Citotoxicidad in vitro del polipéptido antimicrobiano nisina sobre células tumorales sanguíneas / In vitro cytotoxicity of the antimicrobial polypeptide nisin on blood tumor cells

RESUMEN: Las bacteriocinas son péptidos antimicrobianos de síntesis ribosomal secretadas por bacterias. Dentro de estas destaca nisina que posee potenciales usos en terapias antibióticas, como biopreservante de alimentos y probióticos. También se ha descrito que nisina posee citotoxicidad sobre líneas celulares neoplásicas, pero existe poca información de su efecto sobre células tumorales sanguíneas. Debido al potencial uso que presenta nisina, es relevante determinar la toxicidad que presenta sobre líneas celulares tumorales del tipo sanguíneo. Para esto, se realizaron ensayos de actividad hemolítica sobre eritrocitos humanos y de toxicidad sobre células mononucleares de sangre periférica humanas, determinándose que nisina no posee efecto citotóxico sobre este tipo de células normales humanas sanguíneas. Se realizaron también, ensayos de citotoxicidad con líneas celulares tumorales (K562 y U937), con el fin de determinar dosis, tiempo de exposición y selectividad en el efecto tóxico de nisina sobre las células tumorales humanas. Estos ensayos muestran que nisina presenta actividad citotóxica sobre líneas celulares K562 y U937 a las 72 h de exposición, a una concentración de 40 µg/mL, que corresponde a 100 veces la concentración mínima inhibitoria (MIC) usada para su acción sobre bacterias. Al comparar el efecto de nisina sobre células mononucleares de sangre periférica humanas con las líneas tumorales linfoides y mieloides (K562 y U937 respectivamente), se observa un efecto selectivo de nisina sobre las células tumorales sanguíneas.

SUMMARY: Bacteriocins are antimicrobial peptides of ribosomal synthesis secreted by bacteria. Among these, nisin stands out, which has potential uses in antibiotic therapies, as a food bio preservative and probiotics. Nisin has also been reported to have cytotoxicity on neoplastic cell lines, but there is little information on its effect on blood tumor cells. Due to the potential use that nisin presents, it is relevant to determine the toxicity it presents on tumor cell lines of the blood type. For this, hemolytic activity tests were carried out on human erythrocytes and toxicity on human peripheral blood mononuclear cells, determining that nisin does not have a toxic effect on this type of normal human blood cells. Cytotoxicity tests were also carried out with tumor cell lines (K562 and U937), to determine dose, exposure time and selectivity in the toxic effect of nisin on human tumor cells. These tests show that nisin shows cytotoxic activity on K562 and U937 cell lines at 72 h of exposure, at a concentration of 40 µg / mL, which corresponds to 100 times the minimum inhibitory concentration (MIC) used for its action on bacteria. When comparing the effect of nisin on human peripheral blood mononuclear cells with lymphoid and myeloid tumor lines (K562 and U937 respectively), a selective effect of nisin on blood tumor cells is observed.

Antibacterial efficacy of nisin, bacteriophage P100 and sodium lactate against Listeria monocytogenes in ready-to-eat sliced pork ham

ABSTRACT The effectiveness of bacteriophage P100, nisin and sodium lactate, individually and in combination, in inhibiting Listeria monocytogenes in ready-to-eat pork ham slices was assessed. The antimicrobials were applied to the surfaces of ready-to-eat pork ham slices, which were inoculated with a mixture of L. monocytogenes. Among the individual antimicrobial treatments, bacteriophage P100 was the most effective, decreasing L. monocytogenes to undetectable levels at zero and 72 h post-infection. Sodium lactate was the least effective treatment. Treatment with nisin at zero h significantly reduced initial cell density (p < 0.05). However, this pattern was not observed at 72 h of storage. A significant difference (p < 0.05) existed between the results of separate bacteriophage and nisin treatments after refrigerated storage, but not immediately upon inoculation of the bacteria. The results showed that the use of bacteriophage P100 is the method of choice for the control of bacteria.

Inactivation of Listeria monocytogenes ATCC 7644 on fresh-cut tomato using nisin in combinations with organic salts

ABSTRACT The inhibition of Listeria monocytogenes ATCC 7644 on fresh-cut tomato was investigated using nisin alone, and in combinations with organic salts. Nisin at a concentration of 5000 UI/mL was introduced alone or in combination with an organic salt (sodium citrate or sodium acetate each at 3 and 5 g/100 mL each) on fresh-cut tomato previously inoculated with 108 CFU/mL of L. monocytogenes ATCC 7644. Chlorine at 200 ppm was used as a control. The inoculated samples were incubated at different temperatures (4, 10 and 25 °C) and examined at 0, 24, 48 and 72 h. The effects of the antimicrobial treatments on quality parameters of tomato (pH, soluble solids, titratable acidity and vitamin C) were also evaluated, and colour parameters were observed at the lowest storage temperature for 10 days. Both nisin and the organic salts inhibited growth of L. monocytogenes, but the combinations of two compounds were more effective. The nisin-sodium citrate (5%) combination was significantly (p ≤ 0.05) effective, while chlorine was least effective against L. monocytogenes. The quality parameters were substantially retained, especially at 4 °C, suggesting good shelf stability at a low temperature. These results substantiate the use of the cheap and eco-friendly approach to reducing this pathogen of health concern in common fresh produce.

Combined effect of a mixture of tetracycline, acid, and detergent, and Nisin against Enterococcus faecalis and Actinomyces viscosus biofilms

To evaluate the combined effect of a mixture of tetracycline, acid, and detergent [MTAD] and Nisin against Enterococcus faecalis [E. faecalis] and Actinomyces viscosus [A. viscosus] biofilms. This study was conducted between June and December 2013 in collaboration with Dental Caries Research Chair, College of Dentistry, King Saud University, Riyadh, Saudi Arabia. Single-species biofilms [n=9/species/observation period] were generated on membrane filter discs and subjected to 5, 10, or 15 minute incubation with MTADN [MTAD with 3% Nisin], 5.25% sodium hypochlorite [NaOCl], or normal saline. The colony forming units were counted using the Dark field colony counter. A 100% bactericidal effect of 5.25% NaOCl was noted during the 3 observation periods; a significant reduction [p=0.000] in mean survival rates of E. faecalis [77.3+13.6] and A. viscosus [39.6+12.6] was noted after 5 minutes exposure to MTADN compared with normal saline [78000000+5291503] declining to almost no growth after 10 and 15 minutes. The survival rates of the E. faecalis and A. viscosus biofilm were no different after treatment with MTADN and 5.25% NaOCl at the 3 observation periods [p=1.000]. A combination of MTAD and Nisin was as effective as NaOCl against E. faecalis and A. viscosus biofilms

Antibacterial activity of bacteriocin-like substance P34 on Listeria monocytogenes in chicken sausage

The antimicrobial activity of the bacteriocin-like substance (BLS) P34 against Listeria monocytogenes was investigated in chicken sausage. The BLS was applied to chicken sausages (256 AU g-1) previously inoculated with a suspension of 10² cfu g-1 of L. monocytogenes. BLS P34 inhibited the indicator microorganism in situ in all incubation times for up to 10 days at 5 °C. The effectiveness of BLS P34 was increased when it was added in combination with nisin. The bacteriocin was also tested in natural eatable natural bovine wrapping (salty semi-dried tripe) against the same indicator microorganism, also showing inhibitory capability in vitro. BLS P34 showed potential to control L. monocytogenes in refrigerated meat products.

[Effects of nisin and thyme essential oil, individually and in combination, on inoculated populations of Listeria monocytogenes in minced silver carp]

Food poisoning caused by Listeriamonocytogenes results in death in 30% of the cases. Considering the high probability of L. monocytogenes contamination of local fish, the present study aimed at investigating the effects of thyme [Zataria multiflora Boiss] essential oil [EO] and nisin, individually and in combination, on the growth of L. monocytogenes in minced silver carp during refrigerated storage. Minced fish samples were inoculated with 1x10[4] cfu/g of L. monocytogenes. A total of 11 samples were inoculaetd with thyme EO at a concentration [weight/volume]of 0.3%, 0.8% or 1.2%, nisin at a concentration of 500 or 1000 IU/g, or a combination of the two. The treated and control samples were packaged in plastic bags and kept at refrigerator temperature for 12 days.. Samples were cultured on CHROMagarTM Listeria every 2 days and the bacteri counted. Nisin at two different levels [500 and 1000 IU/g]could not inhibit the growth of L.monocytogenes to a level below the acceptable level in raw food [100 cells/g]. The antibacterial activity of nisin decreased during the storage period, while simultaneous use of nisin and thyme EO at a concentartion of 0.8 and 1.2% reduced L. monocytogenes viable count to a level below the acceptable limit during 12 days. A combination of 0.8% thyme [weight/volume]and 1000 IU/g nisin has the best inhibitory effect on growth of L. monocytogenes in minced silver carp during cold [4[degree sign] C] storage

Exploring the use of natural antimicrobial agents and pulsed electric fields to control spoilage bacteria during a beer production process / Exploración del uso de agentes antimicrobianos naturales y de campos eléctricos pulsantes para el control de bacterias contaminantes durante el proceso de elaboración de cerveza

Different natural antimicrobials affected viability of bacterial contaminants isolated at critical steps during a beer production process. In the presence of 1 mg/ml chitosan and 0.3 mg/ml hops, the viability of Escherichia coli in an all malt barley extract wort could be reduced to 0.7 and 0.1% respectively after 2 hour- incubation at 4 °C. The addition of 0.0002 mg/ml nisin, 0.1 mg/ml chitosan or 0.3 mg/ml hops, selectively inhibited growth of Pediococcus sp. in more than 10,000 times with respect to brewing yeast in a mixed culture. In the presence of 0.1mg ml chitosan in beer, no viable cells of the thermoresistant strain Bacillus megaterium were detected. Nisin, chitosan and hops increased microbiological stability during storage of a local commercial beer inoculated with Lactobacillus plantarum or Pediococcus sp. isolated from wort. Pulsed Electric Field (PEF) (8 kV/cm, 3 pulses) application enhanced antibacterial activity of nisin and hops but not that of chitosan. The results herein obtained suggest that the use of these antimicrobial compounds in isolation or in combination with PEF would be effective to control bacterial contamination during beer production and storage.

Diferentes antimicrobianos naturales disminuyeron la viabilidad de bacterias contaminantes aisladas en etapas críticas del proceso de producción de cerveza. En un extracto de malta, el agregado de 1 mg/ml de quitosano y de 0,3 mg ml de lúpulo permitió reducir la viabilidad de Escherichia coli a 0,7 y 0,1%, respectivamente, al cabo de 2 horas de incubación a 4 °C. El agregado de 0,0002 mg/ml de nisina, 0,1 mg/ml de quitosano o de 0,3 mg/ml de lúpulo inhibió selectivamente (10.000 veces más) el crecimiento de Pediococcus sp. respecto de la levadura de cerveza en un cultivo mixto. El agregado de 0,1 mg/ml de quitosano permitió disminuir la viabilidad de una cepa bacteriana termorresistente, Bacillus megaterium, hasta niveles no detectables. Por otra parte, el agregado de nisina, quitosano y lúpulo aumentó la estabilidad microbiológica durante el almacenamiento de cervezas inoculadas con Lactobacillus plantarum y Pediococcus sp. aislados de mosto de cerveza. La aplicación de campos eléctricos pulsantes (CEP) (3 pulsos de 8kV/cm) aumentó el efecto antimicrobiano de la nisina y del lúpulo, pero no el del quitosano. Los resultados obtenidos indicarían que el uso de antimicrobianos naturales en forma individual o en combinación con CEP puede constituir un procedimiento efectivo para el control de la contaminación bacteriana durante el proceso de elaboración y almacenamiento de la cerveza.