The Ltnα and Ltnβ containing fractions were pooled separately and subsequently subjected to rotary evaporation www.selleckchem.com/products/MS-275.html to remove all propan-2-ol before freeze-drying of the peptides. The Ltnα and Ltnβ peptides were weighed in μg quantities using a Mettler UMT2 micro-balance. Antibiotic disc-based assessment of antimicrobial

sensitivity and synergy The sensitivities of S. Typhimurium LT2, C. sakazakii 6440, S. aureus, and E. faecium strains to a variety of JSH-23 in vivo antibiotics were determined by antibiotic disc diffusion assays as described previously [46]. Briefly, stationary-phase cultures (16 h) were diluted to 107 CFU/ml and swabbed onto Mueller Hinton, LB or BHI agar plates. Six PRN1371 mm antibiotic discs (Oxoid) infused with specific antibiotics were placed on the agar plates. On the same plate lacticin 3147 (1.2, 1.9 or 2.5 μg) was added to a second antibiotic-containing

disc and to a blank disc (control). Following overnight incubation (16 h) at 37°C, the resultant zones of inhibition were measured. The antibiotic discs employed included cefotaxime, novobiocin, cefoperazone, teicoplanin, ceftazidime, cefaclor, cephradine, cefaclor (30 μg), bacitracin, imipenem, fusidic acid (10 μg), penicillin G (5 μg), oxacillin (1 μg), colistin sulphate (polymyxin E) (25 μg) and polymyxin B (300U). Minimum inhibitory concentrations MIC determinations were carried out in triplicate in 96 well microtitre plates as previously described by Wiedemann et al., 2006. Briefly, bacterial strains were grown overnight in the appropriate conditions and medium, subcultured into fresh broth and allowed to grow to an OD600nm of ~0.5. Serial two-fold dilutions of the lacticin 3147, polymyxin B or colistin sulphate were made in the growth medium of the respective strain. Bacteria were then diluted and added to each microtitre well resulting in a final concentration of 105 cfu/ml in each 0.2 ml GNA12 MIC test well. After incubation

for 16 h at 37°C, the MIC was read as the lowest peptide concentration causing inhibition of visible growth. Checkerboard assay for combining antimicrobials In order to analyse combinations of two different antimicrobials (e.g. X and Y), the minimum inhibitory concentration of each antimicrobial has to be defined against a specific strain. Once this is known a 2-fold serial dilution of X is made horizontally in broth (50 ul) in a microtitre plate beginning at 8 x MIC for X. In a second microtitre plate, a similar dilution of Y is created and then 50 ul of this is added vertically to the original microtitre plate containing the dilution of X. Bacteria were then added in the same fashion as performed for the singular peptide minimum inhibitory assays described previously. Fractional Inhibitory Concentration (FIC) index is defined by the following equation: FIC = FICX + FICY = (X/MICX) + (Y/MICY).