename: Karl
authors.initials: K
authors.lastname: Lohner
authors.forename: Ignacio
authors.initials: I
authors.lastname: Moriyón
authors.forename: Guillermo
authors.initials: G
authors.lastname: Martínez-de-Tejada
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects* / Q000254:growth & development; D002463:Cell Membrane Permeability / Q000187:drug effects*; D003470:Culture Media / Q000737:chemistry; D007781:Lactoferrin / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests / Q000592:standards*; D050296:Microbial Viability
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D003470:Culture Media; D007781:Lactoferrin