Yes, you better user several layouts to construct your game world. Normaly, when you reload a layout, everything will be reset in it (except if you track those information somewhere in a global variable).
I still haven't use the new save/load feature so If you use it, perhaps you will have to specify on each enemy you want to repawn not to be saved.
I would suggest another thing that will help you : create an empty Layout called, for example, LIBRARY. And put in it one copy of EVERY objects, sfx or whatever you will need several times in your game. That way you can easely add or delete elements in your game level layouts.
The most difficult part I personnaly think, is the creation of the metroidvania minimap system.
Hope that help.