Rapid Detection and Fast Induction of Viable but Nonculturable Vibrio parahaemolyticus and Vibrio cholerae

IF 2.8 4区农林科学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of food protection Pub Date : 2025-10-01 DOI:10.1016/j.jfp.2025.100623

Eleonora Di Salvo , Christopher Zeidler , Tim Bastian Schille , Patrick Mikuni-Mester , Thomas Alter , Stephan Huehn-Lindenbein , Susanne Fleischmann

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引用次数: 0

Abstract

Vibrio (V.) species, such as V. parahaemolyticus and V. cholerae, are commonly associated with foodborne infections and are frequently detected in seafood worldwide. Unfavorable environmental conditions and process-related factors can induce a shift from culturable Vibrio cells into viable but nonculturable (VBNC) cells.

Conventional culture-based detection methods (ISO 21872-1:2023-06) cannot detect bacteria in the VBNC state, even though these cells remain metabolically active and pathogenic due to the expression of toxin−encoding genes. This study aimed to develop a detection method using viable quantitative PCR (vqPCR) to identify viable cells, including those in VBNC state. In parallel, a relatively rapid protocol for inducing the VBNC state to generate VBNC cell controls was established.

The established vqPCR assays included a preliminary step to inhibit dead bacterial cells using a proprietary DNA intercalating dye (Reagent D) in combination with the detection of long gene fragments of groEL (510 bp) for V. parahaemolyticus and ompW (588 bp) for V. cholerae using previously published primers. These assays demonstrated a high sensitivity, detecting as low as 20 fg DNA = 3.5 V. parahaemolyticus cells and 30 fg DNA = 6.9 V. cholerae cells. An induction of Vibrio VBNC cells of ≈ 6.5 Log10 cells/ml was successfully achieved within one hour from an initial 7.3 Log10 viable Vibrio cells/ml by treating the cells with a solution containing 0.5 or 1.0% Lutensol A03 and 0.2 M ammonium carbonate.

The results showed that the established vqPCR methods were able to detect V. parahaemolyticus and V. cholerae in up to 50% (2.6 to 4.2 Log10 cells/g) and 56% (2.8 to 5.2 Log10 cells/g) of retail samples, respectively, that were initially false-negative in culture-based tests.

The use of vqPCR assays along with culture-based tests can significantly enhance the seafood safety assessment by enabling the detection of VBNC cells of the most important foodborne Vibrio pathogens. In addition, the induction assay can be used for a rapid production of VBNC cells to standardize and validate such detection methods.

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活的但不可培养的副溶血性弧菌和霍乱弧菌的快速检测和快速诱导。

弧菌种类，如副溶血性弧菌和霍乱弧菌，通常与食源性感染有关，在世界各地的海产品中经常检测到。不利的环境条件和工艺相关因素可诱导可培养弧菌细胞向可存活但不可培养的（VBNC）细胞转变。传统的基于培养的检测方法（ISO 21872-1:2023-06）无法检测出处于VBNC状态的细菌，即使这些细胞由于毒素编码基因的表达而保持代谢活性和致病性。本研究旨在建立一种活菌定量PCR （vqPCR）检测方法，用于鉴定包括VBNC状态在内的活菌细胞。同时，建立了一种相对快速的诱导VBNC状态生成VBNC细胞对照的方法。建立的vqPCR检测包括使用专有的DNA插入染料（试剂D）抑制死亡细菌细胞的初步步骤，结合使用先前发表的引物检测副溶血性弧菌的groEL （510 bp）长基因片段和霍乱弧菌的ompW （588 bp）长基因片段。这些检测显示出高灵敏度，可检测低至20 fg DNA = 3.5 v的副溶血性细胞和30 fg DNA = 6.9 v的霍乱细胞。用含有0.5或1.0% Lutensol A03和0.2 M碳酸铵的溶液处理细胞，在1小时内成功地从7.3 Log10个活弧菌细胞/ml诱导出了≈6.5 Log10个细胞/ml的VBNC弧菌细胞。结果表明，所建立的vqPCR方法能够分别在高达50% （2.6 ~ 4.2 Log10细胞/g）和56% （2.8 ~ 5.2 Log10细胞/g）的零售样品中检测到副溶血性弧菌和霍乱弧菌，这些样品最初在基于培养的测试中呈假阴性。利用vqPCR检测和基于培养的检测可以检测最重要的食源性弧菌病原体的VBNC细胞，从而显著提高海产品的安全性评估。此外，诱导试验可用于快速生产VBNC细胞，以标准化和验证此类检测方法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of food protection 工程技术-生物工程与应用微生物

CiteScore

4.20

自引率

5.00%

发文量

296

审稿时长

2.5 months

期刊介绍： The Journal of Food Protection® (JFP) is an international, monthly scientific journal in the English language published by the International Association for Food Protection (IAFP). JFP publishes research and review articles on all aspects of food protection and safety. Major emphases of JFP are placed on studies dealing with: Tracking, detecting (including traditional, molecular, and real-time), inactivating, and controlling food-related hazards, including microorganisms (including antibiotic resistance), microbial (mycotoxins, seafood toxins) and non-microbial toxins (heavy metals, pesticides, veterinary drug residues, migrants from food packaging, and processing contaminants), allergens and pests (insects, rodents) in human food, pet food and animal feed throughout the food chain; Microbiological food quality and traditional/novel methods to assay microbiological food quality; Prevention of food-related hazards and food spoilage through food preservatives and thermal/non-thermal processes, including process validation; Food fermentations and food-related probiotics; Safe food handling practices during pre-harvest, harvest, post-harvest, distribution and consumption, including food safety education for retailers, foodservice, and consumers; Risk assessments for food-related hazards; Economic impact of food-related hazards, foodborne illness, food loss, food spoilage, and adulterated foods; Food fraud, food authentication, food defense, and foodborne disease outbreak investigations.