In vitro activity of the antimicrobial peptide AP7121 against the human methicillin-resistant biofilm producers Staphylococcus aureus and Staphylococcus epidermidis.

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.

ENCAPSULACIÓN DEL PÉPTIDO ANTIMICROBIANO AP7121 PARA SU ADMINISTRACIÓN POR VÍA ORAL / Encapsulation of antimicrobial peptide AP7121 for oral administration

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.

Bactericidal Activity and Synergy Studies of Peptide AP-CECT7121 Against Multi-resistant Bacteria Isolated from Human and Animal Soft Tissue Infections.

AP-CECT7121 is an antimicrobial peptide, produced by Enterococcus faecalis CECT7121, with bactericidal activity against Gram-positive bacteria. The aim of this study was to evaluate the bactericidal activity of AP-CECT7121, alone and with gentamicin, against multi-resistant bacteria isolated from human and animals with soft tissue infections. During the period 2014-2015, bacterial strains producing human and animal soft tissue infections were studied. Samples from patients attended at a general hospital and cattle from four dairies in the Province of Buenos Aires (Argentina) were included. Twenty-two methicillin-resistant Staphylococcus aureus (11, human blood samples; 11, cow milk) and five vancomycin-resistant Ent. faecium strains isolated from four mastitic dairy cows were tested. AP-CECT7121 (12 mg/L) potency was assessed by time-kill curves alone or with sub-inhibitory concentrations of gentamicin. All staphylococcal strains were susceptible to gentamicin; enterococci did not show high-level gentamicin resistance. Colony counts were carried out at 0, 2, 4, 8, and 24 h of incubation. AP-CECT7121 showed bactericidal activity against all the enterococcal strains. In addition, AP-CECT7121 had a bactericidal effect on most staphylococci (16/22). Early AP-CECT7121/gentamicin synergy (4-8 h) for all staphylococci was detected. At 24 h, synergy (19/22) and indifference (3/22) were observed. Synergy with gentamicin was detected for staphylococci. AP-CECT7121 constitutes an attractive candidate for its use as a natural therapeutic tool for the treatment of infections produced by multi-resistant Staph. aureus and vancomycin-resistant Ent. faecium isolated from humans and animals.

Clinical and microbiological features of bacteremia caused by Enterococcus faecalis.

INTRODUCTION: Enterococcus faecalis is a frequent etiologic agent of invasive infections in hospitalized patients. The aim of this study was to analyze clinical and microbiological features of bacteremia caused by E. faecalis. METHODOLOGY: Between 2011 and 2013, significant bacteremia caused by E. faecalis in hospitalized patients was studied. Patient characteristics, comorbid conditions, and 14-day mortality were recorded. Virulence genes esp, gelE, and cylA; opsonophagocytosis resistance; resistance to bactericidal effect of normal serum; beta lactamase production; and susceptibility to ampicillin, vancomycin, teicoplanin, gentamicin, and streptomycin were investigated. RESULTS: E. faecalis strains were recovered from 33 bacteremic patients. Polymicrobial bacteremia was diagnosed in 2 patients; 10 patients died. Virulence genes were found in strains from both deceased patients and survivors. Sources of bacteremia included urinary tract infections (36.4%), vascular catheters (15.1%), abscesses (9.1%), and unknown (48.5%). Underlying diseases included cancer (30.3%), diabetes (36.4%), cirrhosis (6.1%), renal (36.4%), and chronic obstructive pulmonary disease (2.0%). Co-morbidities included alcohol use (26.1%); glucocorticoid therapy (19.0%); prior antibiotic therapy (60.6%); and central venous (21.2%), arterial (12.1%), and urinary (63.6%) catheters. Also, 57.6% of patients came from the intensive care unit (ICU); 33.3% had mechanical ventilation. Significant mortality-associated conditions included polymicrobial bacteremia, oncological disease, APACHE II score ≤ 20, ICU stay, renal disease, central venous catheter, and mechanical ventilation. CONCLUSIONS: Outcome of patients was associated with their status and not with the presence of virulence genes in E. faecalis strains. A significant percentage of bacteremia had undetermined origin. An alternate origin may be the gastrointestinal tract, through translocation.

Comparative plasma exposure and lung distribution of two human use commercial azithromycin formulations assessed in murine model: a preclinical study.

Azithromycin (AZM) therapeutic failure and relapses of patients treated with generic formulations have been observed in clinical practice. The main goal of this research was to compare in a preclinical study the serum exposure and lung tissue concentration of two commercial formulations AZM-based in murine model. The current study involved 264 healthy Balb-C. Mice were divided into two groups (n = 44): animals of Group A (reference formulation -R-) were orally treated with AZM suspension at 10 mg/kg of b.w. Experimental animals of Group B (generic formulation -G-) received identical treatment than Group A with a generic formulation AZM-based. The study was repeated twice as Phase II and III. Serum and lung tissue samples were taken 24 h post treatment. Validated microbiological assay was used to determine the serum pharmacokinetic and lung distribution of AZM. After the pharmacokinetic analysis was observed, a similar serum exposure for both formulations of AZM assayed. In contrast, statistical differences (P < 0.001) were obtained after comparing the concentrations of both formulations in lung tissue, being the values obtained for AUC and Cmax (AZM-R-) +1586 and 122%, respectively, than those obtained for AZM-G- in lung. These differences may indicate large differences on the distribution process of both formulations, which may explain the lack of efficacy/therapeutic failure observed on clinical practice.