Spilsberg B, Sekse C, Urdahl Anne M, Nesse Live L, Johannessen Gro S
Section for Molecular Biology, Norwegian Veterinary Institute, Oslo, Norway.
Section for Food Safety and Animal Health Research, Norwegian Veterinary Institute, Oslo, Norway.
Front Microbiol. 2021 Jan 20;11:581575. doi: 10.3389/fmicb.2020.581575. eCollection 2020.
Shiga toxin-producing (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.
产志贺毒素大肠杆菌（STEC）是重要的食源性病原体，志贺毒素是其主要毒力因子。志贺毒素由编码志贺毒素的噬菌体（Stx噬菌体）编码。Stx噬菌体可能以游离噬菌体的形式存在于环境或食物中，也可能以前噬菌体的形式整合到宿主基因组中。从食品安全的角度来看，了解Stx噬菌体在食品中的存活和持久性非常重要，因为如果条件合适，它们可能通过转导整合到细菌宿主中。在此，我们展示了一项研究的结果，该研究调查了一种Stx噬菌体在牛肉碎肉中的存活情况，牛肉碎肉储存在不利于食品储存的温度（8°C）下，并对所应用的方法进行了改进和优化。在储存前，将已知水平的标记Stx噬菌体接种到牛肉碎肉中。噬菌体滤液用于噬菌斑测定和DNA提取，随后进行实时PCR和数字液滴PCR（ddPCR）。初步研究结果表明，最初的DNA提取方案并不理想，在确定最终方案之前测试了几种改进方法。最终的DNA提取方案包括对整个噬菌体滤液进行超速离心以浓缩噬菌体，以及两次酚-氯仿提取。该方案用于两次加标实验。DNA提取方案使得可用于PCR分析的DNA量具有灵活性，最终提高了用于定量样品中噬菌体的方法的灵敏度。所采用的三种定量方法（即噬菌斑测定、实时PCR和ddPCR）在储存期间噬菌体的变化趋势相似，其中ddPCR的优点是在简单的实验设置中能够对DNA拷贝进行绝对定量。结果表明，在本研究使用的储存条件下，Stx噬菌体持续存在并至少在20天内保持感染性。食品中的Stx噬菌体可能对人类构成潜在风险。虽然可以推测在8°C下可能会发生转导并随后形成STEC，但预计这是一个罕见事件。然而，这种事件可能在更适宜的条件下发生，例如食品储存温度升高或在人类胃肠道中。
