Decreased susceptibility to 4'-deoxy-6'-N-methylamikacin (BB-K311) conferred by a mutant plasmid in Escherichia coli

Abstract

Escherichia coli MP6 contains a plasmid that encodes aminoglycoside 3'-phosphotransferase II, which phosphorylates kanamycin and confers high-level kanamycin resistance. Amikacin is a mincr substrate of this enzyme, but MP6 is susceptible to amikacin. Strain MP10 has a spontaneous mutation in the plasmid of MP6 that increases the aminoglycoside 3'-phosphotransferase II activity not only against kanamycin but also against amikacin. This mutation is also responsible for the appearance of resistance to amikacin in MP10. Resistance to 4'-deoxy-6'-N-methylamikacin (BB-K311) by enzymatic modification has not been reported previously. As with amikacin, MP6 was susceptible to BB-K311 and its aminoglycoside 3'-phosphotransferase II did not phosphorylate this amikacin derivative appreciably. We found that the plasmid-borne mutation in MP1O, however, localized by being cloned with a 3.7-megadalton HindIII fragment containing the aminoglycoside 3'-phosphotransferase II gene, resulted in increased phosphorylation of BB-K311 and resistance to it. Thus, the mutation distinguishing MP6 and MP10 has increased the activity of an existing aminoglycoside-modifying enzyme and produced new bacterial resistance to two previously minor substrates of the enzyme.

High-level amikacin resistance in Escherichia coli due to phosphorylation and impaired aminoglycoside uptake

“…From 2 to 5% of colonies which grew after treatment with novobiocin were susceptible to 50 ,ug of kanamycin per ml, 25 ,ug of streptomycin per ml, and 200 ,ug of carbenicillin per ml. Clones representing cured derivatives of each of the strains were saved.…”

“…If the relative level of amikacin resistance depends on the amount of APH(3')-II activity alone, then these transformants should exhibit similar levels of amikacin resistance. The finding that clone MP101 (25), a clone bearing the gene from strain L4(pMP1-2), was more resistant than clone MP61 (25), a clone with the strain L-0(pMP1-1) gene, was inconsistent with this prediction. This discrepancy suggested that another factor besides the amount of APH(3')-II activity was playing a role in the development of clinically significant levels of amikacin resistance in strain L4(pMP1-2).…”

“…The Bryan rate competition model of AG resistance has been modified (4) to account for evidence (14,24) (25) that this enzyme is associated not only with resistance to amikacin, but also with resistance to 4'-deoxy-6'-N-methylamikacin (BB-K 311), a derivative of amikacin designed to be free from attack by the two other enzymes known to modify amikacin and thereby confer resistance to it (9). It is of great interest that mutants with high levels of resistance to amikacin can be selected in strains bearing APH(3')-II by exposing the strains to low levels of an AG such as gentamicin, which is not even a substrate for the enzyme.…”

Bleomycin increases amikacin and streptomycin resistance in Escherichia coli harboring transposon Tn5

“…The results presented above are similar to those reported by De Vos et al (8), who obtained expression of streptomycin resistance in E. coli by setting the str gene under the control of a strong promoter. If mechanisms are identical, higher levels of transcription from the neo-ble-str operon promoter should increase the amounts of APH(3')II, the product of the neo gene, and subsequently, the level of amikacin resistance (20,21). Table 1 shows the amikacin MICs (Bristol Myers Squibb Laboratories, Syracuse, N.Y.) of the strains BM21 (pJB35HH) and BM21(pJB35HB) in the presence of doubling concentrations of bleomycin (19).…”

“…The more patent phenotypic effect of this induction seems to be the increase of resistance to streptomycin and amikacin in E. coli strains containing Tn5. Even if this increase appears to be relatively modest in terms of the MIC, it could be sufficient, under special conditions (20,21), to ensure a prolonged survival of bacterial cells in tissues in which the antibiotic concentrations are limited. These results raise the possibility of a decrease in the antibacterial effects of streptomycin and amikacin in patients treated with bleomycin or other related DNA-damaging agents.…”