Genomic Insight into Natural Inactivation of Shiga Toxin 2 Production in an Environmental Escherichia coli Strain Producing Shiga Toxin 1.

Shiga toxin-producing Escherichia coli (STEC) consists of a group of diverse strains differing greatly in genetic make-up and pathogenicity potential. Here, we investigated production of Shiga toxins (Stxs) in a bovine isolate carrying multiple Shiga toxin genes (stxs) after exposure to several antibiotics commonly used in food animals. Strain RM10809-C3 was co-isolated with a STEC O145:H28 strain from cattle feces near a leafy greens-growing region in California. The genome of RM10809-C3 is composed of a 5,128,479-bp chromosome and a 122,641-bp plasmid, encoding 5108 coding sequences. Strain RM10809-C3 belongs to serotype O22:H8 and is clustered together with two STEC O168:H8 food isolates using either multilocus sequence type or core genome-based phylogenetic analysis. Six intact prophages were identified in the genome of RM10809-C3, among which prophage 4 contained two sets of stx2d; whereas prophage 9 carried one set of stx1a. Increased production of Stx1 was detected in RM10809-C3 after exposure to mitomycin C and enrofloxacin, but not in cells exposed to tetracycline. In contrast, Stx2 remained undetectable in cells treated with any of the antibiotics examined. Comparison of Stx-converting prophages in strain RM10809-C3 with those in strain EDL933 revealed altered stx2 promoters in RM10809-C3, including deletion of the late promoter PR' and the mutations in qut, the binding site of antitermination protein Q. In contrast, both PR' and qut within the promoter of stx1 in RM10809-C3 were identical to the corresponding one in EDL933. Further, the protein Q encoded by Stx1-prophage in RM10809-C3 exhibited >94% identity with either of the two EDL933 protein Q; whereas both protein Q encoded by Stx2-prophage in RM10809-C3 were distantly related to any of the EDL933 protein Q. Natural silence of Stx2 production in strain RM10809-C3 emphasizes that not only the stx coding regions but also their regulatory factors are important in STEC risk assessment.