Are Reactive Oxygen Species (ROS) the Main Mechanism by Which Copper Ion Treatment Degrades the DNA of Mycobacterium avium subsp. paratuberculosis Suspended in Milk?

Background: Mycobacterium avium subsp. paratuberculosis (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. Methodology: 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. Results: In a bacterial load of 104 cells mL−1, blocking effects by both the copper chelators and all the ROS quenchers offered significant protection to MAP DNA. In a concentration of 102 cells mL−1, only D-mannitol and a mix of quenchers significantly protected the viability of the bacteria, and only at a concentration of 106 cells mL−1 was there a lower production of ROS when supplementing milk with gallic acid, quercetin and the mix of quenchers. Conclusion: 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

More Insights about the Efficacy of Copper Ion Treatment on <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> (MAP): A Clue for the Observed Tolerance

Background: Scientific evidence is scarce for the antimicrobial effect of copper on bacteria characterized as more resistant. Using Mycobacterium avium subsp. paratuberculosis (MAP), a highly resistant microorganism, as a pathogen model, copper ion treatment has shown a significant bactericidal effect; however, the sustainability of MAP against copper toxicity was also reported in several studies. Accordingly, the present study aimed to evaluate the impacts of copper on MAP. Methodology: This study considered physicochemical properties and copper concentration in a buffer since it could modulate MAP response during the application of copper treatment. Results: Despite the efficacy of copper ions in significantly reducing the MAP load in Phosphate Buffered Saline, some MAP cells were able to survive. The copper concentration generated by the copper ion treatment device increased significantly with increasing exposure times. MAP bacterial load decreased significantly when treated with copper ions as the exposure times increased. An increase in pH decreased oxygen consumption, and an increase in conductivity was reported after treatment application. Conclusions: Even with higher concentrations of copper, the efficacy of MAP control was not complete. The concentration of copper must be a key element in achieving control of highly resistant microorganisms