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.

Draft Genome Sequence of Enterococcus faecalis Strain CECT7121, a Corn Silage Isolate with Antibacterial Activity against Gram-Positive Pathogens.

Enterococcus faecalis CECT7121 is a corn silage probiotic bacterium that shows antibacterial activity against Gram-positive pathogens from different origins. Its genome sequence is 2.9 Mb long with a G+C content of 37.3%. Genome annotation identified three bacteriocin gene clusters in the genome.

Impact on Public Health of the Spread of High-Level Resistance to Gentamicin and Vancomycin in Enterococci.

Antibiotic resistance has turned into a global public health issue. Enterococci are intrinsically resistant to many antimicrobials groups. These bacteria colonize dairy and meat products and integrate the autochthonous microbiota of mammal's gastrointestinal tract. Over the last decades, detection of vanA genotype in Enterococcus faecium from animals and from food of animal origin has been reported. Vancomycin-resistant E. faecium has become a prevalent nosocomial pathogen. Hospitalized patients are frequently treated with broad-spectrum antimicrobials and this leads to an increase in the presence of VanA or VanB vancomycin-resistant enterococci in patients' gastrointestinal tract and the risk of invasive infections. In humans, E. faecium is the main reservoir of VanA and VanB phenotypes. Acquisition of high-level aminoglycoside resistance is a significant therapeutic problem for patients with severe infections because it negates the synergistic effect between aminoglycosides and a cell-wall-active agent. The aac(6')-Ie-aph (2″)-Ia gene is widely spread in E. faecalis and has been detected in strains of human origin and in the food of animal origin. Enzyme AAC(6')-Ie-APH(2″)-Ia confers resistance to available aminoglycosides, except to streptomycin. Due to the fast dissemination of this genetic determinant, the impact of its horizontal transferability among enterococcal species from different origin has been considered. The extensive use of antibiotics in food-producing animals contributes to an increase in drug-resistant animal bacteria that can be transmitted to humans. Innovation is needed for the development of new antibacterial drugs and for the design of combination therapies with conventional antibiotics. Nowadays, semi-purified bacteriocins and probiotics are becoming an attractive alternative to the antibiotic in animal production. Therefore, a better understanding of a complex and relevant issue for Public Health such as high-level vancomycin and gentamicin resistance in enterococci and their impact is needed. Hence, it is necessary to consider the spread of vanA E. faecium and high-level gentamicin resistant E. faecalis strains of different origin in the environment, and also highlight the potential horizontal transferability of these resistance determinants to other bacteria.

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.

Throat Carriage Rate and Antimicrobial Resistance of Streptococcus pyogenes In Rural Children in Argentina.

OBJECTIVES: The aim of this study was to determine the prevalence of asymptomatic carriers of group A ß-hemolytic streptococci (GAS) in children living in a rural community and to investigate the association between episodes of acute pharyngitis and carrier status. METHODS: Throat swabs were collected from September to November 2013 among children 5-13 years of age from a rural community (Maria Ignacia-Vela, Argentina). The phenotypic characterization of isolates was performed by conventional tests. Antimicrobial susceptibility was assayed for penicillin, tetracycline, chloramphenicol, erythromycin, and clindamycin (disk diffusion). The minimum inhibitory concentration was determined for penicillin, cefotaxime, tetracycline, and erythromycin. RESULTS: The carriage of ß-hemolytic streptococci was detected in 18.1% of participants, with Streptococcus pyogenes in 18 participants followed by S. dysgalactiae ssp. equisimilis in 5. The highest proportion of GAS was found in 8 to 10-year-old children. No significant association between the number of episodes of acute pharyngitis suffered in the last year and the carrier state was detected (p>0.05). Tetracycline resistance (55.5%) and macrolide-resistant phenotypes (11.1%) were observed. Resistance to penicillin, cefotaxime, or chloramphenicol was not expressed in any streptococcal isolate. CONCLUSIONS: The present study demonstrated significant throat carriage of GAS and the presence of group C streptococci (S. dysgalactiae ssp. equisimilis) in an Argentinian rural population. These results point out the need for continuous surveillance of GAS and non-GAS carriage as well as of antimicrobial resistance in highly susceptible populations, such as school-aged rural children. An extended surveillance program including school-aged children from different cities should be considered to estimate the prevalence of GAS carriage in Argentina.

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.

Immunomodulatory properties of cell wall extract from Enterococcus faecalis CECT7121.

The aim of this study was to investigate the immunomodulatory properties of cell wall extract from Enterococcus faecalis CECT7121, measuring the induction of cytokines TNF-α, IL-6, IL-10 and IL-12 from human peripheral blood mononuclear cells (PBMCs). Cell wall extract was prepared from their growth in brain heart infusion broth (18h, 35°C). Subsequently, toxicity of the obtained cell wall extract was tested in Balb-C mice. PBMCs were isolated from buffy coats at the Blood Transfusion Service of Hospital Ramón Santamarina (Tandil, Argentina). PBMCs were purified using standard Ficoll-Paque gradient centrifugation. Aliquots of purified leukocytes were incubated at 37°C for 24h with heat-killed E. faecalis CECT7121 and cell wall extract. Concentrations of IL-6, TNFα, IL-10 and IL-12 (p70) were measured by solid phase sandwich ELISA. Changes in appearance and behavior of mice were evidenced only in the group with the maximal concentration of wall cell extract used (10,000µg). Cell wall extract and heat-killed E. faecalis CECT7121 induced the production of significantly higher amounts of Il-12, IL-6, TNF-α and IL-10 cytokines compared to the nonstimulated PBMCs. These findings provide helpful information on immunomodulation activity by cell wall extract in sight of the application of this compound in controlling certain infectious diseases.

Enterococcus spp. aislados de queso de oveja: resistencia a los antimicrobianos de utilización clínica / Enterococcus spp. isolated from goat cheese: resistance to clinically used antimicrobials

En Enterococcus spp. aislados de quesos de oveja se investigó la resistencia in vitro a antimicrobianos de utilización clínica. Se determinó la concentración inhibitoria mínima (CIM) para gentamicina, estreptomicina, vancomicina, teicoplanina, ampicilina, imipenem, linezolid y tigeciclina. Se detectó la producción de β-lactamasa y se realizaron experimentos de conjugación para transferir resistencia a gentamicina. En un aislamiento de E. faecalis y en 10 de E. faecium se observó resistencia a ampicilina; se demostró la producción de β-lactamasa en un E. faecalis. En E. faecium, 15 aislamientos fueron resistentes a imipenem; 4 a linezolid y 5 a glucopéptidos. En 4 aislamientos de E. faecalis se demostró alto nivel de resistencia plasmídica a gentamicina. No se observaron aislamientos resistentes a tigeciclina. El queso de oveja es un reservorio de enterococos resistentes con potencial diseminación al hombre a través de la cadena alimentaria.

In vitro resistance to clinically used antimicrobials was investigated in Enterococcus spp. isolated from goat cheese. The gentamycin, streptomycin, vancomycin, teicoplanin, ampicillin, imipenem, linezolid and tigecyclin minimum inhibitory concentration (MIC) was determined. Β-lactamase production was detected and conjugation experiments were carried out for transferring gentamycin resistance. In one E. faecalis and in 10 E. faecium isolates there was ampicillin resistance and β-lactamase production was demonstrated in one E. faecalis. Fifteen E. faecalis were imipenenem resistant; 4 linezolid resistant and 5 glucopeptide resistant. There was a high level of plasmidic resistance to tigecyclin in 4 E. faecalis isolates. There were no tigecyclin resistant isolates. Goat cheese is a reservoir of resistant enterococci with potencial human dissemination through the food chain.

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.

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. ß-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.