BIOCOMPATIBILITY AND GENOTOXICITY STUDIES OF SPORE SUSPENSIONS AND SURFACTIN EXTRACTS OF Bacillus spp. WITH POTENTIAL APPLICATION IN ANIMAL HEALTH

DI GIACOMO, A.L; AZCURRA NADIA; GARCIA G; DOGI C.A; GONZÁLEZ PEREYRA M.L

Jornada; XL Reunion Anual de la Sociedad de Biología de Cuyo; 2022

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 though 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 study the biocompatibility of live bacteria and their LPs 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. Selective isolation of Bacillus spp. was performed from pen soil and bovine faeces 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 LP extracts was quantified by HPLC. On the other hand, Bacillus spp. endospores suspensions (ES) (1 x 108 UFC/ml) were obtained from culture in solid medium. ES, purified SF and different dilutions (1:10; 1:50; 1:100; 1:500 and 1:1000) of LP extracts 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 per treatment) administered 0,2 ml of the spore suspensions (containing 108 spores) orally for 10 days. After the experiment, animals were sacrificed and bone marrow samples were collected for the bone marrow erythrocyte micronuclei assay. All isolates produced SF and the quantified extracts contained between 15.96 and 239.96 µg/ml SF. ES suspensions containing 1 x 108 UFC/ml resulted non-cytotoxic to Caco-2 cells showing viability percentages (%V) over 70 % that did not differ significantly from untreated controls. Likewise, SF and all dilutions of LP extracts showed no cytotoxicity over Caco-2 demonstrating not to harm intestinal cells. Only MFF 1.11 isolate show V% < 70 in all dilutions tested. However, SF concentration of this extract was between the safe range that showed no toxicity when testing purified SF (10 ng/ml to 500 µg/ml) suggesting another harmful compound was be produced by this isolate and it will not be selected for future studies. SF concentration of non-cytotoxic LP dilutions tested varied between 0.01 and 239.97 µg/ml. ES of the three isolates tested (MFF 2.2, MFF 1.11 and TC 12) did not show genotoxicity nor cytotoxicity in vivo since micronucleated polychromatic erythrocytes (MNPCE) frequencies and PCE/NCE counts in treated mice did not differ significantly from untreated controls. The present study allowed us to select the safe Bacillus spp. isolates and SF concentrations to test their bneficial and imunostimulant properties and their potential to be used in animal production and health.