Effect of the hexane extract of Achyrocline satureioides on the growth, production of proteases and spore germination of Paenibacillus larvae

PIMENTEL BETANCURT, DIANA; PALETTI ROVEY MF; CASSINA, C.; BEOLETTO V; OLIVA, M.M.; J.M. MARIOLI

Conferencia; 7th International Conference on Microbial Communication for Young Scientists; 2018

PATHOGENESIS // 81 P18: Effect of the hexane extract of Achyrocline satureioides on the growth, production of proteases and spore germination of Paenibacillus larvae Diana C. Pimentel Betancurt 1,2 *, Ma. Fernanda Paletti Rovey 1, Carolina Cassina 1, Viviana Beoletto 1, Ma. De las Mercedes Oliva 1 and Juan Miguel Marioli 2 1 Dept. of Microbiology and Immunology. National University of Río Cuarto, Argentina 2 Dept. of Chemistry. National University of Río Cuarto, Argentina * dpimentelb@exa.unrc.edu.ar Paenibacillus larvae, Gram positive bacillus that affects the larval stage of honey bees, causes the disease known as American Foulbrood (AFB). It is a highly destructive and notifiable disease, which has a negative impact on beekeeping, agriculture and food security [1]. The spores of the pathogen reach the larva through contaminated food provided by the nurse bees, reaching the lumen of the larval intestine, where they germinate and the vegetative cell proliferates massively destroying the peritrophic membrane and the cell-cell junctions until the death of the larva [2]. During infection, P. larvae secretes proteases that could be involved in pathogenicity [3]. For the control of the ABF, antibiotics such as oxytetracycline hydrochloride (OTC) were used to act effectively on the bacterial form, but not on the infectious spore, masking the disease by reducing the symptoms, without completely eradicating it, also generating bacterial resistance and residues of these chemicals in honey [4]. The plant extracts are proposed as an ecological, viable and acceptable alternative for the treatment of the disease in the hives. Achyrocline satureioides is a plant native to southeastern South America, widely used due to its pharmacological and antibacterial properties [5]. In this work, the effect of the hexane extract (HE) of A. satureioides on the growth, the production of proteases and the germination of the spore of P. larvae under different culture conditions was evaluated. For this, the minimum inhibitory concentration (MIC) of the HE was determined by the broth microdilution method [6]. The effect of the HE on the bacterial growth was evaluated by adding the same from the beginning of the test and half of the exponential phase, making measurements of optical density (OD) at 600 nm at different times Samples were taken at different times of the growth curve and subjected to heat treatment in order to analyze the effect of the HE on the germination of the spore.They were seeded on plates with MYPGP agar incubating at 37 °C in microaerophilic for 48 hours, then the colonies were counted. The production of proteases was determined qualitatively in milk agar plates, evidencing the presence or absence of hydrolysis halos of the protein. The MIC of A. satureioides HE was 0.39 μg / ml. The treatments with the HE added from the beginning as well as in the exponential phase, affected the growth of P. larvae, since there were no changes in the OD throughout the experiment, obtaining absorbance values of less than 0.1. After the thermal treatment, scarce or no turbidity was observed in the tubes with the HE, likewise a decrease in the number of colonies was observed in plates with MYPGP agar compared with the control. The production of proteases was affected by the presence of HE, observing haloes of smaller size or absence of them in comparison with the control. In conclusion, the treatment with HE acted directly on the vegetative cell, decreasing the bacterial population and preventing the spore from germinating in the presence of this one. Preliminary results show that there was a decrease in proteolytic activity. Given that germination is the first step of the infection, followed by the production of proteases, the HE of A. satureioides could be used as a preventive and promising measure for the control of AFB. [1[ Djukic, M (2014). How to kill the Honey Bee Larva: Genomic Potential and Virulence Mechanisms of Paenibacillus larvae. Plos one, 9, 1-5. [2] García González E, Genersch E. (2013) Honey bee larval peritrophic matrix degradation during infection with Paenibacillus larvae, the etiological agent of American Foulbrood of honey bees, is a key step in pathogenesis. Environ Microbiol 15, 2894?2901. [3] Antúnez, K; Anido, M; Evans, J. D. & Zunino, P. (2010). Secreted and immunogenic proteins produced by the honeybee bacterial pathogen, Paenibacillus larvae. Vet Microbiol 141, 385?389. [4] Evans, J.D. (2003). Diverse origins of tetracycline resistance en the honey bee bacterial pathogen Paenibacillus larvae. J. Inv. 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