SD standard deviation, n.d. not determined To address additive or synergistic effects of AMPs, we performed a model assay using N. farcinica and a combination of LL-37 and HNP 1-3 (Figure 2). Since the combination of the two AMPs exhibited nocardial killing comparable to each peptide alone at twofold higher concentrations, we found

additive activity PR-171 cell line of the two AMPs. Figure 2 Additive activity of the two AMPs HNP 1-3 and LL-37 in a colony forming unit (CFU) assay against N . farcinica ATCC 3318. A combination of HNP 1-3 and LL-37 exhibited killing comparable to each peptide alone at twofold higher concentrations (i.e. 78.9% CFU reduction by 8 μg/ml HNP 1-3 in combination with 8 μg/ml LL-37 compared to 68.5% CFU reduction by 16 μg/ml LL-37 or 45.6% reduction by 16 μg/ml HNP 1-3 alone). Data are results of a single assay. In contrast to results with N. farcinica and N. nova, hBD-3 and LL-37 did not show

antinocardial activity against N. asteroides ATCC 19247 (Figure 1C). Only human α-defensins HNP 1-3 were found to be active against N. asteroides with LD90 of 32 μg/ml. N. brasiliensis ATCC 19296 proved to be resistant to all human AMPs tested since neither HNP 1-3 nor hBD-3 or LL-37 exhibited killing activity in concentrations up to 64 μg/ml (Figure 1D). Remarkably, stronger growth of N. brasiliensis was observed with all three AMPs investigated. Enhanced growth was not found after incubation with equivalent concentrations of DPY (data not shown). To investigate whether proteolytic degradation of AMPs by N. brasiliensis-derived proteases might play a role, we added a protease inhibitor mix during incubation in CFU assays. Protease inhibitors see more were not able to alter the observed AMP resistance of N. brasiliensis, yet enhanced growth of N. brasiliensis after co-incubation with protease inhibitors could be observed again(data not shown). Activity of bovine AMPs ADP ribosylation factor against Nocardia species CFU-assays revealed activity of all tested bovine AMPs against N. farcinica ATCC 3318 (Figure 3A). Neutrophil-derived indolicidin

and bovine β-defensin LAP showed potent killing with LD90 of 16 μg/ml respectively. Bovine TAP was also active, LD90 proved to be 32 μg/ml. All bovine AMPs revealed at least comparable or greater activity at 32 μg/ml against N. farcinica than levofloxacin. Accordingly, bovine indolicidin exhibited killing activity against N. nova (LD90 8 μg/ml) and N. asteroides (LD90 64 μg/ml) (Table 1). Figure 3 Activity of bovine AMPs TAP, LAP indolicidin and levofloxacin (killing control) against A N. farcinca ATCC 3318 (p < 0.05 for all tested substances), B N. brasiliensis ATCC 19296 (indolicidin and levofloxacin p < 0.05) was investigated using a colony forming unit (CFU) assay. Data are means (percent CFU reduction) of at least two independent sets of experiments with each peptide and each Nocardia species. In contrast to human AMPs, bovine indolicidin exhibited activity against N.