Procedure for in vitro evaluation of antimicrobial compounds against <i>Lawsonia intracellularis</i>

Lawsonia intracellularis is an obligate intracellular bacterium invading the intestinal epithelium. The culture conditions for L. intracellularis make evaluation of antimicrobial compounds challenging. This study demonstrates an in vitro procedure to evaluate antimicrobial efficacy using green tea extract (-)-epigallocatechin-3-gallate (EGCG) as a test compound. To evaluate EGCG's effect on extracellular bacteria, infectivity of L. intracellularis (PHE MN-100) exposed to EGCG or control media was assessed using mouse fibroblast (McCoy) cells. Lawsonia intracellularis in media was quantified by qPCR and in cells by immunostaining at 3- and 5-days post infection (dpi). As no safe dose of EGCG for intestinal epithelial cells is established, enteroids (swine-derived organoids) were exposed to varying concentrations of EGCG. Cell viability indicated a safe dose of ≤500 nM EGCG. To evaluate EGCG's effect on intracellular bacteria, enteroid monolayers were infected. At 3-dpi, enteroids were treated with up to 500 nM EGCG . Lawsonia intracellularis in media was quantified every 48 h by qPCR. Presence of L. intracellularis was evaluated by immunostaining cells at 5-, 7-, and 14-dpi. Although we found no differences in L. intracellularis infection when treated with EGCG, the procedures are useful to evaluate safety and efficacy of antimicrobial compounds on obligate intracellular bacteria in vitro.