Characterization of Lactobacillus Strains Isolated from Apis mellifera L. Bee Intestine

AUDISIO, MC; APELLA, M. C

San Miguel de Tucumán

Simposio; II Internacional Symposium on Lactic Acid Bacteria and First Argentinean LAB Net Meeting International; 2006

CERELA-CONICET

Uno de los mayores problemas de la industria apícola y que produce grandes pérdidas económicas por elevada mortandad, es la infección con Paenibacillus larvae, agente etiológico de la loque americana. El efecto beneficioso que pueden ejercer las bacterias lácticas, utilizadas como suplementos probióticos, en el huésped humano y animal es conocido. Sin embargo, la información científica sobre la presencia de estos microorganismos en la microbiota asociada con la abeja Apis mellifera L. es escasa. Por lo tanto, el objetivo de este trabajo fue aislar, caracterizar fenotípicamente y determinar el potencial efecto antimicrobiano de bacterias del género Lactobacillus del intestino de la abeja. Para tal fin, se analizaron muestras del contenido intestinal de abejas vivas de diversas colmenas localizadas en El Galpón, Salta, Argentina. El intestino se retiró mediante el uso de instrumental estéril y se preparó un homogenato de su contenido con solución fisiológica estéril. Alícuotas de las suspensiones fueron sembradas en profundidad y en superficie en LAPTg agar y MRS agar (selectivo para Lactobacillus). Las placas se incubaron a 35ºC en condiciones de microaerofilia durante 24-72 h. A partir de colonias reaisladas en el medio selectivo, se analizó morfología, respuesta a catalasa, movilidad y tinción de Gram como un paso previo a su tipificación por pruebas bioquímicas (reducción de nitrato, fermentación de esculina, hidrólisis de arginina) y fermentación de azúcares. Por otro lado, se estudió el efecto antagónico de los cultivos libres de células sobre Paenibacillus larvae, Listeria monocytogenes, Bacillus sp., Enterococcus sp.; Staphylococcus aureus y mohos (Ascosphaera apis, Aspergillus niger) por el método de difusión en placa. Una serie de cepas seleccionadas por su efecto inhibitorio fue analizada por microscopia electrónica de barrido y se cuantificó la producción de ácido láctico por HPLC. Se aislaron más de 100 cepas de bacterias en MRS agar las cuales presentaron las siguientes características: bacilos inmóviles, catalasa negativos, Gram-positivos, no esporulados y nitrato negativos. Todas estas propiedades permitieron clasificar a estos aislados en el género Lactobacillus. Ocho cepas se destacaron por su fuerte efecto anti-Listeria y anti-P. larvae, aunque en este caso el efecto fue cepa dependiente. En los sobrenadantes libres de células, el contenido de ácido láctico fue variable y los valores oscilaron entre 3,06 y 6,08 g L-1. La cepa de Lactobacillus spp. A3IO produjo la mayor cantidad de ácido láctico y fue la única que presentó la capacidad de inhibir S. aureus y A. niger debido a la producción del metabolito. Estos resultados indican que Lactobacillus es un constituyente de la microbiota intestinal de la abeja melífera y que es posible la formulación de un suplemento probiótico capaz de inhibir al P. larvae en la protección contra la loque americana. ABSTRACT: Paenibacillus larvae, etiological agent of the American foulbrood, is an infection that produces large economical losses in the apicultural industry by high mortality rate. The beneficial effect that lactic acid bacteria as probiotic suplements can exert on human and animal hosts is well known. However, there is a lack of scientific information concerning the presence of these microorganisms in the microbiota associated with the Apis mellifera L. bee. Therefore, our aim in this work was to isolate, characterize phenotypically and determine the potential antimicrobial effect of bee intestine Lactobacillus strains. To this purpose, intestinal content samples of live bees from several beehives from El Galpón, Salta, Argentina were analyzed.The intestine was pulled out with sterile instruments and an homogenate of its content was prepared with sterile physiological solution. Aliquots of these suspensions were seeded both in depth and surface in LAPTg and MRS agar (selective medium for Lactobacilllus). The plaques were incubated at 35°C under microaerophilic for 24-72 h. Cell morphology was analyzed in colonies reisolated in selective medium. Besides, catalase reaction, motility, Gram tinction previous to biochemical tests (nitrate reduction, esculin fermentation arginine hydrolysis) and carbohydrate fermentation pattern were also studied. The antagonic effect of cell-free cultures on Paenibacillus larvae, Listeria monocytogenes, Bacillus sp., Enterococcus sp.; Staphylococcus aureus and moulds (Ascosphaera apis, Aspergillus niger) was determined according to the well-diffusion assay. A series of strains selected for its antimicriobial activity was analyzed by scanning electron microscope and the lactic acid production was quantified by HPLC. More than 100 strains were isolated from MRS agar. They were immobile rods and catalase negative, Gram-positive, non sporulated and nitrate negative. All these properties classified these strains in the Lactobacillus genera. Also, eight strains exhibited a strong anti-Listeria and anti-P. Larvae effect, but in this case the action was strain dependent. Lactic acid concentration in the cell-free cultures was variable and the values were between 3.06 and 6.08 g L-1. The strain that produced the most lactic acid and the only one that presented an antagonic effect on S. aureus y A. niger, by the production of this metabolite, was Lactobacillus spp. A3IO. These results indicate that Lactobacillus is a constituent of the bee intestinal microbiota, and that it is possible to formulate a probiotic suplement capable of inhibiting P. larvae in the protection against American foulbrood. ABSTRACT: Paenibacillus larvae, etiological agent of the American foulbrood, is an infection that produces large economical losses in the apicultural industry by high mortality rate. The beneficial effect that lactic acid bacteria as probiotic suplements can exert on human and animal hosts is well known. However, there is a lack of scientific information concerning the presence of these microorganisms in the microbiota associated with the Apis mellifera L. bee. Therefore, our aim in this work was to isolate, characterize phenotypically and determine the potential antimicrobial effect of bee intestine Lactobacillus strains. To this purpose, intestinal content samples of live bees from several beehives from El Galpón, Salta, Argentina were analyzed.The intestine was pulled out with sterile instruments and an homogenate of its content was prepared with sterile physiological solution. Aliquots of these suspensions were seeded both in depth and surface in LAPTg and MRS agar (selective medium for Lactobacilllus). The plaques were incubated at 35°C under microaerophilic for 24-72 h. Cell morphology was analyzed in colonies reisolated in selective medium. Besides, catalase reaction, motility, Gram tinction previous to biochemical tests (nitrate reduction, esculin fermentation arginine hydrolysis) and carbohydrate fermentation pattern were also studied. The antagonic effect of cell-free cultures on Paenibacillus larvae, Listeria monocytogenes, Bacillus sp., Enterococcus sp.; Staphylococcus aureus and moulds (Ascosphaera apis, Aspergillus niger) was determined according to the well-diffusion assay. A series of strains selected for its antimicriobial activity was analyzed by scanning electron microscope and the lactic acid production was quantified by HPLC. More than 100 strains were isolated from MRS agar. They were immobile rods and catalase negative, Gram-positive, non sporulated and nitrate negative. All these properties classified these strains in the Lactobacillus genera. Also, eight strains exhibited a strong anti-Listeria and anti-P. Larvae effect, but in this case the action was strain dependent. Lactic acid concentration in the cell-free cultures was variable and the values were between 3.06 and 6.08 g L-1. The strain that produced the most lactic acid and the only one that presented an antagonic effect on S. aureus y A. niger, by the production of this metabolite, was Lactobacillus spp. A3IO. These results indicate that Lactobacillus is a constituent of the bee intestinal microbiota, and that it is possible to formulate a probiotic suplement capable of inhibiting P. larvae in the protection against American foulbrood.