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Isolation, characterization, and cloning of a plasmid-borne gene encoding a phosphotransferase that confers
R Gaynes1, E Groisman, E Nelson
1Department of Internal Medicine, University of Michigan, Ann Arbor 48105.
Abstract:
Clinical isolates of Klebsiella pneumoniae and Serratia marcescens at a hospital that had used amikacin as its principal aminoglycoside for the preceding 42 months demonstrated high-level resistance to amikacin (greater than or equal to 256 micrograms/ml), kanamycin (greater than or equal to 256 micrograms/ml), gentamicin (greater than or equal to 64 micrograms/ml), netilmicin (64 micrograms/ml), and tobramycin (greater than or equal to 16 micrograms/ml). The resistant strains contained an identical 6.8-kilobase plasmid, pRPG101. Transformation of pRPG101 into Escherichia coli produced high-level resistance to amikacin (greater than or equal to 256 micrograms/ml) and kanamycin (greater than or equal to 256 micrograms/ml) but unchanged susceptibilities to gentamicin, netilmicin, and tobramycin. The clinical isolates and transformants produced a novel 3'-phosphotransferase, APH(3'), that modified amikacin and kanamycin in vitro. The location and orientation of the amk gene encoding this APH(3') were determined by analysis of insertions in pRPG101 of the defective gene fusion phage Mu dII1681 (mini-Mulac). Cells containing plasmids with insertions into amk that had the lac operon fused to the amk promoter were selected as Lac+ and amikacin susceptible. A collection of these mini-Mulac insertions was mapped by restriction enzyme analysis. This characterization of amk facilitated its cloning as a 1.8-kilobase EcoRI-Bg/I fragment of pRPG101 into the pUC19 vector. E. coli strains containing this recombinant plasmid had APH(3') activity and demonstrated high-level resistance to amikacin and kanamycin (greater than or equal to 256 micrograms/ml) but were as susceptible to gentamicin, tobramycin, and netilmicin (less than or equal to 1.0 microgram/ml) as the strains harboring the original pRPG101 plasmid.
Insights
High-level amikacin resistance in Klebsiella pneumoniae and Serratia marcescens is linked to a novel plasmid-borne 3'-phosphotransferase, APH(3'). This enzyme confers resistance to amikacin and kanamycin but not other aminoglycosides.
Area of Science:
- Microbiology
- Molecular Biology
- Antimicrobial Resistance
Background:
- Clinical isolates of Klebsiella pneumoniae and Serratia marcescens exhibited high-level resistance to amikacin after 42 months of its use.
- These resistant strains shared an identical 6.8-kilobase plasmid, pRPG101.
Purpose of the Study:
- To investigate the genetic basis of amikacin resistance in clinical isolates.
- To characterize the novel enzyme responsible for amikacin and kanamycin resistance.
Main Methods:
- Plasmid transformation into Escherichia coli.
- Analysis of mini-Mulac insertions to map gene location and orientation.
- Cloning of the amikacin resistance gene (amk).
Main Results:
- Transformation with pRPG101 conferred high-level resistance to amikacin and kanamycin, but not gentamicin, netilmicin, or tobramycin.
- A novel 3 -phosphotransferase, APH(3 ), was identified, modifying amikacin and kanamycin in vitro.
- The amk gene encoding APH(3 ) was cloned and expressed, confirming its role in resistance.
Conclusions:
- A novel plasmid-mediated 3 -phosphotransferase (APH(3 )) is responsible for high-level amikacin and kanamycin resistance in clinical isolates.
- The characterized amk gene provides a target for understanding and combating aminoglycoside resistance.