High abundance and diversity of Bacillus anthracis plasmid pXO1-like replicons in municipal wastewater.

Water from the influent of a municipal wastewater treatment plant as well as soil samples collected from the shoreline of 10 lakes were screened for the presence of the Bacillus anthracis pXO1-like plasmid replicon repX using a PCR assay. Specific PCR products were retrieved from all samples, indicating a widespread presence of pXO1-like plasmid replicons in various environmental settings. Initial screening by restriction enzyme analysis revealed at least two forms of the repX gene in the wastewater sample, which was consequently subjected to further investigation. Nine of 51 Bacillus cereus group strains isolated from the wastewater sample were shown to contain a repX-specific gene sequence. Two of these strains were shown to have repX gene sequences with very high homology to the repX gene of plasmid pXO1. The same two strains also contained replicon-specific sequences with high homology to those of pXO2-like plasmids, but did not contain the pXO1-associated cya and lef virulence genes. Collectively, the sequence information from the isolated strains and PCR products obtained using total genomic DNA as a template suggests the existence of three subgroups of pXO1-like plasmid replicons in the wastewater sample.