Prediction, diversity, and genomic analysis of temperate phages induced from Shiga toxin-producing Escherichia coli strains

Authors: ZHANG, YUJIE - Shanghai Ocean University; Liao, Yen-Te; Salvador, Alexandra; SUN, XIAOHONG - Shanghai Ocean University; Wu, Vivian

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/20/2019
Publication Date: 1/21/2020
Citation: Zhang, Y., Liao, Y., Salvador, A., Sun, X., Wu, V.C. 2020. Prediction, diversity, and genomic analysis of temperate phages induced from Shiga toxin-producing Escherichia coli strains. Frontiers in Microbiology. 10:e3093. https://doi.org/10.3389/fmicb.2019.03093.
DOI: https://doi.org/10.3389/fmicb.2019.03093

Interpretive Summary: Shiga toxin (Stx)-producing Escherichia coli (STEC), including E. coli O157:H7 and other non-O157 pathogenic strains, have been linked to numerous foodborne outbreaks, such as the recent E. coli O157:H7-contaminated romaine lettuce outbreaks in California. The virulence genes—stx genes—fall in a specific sequence region within the STEC genome, also known as Stx prophage. The Stx prophage usually remains silent in the bacterial genome and can be activated through certain external stress and released in the environment. The released phages, also known as Stx-converting phages, pose potential risks of transferring stx gene to other commensal E. coli strains through infection and are likely attributed to emergence of new pathogens. However, the information regarding the diversity of prophage sequences in STEC strains is scarce. Here, we predicted various prophages from different STEC genomes by PHASTER. Eight STEC strains were further selected to study the effect of different stress factors, such as UV irradiation, antibiotics, and common antimicrobial agents used in the food industry, on the release of Stx prophage from the STEC bacterial strains. The results of this study show that an average of 8 to 22 prophages were identified in each STEC genome. Eleven strains contain more than one Stx prophages. In addition, Stx prophages in general were prone to be released after treatment of EDTA and UV irradiation. Stx-converting phages released from the selected strains had different morphologies; one phage, containing stx1 gene, had a capsid with a long tail and the other two phages, containing stx2 gene, had a capsid with a short tail. The findings of this study demonstrate the genomes of STEC strains contain various prophages, which were likely to be released from the host STEC strains under certain stress conditions. Furthermore, Stx-converting phages have the potential to infect and transfer stx genes to the susceptible strains and may contributes to the emergence of new pathogens.

Technical Abstract: Shiga toxin-producing Escherichia coli (STEC) is a notorious foodborne pathogen containing stx genes located in the sequence region of Shiga toxin (Stx) prophages. Stx prophages, as one of the mobile elements, are involved in the transfer of virulence genes to other strains. However, little is known about the diversity of prophages among STEC strains. The objectives of this study were to predict various prophages from different STEC genomes and to evaluate the effect of different stress factors on Stx prophage induction. Forty bacterial whole-genome sequences of STEC strains obtained from NCBI were used for the prophage prediction using PHASTER webserver. Eight of the STEC strains from different serotypes were subsequently selected to quantify the induction of Stx prophages by various treatments, including antibiotics, temperature, irradiation, and antimicrobial agents. After induction, Stx1-converting phage Lys8385Vzw and Stx2-converting phage Lys12581Vzw were isolated and further confirmed for the presence of stx genes using conventional PCR. Phage morphology was observed by transmission electron microscopy. The prediction results showed an average of 8 to 22 prophages, with one or more encoding stx, were predicted from each STEC genome obtained in this study. Additionally, the phylogenetic analysis revealed high genetic diversity of Stx prophages among the 40 STEC genomes. However, the sequences of Stx prophages in the genomes of STEC O45, O111, and O121 strains, in general, shared higher genetic homology than those in other serotypes. Interestingly, most STEC strains with two or more stx genes carried at least one each of Stx1 and Stx2 prophages. The induction results indicated Stx1 and Stx2 prophages from the 8 selected STECs were prone to be induced by EDTA and UV, respectively. Additionally, both Stx-converting phages could infect non-pathogenic E. coli (WG5, DH5a, and MG1655) and form new lysogens. The findings of this study indicate that Stx prophages can be induced by environmental stress, such as exposure to solar radiation, and pose a risk of virulence gene transfer to other commensal strains.