Biocompatibility and genotoxicity studies of spore suspensions and surfactin extracts of Bacillus spp. with potential application in animal health.

DI GIACOMO A.L.; AZCURRA A.; GARCÍA G. R.; DOGI C.A.; GONZÁLEZ PEREYRA, M.L.

Mendoza

Jornada; XL Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2022

Sociedad de Biología de Cuyo

Bacillus species include beneficial strains that are used as probiotic additives to improve animal production. Thermo-resistant spores can be easily administered in water or feed to germinate in the gut and exert their beneficial effect through immunomodulation, reducing inflammation and protecting against enteric pathogens. Surfactin (SF) is a cyclic lipopeptide (LP) produced by Bacillus species with demonstrated anti-inflammatory, anti-microbial, anti-tumoral and Immunomodulatory activity. The aim of the present study was to isolate native SF-producing Bacillus spp. strains and to study the biocompatibility of live bacteria and their LP extracts (LPE) with animal systems in vivo and in vitro to determine their potential to be included in products of veterinary use such as feed additives and/or health-improving treatments. Bacillus spp. were isolated and six isolates were randomly selected. Cell-free culture supernatants of each were extracted 1:1, v/v with n-butanol and SF content of LPE was quantified by HPLC. On the other hand, Bacillus spp. endospores suspensions (ES) (1 x 108 UFC/ml) were obtained from solid medium culture. ES, purified SF and different dilutions (1:10; 1:50; 1:100; 1:500 and 1:1000) of LPE were tested for biocompatibility in vitro on Caco-2 cell line using the MTT colorimetric assay. ES genotoxicity was tested in vivo on BALB/c mice (n=6) administered 0.2 ml of the spore suspensions (108 spores) orally for 10 days. Animals were sacrificed and bone marrowsamples were collected for the bone marrow erythrocyte micronuclei assay. All isolates produced between 15.96 and 239.96 μg/ml SF. ES suspensions containing 108 UFC/ml resulted non-cytotoxic to Caco-2 cells showing viability percentages (%V) over 70 %. Likewise, SF and all dilutions of LPE showed no cytotoxicity over Caco-2 demonstrating not to harm intestinal cells. Only MFF 1.11 isolate showed V% < 70 in all dilutions tested. However, SF concentration of this extract was between the safe range that showed no toxicity for SF (10 ng/ml to 500 μg/ml) suggesting that another harmful compound was be produced by this isolate. SF concentration of non-cytotoxic LPEdilutions tested varied between 0.01 and 239.97 μg/ml. ES of the three tested isolates (MFF 2.2, MFF 1.11 and TC 12) did not show genotoxicity nor cytotoxicity in vivo. The present study allowed us to select the safe Bacillus spp. isolates and SF concentrations to test their beneficial and immunostimulant properties and their potential to be used in animal production and health.