Partial characterization and electroporation of tetracycline resistant plasmids from the Gram-positive spore-forming honey bee pathogen Paenibacillus larvae.

LEON I.E. ,; LOPEZ A.C.; ALIPPI A.M

BARILOCHE

Conferencia; International Plasmid Biology Conference 2010; 2010

IBBM UNLP - CONICET

American Foulbrood of honey bees (AFB) is the most devastating bacterial disease of honeybee brood (Apis mellifera and Apis cerana) worldwide. Its etiological agent is the spore-forming, Gram-positive bacterium Paenibacillus larvae. In areas were disease incidence is high, antibiotic treatment is an alternative to the burning of infected beehives. Oxytetracycline and tylosin are the only antibiotics currently approved to prevent and control of AFB in honeybee colonies; however, tetracycline-resistant P. larvae isolates have been detected in certain areas of the USA, Canada and Argentina. Resistance to tetracycline (Tc) is mainly due to the acquisition of tet determinants frequently associated with mobile elements. Approximately 40 different tet and otr genes have been reported in Gram positive bacteria, whereas in Bacillus and its related species, only the tet(K), tet(L), tet(M), tet(W) and/or otr(A) determinants have been reported. Recently, natural P. larvae plasmids have been shown to confer Tc-resistance by tetK and/or tetL genes, respectively. Therefore, the purposes of this work were 1) to develop an extraction and purification system of plasmid DNA from P. larvae; 2) to transfer plasmids carrying known Tc-resistant determinants from a donor P. larvae strain to a recipient one by electroporation, and 3) to partially characterize these plasmids. According to the results obtained by using the Puriprep-P kit® with modifications, high quality of plasmid DNA from P. larvae strains was obtained and was successfully electroporated. The transfer of different Tc- resistant plasmids (pPL373, pPL374 and pPl395) into a Tc-susceptible P. larvae (NRRL B-14154) strain with a characteristic red phenotype, resulted in the isolation of different P. larvae transformants that exhibited resistance to Tc. Based upon the sequence of plasmid pMA67 (GenBank DQ367664.1) 4 primers were designed, two named CAI/F-oriT homologous to the origin of conjugal transfer region (oriT) and two other, named Mob1/Mob2 homologous to the N-terminus of the MOB protein region. The fragments obtained with each pair of primers were of the expected size, suggesting this that we successfully amplified both plasmid regions. This is the first report of the successful transformation between P. larvae strains with plasmids carrying Tc determinants, such a system might be a useful tool for the elucidation of the genetic system involved in the development of tetracycline resistance in P. larvae. This research was supported by ANPCyT (PICT2411) and CIC.