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

The Waxworm & the P. larvae

From 11-11-14.

Made up another batch of wax worm food from the gerber baby food. Transferred young larva from the colony to five small containers. There was about 12 wax worms and about 10 grams of food in each container. Wax paper was hung from the cap of each container down to the food as done before.

The wax worms were not in the best of health due to overcrowding in their previous containers. Any wax worms that didn't get transferred were frozen in the -20C freezer overnight and disposed of the next day.

The five wax worm containers were placed within another container and incubated in the walk in 30C incubator. I will continue to monitor their growth, but in the future the colony will remain a smaller size due to a lack of a need for them at this current time.

//EWW

Detection of P. larvae in Local Honey

From 12-22-14.

Screened the remaining suspected P. larvae isolates from Dawn D. Created crude genomic lysates of each isolate and performed diagnositc PCR using the AFB primers.

Results:

Not the best image, but the bands (or lack of bands) can be clearly seen. This is actually a photo of a photo of a photo due a corrupt saved file of the image on my USB drive. So, this image is of the printed out picture of the gel.

Top Row
HiLo Marker	18	19	20	21	22	23	24	25	Positive
Bottom Row
HiLo Marker	26	27	28	29	30	31	Neg1	Neg2

Positive control was P. larvae ATCC 9545. Neg1 was the E. coli strain pMarA ran in this experiment, and Neg2 was the E. coli strain ran in the previous experiment. 

Lab Code	EW Code
dd1-6a	18
dd1-8a	19
dd5-J	20
dd5-K	21
dd5-L	22
dd5-M	23
dd5-M	24
dd5-N	25
dd5-Q	26
dd8-55a	27
dd8-63a	28
dd8-86a	29
dd11-2a	30
dd12-22a	31
DH5a	Neg1
pMarA	Neg2

Discussion:
Of all the isolates from Dawn D. that had been screened using the diagnostic PCR AFB primers, only one did not test positive. That one isolate was from the previous study on 12-22-14. The negative result was from isolate dd1-7a (EW code 02). All other isolates were confirmed as being Paenibacillus, specifically P. larvae because the AFB primers amplified a conserved region of the 16S rRNA only found in P. larvae. A complete list will be compiled of all the confirmed isolates and provided to Dawn D. for future studies. It is my understanding that freezer stocks will be made of the positive isolates, at -80C, to be used in future studies.

It was surprising to find that so many honey providers in the area have P. larvae present in their honey. However, I am uncertain on exactly how many isolates were initially screened by Dawn D. that proved to be both Gram positive and Oxidase positive before they were given to me for diagnostic PCR analysis. Also, there wasn't a relatively large presence of the bacteria in any honey source, and it could be that there is just always a low presence of the bacteria in any bee hive. Perhaps that is a route for a future study.

//EWW

Detection of P. larvae in Local Honey

Goal: Identify P. larvae isolates from local honey providers in North Dakota and the Minnesota area. These isolates will be used for future studies as wild type strains of P. larvae. In order to protect the providers of the honey samples, the businesses will not be identified.

Dawn D., lab technician of the Fisher lab, has acquired 12 honey samples (Lab code for honey providers is dd1 to dd12) from a number of states in the country. These honey samples were diluted and cultured on MYPGP agar. Suspected P. larvae isolates were further isolated onto MYPGP. Suspected isolates were then Gram stained and oxidase tested to help confirm their identity as possibly being Paenibacillus.

Of the many isolates recovered, a handful were confirmed to be Gram positive and oxidase positive indicating potential Paenibacillus larvae isolates. The lab code was developed by Dawn D, however my own code was created for simplicity:

Lab Code	EW Code
dd1-5a	01
dd1-7a	02
dd5-4a	03
dd8-55a	04
dd8-63a	05
dd8-67a	06
dd8-70a	07
dd8-78a	08
dd8-82a	09
dd8-83a	10
dd8-85a	11
dd8-85a	12
dd8-86a	13
dd8-91a	14
dd8-95a	15
dd8-99a	16
dd8-119a	17

The code dd# is referring to one of the 12 honey sources, and the last part is referring to a specific isolate that was identified that source. The above list is only a portion of the isolates confirmed to be potential Paenibacillus.

Confirmation of identification of the suspected isolates as being P. larvae was performed by using diagnostic primers developed according to Development of a fast and reliable diagnostic method for American foulbrood disease (Paenibacillus larvae subsp. larvae) using a 16S rRNA gene based PCR (Dobbelaere, 2001). Primer 1 and Primer 2 were used according to the paper (referred to as AFB primers). PCR parameters were as dictated by the paper as well.

AMRESCO Ready PCR Mix, 2x was used for the amplification reaction. A master mix was created and 2 uL of crude genomic lysates were added for each PCR reaction.

Results of PCR:

Top Row
HiLo Marker	01	02	03	04	05	06	07	08	Positive
Bottom Row
HiLo Marker	09	10	11	12	13	14	15	16	17

The bottom row amplicons were mixed with EZ vision loading dye, whereas the top row was not. This explains the difference in band intensities between the top and bottom rows. Positive control was Paenibacillus larvae ATCC 9545 and the negative control was E. coli pMarA, however I ran out of room on this gel to run the negative control. I will the negative control on the subsequent gel. HiLo DNA marker was used.

Interestingly, all the isolates, except for one, tested positive as being P. larvae. The only isolate that didn't amplify using the AFB primers was EW 02 (dd1-7a). I will continue to screen the remaining suspected Paenibacillus isolates.

//EWW

Tripartite Conjugation in P. larvae

The goal of this project is to develop a mutagenesis procedure in P. larvae 9545 that will randomly insert transposons into its genome. This particular method has been shown to be effective in other Bacillus spp..

December 2nd, 2014

Brothed:
P. larvae 9545 into 10 mL of BHI + thiamine (target genome)
EC pMarA into 10 mL of BHI Amp100 and Amp100 + Kan50 (transposon)
EC pRK2013 into 10 mL of BHI Kan 50 (helper plasmid)

//EWW