Aislamiento de cepas de Enterococcus hirae productoras de enterocinas a partir del contenido intestinal de mejillón patagónico (Mytilus edulis platensis) Aislamiento de Enterococcus hirae de mejillón patagónico / Isolation of enterocin-producing Enterococcus hirae strains from the intestinal content of the Patagonian mussel (Mytilus edulis platensis)

Resumen Se aislaron del contenido intestinal del mejillón patagónico dos cepas de bacterias ácido lácticas y se caracterizaron por pruebas fenotípicas y moleculares. Los aislamientos se identificaron como Enterococcus hirae y fueron denominados E. hirae 463Me y 471Me. Por técnicas de PCR se identificó el gen de la enterocina P en ambas cepas, mientras que solamente en la cepa 471Me se detectó la enterocina hiracin JM79. Ambas cepas resultaron sensibles a los antibióticos clínicamente importantes y entre los rasgos de virulencia investigados mediante amplificación por PCR solo se pudieron detectar los genes cylL l y cylL s , sin embargo, no se observó actividad hemolítica en la prueba de agar sangre. Los sobrenadantes libres de células resultaron activos contra todas las cepas de Listeria y Enterococcus ensayadas, contra Lactobacillus plantarum TwLb 5 y contra Vibrio anguilarum V10. En óptimas condiciones de crecimiento, ambas cepas mostraron actividad inhibitoria contra Listeria innocua ATCC 33090 después de 2h de incubación. E. hirae 471Me alcanzó una actividad inhibitoria máxima de 163.840UA/ml después de 6h de incubación, mientras que el mismo valor se registró para E. hirae 463Me después de 8h. En ambos casos, la actividad antagonista alcanzó su máximo antes de lograr la fase estacionaria y permaneció estable hasta las 24h de incubación. En nuestro conocimiento, este es el primer informe de aislamiento de cepas bacteriocinogénicas de E. hirae de mejillón patagónico. La alta actividad inhibitoria y la ausencia de rasgos de virulencia indican que estos microorganismos podrían aplicarse en áreas biotecnológicas como la biopreservación de alimentos o las formulaciones probióticas.

Abstract Two bacteriocin-producing lactic acid bacterial strains were isolated from the intestinal content of the Patagonian mussel and characterized by phenotypic and molecular tests. The isolates were identified as Enterococcus hirae and named E. hirae 463Me and 471Me. The presence of the enterocin P gene was identified in both strains by PCR techniques, while enterocin hiracin JM79 was detected only in the 471Me strain. Both strains were sensitive to clinically important antibiotics and among the virulence traits investigated by PCR amplification, only cylL l and cylL s could be detected; however, no hemolytic activity was observed in the blood agar test. Cell free supernatants were active against all Listeria and Enterococcus strains tested, Lactobacillus plantarum TwLb 5 and Vibrio anguilarum V10. Under optimal growth conditions, both strains displayed inhibitory activity against Listeria innocua ATCC 33090 after 2h of incubation. E. hirae 471Me achieved a maximum activity of 163840AU/ml after 6h of incubation, while the same value was recorded for E. hirae 463Me after 8h. In both cases, the antagonist activity reached its maximum before the growth achieved the stationary phase and remained stable up to 24h of incubation. To our knowledge, this is first report of the isolation of bacteriocinogenic E. hirae strains from the Patagonian mussel. The high inhibitory activity and the absence of virulence traits indicate that they could be applied in different biotechnological areas such as food biopreservation or probiotic formulations.

[Isolation of enterocin-producing Enterococcus hirae strains from the intestinal content of the Patagonian mussel (Mytilus edulis platensis)]. / Aislamiento de cepas de Enterococcus hirae productoras de enterocinas a partir del contenido intestinal de mejillón patagónico (Mytilus edulis platensis).

Two bacteriocin-producing lactic acid bacterial strains were isolated from the intestinal content of the Patagonian mussel and characterized by phenotypic and molecular tests. The isolates were identified as Enterococcus hirae and named E. hirae 463Me and 471Me. The presence of the enterocin P gene was identified in both strains by PCR techniques, while enterocin hiracin JM79 was detected only in the 471Me strain. Both strains were sensitive to clinically important antibiotics and among the virulence traits investigated by PCR amplification, only cylLl and cylLs could be detected; however, no hemolytic activity was observed in the blood agar test. Cell free supernatants were active against all Listeria and Enterococcus strains tested, Lactobacillus plantarum TwLb 5 and Vibrio anguilarum V10. Under optimal growth conditions, both strains displayed inhibitory activity against Listeria innocua ATCC 33090 after 2h of incubation. E. hirae 471Me achieved a maximum activity of 163840AU/ml after 6h of incubation, while the same value was recorded for E. hirae 463Me after 8h. In both cases, the antagonist activity reached its maximum before the growth achieved the stationary phase and remained stable up to 24h of incubation. To our knowledge, this is first report of the isolation of bacteriocinogenic E. hirae strains from the Patagonian mussel. The high inhibitory activity and the absence of virulence traits indicate that they could be applied in different biotechnological areas such as food biopreservation or probiotic formulations.

Distribution and growth of Vibrio parahaemolyticus in southern Chilean clams (Venus antiqua) and blue mussels (Mytilus chilensis).

We evaluated the distribution and growth of Vibrio parahaemolyticus in the inland sea of southern Chile, where the world's largest foodborne gastroenteritis outbreak by the pandemic strain O3:K6 occurred in 2005. Intertidal samples of Mytilus chilensis and Venus antiqua were collected around port towns between 41°28'S and 43°07'S, during April to May 2011 and January to March 2012. We used most probable number real-time polymerase chain reaction (MPN-PCR) for enumeration of the tlh, tdh, and trh genes in freshly harvested bivalves and after a controlled postharvest temperature abuse. Pathogenic markers (tdh+ or trh+) were not detected. Total V. parahaemolyticus (tlh+) in freshly harvested samples reached up to 0.38 and 3.66 log MPN/g in 2011 and 2012, respectively, with values close to or above 3 log MPN/g only near Puerto Montt (41°28'S, 72°55'W). Enrichments by temperature abuse (>2 log MPN/g) occurred mainly in the same zone, regardless of the year, suggesting that both natural or anthropogenic exposure to high temperatures were more critical. Lower salinity and higher sea surface temperature in Reloncaví Sound and Reloncaví Estuary were consistent with our observations and allowed confirmation of the existence of a high-risk zone near Puerto Montt. Based on the results, a strategy focused on risk management inside this defined hazard zone is recommended.

Immunological responses, histopathological finding and disease resistance of blue mussel (Mytilus edulis) exposed to treated and untreated municipal wastewater.

This study provides new information on the response of the immune system of Mytilus edulis exposed to untreated and treated sewage, linking immune response to ecologically relevant endpoints, such as disease resistance. Our goal was to assess the potential effects of sewage on the immune system (phagocytic activity and production of cytotoxic metabolites, disease resistance) and gills (light microscope) of mussels through a bioassay and field study in an estuarine receiving environment (RE). A semi-static experiment was developed in a wastewater treatment plant in New Glasgow, NS Canada. Mussels were exposed for 21 days to 12.5%, 25%, 50% and 100% of untreated sewage influent and artificial seawater control. Sampling occurred after 7, 14 and 21 days of exposure. In the field study, eight sites were selected in East River and Pictou Harbour, NS, positioned upstream and downstream of sewage effluents outfalls. Caged mussels were exposed to the RE for 90 days (May-July 2005). Mussels were challenged to test their efficiency at eliminating the bacteria, Listonella anguillarium in the bioassay and field studies. The bioassay results showed that higher concentrations of untreated sewage could modulate the immune system of mussels through increased of phagocytic activity (PA), nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) production during 14 days of exposure, and decreased activity and production at 21 days, with the exception of H(2)O(2) production which was high even at 21 days. Mussels exposed to untreated sewage RE also presented a high PA, NO and H(2)O(2) production and lower number of haemocytes compared to mussels from reference sites. In the bacterial challenge, mussels pre-exposed to 100% sewage died 24h after being infected with L. anguillarium, while mussels pre-exposed to 50% eliminated bacteria had a mortality rate of 30%. Mussels from the control, 12.5% and 25% groups eliminated bacteria and no mortality was observed. No significant difference was observed in bacterial clearance in mussels exposed to effluents in the RE. The lesions observed in gills in both studies were: infiltration of haemocytes in the tissue, epithelium proliferation, lamellar fusion and dilated haemolymphatic sinus. In summary, untreated municipal wastewater affected the immune system of blue mussels during 21 days of exposure and the effects were reflected in their capability to resist pathogens. And an immune modulation was observed in mussels exposed to untreated sewage in a RE, but this modulation was not reflected in the mussel's capability in eliminating pathogens.