Persistence of a Stx-Encoding Bacteriophage in Minced Meat Investigated by Application of an Improved DNA Extraction Method and Digital Droplet PCR

Abstract

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens with Shiga toxins as the main virulence factor. Shiga toxins are encoded on Shiga toxin-encoding bacteriophages (Stx phages). Stx phages may exist as free bacteriophages in the environment or in foods or as prophages integrated into the host genome. From a food safety perspective, it is important to have knowledge on the survival and persistence of Stx phages in food products since these may integrate into the bacterial hosts through transduction if conditions are right. Here, we present the results from a study investigating the survival of a Stx phage in minced meat from beef stored at a suboptimal temperature (8°C) for food storage along with modifications and optimizations of the methods applied. Minced meat from beef was inoculated with known levels of a labeled Stx phage prior to storage. Phage filtrates were used for plaque assays and DNA extraction, followed by real-time PCR and digital droplet PCR (ddPCR). The results from the pilot study suggested that the initial DNA extraction protocol was not optimal, and several modifications were tested before a final protocol was defined. The final DNA extraction protocol comprised ultra-centrifugation of the entire phage filtrate for concentrating phages and two times phenol–chloroform extraction. The protocol was used for two spiking experiments. The DNA extraction protocol resulted in flexibility in the amount of DNA available for use in PCR analyses, ultimately increasing the sensitivity of the method used for quantification of phages in a sample. All three quantification methods employed (i.e., plaque assays, real-time PCR, and ddPCR) showed similar trends in the development of the phages during storage, where ddPCR has the benefit of giving absolute quantification of DNA copies in a simple experimental setup. The results indicate that the Stx phages persist and remain infective for at least 20 days under the storage conditions used in the present study. Stx phages in foods might represent a potential risk for humans. Although it can be speculated that transduction may take place at 8°C with subsequent forming of STEC, it can be expected to be a rare event. However, such an event may possibly take place under more optimal conditions, such as an increase in storage temperature of foods or in the gastrointestinal tract of humans.

Droplet-based methodology for investigating bacterial population dynamics in response to phage exposure

“…As combination phage-antibiotic treatments have been considered the most efficient type of phage therapy (Suh et al, 2022;Van Nieuwenhuyse et al, 2022), dropletbased screening platforms could be employed to better understand phage-antibiotic interactions and to identify mechanisms that drive those interactions. In addition, such platforms could implement the existing droplet digital PCR (ddPCR) technology, to understand molecular alongside cellular aspects of bacteria-phage dynamics (Morella et al, 2018;Spilsberg et al, 2021). ddPCR could quantify DNA phage or RNA phage densities over time (by measuring their DNA or RNA content respectively), and also quantify bacterial and DNA phage densities simultaneously.…”

Enrichment Culture but Not Metagenomic Sequencing Identified a Highly Prevalent Phage Infecting Lactiplantibacillus plantarum in Human Feces

“…Next, we amplified and cultured the purified phage in the host strain L. plantarum CNGBCC 1800069. After 6 h of incubation, the phage genome was extracted by following a standard phenol-chloroform protocol ( 57 , 58 ). Briefly, cultures were centrifuged twice at 8,000 × g for 15 min at 4°C and filtered with a 0.22-μm filter membrane.…”

Isolation and identification of the broad-spectrum high-efficiency phage vB_SalP_LDW16 and its therapeutic application in chickens

“…The buffer was stored at − 20 °C until further use. The whole bacteriophage genome was extracted using the phenol–chloroform extraction method [ 50 ]. The whole bacteriophage genomic was then digested using the restriction enzymes DNase I (Hunan Accurate Biotechnology, China), RNase A (Hunan Accurate Biotechnology, China), and Mung Nuclease, respectively.…”