Characterization of polyvalent Escherichia phage Sa157lw for the biocontrol potential of Salmonella Typhimurium and Escherichia coli O157:H7 on contaminated mung bean seeds

Authors: Liao, Yen-Te; ZHANG, YUJIE - Oak Ridge Institute For Science And Education (ORISE); Salvador, Alexandra; HO, KAN-JU - Forest Service (FS); Cooley, Michael; Wu, Vivian

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/26/2022
Publication Date: 11/10/2022
Citation: Liao, Y., Zhang, Y., Salvador, A., Ho, K., Cooley, M.B., Wu, V.C. 2022. Characterization of polyvalent Escherichia phage Sa157lw for the biocontrol potential of Salmonella Typhimurium and Escherichia coli O157:H7 on contaminated mung bean seeds. Frontiers in Microbiology. 13. Article 1053583. https://doi.org/10.3389/fmicb.2022.1053583.
DOI: https://doi.org/10.3389/fmicb.2022.1053583

Interpretive Summary: Sprouts are prone to bacterial contamination during production. Although chemical-based interventions, such as chlorinated compounds, are commonly used in the sprout industry, the yield and quality of sprouts can be affected by these antimicrobial treatments. Yet, unlike other intervention technologies that kill both good and harmful bacteria to humans, bacteriophage (viruses that kill bacteria) applications can specifically target certain bacterial species that are human pathogens. Additionally, phage application is eco-friendly and can also prevent the occurrence of antimicrobial-resistant bacteria. To our knowledge, there is limited information concerning the characterization of a lytic phage that can infect both pathogenic E. coli and Salmonella. This study focused on understanding the genomic and biological features of a lytic bacteriophage Sa157lw and determining its biocontrol potential of pathogenic E. coli and Salmonella strains on contaminated seeds. The results show that phage Sa157lw is a new member of the bacteriophage with a contractile tail belonging to the Kuttervirus genus. The bacteriophage does not contain unwanted genes (virulence and lysogenic genes), which could diminish the safety of phage application. Additionally, genomic analysis supported that Sa157lw has a broad host range, which can infect different bacterial species, including Salmonella and E. coli O157:H7 strains. The biological features indicate that bacteriophage Sa157lw has the same single replication period (30 min) for both E. coli O157:H7 and Salmonella Typhimurium to produce 130 and 220 phage particles per infected bacterial cell, respectively. Additionally, Sa157lw demonstrated significant antimicrobial effects against both E. coli O157:H7 and Salmonella Typhimurium upon application to the contaminated mung bean seeds, with a higher reduction in Salmonella. The findings of this study confirm the potential for using bacteriophage Sa157lw as a biocontrol agent to prevent Salmonella Typhimurium and E. coli O157:H7 from the contamination of mung bean seeds.

Technical Abstract: Seeds are one of the primary sources of contamination with foodborne pathogens, such as pathogenic E. coli, and various Salmonella serovars, for produce, particularly sprouts. Due to the susceptibility of sprout growth to chemical-based antimicrobials and the rising issue of antimicrobial resistance, developing innovative antimicrobial interventions is an urgent need. Therefore, the objective of this study was to characterize Escherichia phage Sa157lw (or Sa157lw) for the biocontrol potential of Salmonella Typhimurium and E. coli O157:H7 on contaminated mung bean seeds. Phage Sa157lw was subjected to whole-genome sequencing and biological characterization, including morphology, one-step growth curve, and stress stability tests. Later, antimicrobial activity was determined in vitro and upon application on the mung bean seeds artificially contaminated with E. coli O157:H7 or Salmonella Typhimurium. Sa157lw possessed a contractile tail and belonged to the Kuttervirus genus under the Ackermannviridae family, sharing a close evolutionary relationship with E. coli phage ECML-4 and Kuttervirus ViI; however, tail spike genes (ORF_102 and ORF_104) were the primary region of difference. Comparative genomics showed that Sa157lw encoded a cluster of tail spike genes—including ORF_101, ORF_102, and ORF_104—sharing high amino acid similarity with the counterfeits of various Salmonella phages. Additionally, Sa157lw harbored a unique tail fiber (ORF_103), possibly related to the receptors binding of O157 strains. The genomic evidence accounted for the polyvalent effects of Sa157lw against E. coli O157:H7 and various Salmonella serovars (Typhimurium, Enteritidis, Agona, Saintpaul, and Heidelberg). Furthermore, the phage did not contain any virulence, antibiotic-resistant, or lysogenic genes. Sa157lw had a 30-min latent period on both E. coli O157:H7 and Salmonella Typhimurium, with an estimated burst size of 130 and 220 PFU/CFU, respectively, and was stable at a wide range of temperatures (4'C to 60'C) and pH (pH4 to pH10). The phage application demonstrated a strong anti-E. coli O157:H7 and anti-Salmonella Typhimurium effects in 1.1 and 1.8 log reduction on the contaminated mung bean seeds after overnight storage at 22'C. These findings provide valuable insights into the polyvalent Sa157lw as a potential biocontrol agent of Salmonella Typhimurium and E. coli O157:H7 on sprout seeds.