RESUMEN
In canine nutrition, the use of goat nutraceutical dairy products is an innovative proposal. Therefore, the objective of this study was to prepare fermented goat milk with probiotic potential in dogs in an in vitro model. A total of 40 lactic acid bacteria (LAB) species were grown, of which 30 were CAP isolates originally from goat milk and 10 were CAN isolates originally from fecal material of newborn dogs. The isolates were selected based on resistance to the simulated canine gastrointestinal condition and acidifying ability. After this preliminary screening, the analyses were performed regarding ß-galactosidase and exopolysaccharide formation, diacetyl production, adhesion proteins Mub and mapa, hydrophobicity, DPPH assay, virulence and antibiotic resistance. With these evaluations, four LAB isolates were identified using sequencing of the 16S rRNA gene. These were identified as Enterococcus hirae and were used to produce fermented goat milk. For statistical analysis, the data were analyzed using the Scott-Knott test and also submitted to analysis of variance and the Tukey test (P < 0.05). In the evaluation of goat milk fermented with E. hirae and control, over the 36-day storage period there was a reduction in pH and an increase in acidity, and higher levels of LAB were observed in goat milk fermented with E. hirae. Therefore, both these E. hirae isolates and the fermented goat milk produced showed satisfactory results in vitro, demonstrating probiotic efficiency and food safety for dogs.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Perros/microbiología , Enterococcus hirae/química , Tracto Gastrointestinal/microbiología , Cabras/microbiología , Leche/microbiología , Probióticos/administración & dosificación , Animales , Perros/fisiología , Heces/microbiología , Fermentación , Tracto Gastrointestinal/fisiologíaRESUMEN
BACKGROUND: An increasing rate of antibiotic resistance among Gram-negative bacterial pathogens has created an urgent need to discover novel therapeutic agents to combat infectious diseases. Use of bacteriocins as therapeutic agents has immense potential due to their high potency and mode of action different from that of conventional antibiotics. RESULTS: In this study, a novel bacteriocin E20c of molecular weight 6.5 kDa was purified and characterized from the probiotic strain of Enterococcus hirae. E20c had bactericidal activities against several multidrug resistant (MDR) Gram-negative bacterial pathogens. Flow cytometry and scanning electron microscopy studies showed that it killed the Salmonella enterica cells by forming ion-permeable channels in the cell membrane leading to enhanced cell membrane permeability. Further, checkerboard titrations showed that E20c had synergistic interaction with antibiotics such as ampicillin, penicillin, ceftriaxone, and ciprofloxacin against a ciprofloxacin- and penicillin-resistant strain of S. enterica. CONCLUSION: Thus, this study shows the broad spectrum antimicrobial activity of novel enterocin E20c against various MDR pathogens. Further, it highlights the importance of bacteriocins in lowering the minimum inhibitory concentrations of conventional antibiotics when used in combination.
Asunto(s)
Antibacterianos/farmacología , Bacteriocinas/farmacología , Ciprofloxacina/farmacología , Salmonella enterica/efectos de los fármacos , beta-Lactamas/farmacología , Bacteriocinas/aislamiento & purificación , Hidrocarburos Aromáticos con Puentes/aislamiento & purificación , Hidrocarburos Aromáticos con Puentes/farmacología , Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple , Sinergismo Farmacológico , Enterococcus hirae/química , Enterococcus hirae/metabolismo , Bacterias Gramnegativas/efectos de los fármacos , Pruebas de Sensibilidad MicrobianaRESUMEN
AIMS: To understand the bactericidal action of enterocin LD3 against Gram-negative bacteria. METHODS AND RESULTS: Minimum inhibitory concentration (MIC) of enterocin LD3 against Micrococcus luteus MTCC 106 and Escherichia coli NCDC 135 was 80 and 112 µg ml-1 , and minimum bactericidal concentration (MBC) was 128 and 180 µg ml-1 , respectively. The efflux of potassium ion (K+ ) was 14 and 13 ppm and electrical conductivity 10·5 and 8·3 mS cm-1 in cell-free supernatant of MIC-treated cells of M. luteus and E. coli respectively. The increased absorbance (OD260/280 ) 0·422/0·260 and 0·110/0·075 in the bacteriocin-treated cells of M. luteus MTCC 106 and E. coli, NCDC 135, respectively, suggested the release of nucleic acids and proteins. The higher infrared absorbance at 1451·82 and ~1094·30 cm-1 further suggested its interaction with cell membrane and nucleic acids of the target bacteria. The interaction of bacteriocin with nucleic acids was also confirmed using gel retardation assay. Transmission electron microscopy of the bacteriocin-treated cells revealed disruption of cell membrane and leakage of cytoplasmic contents. CONCLUSIONS: Enterocin LD3 demonstrates bactericidal activity against Gram-negative bacteria interacting with cell membrane and nucleic acids. SIGNIFICANCE AND IMPACT OF THE STUDY: The study discloses the possible mechanism of action of enterocin LD3 against Gram-negative bacteria which is a rare phenomenon.
Asunto(s)
Antibacterianos/farmacología , Enterococcus hirae/química , Bacterias Gramnegativas/efectos de los fármacos , Iones/metabolismo , Transporte Biológico/efectos de los fármacos , Hidrocarburos Aromáticos con Puentes/farmacología , Membrana Celular/efectos de los fármacos , Bacterias Gramnegativas/metabolismo , Pruebas de Sensibilidad Microbiana , Ácidos Nucleicos/metabolismoRESUMEN
A low-intensity electromagnetic field of extremely high frequency has inhibitory and stimulatory effects on bacteria, including Enterococcus hirae. It was shown that the low-intensity (the incident power density of 0·06 mW cm-2 ) electromagnetic field at the frequencies of 51·8 GHz and 53 GHz inhibited E. hirae ATCC 9790 bacterial growth rate; a stronger effect was observed with 53 GHz, regardless of exposure duration (0·5 h, 1 h or 2 h). Scanning electron microscopy analysis of these effects has been done; the cells were of spherical shape. Electromagnetic field at 53 GHz, but not 51·8 GHz, changed the cell size-the diameter was enlarged 1·3 fold at 53 GHz. These results suggest the difference in mechanisms of action on bacteria for electromagnetic fields at 51·8 GHz and 53 GHz. SIGNIFICANCE AND IMPACT OF THE STUDY: A stronger inhibitory effect of low-intensity electromagnetic field on Enterococcus hirae ATCC 9790 bacterial growth rate was observed with 53 GHz vs 51·8 GHz, regardless of exposure duration. Scanning electron microscopy analysis showed that almost all irradiated cells in the population have spherical shapes similar to nonirradiated ones, but they have increased diameters in case of irradiated cells at 53 GHz, but not 51·8 GHz. The results are novel, showing distinguishing effects of low-intensity electromagnetic field of different frequencies. They could be applied in treatment of food and different products in medicine and veterinary, where E. hirae plays an important role.
Asunto(s)
Enterococcus hirae/crecimiento & desarrollo , Enterococcus hirae/efectos de la radiación , Radiación Electromagnética , Enterococcus hirae/química , Enterococcus hirae/ultraestructura , Cinética , Microscopía Electrónica de RastreoRESUMEN
Enterocin LD3 was purified using activity-guided multistep chromatography techniques such as cation-exchange and gel-filtration chromatography. The preparation's purity was tested using reverse-phase ultra-performance liquid chromatography. The specific activity was tested to be 187.5 AU µg(-1) with 13-fold purification. Purified enterocin LD3 was heat stable up to 121 °C (at 15 psi pressure) and pH 2-6. The activity was lost in the presence of papain, reduced by proteinase K, pepsin and trypsin, but was unaffected by amylase and lipase, suggesting proteinaceous nature of the compound and no role of carbohydrate and lipid moieties in the activity. MALDI-TOF/MS analysis of purified enterocin LD3 resolved m/z 4114.6, and N-terminal amino acid sequence was found to be H2NQGGQANQ-COOH suggesting a new bacteriocin. Dissipation of membrane potential, loss of internal ATP and bactericidal effect were recorded when indicator strain Micrococcus luteus was treated with enterocin LD3. It inhibited Gram-positive and Gram-negative bacteria including human pathogens such as Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Listeria monocytogenes, Escherichia coli O157:H7, E. coli (urogenic, a clinical isolate) and Vibrio sp. These properties of purified enterocin LD3 suggest its applications as a food biopreservative and as an alternative to clinical antibiotics.