Transformation and characterization of tetracycline resistant plasmids from Paenibacillus Larvae the causal agent of American Foulbrood Disease

LEÓN I.E ; LOPEZ A.C,; ALIPPI A.M

Congreso; 42° International Apicultural congress APIMONDIA 2011; 2011

American Foulbrood of honey bees (AFB) is the most serious bacterial disease affecting honeybees worldwide and is caused by the spore-forming, Gram-positive bacterium Paenibacillus larvae. Oxytetracycline is currently used to prevent and control AFB in honeybee colonies as an alternative to the burning of infected beehives in areas where disease incidence is high. However, tetracycline-resistant (TcR) P. larvae isolates have been detected in USA, Canada and Argentina. Resistance to tetracycline is mainly due to the acquisition of tet determinants frequently associated with mobile elements. Horizontal gene transfer of genetic information between bacterial cells is an integral factor in the generation of genetic variability and evolution in bacteria. Natural P. larvae plasmids have been shown to confer TcR by tetK and/or tetL genes, respectively. Therefore the purposes of this work were to characterize 3 different natural TcR plasmids and to electroporate those plasmids into a tetracycline-susceptible (Tcs) P. larvae recipient strain. By using the Puriprep-P kit® with modifications, high quality of plasmidic DNA from P. larvae was obtained. The transfer of 3 different TcR purified plasmids (pPL373, pPL374 and pPl395) into a Tcs P. larvae strain was made by electroporation. The strain NRRLB-14154 with a characteristic red phenotype was used as recipient and TcR P. larvae transformants exhibiting red color were obtained. All the transformants were cured by heat treatment combined with acridine orange. The cured transformants recovered their original Tcs phenotype; in addition, BOX-PCR fingerprinting was used to confirm the identity of the recipient, transformants, and cured strains. DNA sequence comparisons suggested the presence of the same plasmid transfer origin-like site (OriT) an also mobilization (mob) genes similar to those founded on pMV158-superfamily of plasmids. We reported a successful transformation of plasmids pPL373, pPL374 and pPl395 containing known TcR determinants into a Tcs P. larvae.