Isomeric DNA ladder formation of a plasmid encoding tobramycin resistance from Escherichia coli

Abstract

During a 20-month survey of resistance to three aminoglycosides (gentamicin, tobramycin, and amikacin) in Escherichia coli at a university hospital, six tobramycin-, kanamycin-resistant isolates containing a 50 kilobase conjugative R-plasmid which encoded an aminoglycoside phosphotransferase (APH-(3')) were isolated. The APH-(3') conferred resistance to kanamycin (MIC = 100 mg/L) but not to tobramycin (MIC = 20 mg/L). In both the original isolates and transconjugants the six R-plasmids demonstrated an isomeric ladder in the range of 50-112 kb, which was enhanced by exposure of the bacterial cultures to tobramycin. pJFJ2522 is the prototype for this group of plasmids. Bacterial DNA gyrase reversed the isomeric DNA ladder in pJFJ25222 by increasing the supercoiling of the plasmid DNA. Regardless of the level of supercoiling, the plasmids produced indistinguishable restriction endonuclease fragment patterns. The clinical isolates containing these plasmids demonstrated different restriction fragment length polymorphism (RFLP) of their EcoRI digested genomic DNA using E. coli rRNA as a probe. Ladder formation was plasmid specific since other tobramycin R-plasmids did not form a ladder, but it was not host specific. pJFJ25222 formed a ladder in a recA- host and displayed the same restriction pattern in a recA- as in a recA+ environment. In conclusion, pJFJ2522 contains a new tobramycin resistance gene whose mechanism of resistance is not known and whose product probably influences the isomerization of the plasmid.