Native Pig Neutrophil Products: Insights into Their Antimicrobial Activity

Abstract

Cationic antimicrobial peptides are molecules with potential applications for treating infections due to their antimicrobial and immunomodulatory properties. The aim of this work was to explore the antimicrobial activity and mechanisms of action of a porcine neutrophil cathelicidin mixture (MPPN). Gram-positive and Gram-negative bacteria were used to determine the minimum inhibitory concentration (MIC) and experiments of both time-kill kinetics and effects on growth curves were performed. Planar black lipid bilayer conductance was measured to analyze the interaction of MPPN with lipid bilayers. Visualization of bacterial surfaces and membrane alterations was achieved using atomic force microscopy and transmission electron microscopy. The effects on the activity of efflux pumps (EPs) were studied with an intracellular accumulation of acridine orange (AO) assay. In E. coli, MPPN behaves as a bactericide at high concentrations and as a bacteriostatic at lower concentrations. The bacteriostatic effect was also observed for slightly shorter periods in S. enterica. The mixture was not active on S. aureus. The increase in AO accumulation in the presence of MPPN indicates that, at least in E. coli, the mixture causes inhibition of the EP function. Observed and detected variable conductance events demonstrate a strong MPPN effect on lipid bilayers. Damage to the structure of treated E. coli indicates that MPPN induces alterations in the bacterial surface. The use of AMPs capable of inhibiting EP can be seen as a good tool to combat antimicrobial resistance since they could be used alone or in combination with other conventional antibiotics to which bacteria have become resistant.

Keywords: antimicrobial peptides; atomic force microscopy; black lipid bilayer; cathelicidins; efflux pumps; membrane disruption; transmission electron microscopy.

Figure 1

Growth curves of (a) E. coli ATCC 25922 and (b) S. enterica ATCC 13076 treated with 68.7 μg/mL (MIC) (red line), 34.4 μg/mL (1/2 MIC) (blue line). Control without MPPN (green line).

Figure 2

Time–kill curves. (a) E. coli ATCC 25922 and (b) S. enterica ATCC 13076 treated with 68.7 μg/mL (MIC) (red line) or 34.4 μg/mL (1/2 MIC) (blue line). Control without MPPN (green line).

Figure 3

Percentage of acridine orange (AO) accumulation in the presence of the efflux inhibitor PaβN and sub-inhibitory concentrations of MPPN in E. coli (a) and in S. enterica (b). Control: bacteria plus AO.

Figure 4

Records of the membrane conductance events that occurred upon addition of MPPN to the solution (1 M KCl) bathing a black planar lipid bilayer at a voltage of −50 mV. Concentration of 41.13 ng/mL. The scale to interpret the registers is represented as time (minutes) on the x-axis and conductance (nS) on the y-axis.

Figure 5

Amplitude and topography AFM images of E. coli obtained at a 25 μm² scan size: (a) untreated E. coli, (b) E. coli treated at MIC (68.7 μg/mL) of MPPN, and (c) E. coli treated with 1/2 MIC (34.4 μg/mL) of MPPN. Black arrow indicates a ghost bacterium.

Figure 6

TEM images of the E. coli ATCC 25922 (a) untreated and (b,c) treated at the MIC of MPPN. In (b), the arrow indicates a waved outer membrane and, in (c), the arrow indicates cell envelope disruptions.