Native Pig Neutrophil Products: Insights into Their Antimicrobial Activity

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.


Introduction
The increase in bacterial resistance to antimicrobials is a relevant threat to public health, and the treatment of infections caused by multidrug-resistant bacteria (MDR) is a pressing need. Among the essential objectives of research in this field is the search for new antimicrobial agents. Antimicrobial peptides (AMPs) (natural and synthetic) have emerged as potential candidates and constitute an inexhaustible source of new antimicrobial molecules [1,2]. Some of these molecules, present in most organisms as an important part of the immune innate response [3], play key roles in cellular differentiation processes, being the first defense line against a large number of pathogens. Natural peptides are also involved in physiological processes such as angiogenesis, cellular signaling and inflammatory responses [4,5]. In terms of their antimicrobial capabilities, many AMPs exhibit MPPN displayed significant antimicrobial activity on E. coli ATCC 25922 and S. enterica ATCC 13076, but not on S. aureus ATCC 29313. MPPN induced morphology alterations on the surface of E. coli, caused inhibition of the efflux pumps' function, and altered the conductance and stability of artificial lipid membranes even through formation of pores, suggesting that the mechanisms of action of MPPN involve membranes and inhibition of the efflux pumps.
A mixture of natural peptides from a crude extract of porcine neutrophils was obtained according to the method described by Wessely-Szponder et al. [13] and then lyophilized and stored at −80 • C. Peptides present in the mixture were identified using MALDI-TOF MS analysis (RTOF MS-built in the Institute of Physics, Division of Molecular Physics, UMCS Lublin, Poland, with an ion source MALDI). Parameters were previously described by Głuch et al., 2001 andGruszecka et al., 2008 [25,26]. Table 1 details the structure, sequences and size of the peptides [15]. Herein, we explore the antimicrobial activity and the mechanisms of action of a mixture of natural peptides belonging to the cathelicidin family, directly obtained from porcine neutrophils (MPPN), as previously described by Wessely et al. [14].
MPPN displayed significant antimicrobial activity on E. coli ATCC 25,922 and S. enterica ATCC 13076, but not on S. aureus ATCC 29313. MPPN induced morphology alterations on the surface of E. coli, caused inhibition of the efflux pumps' function, and altered the conductance and stability of artificial lipid membranes even through formation of pores, suggesting that the mechanisms of action of MPPN involve membranes and inhibition of the efflux pumps.
A mixture of natural peptides from a crude extract of porcine neutrophils was obtained according to the method described by Wessely-Szponder et al. [13] and then lyophilized and stored at −80 °C. Peptides present in the mixture were identified using MALDI-TOF MS analysis (RTOF MS-built in the Institute of Physics, Division of Molecular Physics, UMCS Lublin, Poland, with an ion source MALDI). Parameters were previously described by Głuch et al., 2001 andGruszecka et al., 2008 [25,26]. Table 1 details the structure, sequences and size of the peptides [15].

Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)
The antimicrobial activity of MPPN was initially evaluated by determining the MIC using the reference broth microdilution method in 96-well microtiter plates as recommended by CLSI [27]. In addition, the MBC of MPPN was determined by plating 100 µL from each well where no visible growth on tryptone soy agar (TSA) (Condalab, Madrid, Spain) was observed. Plates were incubated at 37 °C for 24 h. All experiments were performed in triplicate.

Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)
The antimicrobial activity of MPPN was initially evaluated by determining the MIC using the reference broth microdilution method in 96-well microtiter plates as recommended by CLSI [27]. In addition, the MBC of MPPN was determined by plating 100 µL from each well where no visible growth on tryptone soy agar (TSA) (Condalab, Madrid, Spain) was observed. Plates were incubated at 37 • C for 24 h. All experiments were performed in triplicate.

Growth Curves
A starting inoculum of 1 × 10 6 CFU/mL was used to determine the effect of MPPN on the growth. Briefly, MPPN at MIC and 1/2 MIC was added to cultures in cationadjusted Mueller-Hinton (CAMHB) and incubated in real-time reverse spin bioreactors RTS-1 (Biosan SIA, Riga, Latvia) at 37 • C for 24 h. Growth was measured non-invasively at an optical density of 850 nm every 15 min for 24 h. All measurements were performed in triplicate.

Time-Kill Curves
MPPN at concentrations identical to the determined MIC value and 1/2 MIC was added to the bacterial cultures (1 × 10 6 CFU/mL) and incubated for 24 h at 37 • C, with shaking at 200 rpm. Samples were aseptically obtained at 0, 1, 2, 4, 6 and 8 h, serially diluted in Ringer 1/4 and plated on TSA for colony counting. Plates were incubated for 24 h at 37 • C. The response of the strains to MPPN was determined based on a logarithmic decrease in viable bacteria. Time-kill assays were performed in triplicate.

Acridine Orange Intracellular Accumulation
To investigate the inhibition of the efflux machinery in the presence of different concentrations of MPPN, the accumulation of acridine orange (AO) in bacteria was calculated as previously described by Armengol et al. [28]. Briefly, assays were performed in 96-well flatbottomed microtiter plates. Overnight cultures were diluted in Ringer 1/4 solution to an OD 520nm of 1.5 and 100 µL was added to the wells previously filled with 100 µL of Ringer's solution. AO at 0.25 µg/mL was added and then the inoculated plates were incubated for 1 h with shaking, after which the fluorescence was measured in a FLUOstar OPTIMA fluorescence microplate reader (BMG Labtech, Ortenberg, Germany). The AO uptake was determined in the presence of 20 µg/mL of the efflux pump inhibitor PaβN, in the presence of sub-inhibitory concentrations of MPPN (1/2 MIC, 1/4 MIC, 1/8 MIC), and untreated bacteria was used as the control. The results were expressed as the percentage increase in fluorescence with respect to the control. All experiments were performed in triplicate.

Planar Lipid Bilayer Assays
To analyze a possible interaction of the peptides with lipid bilayers, artificial membranes were reconstituted from 1% (w/v) diphytanoylphosphatidylcholine (DiPhPC), a zwitterionic lipid, in n-decane. The bilayers were painted over a 0.8 mm diameter hole in a Teflon partition separating two compartments containing 5 mL each of 1 M KCl. Voltages were applied across this membrane via Ag/AgCl electrodes connected by a salt bridge, and the resulting current was amplified 10 9 -fold by a current amplifier and recorded on a Rikadenki R-01 strip chart recorder [29,30].

Atomic Force Microscopy (AFM)
AFM was used to visualize the bacterial surfaces of untreated and treated strains after exposure to MPPN at the MIC and 1 2 MIC. Bacteria were collected by centrifugation at 2000× g for 3 min and the pellet was resuspended in sterile water. A volume of 10 µL was applied to the MICA surface, dried at room temperature and then imaged in air by using an atomic force microscope XE-70 (Park Systems, Korea) [31]. All images were collected in non-contact mode using pyramidal-shaped silicon cantilevers with a spring constant of ±40 N/m and a resonance frequency of ±300 kHz, with the upper side coated with aluminum to increase the reflectivity of the laser beam [32]. Data collected during the surface scanning were converted into topography and amplitude images and analyzed using XEP and XEI software version 1.8.0 (Park Systems, Korea) to analyze the shape, structure and surface of the bacteria. AFM images were acquired with a scan size of 25 µm 2 at a scan rate of 0.3-0.6 Hz [33]. phosphate buffer for 30 min at room temperature. The fixed samples were centrifuged at 2000× g for 5 min and the pellet was resuspended in the fixing solution and stored at 4 • C. Bacteria were postfixed with osmium tetroxide (Sigma-Aldrich, Schnelldorf, Germany), dehydrated with acetone (Panreac, Barcelona, Spain), embedded in resin and sectioned with an ultramicrotome (Ultramicrotome UC7, Leica, Viena, Austria). Ultrathin sections (50-70 nm) were stained with 2% uranyl acetate (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min, a lead staining solution for 5 min and finally analyzed on a JEOL 1010 transmission electron microscope with a CCD Orius digital camera (Gatan) (400 kV of maximum TEM operating voltage; CCD active area 15 × 15 mm; readout speed 30/5 MHz; frame rate of ≤30 frames per second and exposure setting of 0.001-100 s).

Antimicrobial Activity (MIC and MBC)
MPPN had an MIC of 68.7 µg/mL in E. coli and S. enterica, while higher MICs (>275 µg/mL) were found in P. aeruginosa, A. baumannii, K. pneumoniae, E. faecalis and S. aureus. The MBC was >275 µg/mL for all strains, except for E. coli (137 µg/mL). Results are shown in Table 2.

Effect of MPPN on the Microbial Growth
As can be seen in Figure 1a

Time-kill Curves
In E. coli at the MIC, a reduction of 4 logs was observed and was maintained for up to eight hours. At ½ MIC, a reduction of approximately one log was observed after two hours and was maintained up to eight hours (Figure 2a). The effect on S. enterica at both MIC and ½ MIC is shown in Figure 2b.

Time-Kill Curves
In E. coli at the MIC, a reduction of 4 logs was observed and was maintained for up to eight hours. At 1/2 MIC, a reduction of approximately one log was observed after two hours and was maintained up to eight hours (Figure 2a). The effect on S. enterica at both MIC and 1/2 MIC is shown in Figure 2b.

Time-kill Curves
In E. coli at the MIC, a reduction of 4 logs was observed and was maintained for up to eight hours. At ½ MIC, a reduction of approximately one log was observed after two hours and was maintained up to eight hours (Figure 2a). The effect on S. enterica at both MIC and ½ MIC is shown in Figure 2b.

Acridine Orange Intracellular Accumulation
As AO is a good substrate for efflux pumps, and is a well-known resistance mechanism; its intracellular accumulation is enhanced by the inhibition of efflux pumps. Changes in AO accumulation in the presence of MPPN by both E. coli and S. enterica are shown in Figure 3. In this experiment, the control (untreated bacteria) was considered as 100% AO accumulation. In the experiments performed in E. coli, the presence of the efflux pump inhibitor PaβN caused an increase in AO accumulation by nearly 200%; MPPN at sub-inhibitory concentrations led to increases in AO accumulation by nearly 160% at ½ MIC, 180% at ¼ MIC and 120% at ⅛ MIC. (Figure 3a

Acridine Orange Intracellular Accumulation
As AO is a good substrate for efflux pumps, and is a well-known resistance mechanism; its intracellular accumulation is enhanced by the inhibition of efflux pumps. Changes in AO accumulation in the presence of MPPN by both E. coli and S. enterica are shown in Figure 3. In this experiment, the control (untreated bacteria) was considered as 100% AO accumulation. In the experiments performed in E. coli, the presence of the efflux pump inhibitor PaβN caused an increase in AO accumulation by nearly 200%; MPPN at subinhibitory concentrations led to increases in AO accumulation by nearly 160% at 1/2 MIC, 180% at 1/4 MIC and 120% at 1/8 MIC (Figure 3a

Planar Lipid Bilayer Assays
MPPNs were investigated for their ability to cause conductance events in an artificial planar lipid bilayer. The initial purpose was to explore if they were able to form transmembrane channels. As expected, and shown in Figure 4, some membrane conductance events were observed upon addition of MPPN at −50 mV. Furthermore, many of the events observed resulted in rapid conductance changes of variable magnitude and duration, often with short lifetimes followed by a return to baseline. The observed changes in membrane conductance suggest that MPPNs are membrane-active antimicrobial peptides but that they produce small transient transmembrane channels whose life is short. Moreover,

Planar Lipid Bilayer Assays
MPPNs were investigated for their ability to cause conductance events in an artificial planar lipid bilayer. The initial purpose was to explore if they were able to form transmembrane channels. As expected, and shown in Figure 4, some membrane conductance events were observed upon addition of MPPN at −50 mV. Furthermore, many of the events observed resulted in rapid conductance changes of variable magnitude and duration, often with short lifetimes followed by a return to baseline. The observed changes in membrane conductance suggest that MPPNs are membrane-active antimicrobial peptides but that they produce small transient transmembrane channels whose life is short. Moreover, longer lifetimes occurred only occasionally. In contrast, no electrophysiological events were observed at +50 mV. This result is consistent with the positive charge of the peptides. In fact, all transmembrane conductance events were often completely lost when the voltage was switched to positive.

Planar Lipid Bilayer Assays
MPPNs were investigated for their ability to cause conductance events in planar lipid bilayer. The initial purpose was to explore if they were able to membrane channels. As expected, and shown in Figure 4, some membrane c events were observed upon addition of MPPN at −50 mV. Furthermore, many o observed resulted in rapid conductance changes of variable magnitude and d ten with short lifetimes followed by a return to baseline. The observed chang brane conductance suggest that MPPNs are membrane-active antimicrobial p that they produce small transient transmembrane channels whose life is short longer lifetimes occurred only occasionally. In contrast, no electrophysiolo were observed at +50 mV. This result is consistent with the positive charge of th In fact, all transmembrane conductance events were often completely lost wh age was switched to positive.

Atomic Force Microscopy (AFM)
Amplitude and topography AFM images obtained after treatment of MPPN at MIC, ½ MIC and untreated bacteria are shown in Figure 5. Untrea appeared as typically rod-shaped (Figure 5a), whereas MPPN-treated bacte clearly visible morphological changes. Damage to the overall structure of m treated E. coli with leakage of cell contents was observed after exposure (Figu

Atomic Force Microscopy (AFM)
Amplitude and topography AFM images obtained after treatment of E. coli with MPPN at MIC, 1 2 MIC and untreated bacteria are shown in Figure 5. Untreated bacteria appeared as typically rod-shaped (Figure 5a), whereas MPPN-treated bacteria showed clearly visible morphological changes. Damage to the overall structure of most MPPN-treated E. coli with leakage of cell contents was observed after exposure (Figure 5b,c).

Transmission Electron Microscopy (TEM)
TEM observations of ultrathin segments of E. coli are shown in Figure 6. Untreated E. coli showed the typical bacterial shape without structural damage (Figure 6a). A waved outer membrane and some cell envelope disruptions were detected in bacterial cells after treatment of E. coli at the MIC (Figure 6b,c).

Transmission Electron Microscopy (TEM)
TEM observations of ultrathin segments of E. coli are shown in Figure 6. Untreated E. coli showed the typical bacterial shape without structural damage (Figure 6a). A waved outer membrane and some cell envelope disruptions were detected in bacterial cells after treatment of E. coli at the MIC (Figure 6b,c).

Discussion
Traditional antibiotics are becoming less effective against many bacterial infections due to the growing emergence of multidrug-resistant bacteria, leading to increased morbidity, mortality, and healthcare costs [34]. In this context, the discovery of new therapeutic agents is a worldwide necessity.
Several studies have confirmed that cationic antimicrobial peptides, such as cathelicidins, kill or inhibit microorganisms [35]. Among them, porcine cathelicidins from neutrophils have mainly been studied individually against Gram-positive and Gram-negative bacteria [9,14,36]. In addition, Scapinello et al. [37] found antimicrobial activity of unfractioned porcine neutrophil secretions against relevant swine pathogens (Actinobacillus suis, Streptococcus suis and Pasteurella multocida). Thus, the aim of this work was to explore the antimicrobial activity and mechanisms of action of a mixture of cathelicidins extracted from porcine neutrophils using some human pathogens of clinical relevance. The antimicrobial activity of the mixture of cathelicidins (MPPN) was better, albeit low if one considers MIC values, against the Gram-negative bacteria E. coli and S. enterica than against the Gram-positive bacteria E. faecalis and S. aureus. The MIC determination showed values that should be considered elevated (>64 µg/mL). However, MPPN showed good results when considering other ways of evaluating antimicrobial activity, particularly in E. coli. MPPN behaved as a bactericide at the MIC and as a bacteriostatic at lower concentrations. The bacteriostatic effect was observed for slightly shorter periods in S. enterica. The ability of the peptide mixture to inhibit growth and kill bacteria for limited periods of time at these concentrations may still be relevant, as it could prevent bacterial proliferation for periods of time long enough to confer advantages to the immune system to clear the infection [38]. The results of the time-kill curves confirmed the initial interpretations, since in E. coli at the MIC, the product behaves as an effective bactericide, whereas at lower concentrations (½ MIC) or in S. enterica, it behaves as a bacteriostatic. Previous studies have reported that individual cathelicidins such as prophenin 1 and 2 [39,40]; the antibacterial protein PR-39 [41]; and protegrin 1, 2 and 3 [42][43][44] are effective as microbicides, mainly against E. coli. Despite the lack of experimental evidence, a plausible reason may reside in the fact that Gram-negative bacteria are protected from their environment by an outer membrane that is primarily composed of lipopolysaccharides (LPSs). Under stress, pathogenic serotypes of Salmonella enterica remodel their LPSs through the PhoPQ twocomponent regulatory system, which has been shown to determine resistance to both conventional antibiotics and antimicrobial peptides (AMPs), including colistin [45].
The increase in AO accumulation in the presence of MPPN strongly indicates that, at least in E. coli, the mixture causes an inhibition of the efflux pumps' function. This is not the first report of this, since it has been demonstrated that other cationic peptides may

Discussion
Traditional antibiotics are becoming less effective against many bacterial infections due to the growing emergence of multidrug-resistant bacteria, leading to increased morbidity, mortality, and healthcare costs [34]. In this context, the discovery of new therapeutic agents is a worldwide necessity.
Several studies have confirmed that cationic antimicrobial peptides, such as cathelicidins, kill or inhibit microorganisms [35]. Among them, porcine cathelicidins from neutrophils have mainly been studied individually against Gram-positive and Gramnegative bacteria [9,14,36]. In addition, Scapinello et al. [37] found antimicrobial activity of unfractioned porcine neutrophil secretions against relevant swine pathogens (Actinobacillus suis, Streptococcus suis and Pasteurella multocida). Thus, the aim of this work was to explore the antimicrobial activity and mechanisms of action of a mixture of cathelicidins extracted from porcine neutrophils using some human pathogens of clinical relevance. The antimicrobial activity of the mixture of cathelicidins (MPPN) was better, albeit low if one considers MIC values, against the Gram-negative bacteria E. coli and S. enterica than against the Gram-positive bacteria E. faecalis and S. aureus. The MIC determination showed values that should be considered elevated (>64 µg/mL). However, MPPN showed good results when considering other ways of evaluating antimicrobial activity, particularly in E. coli. MPPN behaved as a bactericide at the MIC and as a bacteriostatic at lower concentrations. The bacteriostatic effect was observed for slightly shorter periods in S. enterica. The ability of the peptide mixture to inhibit growth and kill bacteria for limited periods of time at these concentrations may still be relevant, as it could prevent bacterial proliferation for periods of time long enough to confer advantages to the immune system to clear the infection [38]. The results of the time-kill curves confirmed the initial interpretations, since in E. coli at the MIC, the product behaves as an effective bactericide, whereas at lower concentrations (1/2 MIC) or in S. enterica, it behaves as a bacteriostatic. Previous studies have reported that individual cathelicidins such as prophenin 1 and 2 [39,40]; the antibacterial protein PR-39 [41]; and protegrin 1, 2 and 3 [42][43][44] are effective as microbicides, mainly against E. coli. Despite the lack of experimental evidence, a plausible reason may reside in the fact that Gram-negative bacteria are protected from their environment by an outer membrane that is primarily composed of lipopolysaccharides (LPSs). Under stress, pathogenic serotypes of Salmonella enterica remodel their LPSs through the PhoPQ two-component regulatory system, which has been shown to determine resistance to both conventional antibiotics and antimicrobial peptides (AMPs), including colistin [45].
The increase in AO accumulation in the presence of MPPN strongly indicates that, at least in E. coli, the mixture causes an inhibition of the efflux pumps' function. This is not the first report of this, since it has been demonstrated that other cationic peptides may exert a similar effect [28]. Drug efflux is a common mechanism of antibiotic resistance, particularly in Gram-negative bacteria, which allows bacteria to extrude harmful substances, such as antimicrobial agents, to keep bacterial concentrations below toxic levels [46,47]. A paradoxical result was obtained as MPPN at 1/4 MIC was more active than at twice the concentration. Despite this, we lack experimental data; this may be due to the fraction of killed bacteria at 1/2 MIC that is not caused by lower concentrations. However, some other reason may not be ruled out.
Despite this, efflux pumps have been shown to be active in extruding antimicrobial peptides, although they do not have any critical role in the tested pathogens as a strategy to increase virulence by circumventing the antimicrobial action of innate defense. Our results demonstrate that (at least in E. coli) the peptide mixture may effectively inhibit the efflux machinery. Efflux pump inhibitors increase the cellular concentration of antibiotics to potentiate their function and should be regarded as a very interesting source of novel antibiotic adjuvant scaffolds, which may revert the effects of multidrug resistance.
Another approach to better understand the mechanism of action of novel peptides is to examine membrane damage by electrophysiological experiments. The variable conductance increments, both in magnitude and lifetime, observed in our study demonstrated the ability of MPPN to act on lipid membranes, including the induction of unstable and variable pore formation. Cationic peptides interact with membranes; this is simply derived from their cationic nature. Some of them have been studied using similar or identical methods, such as colistin [48], gramicidin S and bactenecin [30]. In all of these cases, AMPs interact with membranes, causing permeability alterations and sometimes membrane disruption. Despite this, black lipid bilayer conductance measurements are probably not the best manner to study this. The variability of conductance events observed in the present work demonstrates a strong effect of the peptide mixture with the bilayers, although we cannot rule out that some pore-forming molecule may be present in the MPPN, or that some aggregates may have such an effect [49]. This observation also suggests that different cationic peptide molecules could intercalate into the membrane, as has been previously described for peptide NK-lysin [50], CAP18-derived peptides [51] and eumenitin [52].
Since some peptides induce changes in the bacterial surface, AFM studies allowed the visualization of the changes induced by MPPN-treated bacteria. Damage to the overall structure, leakage of cell contents, increased roughness and presence of empty ghost cells were observed in treated E. coli. These results indicate that MPPN induces bacterial surface alterations. Although it is not identical, this phenomenon is the same as that seen with polymyxin B, which acts on the envelope of Gram-negative bacteria by disrupting and disorganizing the outer membrane [48]. Other studies have shown that some AMPs are able to promote membrane perturbations that ultimately lead to the lysis of the bacteria [53]. Finally, TEM images showed that the envelope of E. coli was affected by the presence of MPPN in a way similar to that observed in other cases [54]. MPPN caused the surface of E. coli to wrinkle, resulting in increased surface roughness.
In summary, MPPN is active against E. coli and S. enterica, but not S. aureus, and the main mechanisms of action involve membrane alterations (including disruption, disorganization and formation of pores) and inhibition of efflux pumps. The use of AMPs capable of inhibiting these efflux systems can be considered as a good tool to combat antimicrobial resistance, as they could be used alone or in combination with other conventional antibiotics to which bacteria have become resistant.