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.

Molecular cloning of amikacin resistance determinants from a Klebsiella pneumoniae plasmid

“…However, we cannot rule out the possibility of the presence of two separate genes or two genes that overlap. The existence of a plasmid-mediated acetyltransferase that inactivates amikacin was demonstrated in Pseudomonas aeruginosa (19), and a phosphotransferase activity mediating Ak in E. coli has also been described (5). The fact that extracts obtained from cells harboring the recombinant clones encoding Ak, Km, and Tm but not Gm were able to acetylate all four aminoglycosides can be explained considering that resistance to an antibiotic is the result of a competition between two factors: rate of transport of the antibiotic inside the cell and modification of the drug (15).…”

“…This aminoglycoside is refractory to most inactivating enzymes (31). However, chromosome-and plasmid-encoded resistance to amikacin has already been reported (5,19,20,24,(28)(29)(30)36). In one case, amikacin resistance determinants were carried by transposon Tn2424 (28).…”

“…Antibiotic-resistant strains of K. pneumoniae are frequently found in many different geographical locations, with aminoglycoside resistance becoming a growing clinical problem (5,9,15,16,19,20,24,29). The aminoglycoside amikacin, introduced in 1976, is a semisynthetic derivative of kanamycin A, obtained through acylation with the L(-)-y-amino-a-hydroxybutyryl side chain at the C-1 amino group of the deoxystreptamine moiety (21).…”

Plasmid-encoded amikacin resistance in multiresistant strains of Klebsiella pneumoniae isolated from neonates with meningitis

“…Amikacin (Ak), a semisynthetic derivative of kanamycin A which was introduced in 1976 (18), is the most resistant to enzyme inactivation of all the aminoglycosides currently available for therapeutic use (29). However, there have recently been reports of chromosome-and plasmid-mediated resistance to amikacin (3,17,19,23,24,28). It seems inevitable that a higher incidence of resistance will occur in the United States as amikacin and other aminoglycosides are increasingly used (19).…”

“…As a result, aminoglycosides have been used sparingly to reduce the selection of resistant strains. Despite these precautions, resistance to these antibiotics is now being identified in a number of isolates from different geographical locations (3,5,13,17,19,24). Amikacin (Ak), a semisynthetic derivative of kanamycin A which was introduced in 1976 (18), is the most resistant to enzyme inactivation of all the aminoglycosides currently available for therapeutic use (29).…”