Villegas Marcela, Tejeda Carlos, Umaña Reydoret, Iranzo Esperanza C, Salgado Miguel
Instituto de Medicina Preventiva Veterinaria, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Saelzer Building 5° Floor, Campus Isla Teja, Valdivia P.O. Box 567, Chile.
Facultad de Ciencias, Escuela de Graduados, Universidad Austral de Chile, Valdivia P.O. Box 567, Chile.
Microorganisms. 2022 Nov 16;10(11):2272. doi: 10.3390/microorganisms10112272.
subsp. (MAP) is the causal agent of paratuberculosis. This pathogen is able to survive adverse environmental conditions, including the pasteurization process. Copper, a well-studied metal, is considered an important antibacterial tool, since it has been shown to inactivate even MAP in treated milk through unknown mechanisms. The aim of the present study is to show the effect of copper ions, and reactive oxygen species (ROS) generated in response to oxidative stress, on the damage to MAP DNA when exposed to a copper ion challenge in cow's milk.
Spiked milk with different MAP bacterial loads was supplemented with blocking agents. These were either the copper chelators ethylenediaminetetraacetic acid (EDTA) and batocuproin (BCS) or the ROS quenchers D-mannitol, gallic acid and quercetin. The DNA protection, MAP viability and ROS production generated after exposure to a copper challenge were then measured.
In a bacterial load of 10 cells mL, blocking effects by both the copper chelators and all the ROS quenchers offered significant protection to MAP DNA. In a concentration of 10 cells mL, only D-mannitol and a mix of quenchers significantly protected the viability of the bacteria, and only at a concentration of 10 cells mL was there a lower production of ROS when supplementing milk with gallic acid, quercetin and the mix of quenchers.
Based on these findings, it may be concluded that MAP DNA damage can be attributed to the combined effect of the direct copper ions and ROS generated. Nevertheless, taking into account the antioxidant environment that milk provides, the direct effect of copper could play a prominent role.
副结核分枝杆菌亚种（MAP）是副结核病的病原体。这种病原体能够在包括巴氏杀菌过程在内的不利环境条件下存活。铜是一种研究充分的金属，被认为是一种重要的抗菌工具，因为已表明它能通过未知机制使经处理牛奶中的MAP失活。本研究的目的是展示铜离子以及氧化应激产生的活性氧（ROS）对牛奶中暴露于铜离子挑战时MAP DNA损伤的影响。
向添加了不同MAP细菌载量的牛奶中添加阻断剂。这些阻断剂要么是铜螯合剂乙二胺四乙酸（EDTA）和联苯三氮唑（BCS），要么是ROS淬灭剂D - 甘露醇、没食子酸和槲皮素。然后测量暴露于铜挑战后产生的DNA保护、MAP活力和ROS产生情况。
在细菌载量为10⁶个细胞/毫升时，铜螯合剂和所有ROS淬灭剂的阻断作用都为MAP DNA提供了显著保护。在浓度为10⁵个细胞/毫升时，只有D - 甘露醇和淬灭剂混合物显著保护了细菌的活力，并且只有在浓度为10⁴个细胞/毫升时，用没食子酸、槲皮素和淬灭剂混合物补充牛奶时ROS产生量较低。
基于这些发现，可以得出结论，MAP DNA损伤可归因于直接的铜离子和产生的ROS的联合作用。然而，考虑到牛奶提供的抗氧化环境，铜的直接作用可能起主要作用。
