However, when using concentrations above the MIC, LP5 targets the bacterial membrane leading to disruption of the bacterial membrane. Results and discussion Determination of MIC of LP5 against S. aureus Given PKC412 in vitro that the lysine-peptoid LP5 has antimicrobial activity toward a number of bacterial and fungal pathogens, we investigated how LP5 interacts with and affects the pathogenic bacterium S. aureus. We tested the MIC of LP5 against two S. aureus strains, 8325–4, a laboratory strain of
human origin , and the clinically relevant community acquired strain USA300 . MIC was in the range of 16 to 32 μg/ml for both strains. Permeabilization of the S. aureus membrane by LP5 is concentration dependent Selleckchem ARRY-162 Many AMPs interact with the bacterial membrane, leading to pore-formation and subsequently leakage of intracellular components . Therefore, to determine whether LP5 influences S. aureus membrane Evofosfamide manufacturer structure, we investigated membrane integrity
by measuring ATP leakage. We found that increasing concentrations of LP5 added to S. aureus 8325–4 at time-point 0, lead to a gradual increase in ATP leakage from the cells (Figure 2). The addition of 1000 μg/ml of LP5 most likely resulted in an abrupt destruction of the bacterial membrane, since no intracellular ATP was detectable and an immediate increase in extracellular ATP was detected. However, at low concentrations of LP5 only limited leakage of ATP was observed, showing that the leakage of ATP is concentration dependent. Thus, in this experiment we find
that LP5 targets the membrane at high concentrations whereas little effect on the membrane was seen at low concentrations. Figure 2 Measurement of ATP leakage from S . aureus 8325–4 after treatment with LP5. Measurement of intracellular (IC) and extracellular (EC) ATP after treatment with increasing concentrations of LP5 (0–1000 μg/ml). These observations agree well with the killing kinetics Methocarbamol of LP5 against S. aureus (Figure 3). Here, we performed dose-dependent time-kill assays at two concentrations representing 1 × MIC and 5 × MIC (Figure 3). LP5 reduced the colony forming unit (CFU) counts by 2 log units during the first 30 min of the experiment at 5 × MIC. Thereafter, the killing rate gradually decreased and after the 5 h time course approached a total reduction of CFU count by 4 log units. At 1 × MIC LP5 did not reduce the CFU within the 5 h of exposure (Figure 3) and the exposed bacteria resumed growth when transferred to media without LP5 (data not shown). Thus, at this concentration LP5 does not to kill S. aureus, instead it prevents growth, indicating that LP5 does not affect the cell membrane but rather has an intracellular target. This notion is supported by the finding that concentrations several fold above the MIC is needed to see ATP leakage.