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Amikacin resistance associated with a plasmid-borne aminoglycoside phosphotransferase in Escherichia coli
Abstract:
Enzymatic phosphorylation of amikacin has not been reported previously in gram-negative bacteria. We found that extracts of MP1, a mutant of Escherichia coli JR66/W677 that is resistant to amikacin, were able to phosphorylate this aminoglycoside more rapidly than were extracts of the parental strain. Conjugal transfer of resistance from MP1 to a recipient strain was accompanied by acquisition in the transconjugants of amikacin phosphotransferase activity and of a 57-megadalton plasmid present in the donor. Partial purification of the phosphotransferase activity on amikacin-Sepharose 4B yielded an enzyme with a substrate spectrum similar to that of the 3'-neomycin-kanamycin phosphotransferase II found E. coli, except that it was also active against amikacin. A mutant of MP1, MP5, had increased susceptibility to amikacin and reduced phosphotransferase activity. MP9, a mutant MP5, was more resistant to amikacin and had increased phosphotransferase activity. The mutations leading to these alterations of amikacin susceptibility and amikacin phosphotransferase activity were transferable with the same plasmid that was associated with amikacin resistance and phosphotransferase activity in MP1. These studies demonstrate that resistance to amikacin in a laboratory strain of E. coli is due to an aminoglycoside phosphotransferase coded by a transferable plasmid-borne gene.
Insights
Amikacin resistance in Escherichia coli is caused by an aminoglycoside phosphotransferase enzyme. This enzyme is encoded by a transferable plasmid gene, enabling resistance spread in gram-negative bacteria.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Enzymatic phosphorylation of amikacin in gram-negative bacteria was previously unreported.
- Amikacin is an important aminoglycoside antibiotic used to treat infections.
Purpose of the Study:
- To investigate the mechanism of amikacin resistance in a mutant strain of Escherichia coli.
- To identify the enzyme responsible for amikacin phosphorylation and its genetic basis.
Main Methods:
- Comparative analysis of phosphotransferase activity in amikacin-resistant and susceptible E. coli strains.
- Conjugal transfer experiments to assess the heritability of resistance and enzyme activity.
- Enzyme purification using amikacin-Sepharose 4B chromatography.
- Characterization of enzyme substrate specificity.
Main Results:
- Amikacin-resistant E. coli extracts showed higher phosphotransferase activity compared to the parental strain.
- Amikacin resistance and phosphotransferase activity were transferable via a 57-megadalton plasmid.
- A partially purified enzyme exhibited phosphotransferase activity against amikacin, similar to known neomycin-kanamycin phosphotransferases but with broader substrate activity.
- Mutations affecting amikacin susceptibility correlated with changes in phosphotransferase activity and were plasmid-associated.
Conclusions:
- Amikacin resistance in this E. coli strain is mediated by an aminoglycoside phosphotransferase.
- The gene encoding this enzyme is located on a transferable plasmid, suggesting a mechanism for the spread of amikacin resistance in gram-negative bacteria.