Low Frequencies of Resistance among Staphylococcus and Enterococcus Species to the Bactericidal DNA Polymerase Inhibitor N ³ -Hydroxybutyl 6-(3′-Ethyl-4′-Methylanilino) Uracil

Abstract

The 6-anilinouracils (AUs) constitute a new class of bactericidal antibiotics selective against gram-positive (Gr ؉ ) organisms. The AU family of compounds specifically inhibits a novel target, replicative DNA polymerase Pol IIIC. Like other antibiotics, AUs can be expected to engender the development of resistant bacteria. We have used a representative AU and clinically relevant strains of Staphylococcus aureus and Enterococcus to determine the frequency and mechanism(s) of resistance development. The frequency of resistance was determined by using N 3 -hydroxybutyl 6-(3-ethyl-4-methylanilino) uracil (HBEMAU) and commercially available antibiotics at eight times the MICs. For all five Gr ؉ organisms tested, the frequency of resistance to HBEMAU ranged from 1 ؋ 10 ؊8 to 3 ؋ 10 ؊10 . The frequencies of resistance to the antibiotics tested, including rifampin, gentamicin, and ciprofloxacin, were either greater than or equal to those for HBEMAU. In order to understand the mechanism of resistance, HBEMAU-resistant organisms were isolated. MIC assays showed that the organisms had increased resistance to AU inhibitors but not to other families of antibiotics. Inhibition studies with DNA polymerases from HBEMAU-sensitive and -resistant strains demonstrated that the resistance was associated with Pol IIIC. DNA sequence analysis of the entire polC genes from both wild-type and resistant organisms revealed that the resistant organisms had a sequence change that mapped to a single amino acid codon in all strains examined.

Biological Characterization of Novel Inhibitors of the Gram-Positive DNA Polymerase IIIC Enzyme

“…Low frequencies of resistance to HBEMAU were observed (8), which seems to be in accordance with the fact that probably only a single allowable mutation leads to resistance to the anilinouracil-based compound class. However, to estimate the clinical relevance of this resistance phenomenon, more detailed studies with more strains should be performed.…”

“…The region of this mutation is described as the C-terminal domain of the Pol IIIC enzyme required for dNTP binding and as the known aryl-binding region in B. subtilis (4,18). The Pol IIIC enzymes from resistant organisms displayed the same polymerase activities as their wild-type counterparts, but they showed higher K i values for AU compounds (8). Thus, resistance to the AU compound class is based on the lower binding affinities of the inhibitory molecules to the target enzyme.…”

“…The mutation that caused resistance to compound 1 was located at position 1261 of the Pol IIIC enzyme, which resulted in a change of phenylalanine to leucine. This conserved phenylalanine was also changed into a leucine residue in different gram-positive organisms resistant to 3(4Ј-hydroxybutyl)-6-(3Ј-ethyl-4Ј-methylanilino) uracil (HBEMAU) (8). A Pol IIIC enzyme from B. subtilis resistant to p-hydroxyphenyl azouracil has been described to be the result of a mutation in azp-12, which contains a single base change that results in the replacement of the serine at position 1175 by alanine (18).…”

Antibacterial Activity and Mechanism of Action of a Novel Anilinouracil-Fluoroquinolone Hybrid Compound

“…The frequency of development of spontaneous (single step) resistance to 251D was estimated using four representative gram-positive organisms. Table 6 summarizes the results and includes for comparison results for 340E and HB-EMAU (also reported previously [12]). At 8؋ MIC, mutation frequencies for HB-EMAU were in the range of 10 Ϫ9 to 10…”

“…E. coli XL1-Blue was purchased from Stratagene (La Jolla, CA). HB-EMAU-resistant S. aureus was obtained as described previously (12). HB-EMAU-resistant and ciprofloxacin-resistant S. aureus was generated by isolating HB-EMAU-resistant colonies (12) from the ciprofloxacin-resistant strain provided by D. Hooper.…”