Tripartite Conjugation in P. larvae

From 12-3-14.

Transposon insert, pMarA, was from this paper. This process was successful in Bacillus spp., however, it has not been attempted in P. larvae ATCC 9545. It is highly likely that this process should work in P. larvae, which is why I am attempting it.

Ran into a few complications regarding antibiotics for the tripartide conjugation between P. larvae, pMarA, and pRK2013. Either the bacteria's resistances are not what I originally thought they were based on literature review and previous experiments, or our antibiotic stocks are no longer functioning properly. I believe the latter to be the case as upon further inspection some of the antibiotics (like ampicillin) were created in April of this year. It is now December.

This experiment will be done to determine the antibiotic resistance/susceptibilities of each of the bacteria that will be used in the tripartide conjugation. From plate cultures, bacteria were inoculated into 10mL of BHI broth spiked with antibiotic concentrations:

Bacteria	Abx	Growth?
pMarA	Kan50 Amp100	Yes
pMarA	Poly60	No
pRK2013	Kan50	Yes
pRK2013	Amp100	No
pRK2013	Poly60	No
P. larvae	Poly60	NO
P. larvae	Kan50 Amp100	No

Visual growth was observed after 24 hours, shaking at 225 rpm at 37C, on those that are marked as "Yes" growth. It is odd that P. larvae was not resistant to Polymyxin at a concentration of 60 ug/ml, because it was shown to be able to grow under such conditions on 10-8-14. It is my understanding that the polymyxin antibiotic used in this experiment was the exact same tube used in the previous experiment. It is known that P. larvae is a slower grower than the other E. coli strains and that may be the reason no growth was observed in the P. larvae tubes. For that reason I will continue to incubate those two tubes and observe them at a later date.

Another reason that P. larvae didn't grow under the Polymyxin condition may be because it was an older plate culture that had been stored at 4C for a few days. However, viable spores should have still be present even if there was no present viable vegetative cells. Perhaps the spores are unable to germinate in the presence of Polymyxin, which it is ordinarily resistant to as a vegetative cell? This might be a route for a quick pilot study in the future...

Obviously, a negative control for each bacterial strain would have been appropriate in this experiment, however the lab is on short supply on conical tubes and there was a limited supply of BHI broth available at the time. In essence, I did have negative controls, just not in the traditional sense. I was fairly confident that the bold antibiotics used in the table above would result in bacterial growth since it had been observed previously.

The antibiotic resistances was confirmed in both pMarA and pRK2013, Kan50Amp100 and Kan50, respectively. It is also good that both pMarA and pRK2013 are sensitive to Polymyxin, since that is what will be used as a counter selection for P. larvae. Below is a visual display of how the tripartide conjugation will be set up using suspension:

Broth tripartide conjugation visual display

I will also attempt a solid conjugation using filter discs on agar plates... If I can locate where our remaining sterile filter discs are.

//EWW