An improved technique for direct visualization of plasmid patterns in strains of Paenibacillus larvae

Abstract

American Foulbrood disease (AFB) caused by Paenibacillus larvae is the most devastating bacterial disease affecting honey bee brood worldwide. Beekeepers have been using oxytetracycline for decades to prevent and control AFB. In some countries with high disease incidence, antibiotics are still employed with the consequent risks of the appearance of tetracycline-resistant phenotypes associated with small plasmids carrying a tetracycline-resistance gene. Techniques for plasmid visualization in Gram-positive bacteria, particularly spore-forming, are not always successful. The present study aims to improve a method for direct visualization of plasmid patterns in P. larvae and other spore-forming bacteria isolated from apiarian sources. We tested 51 P. larvae strains from different geographical areas, including tetracycline-sensitive and resistant ones. The results revealed six different plasmid profiles, named I, II, III, IV, and V. Within tetracycline-resistant strains, four strains showed pattern IV with two plasmids of 5,000 and 8,000 bp, and two strains exhibited pattern V, with bands of about 5,000, 7,000, and 8,000 bp. The plasmid band of 5,000 bp present in the six resistant strains corresponds to the tetracycline-resistance mobilizable plasmid reported for P. larvae. Within the tetracycline-sensitive phenotypes, 33 strains showed no plasmids (pattern I); while two additional profiles were detected in the rest, i.e., pattern III with three plasmids in one strain and pattern VI showing two plasmids in six strains. Patterns II, III, and VI are probably related to the presence of cryptic plasmids. In conclusion, the improved technique allows us to identify plasmid patterns in P. larvae, ensuring their reproducibility and feasibility.