Department of Entomology and Plant Pathology, National Institute for Microbial Forensics and Food and Agricultural Biosecurity, Oklahoma State University, Stillwater, Oklahoma, United States of America.
PLoS One. 2011 Apr 4;6(4):e18083. doi: 10.1371/journal.pone.0018083.
Many food safety-related studies require tracking of introduced foodborne pathogens to monitor their fate in complex environments. The green fluorescent protein (GFP) gene (gfp) provides an easily detectable phenotype so has been used to label many microorganisms for ecological studies. The objectives of this study were to label major foodborne pathogens and related bacteria, including Listeria monocytogenes, Listeria innocua, Salmonella, and Escherichia coli O157:H7 strains, with GFP and characterize the labeled strains for stability of the GFP plasmid and the plasmid's effect on bacterial growth. GFP plasmids were introduced into these strains by a CaCl(2) procedure, conjugation or electroporation. Stability of the label was determined through sequential propagation of labeled strains in the absence of selective pressure, and rates of plasmid-loss were calculated. Stability of the GFP plasmid varied among the labeled species and strains, with the most stable GFP label observed in E. coli O157:H7. When grown in nonselective media for two consecutive subcultures (ca. 20 generations), the rates of plasmid loss among labeled E. coli O157:H7, Salmonella and Listeria strains ranged from 0%-30%, 15.8%-99.9% and 8.1%-93.4%, respectively. Complete loss (>99.99%) of the plasmid occurred in some labeled strains after five consecutive subcultures in the absence of selective pressure, whereas it remained stable in others. The GFP plasmid had an insignificant effect on growth of most labeled strains. E. coli O157:H7, Salmonella and Listeria strains can be effectively labeled with the GFP plasmid which can be stable in some isolates for many generations without adversely affecting growth rates.
许多食品安全相关的研究需要追踪引入的食源性病原体，以监测其在复杂环境中的命运。绿色荧光蛋白（GFP）基因（gfp）提供了一种易于检测的表型，因此已被用于标记许多微生物进行生态研究。本研究的目的是用 GFP 标记主要食源性病原体和相关细菌，包括李斯特菌、无害李斯特菌、沙门氏菌和大肠杆菌 O157:H7 菌株，并对标记菌株进行 GFP 质粒稳定性和质粒对细菌生长影响的特征分析。通过氯化钙程序、接合或电穿孔将 GFP 质粒引入这些菌株。通过在无选择压力的情况下连续传代标记菌株来确定标记的稳定性，并计算质粒丢失率。标记物种和菌株的 GFP 质粒稳定性差异很大，在大肠杆菌 O157:H7 中观察到的 GFP 标记最稳定。当在非选择性培养基中连续传代两次（约 20 代）时，标记的大肠杆菌 O157:H7、沙门氏菌和李斯特菌菌株的质粒丢失率分别为 0%-30%、15.8%-99.9%和 8.1%-93.4%。在无选择压力的情况下，经过连续五次传代，一些标记的菌株中质粒完全丢失（>99.99%），而其他菌株则稳定存在。GFP 质粒对大多数标记菌株的生长影响不大。大肠杆菌 O157:H7、沙门氏菌和李斯特菌菌株可以有效地用 GFP 质粒标记，在一些分离株中可以稳定存在数代而不影响生长速率。
