5). The cultures were diluted to a concentration of 1 × 105 cfu/ml in a 0.2 ml volume. The purified peptides were resuspended in BHI broth to a stock concentration of 60 μM and adjusted to a 15 μM starting concentration. Two-fold dilutions of the peptides were made in the 96-well plates, which were subsequently inoculated
with the bacterial strains and incubated at 37°C for 16 hours. The minimum inhibitory concentration (MIC) was read as the minimum peptide concentration inhibiting visible growth of the bacterial strains. Inoculum preparation L. monocytogenes EGDe was grown overnight in BHI broth at 37°C from an isolated colony growing on a BHI agar plate containing 7.5 mg/L chloramphenicol. The overnight culture was diluted in order to facilitate its administration in a dose of 1 × 105 cfu/200 μl PBS. Mouse model All procedures Pembrolizumab in vitro involving animals were approved by the UCC Animal Experimentation Ethics Committee and carried out by a licensed individual with an ethical approval number of 2011/017. For the L. monocytogenes murine model, 15 Balb/c female mice (7 weeks this website old, 15 g ± 2 g in weight) were divided into three groups (A, B and C) with each group containing 5 mice. At T0 on day 1, all groups were infected with 1 × 105 viable cells of L. monocytogenes EDGe::pPL2luxpHELP in a 200 μl dose of PBS via the intraperitoneal (I.P.) route. At T0.5hrs, mice in group A
were administered PBS (control), group B were treated with nisin A (58.82 mg/kg) and
group C treated with nisin V (58.82 mg/kg). Both PBS and the nisin peptides were administered in 200 μl doses via the I.P. route. On day 3, the mice were anaesthetised using a mixture of aerosolised isoflurane and oxygen. Bioluminescence was monitored using an IVIS® Imaging System 100 series (Xenogen Corporation, Almeda, CA) with a 5 minute exposure time. Immediately afterward, the mice were euthanized and the livers and spleens were extracted. The organs were mechanically disrupted and serial dilutions Cytidine deaminase made which were subsequently plated in 100 μl volumes on BHI agar plates containing chloramphenicol 7.5 mg/L in order to enumerate L. monocytogenes present in each organ. Luminescence quantification IVIS imaging software was used to carry out quantification of luminescence. Bioluminescence emitted from the infection site was measured as total counts across the region of interest (designated relative light units – “RLU”) and was averaged across all groups of mice. The reduction in luminescence was quantified and represents a comparison with the luminescence from mice administered PBS control at the same time point. Statistical analysis CFU and RLU data was transformed to log10 prior to analysis. All comparisons were based on the mean ± standard error of the mean (SEM). Parametric data was analysed using one way analysis of variance (ANOVA) with post hoc comparison using the Student-Newman-Keuls method.