Diversity of metal and antibiotic resistance genes in Enterococcus spp. from the last century reflects multiple pollution and genetic exchange among phyla from overlapping ecosystems

Abstract

Arsenic (As), mercury (Hg), and copper (Cu) are among the major historical and contemporary metal pollutants linked to global anthropogenic activities. Enterococcus have been considered indicators of fecal pollution and antibiotic resistance for years, but its largely underexplored metallome precludes understanding their role as metal pollution bioindicators as well. Our goal was to determine the occurrence, diversity, and phenotypes associated with known acquired genes/operons conferring tolerance to As, Hg or Cu among Enterococcus and to identify their genetic context (381 field isolates from diverse epidemiological and genetic backgrounds; 3547 enterococcal genomes available in databases representing a time span during 1900–2019). Genes conferring tolerance to As (arsA), Hg (merA) or Cu (tcrB) were used as biomarkers of widespread metal tolerance operons. Different variants of metal tolerance (MeT) genes (13 arsA, 6 merA, 1 tcrB) were more commonly recovered from the food-chain (arsA, tcrB) or humans (merA), and were shared with 49 other bacterial taxa. Comparative genomics analysis revealed that MeT genes occurred in heterogeneous operons, at least since the 1900s, with an increasing accretion of antibiotic resistance genes since the 1960's, reflecting diverse antimicrobial pollution. Multiple MeT genes were co-located on the chromosome or conjugative plasmids flanked by elements with high potential for recombination, often along with antibiotic resistance genes. Phenotypic analysis of some isolates carrying MeT genes revealed up to 128× fold increase in the minimum inhibitory concentrations to metals. The main distribution of functional MeT genes among Enterococcus faecium and Enterococcus faecalis from different sources, time spans, and clonal lineages, and their ability to acquire diverse genes from multiple taxa bacterial communities places these species as good candidates to be used as model organisms in future projects aiming at the identification and quantification of bioindicators of metal polluted environments by anthropogenic activities.