In this paper we describe a biological indicator which can be used to study the behavior of Vibrio vulnificus, an important molluscan shellfish-associated human pathogen. A V. vulnificus ATCC 27562 derivative that expresses green fluorescent protein (GFP) and kanamycin resistance was constructed using conjugation. Strain validation was performed by comparing the GFP-expressing strain (Vv-GFP) and the wild-type strain (Vv-WT) with respect to growth characteristics, heat tolerance (45 degrees C), freeze-thaw tolerance (-20(o) and -80 degrees C), acid tolerance (pH 5.0, 4.0, and 3.5), cold storage tolerance (5 degrees C), cold adaptation (15 degrees C), and response to starvation. Levels of recovery were evaluated using nonselective medium (tryptic soy agar containing 2% NaCl) with and without sodium pyruvate. The indicator strain was subsequently used to evaluate the survival of V. vulnificus in oysters exposed to organic acids (citric and acetic acids) and various cooling regimens. In most cases, Vv-GFP was comparable to Vv-WT with respect to growth and survival upon exposure to various biological stressors; when differences between the GFP-expressing and parent strains occurred, they usually disappeared when sodium pyruvate was added to media. When V. vulnificus was inoculated into shellstock oysters, the counts dropped 2 log(10) after 11 to 12 days of refrigerated storage, regardless of the way in which the oysters were initially cooled. Steeper population declines after 12 days of refrigerated storage were observed for both iced and refrigerated products than for slowly cooled product and product held under conservative harvest conditions. By the end of the refrigeration storage study (22 days), the counts of Vv-GFP in iced and refrigerated oysters had reached the limit of detection (10(2) CFU/oyster), but slowly cooled oysters and oysters stored under conservative harvest conditions still contained approximately 10(3) and >10(4) CFU V. vulnificus/oyster by day 22, respectively. The Vv-GFP levels in the oyster meat remained stable for up to 24 h when the meat was exposed to acidic conditions at various pH values. Ease of detection and comparability to the wild-type parent make Vv-GFP a good candidate for use in studying the behavior of V. vulnificus upon exposure to sublethal stressors that might be encountered during postharvest handling of molluscan shellfish.

译文

在本文中,我们描述了一种生物学指标,可用于研究创伤弧菌 (一种与软体动物贝类相关的重要人类病原体) 的行为。使用缀合物构建表达绿色荧光蛋白 (GFP) 和卡那霉素抗性的创伤弧菌ATCC 27562衍生物。通过比较表达GFP的菌株 (vv-gfp) 和野生型菌株 (vv-wt) 在生长特性,耐热性 (45 ℃),冻融耐受性 (-20(o) 和-80 ℃) 方面进行菌株验证,耐酸性 (pH 5.0、4.0和3.5) 、耐冷性 (5 ℃) 、冷适应 (15 ℃) 和对饥饿的响应。使用含和不含丙酮酸钠的非选择性培养基 (含2% NaCl的胰蛋白酶大豆琼脂) 评估回收率水平。随后使用指示菌株评估暴露于有机酸 (柠檬酸和乙酸) 和各种冷却方案的牡蛎中vulnificus的存活率。在大多数情况下,Vv-GFP在暴露于各种生物应激源后的生长和存活方面与Vv-WT相当; 当表达GFP的菌株和亲本菌株之间发生差异时,当向培养基中添加丙酮酸钠时,它们通常消失。当将vulnificus接种到贝壳类牡蛎中时,冷藏储存11至12天后,计数下降了2 log(10),而与最初冷却牡蛎的方式无关。冷藏和冷藏产品在冷藏12天后的种群数量下降幅度均大于缓慢冷却的产品和在保守收获条件下保存的产品。到冷藏储存研究结束时 (22天),冰镇牡蛎和冷藏牡蛎中Vv-GFP的计数已达到检测极限 (10(2) CFU/牡蛎),但是缓慢冷却的牡蛎和在保守收获条件下储存的牡蛎到第22天仍然分别含有大约10(3) 和> 10(4) CFU V. vulnificus/牡蛎。当将肉暴露于各种ph值的酸性条件下时,牡蛎肉中的Vv-GFP水平保持稳定长达24小时。易于检测和与野生型亲本的可比性使vv-gfp成为研究暴露于软体动物贝类收获后可能遇到的亚致死性应激源时的创伤弧菌行为的良好候选者。

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