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.

More Insights about the Efficacy of Copper Ion Treatment on Mycobacterium avium subsp. paratuberculosis (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.

Understanding the antibacterial mechanisms of copper ion treatment on Mycobacterium avium subsp. paratuberculosis.

Copper and its alloys are natural and very well-proven antimicrobial materials. The mechanisms of action through which copper is highly effective have been described at the molecular and cellular level. However, both the design of the studies carried out and the nature of the microorganisms studied have meant that this research has been of limited scope. In the present study, we examined the action mechanisms of a copper ion treatment on the integrity of Mycobacterium avium subsp. paratuberculosis (MAP), a highly resistant animal pathogen. The copper ion treatment applied to MAP cells, resulted in nucleic acid degradation and disintegration, increased ROS production and protein alteration. However, the observed susceptibility of MAP to copper-based treatment was dose-dependent. Finally, it had no effect on the integrity of the MAP cell wall. This new evidence about the observed tolerance in the MAP cell wall against the copper ions, may help us to understand how we can improve the proposed copper-based treatment, and finally achieve a totally effective alternative to control MAP in calf´s milk.

Niche segregation between wild and domestic herbivores in Chilean Patagonia.

Competition arises when two co-occuring species share a limiting resource. Potential for competition is higher when species have coexisted for a short time, as it is the case for herbivores and livestock introduced in natural systems. Sheep, introduced in the late 19(th) century in Patagonia, bear a great resemblance in size and diet to the guanaco, the main native herbivore in Patagonia. In such circumstances, it could be expected that the two species compete and one of them could be displaced. We investigated spatial overlap and habitat selection by coexisting sheep and guanaco in winter and in summer. Additionally, we studied habitat selection of the guanaco in a control situation free from sheep, both in summer and winter. We also determined overlap between species in areas with different intensity of use (named preferred and marginal areas) in order to further detect the potential level of competition in the case of overlapping. Guanaco and sheep showed significantly different habitat preferences through all seasons, in spite of their spatial overlap at landscape scale. Additionally, the habitat used by guanaco was similar regardless of the presence or absence of livestock, which further indicates that sheep is not displacing guanaco where they coexist. These results suggest that habitat segregation between guanaco and sheep is due to a differential habitat selection and not to a competitive displacement process. Therefore, the potential for competition is considered low, contrary to what has been previously observed, although this could be a density-dependent result.