Submitted to: Journal of Apicultural Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/25/2006
Publication Date: 12/20/2006
Citation: Murray, K.D., Aronstein, K.A. 2006. Oxytetracycline-resistance in the honey bee pathogen Paenibacillus larvae is encoded on novel plasmid pMa67. Journal of Apicultural Research. 45(4):207-214. Interpretive Summary: American foulbrood (AFB) is the most serious infectious disease of honey bees. It is caused by bacteria of a species called Paenibacillus larvae subsp. larvae (formerly called Bacillus larvae). Infected bee larvae die, and in some cases entire colonies are lost. Beekeepers have controlled the disease for decades by supplying their bees with Terramycin, whose active ingredient is the antibiotic oxytetracycline (OTC). However, in the past few years, there have been several reports of loss of effectiveness of Terramycin against AFB. The reason for this is that some bacteria have become resistant to the OTC antibiotic. The present study announces the discovery of a gene in the bacteria which is responsible for the OTC-resistance. We studied 36 different strains of the bacteria collected mostly from the western and Midwestern U.S. and found this gene is present in all OTC-resistant strains, but is absent in the strains which are susceptible to OTC. We transferred the gene to another bacterial species in the laboratory, and found that it also conferred OTC-resistance to that bacterium. We have found that this gene is not on the bacterial chromosomal DNA, but instead resides on a small DNA molecule called a plasmid. We determined the DNA sequence of the entire plasmid and the results revealed that this plasmid has the ability to be naturally transferred to other bacterial cells in a process known as plasmid mobilization. This process can lead to the spreading of antibiotic resistance among different bacterial species in nature. We believe that such knowledge of the basic mechanisms of antibiotic resistance can aid in planning future strategies to combat honey bee infectious agents. This study is also the first report of any tetracycline-resistance gene found in any Paenibacillus bacteria.
Technical Abstract: The gram-positive bacterium, Paenibacillus larvae subsp. larvae, causes a serious honey bee disease called American Foulbrood. For several decades, this disease has been controlled by commercial and hobbyist beekeepers by the use of the antibiotic oxytetracycline. However, in the past few years, there have been several reports of oxytetracycline-resistant P. larvae subsp. larvae. In this study, we report that the reason for the oxytetracycline resistance in P. larvae subsp. larvae is the presence of a novel mobilizable plasmid carrying a tetracycline resistance gene –tetL. In 36 strains of P. larvae subsp. larvae from the U.S. and Canada that we tested, this plasmid was found in all oxytetracycline-resistant strains and in none of the oxytetracycline-sensitive strains. We cloned and expressed the P. larvae subsp. larvae tetL gene in Escherichia coli and showed it was functional. Sequencing of the entire plasmid, which we named pMA67, revealed that it is a mobilizable rolling-circle replication plasmid. We suggest that pMA67 is responsible for most, if not all of the tetracycline resistance seen in P. larvae subsp. larvae in North America in recent years. This work provides the first sequence information for any P. larvae subsp. larvae plasmid, and the first tetracycline-resistance gene found in the entire Paenibacillus genus.