Resistance and Tolerance to Tropodithietic Acid, an Antimicrobial in Aquaculture, Is Hard To Select

Abstract

The antibacterial compound tropodithietic acid (TDA) is produced by bacteria of the marine Roseobacter clade and is thought to explain the fish probiotic properties of some roseobacters. The aim of the present study was to determine the antibacterial spectrum of TDA and the likelihood of development of TDA resistance. A bacterial extract containing 95% TDA was effective against a range of human-pathogenic bacteria, including both Gram-negative and Gram-positive bacteria. TDA was bactericidal against Salmonella enterica serovar Typhimurium SL1344 and Staphylococcus aureus NCTC 12493 and killed both growing and nongrowing cells. Several experimental approaches were used to select mutants resistant to TDA or subpopulations of strains with enhanced tolerance to TDA. No approach (single exposures to TDA extract administered via different methods, screening of a transposon library for resistant mutants, or prolonged exposure to incremental concentrations of TDA) resulted in resistant or tolerant strains. After more than 300 generations exposed to sub-MIC and MIC concentrations of a TDA-containing extract, strains tolerant to 2؋ the MIC of TDA for wild-type strains were selected, but the tolerance disappeared after one passage in medium without TDA extract. S. Typhimurium mutants with nonfunctional efflux pump and porin genes had the same TDA susceptibility as wild-type strains, suggesting that efflux pumps and porins are not involved in innate tolerance to TDA. TDA is a promising broad-spectrum antimicrobial in part due to the fact that enhanced tolerance is difficult to gain and that the TDA-tolerant phenotype appears to confer only low-level resistance and is very unstable.

Global occurrence and heterogeneity of the Roseobacter-clade species Ruegeria mobilis

“…Specifically, strains belonging to the species Ruegeria mobilis and Phaeobacter inhibens are potent antagonists of the fish pathogens (Planas et al, 2006;Porsby et al, 2008;D'Alvise et al, 2010) primarily caused by production of the antibacterial compound tropodithietic acid (TDA) (D'Alvise et al, 2010(D'Alvise et al, , 2012. TDA is also bactericidal against human pathogens including Salmonella enterica and Staphylococcus aureus (Porsby et al, 2011) and it has been recently been demonstrated that TDA acts by disrupting the proton motive force (Wilson et al, 2016). TDA is produced by R. mobilis, P. inhibens and Phaeobacter gallaeciensis, as well as by other alphaproteobacteria belonging to Pseudovibrio (Porsby et al, 2008;Geng and Belas, 2010;Penesyan et al, 2011;Harrington et al, 2014).…”

Vibrio anguillarum Is Genetically and Phenotypically Unaffected by Long-Term Continuous Exposure to the Antibacterial Compound Tropodithietic Acid

“…TDA is a broad-spectrum antibacterial that is bactericidal and inhibits the growth of several human and fish pathogens (16,(21)(22)(23) but has very low toxicity in Caenorhabditis elegans and Artemia (24). Recently, it was demonstrated that TDA is cytotoxic against N2a cells and OLN-93 cells of the mammalian nervous system and several cancer cell lines, respectively (25,26).…”

“…Since TDA has pronounced antibacterial activity, the development of resistance to TDA could be a concern, especially following long-term use. Attempts to select for TDA resistance and tolerance in pathogenic bacteria have not been successful (22), suggesting that TDA resistance does not arise easily, likely due to a highly conserved target (or targets). Very recently, Wilson et al demonstrated that cell membranes and the proton motive force (PMF) were targets of TDA, and TDA was suggested to function as an electroneutral proton antiporter, facilitating a one-to-one exchange of H ϩ for a single charged metal ion and thus disrupting the transmembrane proton gradient (⌬pH) (26).…”

Tropodithietic Acid Production in Phaeobacter gallaeciensis Is Regulated by N-Acyl Homoserine Lactone-Mediated Quorum Sensing

“…in turbot larval farms was found to suppress growth of marine pathogens such as Vibrio anguillarum (37) and enhanced survival of the larvae (45). Thus, the use of these roseobacters as probiotics in aquaculture facilities (25,36,38) or as antifouling agents in marine systems (15,40) is of great interest.…”