Location: Produce Safety and Microbiology ResearchTitle: Genomic insight into natural inactivation of Shiga toxin 2 production in an environmental Escherichia coli strain producing Shiga toxin 1
Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2020
Publication Date: 3/4/2020
Citation: Carter, M.Q., Pham, A.C., He, X., Hnasko, R.M. 2020. Genomic insight into natural inactivation of Shiga toxin 2 production in an environmental Escherichia coli strain producing Shiga toxin 1. Foodborne Pathogens and Disease. 17(9):555-567. https://doi.org/10.1089/fpd.2019.2767.
Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) are a diverse group of strains that are implicated in over 270,000 cases of human illness annually in the United States alone. Cattle are the main reservoirs for STEC. Addition of sub-therapeutic antibiotics to the feed of food-producing animals for growth promotion and disease prevention is a common agricultural practice in the U.S. Such a practice may not only affect antibiotic resistance, but also facilitate the dissemination of virulence factors such Shiga toxins since genes encoding Shiga toxins are often located in the genomes of temperate lambdoid bacteriophages. In this study, we investigated the genomic signatures of a bovine isolate RM10809-C3 and of the Stx-converting prophages in this strain by comparative genomics analyses. We further determined if the antibiotics that are commonly used in livestock could induce the Stx-converting prophages. Unexpectedly, Stx2 was not detected in control cells or cells treated with any of the antibiotics examined in this study. Further examination of regulatory cascade of stx2 in RM10809-C3 revealed several alterations in both stx2 promoters, including deletion of late promoter PR’ and the mutations in the binding site of anti-termination protein Q. As a result, the expression of stx2 was repressed constantly. Increased production of Stx1 was detected in RM10809-3 following exposure to mitomycin C and enrofloxacin, but not in cells exposed to tetracycline or monensin. Induction Stx-prophages by certain growth promoting antibiotics would lead to transfer of Shiga toxin genes to other bacteria by transduction; thus promote emergence of new Shiga toxin-producing strains in natural environments.
Technical Abstract: 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 in a bovine isolate that carries multiple Shiga toxin genes following exposure to several antibiotics commonly used in food animals. Strain RM10809-3 was co-isolated with a STEC O145:H28 strain from cattle feces near a leafy greens-growing region in California. The genome of RM10809-3 is composed of a 5,128,479-bp chromosome and a 122,641-bp plasmid, encoding 5,116 CDSs. Strain RM10809-3 belonged to O22:H8 and was clustered together with two STEC O168:H8 food isolates using either MLST or core genome-based phylogeny construction. There were six intact prophages in the genome of RM10809-3, among which, prophage 4 contains two sets of stx2; while prophage 9 carries one set of stx1. Increased production of Stx1 was detected in RM10809-3 following exposure to mitomycin C and enrofloxacin, but not in cells exposed to tetracycline or monensin. In contrast, Stx2 remained undetectable in cells treated with any of the antibiotics examined. Comparison of Shiga toxin-converting prophages in strain RM10809-3 with those in strain EDL933 revealed altered stx2 promoters in RM10809-3, including deletion of the late promoter PR' and the mutations in qut, the binding site of anti-termination protein Q. In contrast, both PR' and qut within the promoter of stx1 in RM10809-3 were identical to the corresponding one in EDL933. Furthermore, the protein Q encoded by Stx1-prophage in RM10809-C3 exhibited >94% identity with either of the two EDL933 protein Q; where as both protein Q encoded by Stx2-prophage in RM10809-C3 were distantly related to any of the EDL933 protein Q. Induction of Stx1 by enrofloxacin, a common antibiotic used in cattle, reveals potential impact of agricultural practice on dissemination of stx in natural environments.