RESUMEN

In vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis biofilm producers from blood cultures of patients with prosthetic hip infections was evaluated. The Minimum Inhibitory Concentration (MIC) for AP7121 was determined and the bactericidal activity of AP7121 (MICx1, MICx4) against planktonic cells was studied at 4, 8 and 24 h. The biofilms formed were incubated with AP7121 (MICx1, MICx4) for 1 and 24 h. The anti-adhesion effect of an AP7121-treated inert surface over the highest MIC isolate was studied with scanning electron microscopy (SEM). The bactericidal activity of AP7121 against all the planktonic staphylococcal cells was observed at 4 h at both peptide concentrations. Dose-dependent anti-biofilm activity was detected. AP7121 (MICx4) showed bactericidal activity at 24 h in all isolates. SEM confirmed prevention of biofilm formation. This research showed the in vitro anti-biofilm activity of AP7121 against MRSA and S. epidermidis and the prevention of biofilm formation by them on an abiotic surface.

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RESUMEN

El péptido antimicrobiano AP7121, producido por Enterococcus faecalis CECT7121, presenta actividad bactericida sobre patógenos Gram positivos. Sin embargo, su vía de administración oral está inhibida por la acidez gástrica y enzimas proteolíticas digestivas. El objetivo del presente trabajo fue analizar la eficacia de la encapsulación de AP7121 para su administración por vía oral. Para ello, se realizó la encapsulación del péptido antimicrobiano mediante la formación de gotas de alginato de sodio estéril 2,2% conteniendo AP7121 (30,0 mg/L) y circulando en dispositivo extrusor. Se evaluó la actividad inhibitoria de cápsulas obtenidas mediante la determinación de la Concentración Inhibitoria Mínima de AP7121 (CIMAP7121), con Listeria monocytogenes ATCC 19111 (LM, CIMAP7121: 0,8 mg/L). Asimismo, se investigó la estabilidad del péptido antimicrobiano frente a las enzimas proteolíticas tripsina, α-quimotripsina, proteinasa K y pronasa E (1 mg/mL) y se evaluó el efecto del pH utilizando una solución de HCl, pH=2,0. Las cápsulas obtenidas fueron uniformes y se obtuvo una concentración final de AP7121 de 29,7±0,3 mg/L con una CIMAP7121: 0,8 mg/L para LM. Frente a enzimas proteolíticas, no se observó descenso de actividad, permaneciendo inalterable en las cápsulas (CIMAP7121 de LM: 0,8 mg/L). Luego de la exposición a pH=2,0, se observó pérdida significativa de actividad a las 4 h de exposición. Los resultados obtenidos habilitarían la utilización de AP7121 en cápsulas para su administración por vía oral, dada su resistencia al pH ácido estomacal y enzimas proteolíticas, factores limitantes para su uso sin protección de su actividad.

The antimicrobial peptide AP7121, produced by Enterococcus faecalis CECT7121, presents bactericidal activity on Gram-positive pathogens. However, its administration via oral route is inhibited by gastric acidity and proteolytic digestive enzymes. The objective of this work was to analyze the effectiveness of the encapsulation of AP7121 for oral administration. In order to do this, the encapsulation of the antimicrobial peptide was performed by forming sterile 2.2% sodium alginate drops containing AP7121 (30.0 mg / L) and circulating in an extruder device. The inhibitory activity of the obtained capsules was evaluated by determining the Minimum Inhibitory Concentration of AP7121 (MICAP7121), with Listeria monocytogenes ATCC 19111 (LM, MICAP7121: 0.8 mg / L). Likewise, the stability of the antimicrobial peptide was investigated against trypsin, α-chymotrypsin, proteinase K and pronase E (1 mg / mL) proteolytic enzymes, and the effect of pH was evaluated using an HCl, pH = 2.0 solution. The capsules obtained were uniform and a final AP7121 concentration of 29.7 ± 0.3 mg / L with a MIC AP7121: 0.8 mg / L for ML was obtained. Against proteolytic enzymes, no decrease in activity was observed, remaining unchanged in the capsules (MICAP7121 of LM: 0.8 mg / L). After exposure to pH = 2.0, a significant loss of activity was observed after 4 h of exposure. The results obtained would enable the use of AP7121 in capsules for oral administration given its resistance to stomach acid pH and proteolytic enzymes, factors that limit the use without protection of its activity.

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