Experimental evidence of the anti-bacterial activity pathway of copper ion treatment on Mycobacterium avium subsp. paratuberculosis.

Copper causes significant damage to the integrity of many bacteria, mainly at the DNA level, through its redox states, as well as its reactive oxygen species (ROS) generating capacity at the cellular level. But whether these mechanisms also apply to Mycobacterium avium subsp. paratuberculosis (MAP) is unknown. In the present study, we have evaluated whether copper ions produce damage at the DNA level of MAP, either through their redox states or through ROS production. MAP-spiked PBS was first supplemented with different copper chelators (2) and ROS antioxidants (3), followed by treatment with copper ions at 942 ppm. MAP DNA integrity (qPCR, magnetic phage separation) was then evaluated. We found that bathocuproine (BCS), as a chelator, and D-mannitol, as an antioxidant of hydroxyl radicals, had a significant protective effect (P < 0.05) on DNA molecules, and that EDTA, as a chelator, and D-mannitol, as an antioxidant had a significant positive effect (P < 0.05) on the viability of this pathogen in contrast to the control and other chelators and anti-oxidants used. In light of the reported findings, it may be concluded that copper ions within MAP cells are directly related to MAP DNA damage.

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.