Characterization of a T4-like bacteriophage vB_EcoM-Sa45lw as a potential biocontrol agent for Shiga toxin-producing Escherichia coli O45 contaminated on Mung Bean seeds

Authors: Liao, Yen-Te; ZHANG, YUJIE - Oak Ridge Institute For Science And Education (ORISE); Salvador, Alexandra; Harden, Leslie - Les; Wu, Vivian

Submitted to: Microbiology Spectrum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/29/2021
Publication Date: 2/2/2022
Citation: Liao, Y., Zhang, Y., Salvador, A., Harden, L.A., Wu, V.C. 2022. Characterization of a T4-like bacteriophage vB_EcoM-Sa45lw as a potential biocontrol agent for Shiga toxin-producing Escherichia coli O45 contaminated on Mung Bean seeds. Microbiology Spectrum. 10(1). Article e02220-21. https://doi.org/10.1128/spectrum.02220-21.
DOI: https://doi.org/10.1128/spectrum.02220-21

Interpretive Summary: Lytic bacteriophages (or phages) have been considered as an alternative to antibiotics to control bacterial pathogens due to antibiotic resistance issues. Shiga toxin-producing E. coli (STEC) strains have been contributed to many produce-associated outbreaks around the world. Although more and more phages lytic against various STEC strains have been isolated and studied for their biocontrol effects, the related information regarding STEC O45-infecting phages is scarce. This study focused on understanding the genomic and biological features of a lytic bacteriophage—vB_EcoM-Ro45lw (or Sa45lw)—and determining the antimicrobial activity to control STEC O45 strains on the contaminated seeds. The results show that phage Sa45lw is a new tailed-phage member classified as the Tequatrovirus genus, sharing a close evolutionary relationship to Escherichia phage AR1. The phage does not contain virulence and lysogenic genes, which could diminish the safety of phage application. The biological features indicate that phage Sa45lw has a single replication period of 27min and a burst size of 80 phage particles per infected bacterial cell. The phage has a wide host range, showing a stronger lytic activity against STEC O45 and generic E. coli ATCC13706 than STEC O157. Additionally, Sa45lw was able to reduce more than 90% of STEC O45 strains in liquid media and on the contaminated seeds at room temperature within 6 h. The findings of this study demonstrate that phage Sa45lw is a promising biocontrol agent to combat STEC O45 and could be used in the produce pre-harvest environment.

Technical Abstract: Lytic bacteriophages (or phages) have been considered as an alternative to antibiotics to control bacterial pathogens due to antibiotic resistance issues. Shiga toxin-producing E. coli (STEC) strains, including the serogroups of O157 and the top six non-O157, have been contributed to many produce-associated outbreaks around the world, and the contaminated seeds are commonly found to introduce the pathogens into the farm-to-fork production line. Although there are increasing numbers of phages isolated and characterized to combat these STEC pathogens, the efforts on STEC O45-infecting phages are relatively scarce. The objectives of this study were to characterize a T4-like Escherichia phage vB_EcoM-Sa45lw (or Sa45lw) and to determine the antimicrobial activity against STEC O45 on mung bean seeds. The results indicated that Phage Sa45lw, previously isolated from surface water in a produce-growing area, is classified to the Tequatrovirus genus under the Myoviridae family, showing the highest evolutionary relationship with Escherichia phage AR1. The phylogenetic results showed that Sa45lw contained the gene of long-tail fiber (gp37) similar to that of Escherichia phage KIT03, even though the host ranges between phages Sa45lw (O157, O45, and ATCC13706) and KIT03 (O157 and Salmonella enterica) were different. No stx, antibiotic resistance, and lysogenic genes were found. Sa45lw has a latent period of 27 min with an estimated burst size of 80 PFU/CFU. The phage was stable at a wide range of pH (pH 3 to pH 10.5) and temperature (-80'C to 50'C). Phage Sa45lw (MOI=10) had a stronger antimicrobial effect against E. coli O45:H16 than E. coli O157:H7, reducing the STEC O45 by approximately 5 logs in lysogeny broth after 6 h at 25'C. Additionally, after phage treatment (MOI=1,000) for 15 min, approximately 1 log reduction of the STEC O45 on the contaminated seeds was observed compared to the untreated group within 6 h at room temperature. These findings indicate phage Sa45lw is a promising antimicrobial agent and can be used for the development of phage cocktails or hurdle interventions to enhance the biocontrol of STEC on contaminated seeds.