Resistance of bacteria to the newer aminoglycoside antibiotics: An epidemiological and enzymatic study.

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.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Bongaerts¹,

Molendijk²

1984

“…Until now, only one type of aminoglycoside 2"-adenylyltransferase [AAD(2")] has been described (2,4,11), and its substrate range includes kanamycin, gentamicin, and tobramycin. This paper reports the isolation, purification, and characterization of a new plasmid-mediated AAD(2") from Klebsiella pneumoniae, Escherichia coli, Serratia marcescens, and Proteus vulgaris.…”

mentioning

confidence: 99%

New plasmid-mediated aminoglycoside adenylyltransferase of broad substrate range that adenylylates amikacin

Coombe¹,

George²

1981

The same aminoglycoside 2"-adenylyltransferase was isolated from four gram-negative species which were among a random group of gentamicin-resistant isolates from the same hospital. The enzyme was partially purified from a crude extract which also contained a second modifying enzyme identified as APH(3')-I. The substrate range of the new aminoglycoside 2"-adenylyltransferase included the newer aminoglycosides sisomicin and amikacin, but showed much-reduced activity against gentamicins C2 and Cla. The pH optimum was 7.8 to 8.0 for every substrate, and the molecular weight of the enzyme molecule was estimated at approximately 29,000. Genetic experiments clearly established that both enzymes were expressed by a conjugative plasmid.

“…These enzyme-specific substrate profiles are usually considered adequate for proper identification. In the past, profiles were derived from enzymatic measurements at one fixed substrate concentration (5,8,10,14). However, in a previous study (4) we observed that ANT(2") activities against various aminoglycosides appeared to be concentration dependent.…”

mentioning

confidence: 99%

Effect of aminoglycoside concentration on reaction rates of aminoglycoside-modifying enzymes

Bongaerts¹,

Vliegenthart²

1988

Reaction rates of several reference aminoglycoside-modifying enzymes were studied at various substrate concentrations. The resulting concentration-response curves showed wide variation in threshold concentration, in curve slope, in enzyme saturation, and in substrate inhibition. Together, the curves of a defined aminoglycoside panel yielded more specific information for each individual aminoglycoside-modifying enzyme tested than did conventional substrate profiles obtained at a single substrate concentration.Enzymatic aminoglycoside resistance arises from the presence of one or more aminoglycoside phosphotransferases (APHs), aminoglycoside nucleotidyltransferases (ANTs), or aminoglycoside acetyltransferases (AACs). To determine to which group an aminoglycoside-modifying enzyme (AME) belongs, specific radioactively labeled coenzymes,]acetyl coenzyme A, are used as cosubstrate in enzymatic reactions. With regard to the aminoglycoside modification site, several specific types can be distinguished within each group. The modification site is determined by examining the chemical structure of the modified aminoglycoside. For isoenzyme typing, discriminative enzymology is required.In practice, AME identification takes place by comparison of the substrate profile of an unknown enzyme with those of reference AMEs. The substrate profiles are defined as sets of relative rates of modification of various aminoglycosides. These enzyme-specific substrate profiles are usually considered adequate for proper identification. In the past, profiles were derived from enzymatic measurements at one fixed substrate concentration (5,8,10,14). However, in a previous study (4) we observed that ANT(2") activities against various aminoglycosides appeared to be concentration dependent. Substrate inhibition occurred in several cases and may produce a falsely low index of activity against a specific substrate, even one against which the enzyme confers resistance at the lower concentrations encountered in vivo. There is, moreover, an even more common problem with the usual practice of determining substrate activity profiles. With excess substrate, i.e., concentrations at which the enzyme is saturated, the activity reflects the Vmax and is proportional to the amount of enzyme. At lower concentrations, near the K,, and below, the rate of activity should be proportional to substrate concentration. Most investigators characterize activity against a substrate profile by using a single concentration which is in excess. Thus, as pointed out by Bongaerts and Kaptijn (3) and Perlin and Lerner (15) with regard to APH(3')-II-mediated modification of amikacin, and by Bongaerts and Molendijk (4) and DeHertogh and Lerner (7) with regard to ANT(2")-mediated modification of netilmicin, excess substrate may result in greater activity against a high-* Corresponding author. t Present address: