Relation between aminoglycoside 2"-O-nucleotidyltransferase activity and aminoglycoside resistance

Abstract

The relationship between plasmid-coded aminoglycoside 2"-O-nucleotidyltransferase [ANT(2")] activity and the minimum inhibitory concentration for aminoglycosides was studied. ANT(2") was very unstable, and therefore, procedures for handling this enzyme were optimized. Escherichia coli L58058.1 producing ANT(2") was resistant to gentamicin, sisomicin, kanamycin, tobramycin, and dibekacin but susceptible to netilmicin, amikacin, and neomycin. However, only amikacin and neomycin were not modified by ANT(2"). A correlation between the substrate profile of the enzyme tested at 0.15 mM and the minimum inhibitory concentrations of the aminoglycosides could not be demonstrated. The substrate concentration-dependent enzyme activity curves proved to be a better alternative than a substrate profile based on tests at one concentration. At concentrations of >10-2 mM, ANT(2") demonstrated substrate inhibition that explains the discrepancy between ANT(2") activity and the minimum inhibitory concentrations. A close correlation was found between the physiological efficiency (VmaxlKm ratio) of ANT(2") and the minimum inhibitory concentrations for the enzyme-producing strain.

Resolving the Antibiotic Paradox

“…Aminoglycosides yielding kea/KIn of 10 6 M-'s-I also showed lower MIC values (::;; 8 f.lg/mL) while substrates with kea/Kill of 107--1l M-'s-' gave rise to MICs of> 1024 f.lg/mL. This parallels studies with other aminoglycoside-resistance enzymes, notably APH(3 ')-IIIa (McKay et a!., 1994b), ANT(2") (Bongaerts and Molendijk, 1984;Dettertogh and Lerner, 1985), and AAC(6')-Ib (Radika and Northrop, 1984d). The biological significance of these observations is that aminoglycoside-modifying enzymes are optimally effective at low concentrations (sub Kill) of antibiotic, a scenario which is ideally suited to a detoxifying mechanism.…”

Inhibition of the ANT(2″)-Ia resistance enzyme and rescue of aminoglycoside antibiotic activity by synthetic α-hydroxytropolones

“…4 One of these enzymes is the aminoglycoside-2″-O-nucleotidyltransferase [ANT(2″)-Ia], which catalyzes the adenylation of several clinically used aminoglycoside antibiotics, such as gentamicin and tobramycin, rendering them inactive (Scheme 1). 5 ANT(2″)-Ia is highly prevalent among pathogenic Gram-negative bacteria, and has been classified along with N-acetyltransferase-6′ [AAC-(6′)] as the most common determinant of enzyme-dependant aminoglycoside resistance in Pseudomonas aeruginosa . 6 Thus, therapeutically viable inhibitors of ANT(2″)I-a could find use in combination with ANT(2″)-Ia susceptible aminoglycoside antibiotics.…”

Correlation of aminoglycoside resistance with the KmS and Vmax/Km ratios of enzymatic modification of aminoglycosides by 2''-O-nucleotidyltransferase

“…For ANT(2"), 3-N-acetyltransferase [AAC(3)-I], and 2'-N-acetyltransferase [AAC(2')], they showed that aminoglycosides which had low MICs had high Kms. However, Radika and Northrop (8) and Bongaerts and Molendijk (2) reported a poor correlation between MICs and the Km values for 6'-N-acetyltransferase [AAC(6')] and ANT(2") enzymes, respectively. These investigators maintained that only the Vmax/Km ratio was predictive of the MICs.…”