It could have, though the general belief is that most of your beneficial bacteria resides in your filter, where water flow is greater, than in the gravel. There's certainly some in your gravel, but washing your media in tap water would do a lot more damage, I would think, than over-vacuuming. How long has this problem with ammonia been going on? That will sort of direct you to when the cycle got interrupted. Is there any chance you forgot to add dechlorinator during a water change? This could have killed off some of your bacteria as well. You haven't dosed with any sort of antibacterial for illness, have you? Cutting back on feeding is going to help, also, but unless you have somewhere to put these fish, or a friend that could hold them for a little while, you'll have to complete this mini-cycle with the fish in the tank, which is going to be rough for them. One idea is to either add cycled media from another tank to jump-start the cycle or to add a real beneficial-bacteria-in-a-bottle. I say "real" because not all of them work. My personal favorite is Dr. Tim's One-And-Only, but I have also heard good things about Seachem's Stability (though I haven't used that one). These will add the beneficial bacteria immediately, without your having to grow it in order to begin the cycle.
Edit: Sorry, I missed that you just got the test kit yesterday. Is this the first time the tank has been tested, or did you have an LFS or a friend test it prior to that? If it's the first time, then obviously re-tracing steps to determine where the cycle was interrupted won't work.
Edit: Sorry, I missed that you just got the test kit yesterday. Is this the first time the tank has been tested, or did you have an LFS or a friend test it prior to that? If it's the first time, then obviously re-tracing steps to determine where the cycle was interrupted won't work.