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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #359835

Research Project: Ecology and Detection of Human Pathogens in the Produce Production Continuum

Location: Produce Safety and Microbiology Research

Title: Effects of lyophilization on the stability of bacteriophages against different serogroups of Shiga toxin-producing Escherichia coli

Author
item Lavenburg, Valerie
item Liao, Yen-te
item Salvador, Alexandra
item Wu, Vivian

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 1/29/2019
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Preservation is a critical process to ensure long-term physiological viability and genetic stability of the purified microorganisms to facilitate future research or application. Lyophilization is one of the most efficient methods to preserve prokaryote cells and some bacteriophages, such as T-even bacteriophages, even though it has been indicated that various bacteriophages may react differently to the similar lyophilization parameters. However, information regarding the stability of numerous bacteriophages specific to different serogroups of O157 and non-O157 STEC strains preserved using lyophilization is scarce. The objective of this study was to evaluate the preservation effects of different cryoprotectants on various Shiga toxin-producing E. coli-specific bacteriophages during lyophilization. The phage Ro111lw (9.2 log pfu/ml), previously isolated from a non-fecal compost, was used to optimize lyophilization parameters, including the temperature (-20°C to -40°C) used at primary drying and the drying time (490 to 610 minutes) used at the secondary drying step. Following optimization, an aliquot of 250ml of the phages Ro111lw and Pr103g (8.2 log pfu/ml), isolated from goat feces, were mixed with equal volume of different cryoprotectants, including trehalose (0.1M & 0.5M), sucrose (0.1M & 0.5M), SM buffer and 1% PEG solution, for lyophilization. The preservation effects of these cryoprotectants were evaluated by quantifying the treated phages using double-layer agar plaque assay after additional storage at 4°C for 6 days. Results showed that the use of -20°C at the primary drying cycle in combination with 610-minute drying time at the secondary drying step rendered the best preservation on phage Ro111lw mixed with SM buffer. Among the selected cryoprotectants, both phages responded differently to each individual cryoprotectant. However, 0.5M sucrose and 0.5M trehalose were attributed to better preservation effects on the phages Pr103g and Ro111lw, respectively, both with 0.61 log pfu/ml reduction after the lyophilization and refrigeration processes. The findings of this study indicate that different phages that are from different environments and targeting different serogroups of STEC strains respond differently to the use of same cryoprotectants. Additionally, higher concentration of sugar-based cryoprotectants, such as sucrose and trehalose, may provide better preservation effects.