Reversible monensin adaptation in Enterococcus faecium, Enterococcus faecalis and Clostridium perfringens of cattle origin: potential impact on human food safety

To determine the stability/reversibility and mechanism of monensin adaptation in monensin-treated cattle isolates compared with reference bacterial isolates, exposed in vitro to high monensin concentrations. We evaluated the potential for cattle-origin strains of Clostridium perfringens, Enterococcus faecium and Enterococcus faecalis exposed to monensin in vivo (in vivo monensin-exposed isolates) to maintain or achieve the ability to grow in the presence of high monensin concentrations (in vitro monensin-adapted isolates). Twenty-one consecutive subcultures of the in vitro monensin-adapted strains were performed, and monensin MICs were determined for the 3rd, 7th, 14th and 21st subcultures (subcultured isolates). SDSPAGE and transmission electron microscopy (TEM) were used to determine protein expression and visualize extracellular morphology changes. Monensin-non-exposed isolates did not display monensin adaptation during in vitro monensin exposure. In contrast, in vivo monensin-exposed isolates displayed monensin adaptation enabling growth at 32 MIC. Upon consecutive subculturing, monensin MICs returned to baseline, or one dilution above, for the monensin-adapted strains. SDSPAGE identified overexpression of a 14 kDa protein (C. perfringens) and a 20.5 kDa protein (E. faecium and E. faecalis) in the monensin-adapted isolates. TEM demonstrated that in vitro monensin-adapted strains had a significantly thicker cell wall or glycocalyx compared with in vivo monensin-exposed or subcultured isolates. In vivo monensin-exposed isolates of C. perfringens, E. faecium and E. faecalis have the ability to grow in the presence of high monensin concentrations in vitro. This is associated with an increased thickening of the cell wall or glycocalyx that is reversible upon serial passage, suggesting a phenotypically expressed, but not genetically stable, trait.