Mónica Sparo

High-level resistance to gentamicin: genetic transfer between Enterococcus faecalis isolated from food of animal origin and human microbiota.

Aims:  To investigate the in vivo gene transfer of high‐level gentamicin resistance (HLRG) from Enterococcus faecalis isolated from the food of animal origin to a human isolate, using a mouse model of intestinally colonized human microbiota.

In vitro and in vivo effects of Enterococcus faecalis CECT7121 on Toxocara canis

The aim of the present paper was to evaluate the larvicidal effect of Enterococcus faecalis CECT7121 (Ef7121) on the Toxocara canis cycle both in vitro and in vivo. For the in vitro experiments, T. canis larvae were incubated with the supernatants of Ef7121 (EI) and mutant Ef7121 (EIm), in a pre-culture of Ef7121 (EII) and in a fresh culture with Ef7121 (EIII) and the Ef7121 mutant strain (EIIIm). The viability of the larvae was calculated after a 48 h incubation. A significant reduction of the viability of T. canis larvae was observed in EI, EII and EIII. A decrease of this inhibitory effect was observed in EIm and EIIIm (p = 0.008). In the in vivo experiments, mice were orally inoculated with three doses of Ef7121. To study the probiotic persistence in the intestine, the animals were sacrificed every four days and their intestines were dissected. The initial average bacterial levels were 9.7 x 10(4) for Ef7121 (colony forming units/g). At the end of the assay the levels were 1.46 x 10(4). No bacterial translocation was detected in mesenteric lymphatic nodules and spleen. Ef7121 interference with the biological cycle was evaluated in mice challenged with T. canis. The interference was significant when the mice were challenged with probiotic and T. canis simultaneously (p = 0.001), but it was not significant when the challenge was performed 15 days after administration of the bacterial inoculum (p = 0.06). In conclusion, Ef7121 possessed in vitro and in vivo larvicidal activity.

Assessment of the in vitro Efficacy of the Novel Antimicrobial Peptide CECT7121 against Human Gram-Positive Bacteria from Serious Infections Refractory to Treatment

Background: Resistant Gram-positive bacteria are causing increasing concern in clinical practice. This work investigated theefficacy of AP-CECT7121 (an antimicrobial peptide isolated from an environmental strain of Enterococcus faecalis CECT7121) against various pathogenic Gram-positive bacteria. Methods: Strains were isolated from intensive care unit patients unresponsive to standard antibiotic treatments. Inhibitory activity of AP-CECT7121 was assessed using the agar-well diffusion method. The most resistant isolates from each species screened (Enterococcus faecium, Enterococcus faecalis,Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Clostridium perfringens and Clostridium difficile) were further examined in time-killing curve studies. Results: These bactericidal kinetic experiments demonstrated a rapid killing effect with no viable bacteria being detected within 30 and 90 min for enterococcal and streptococcal strains and 180 min for community-acquired methicillin-resistant S. aureus and C. perfringens: viable counts for C. difficile were threefold decreased after 90 min. Conclusions: AP-CECT7121 may provide a novel strategy for treating potentially fatal clinical infections in hospitalized patients.

Enrofloxacin‐based therapeutic strategy for the prevention of endometritis in susceptible mares

Enrofloxacin (EFX) is often used empirically to prevent uterine infections in mares in order to improve efficiency on Commercial Embryo Transfer Farms. This study investigated the uterine distribution of EFX and its metabolite ciprofloxacin (CFX) in mares and assessed the minimal inhibitory concentrations (MIC) of EFX against various common pathogens as a basis for establishing a rational dosing schedule. Plasma and uterine pharmacokinetic (PK) studies were performed in two groups (n = 5) of healthy mares following intravenous (i.v.) administration of EFX at either 2.5 and at 5 mg/kg bodyweight. Plasma and endometrial tissue samples, taken before for up to 48 h after treatment were analysed by Reverse Phase HPLC. MIC values for wild strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (beta-haemolytic streptococci) ranged from 0.25-2 and 1.5-3.0 microg/mL respectively. In terms of tissue distribution, the sum of the endometrial concentrations of the parent drug (EFX) and its active metabolite (CFX) (in terms of AUC), exceeded those in plasma by 249% and 941% following administration of EFX at 2.5 and 5 mg/kg respectively. After i.v. treatment with EFX at 5 mg/kg, endometrial concentrations of EFX and CFX above the MIC value were detected for 36-48 and 22-43 h posttreatment for Gram-negative and -positive isolates respectively. Concentrations above MIC were maintained for much shorter periods at the lower (2.5 mg/kg) treatment dose. Based on these results, a conventional dose (5 mg/kg) of EFX given prebreeding followed by two further doses at 36-48 h postbreeding are proposed as a rational strategy for using of EFX as a preventative therapy against a variety of common bacterial strains associated with equine endometritis.

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.

Antimicrobial Resistance Profiles of Enterococcus faecalis and Enterococcus faecium Isolated from Artisanal Food of Animal Origin in Argentina

Enterococci are part of the indigenous microbiota of human gastrointestinal tract and food of animal origin. Enterococci inhabiting non-human reservoirs play a critical role in the acquisition and dissemination of antimicrobial resistance determinants. The aim of this work was to investigate the antimicrobial resistance in Enterococcus faecalis and Enterococcus faecium strains recovered from artisanal food of animal origin. Samples of goat cheese (n = 42), cow cheese (n = 40), artisanal salami (n = 30), and minced meat for the manufacture of hamburgers (n = 60) were analyzed. Phenotypic and genotypic tests for species-level identification of the recovered isolates were carried out. Minimum inhibitory concentration (MIC) study for in vitro quantitative antimicrobial resistance assessment was performed, and 71 E. faecalis and 22 E. faecium were isolated. The recovered enterococci showed different multi-drug resistance patterns that included tretracycline, erythromycin, ciprofloxacin, linezolid, penicillin, ampicillin, vancomycin, teicoplanin, gentamicin (high-level resistance), and streptomycin (high-level resistance). VanA-type E. faecium were detected. β-lactamase activity was not observed. Artisanal foods of animal origin act as a non-human reservoir of E. faecalis and E. faecuim strains, expressing multi-resistance to antimicrobials. In conclusion, the implementation of a continuous antimicrobial resistance surveillance in enterococci isolated from artisanal food of animal origin is important.

Bio-preservation of ground beef meat by Enterococcus faecalis CECT7121

Meat and particularly ground beef is frequently associated with Food Poisoning episodes and breeches in Food Safety. The main goal of this research was to evaluate the bactericide effect of the probiotic Enterococcus faecalis CECT7121, against different pathogens as: Escherichia coli O157:H7, Staphylococcus aureus, Clostridium perfringens and Listeria monocytogenes, inoculated in ground beef meat. Three studies were performed to evaluate the inhibition of E. faecalis CECT7121 on ground beef meat samples inoculated with pathogens: Study I: Samples (100 g meat) were inoculated with pathogens (103 CFU/g)) and E. faecalis CECT7121 (104 CFU/g) simultaneously. Study II: Samples were inoculated with E. faecalis CECT7121 24 h before the pathogens. Study III: E. faecalis CECT7121were inoculated 24 h after pathogens. The viable counts were performed at 0, 24, 48 and 72 h post-inoculation. The simultaneous inoculation of E. faecalis CECT7121 with E. coli O157:H7 strains resulted in the absence of viable counts of bacteria at 72 h post-treatment. However, when the probiotic was added 24 h before and 24 h after the pathogen E. coli O157:H7, viable cells were not detected at 24 h and 48 h post-treatment, respectively. Consistently, neither S. aureus nor Cl. perfringens viable bacteria were detected at 48 h in whole assays when inoculated with E. faecalis CECT7121. The same trend than described before was obtained after applying the 3 models assayed for L. monocytogenes. The current assays demonstrated the bactericide activity of E. faecalis CECT7121 strain on bacterial pathogens in ground beef meat.

In vitro efficacy of the novel peptide CECT7121 against bacteria isolated from mastitic dairy cattle.

Aims:  To evaluate the in vitro bactericidal activity of the novel antimicrobial peptide (AP) CECT7121 against Gram‐positive bacteria from mastitic dairy cattle.

Probiotic activity of Enterococcus faecalis CECT7121: effects on mucosal immunity and intestinal epithelial cells

To analyse the effect of Enterococcus faecalis CECT7121 on intestinal epithelial cells (IECs) and its effects on the mucosal immune response.

Bacterial antagonist mediated protein molecules

Bacterial antagonism mediated by ribosomally synthesised peptides has gained considerable attention in recent years because of its potential applications in the control of undesirable microbiota. These peptides, generally referred to as bacteriocins, are defined as a heterogeneous group of ribosomally synthesised, proteinaceous substances (with or without further modifications) extracellularly secreted by many Gram-positive and some Gram-negative bacteria. Their mode of activity is primarily bactericidal and directed against closely related strains and species. These peptides are nearly all cationic and very often amphyphilic, which is reflected in the fact that many of these peptides kill their target cells by accumulation in the membrane causing increasing permeability and loss of barrier functions . Bacteriocins have been explored primarily as natural food preservatives, but there is much interest in exploring the application of these therapeutic peptides as antimicrobial agents since many of them exhibit antimicrobial activity against various important human pathogens.The suitability of bacteriocins such as pharmaceuticals is explored through measures of cytotoxicity, effects on the natural microbiota, and in vivo efficacy in mouse models. Bacteriocins are promising therapeutic agents.