Interaction of Chromobacterium species with Batrachochytrium dendrobatidis at different Temperatures

Abstract

Chytrid fungus, Batrachochytrium dendrobatidis (Bd) has been identified as one of the major culprits in amphibian population declines worldwide. This fungus grows upon and colonizes the keratin layer of the amphibian skin. Amphibian skin also harbors a rich diversity of bacteria species, and some of these bacteria have been found to inhibit Bd growth and colonization. Janthinobacterium lividum, a violacein-producing bacteria species is one of the most studied anti-Bd bacteria, but there are Chromobacterium species that also produce similar metabolites but have received less attention regarding their efficacy against Bd. Violacein produced by J. lividum has been reported to be antifungal, but the effect of temperature on the efficacy of this bacterial metabolite has not been examined. Also, the anti-Bd properties of Chromobacterium spp. has not been tested. In this study, we isolated and identified four Chromobacterium spp. from the skin of cricket frogs (Acris gryllus) in ponds around Tuskegee, AL. We grew the different species (C. amazonesis, C. substugae, C. etc) in a tryptone broth culture for three days and collected purple pigmented, cell-free supernate of the cultures (which contained violacein, confirmed by mass spectrometry). Using a 96 well plate, each identified bacterial species supernate as well as the supernate from J. lividum (YO8846) was tested against three strains of Bd (JEL 423, JEL 310 and JEL 197), that have been documented to be highly pathogenic to frogs at a range of environmentally relevant temperatures (12, 16, 22 and 24℃). Each well absorbance was recorded every other day for 27 days, a inhibition score calculated, o analyze the degree of inhibition of each bacterial ribotype (the 4 Chromobacterium and J. lividum) for each Bd strain. We found that temperature had a significant effect on the strength of Bd inhibition by all bacteria species, with significantly more inhibition observed at the lower tested temperatures. Also, all tested species of Chromobacterium significantly inhibited Bd growth more than J. lividum at almost all tested temperatures. We also tested the effect of temperature on proliferation of the isolated Chromobacterium spp. and characterized the potency of violacein produced from the different bacterial isolates We found the tested Chromobacterium spp. grew faster the J. lividum at all tested temperatures and produced violacein at all these temperatures, though in varying quantity. In contrast, J. lividum did not produce violacein at 12℃ and 30℃. We also found that all Chrombacterium to have significantly higher inhibitory ability than J. lividum at equivalent dilution factors, only becoming equivalent to J. lividum at the 1:1000 fold dilution. v. Overall, this study shows that these set of violacein-producing bacteria are abundant in majority of sampled frogs and could provide a vital protection against Bd infection.