| Summary: | <i>Listeria monocytogenes,</i> the bacterial foodborne pathogen responsible for the severe disease listeriosis, frequently exhibits heavy metal resistance. Concurrent resistance to cadmium and arsenic in <i>L. monocytogenes</i> is strongly associated with the 35-kb chromosomal island LGI2. LGI2 has been encountered repeatedly among <i>L. monocytogenes</i> serotype 4b hypervirulent clones but, surprisingly, not among non-pathogenic <i>Listeria</i> spp. Here we describe a novel LGI2 variant, LGI2-3, in two <i>L. welshimeri</i> strains from an urban aquatic environment. Whole genome sequence analysis revealed that the genomes were closely related except for one prophage region and confirmed a chromosomally integrated LGI2-3. It harbored a cystathionine beta-lyase gene previously only encountered in LGI2-1 of <i>L. monocytogenes</i> clonal complex 1 but was otherwise most closely related to LGI2. LGI2-3 harbored a novel <i>cadAC</i> cassette (<i>cadA7C7)</i> that, like LGI2′s <i>cadA4C4</i>, was associated with lower-level tolerance to cadmium (MIC 50 μg/mL) than other <i>cadAC</i> cassettes (MIC ≥ 140 μg/mL). CadA sequence analysis identified two amino acids that may be important for mediating different levels of cadmium tolerance. Our findings clearly demonstrated the potential for LGI2-like islands to be harbored by non-pathogenic <i>Listeria</i> spp. and generate intriguing hypotheses on the genetic diversity mediated by this island and its transfer among <i>Listeria</i> spp.
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