Related Experiment Videos
Aminoglycoside phosphotransferase-II-mediated amikacin resistance in Escherichia coli
Abstract:
An Escherichia coli strain with a plasmidic amikacin resistance has been selected for which the evidence strongly indicates that resistance is mediated by aminoglycoside phosphotransferase [APH(3')-II]: (i) this resistance was coupled with resistance against kanamycin and neomycin; (ii) partially purified APH(3')-II[APH(3") free] modified amikacin by phosphorylation; (iii) the product of the APH(3')-II mediated reaction (i.e., 3'-O-phosphoryl-amikacin) lost its antibacterial activity; and (iv) the amikacin-modifying APH(3')-II activity increased 5- to 10-fold after adaptation of the cells to higher concentrations of amikacin. The substrate spectrum of this enzyme showed a low activity against amikacin as compared with neomycin. It is argued that the enzyme level rather than its substrate spectrum is important for enzyme-mediated resistance. The increase in enzyme levels was found to be correlated with an increase in copy number of a 110-Megadalton plasmid (pBN66) which coded for the APH(3')-II and the APH(3") activity. The increase in copy number was irreversible, and therefore this phenomenon is ascribed to a mutation of a gene which affects the copy number. In transconjugants, the original low copy number was present, and therefore the mutation must be located on the chromosome and not on the plasmid.
Insights
This study identifies aminoglycoside phosphotransferase [APH(3')-II] as the enzyme conferring amikacin resistance in Escherichia coli. Increased enzyme levels, driven by plasmid copy number mutations, are key to this antibiotic resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Antibiotic resistance is a growing global health concern.
- Aminoglycoside antibiotics, like amikacin, are crucial for treating bacterial infections.
- Understanding the mechanisms of antibiotic resistance is vital for developing new therapeutic strategies.
Purpose of the Study:
- To elucidate the mechanism of amikacin resistance in a selected Escherichia coli strain.
- To characterize the enzyme responsible for amikacin resistance.
- To investigate the genetic basis for increased enzyme activity and resistance.
Main Methods:
- Enzyme purification and characterization.
- In vitro enzymatic assays using amikacin, kanamycin, and neomycin.
- Plasmid analysis and copy number determination.
- Bacterial adaptation studies.
- Transconjugant analysis.
Main Results:
- Amikacin resistance was mediated by aminoglycoside phosphotransferase [APH(3 étaire')-II].
- The enzyme phosphorylated amikacin, rendering it inactive.
- Enzyme activity increased significantly after cellular adaptation to amikacin.
- Increased APH(3 étaire')-II levels correlated with elevated copy numbers of plasmid pBN66.
- A chromosomal mutation affecting plasmid copy number was identified as the cause of irreversible resistance.
Conclusions:
- The level of APH(3 étaire')-II enzyme, rather than its substrate spectrum, is critical for amikacin resistance.
- Chromosomal mutations influencing plasmid copy number can lead to stable, high-level antibiotic resistance.
- This study provides insight into the molecular mechanisms of plasmid-mediated antibiotic resistance in bacteria.