Five copper homeostasis gene clusters encode the Cu-efflux resistome of the highly copper-tolerant Methylorubrum extorquens AM1

BACKGROUND: In the last decade, the use of copper has reemerged as a potential strategy to limit healthcare-associated infections and to control the spread of multidrug-resistant pathogens. Numerous environmental studies have proposed that most opportunistic pathogens have acquired antimicrobial resistance in their nonclinical primary habitat. Thus, it can be presumed that copper-resistant bacteria inhabiting a primary commensal niche might potentially colonize clinical environments and negatively affect the bactericidal efficacy of Cu-based treatments. The use of copper in agricultural fields is one of the most important sources of Cu pollution that may exert selection pressure for the increase of copper resistance in soil and plant-associated bacteria. To assess the emergence of copper-resistant bacteria in natural habitats, we surveyed a laboratory collection of bacterial strains belonging to the order Rhizobiales. This study proposes that Methylorubrum extorquens AM1 is an environmental isolate well adapted to thrive in copper-rich environments that could act as a reservoir of copper resistance genes. METHODS: The minimal inhibitory concentrations (MICs) of CuCl(2) were used to estimate the copper tolerance of eight plant-associated facultative diazotrophs (PAFD) and five pink-pigmented facultative methylotrophs (PPFM) belonging to the order Rhizobiales presumed to come from nonclinical and nonmetal-polluted natural habitats based on their reported source of isolation. Their sequenced genomes were used to infer the occurrence and diversity of Cu-ATPases and the copper efflux resistome of Mr. extorquens AM1. RESULTS: These bacteria exhibited minimal inhibitory concentrations (MICs) of CuCl(2) ranging between 0.020 and 1.9 mM. The presence of multiple and quite divergent Cu-ATPases per genome was a prevalent characteristic. The highest copper tolerance exhibited by Mr. extorquens AM1 (highest MIC of 1.9 mM) was similar to that found in the multimetal-resistant model bacterium Cupriavidus metallidurans CH34 and in clinical isolates of Acinetobacter baumannii. The genome-predicted copper efflux resistome of Mr. extorquens AM1 consists of five large (6.7 to 25.7 kb) Cu homeostasis gene clusters, three clusters share genes encoding Cu-ATPases, CusAB transporters, numerous CopZ chaperones, and enzymes involved in DNA transfer and persistence. The high copper tolerance and the presence of a complex Cu efflux resistome suggest the presence of relatively high copper tolerance in environmental isolates of Mr. extorquens.