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Characterization of Salmonella Bacteriophages Isolated from Farm Environments for Use in Decontamination of Liquid Whole Egg

Yi, Yue
http://rave.ohiolink.edu/etdc/view?acc_num=osu1566222931949046

Abstract Details

2019, Master of Science, Ohio State University, Food Science and Technology.
Salmonella enterica is the most prevalent bacterial pathogen causing foodborne illnesses. Globally, contamination of eggs has been identified as a major vehicle for transmitting foodborne salmonellosis. Two S. enterica serovars, Typhimurium and Enteritidis, are efficient colonizers of the reproductive organs of hens, hence, these two serovars are closely associated with salmonellosis transmitted by eggs. Current decontamination procedures for the production of microbiologically safe eggs rely on thermal treatments, but these adversely affect egg quality. Hence, research is needed to explore effective decontamination methods that do not damage egg quality and functionality. The objectives of this research were (1) to isolate and select Salmonella bacteriophages from environmental samples; (2) to characterize selected phage isolates and assess their lytic activities; and (3) to evaluate the effectiveness of promising phage isolates against Salmonella in liquid whole eggs. To achieve these objectives, the following procedure was followed. Environmental samples (water, animal feces, feather, etc.) were collected from animal farms in Ohio. Samples were inoculated with a cocktail of S. Typhimurium, S. Enteritidis and S. Heidelberg and incubated to allow for the amplification of potential phages. Incubated samples were micro-filtered to remove bacterial cells and large particles. The presence of bacteriophages was determined by spotting 10 µl of filtrates onto Salmonella-embedded soft agar, followed by isolation and purification of bacteriophages from the filtrates by double agar overlay technique. The most promising phage candidates were selected based on their host range which was determined against 37 Salmonella strains. Two phage isolates, OSY-STA and OSY-SHC, were characterized by determining replication kinetics, measuring pH and thermal stability, examining phage morphology, estimating phage genome size and evaluating phage lytic ability against Salmonella in Tryptic Soy Broth (TSB). For determination of replication kinetics, titer of phages in the presence of their host at each treatment were enumerated during incubation at 37 ℃. The phages had similar latent periods and generation times, while burst size of OSY-STA and OSY-SHC were 176 and 243 PFU/cell, respectively. Both OSY-STA and OSY-SHC were stable while holding in media at pH 4 – 12 and during storage at temperatures of 4, 25, 37 and 55 ℃. Phage morphology was examined by Transmission Electron Microscope (TEM), and results showed that OSY-STA and OSY-SHC are members of family Siphoviridae of tailed bacteriophages. Phage OSY-STA was subjected to DNA sequencing considering its broad host range. Sequencing results revealed a 111,373 bp dsDNA genome containing 169 ORFs organized into four major categories: DNA replication and nucleotides metabolism-related proteins, structural proteins, regulator proteins, and cell lysis proteins. Lytic activity of phage was determined at 4 ℃ or 25 ℃ by enumerating viable Salmonella after phage treatments. At 25 ℃, application of the mixture of two isolated phages significantly reduced Salmonella population after 24 h (Tukey’s test, P<0.05). Furthermore, the growth of Salmonella Enteritidis was significantly (Tukey’s test, P < 0.05) inhibited after 3-day incubation at 4 ℃. Lysis results deliver valuable information to assess the potential of the phage cocktail as a biocontrol agent against diverse Salmonella serovars in contaminated food products. Liquid egg samples were inoculated with 100 µl of 105 CFU/ml Salmonella Typhimurium or Enteritids in 10 ml liquid whole egg sample, and treated with 100 µl of the phage cocktail (OSY-STA + OSY-SHC) at a concentration of 107 PFU/ml. Therefore, the final concentration of Salmonella and phage in liquid egg sample were 103 CFU/ml and 105 PFU/ml, respectively (i.e., MOI of 100). Efficiency of the phage cocktail was determined by counting viable Salmonella population after 24 h incubation at 4 ℃. Significant reduction (Tukey’s test, P<0.05) in bacterial counts was only observed in Salmonella. Enteritidis-inoculated samples. Overnight phage cocktail treatment (at 4 ℃) following heating at 55 ℃ showed higher efficiency in reducing bacterial counts than when the phage or thermal treatment was applied individually. There was at least 2.8 log10 CFU/ml reduction in the population of S. Enteritidis when contaminated liquid egg was treated with a combination of phage cocktail, followed by 13-min of heating. This study illustrated the characteristics of isolated phages and provided a basis for phage application in food processing. The study specifically demonstrated the applicability of phage in conjunction with thermal treatment to pasteurize liquid eggs.
Yousef Ahmed, Dr. (Advisor)
Jiyoung Lee, Dr. (Committee Member)
Abigail Snyder, Dr. (Committee Member)
166 p.
Food Science
Salmonella enterica serovar; Bacteriophages; Thermal treatments; Liquid whole egg

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Citations

  • Yi, Y. (2019). Characterization of Salmonella Bacteriophages Isolated from Farm Environments for Use in Decontamination of Liquid Whole Egg [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1566222931949046

    APA Style (7th edition)

  • Yi, Yue. Characterization of Salmonella Bacteriophages Isolated from Farm Environments for Use in Decontamination of Liquid Whole Egg. 2019. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1566222931949046.

    MLA Style (8th edition)

  • Yi, Yue. "Characterization of Salmonella Bacteriophages Isolated from Farm Environments for Use in Decontamination of Liquid Whole Egg." Master's thesis, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1566222931949046

    Chicago Manual of Style (17th edition)

osu1566222931949046
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