TABLE 3

Clones selected on ATM, related MICs, in vitro-specific hydrolytic activities of the tested antibiotics, and IC50s of clavulanic acid

Variant or constructaAmino acid changes at positionb:MIC (μg/ml)cIn vitro-specific hydrolytic activity (nmol min−1 μg−1 extract)dIC50 of clavulanic acid (μM)e
6104237243CTXATMCAZIPMETPFOXPENCTXATMCAZIPMFOX
GES-1 (wild type)AETG0.750.25120.250.00643.4 ± 0.61.0 ± 0.2<0.1<0.1<0.1<0.17.7 ± 0.5
Round 1
    GES-A1K1.53>2560.250.0084NDNDNDNDNDNDND
    GES-A2A331280.380.0123NDNDNDNDNDNDND
Round 2
    GES-A3KA432>2560.250.01241.3 ± 0.24.1 ± 0.64.7 ± 0.51.4 ± 0.3<0.1<0.1ND
Round 3
    GES-A4KAA2>256>2560.380.02548.7 ± 2.34.5 ± 1.114.2 ± 3.04.0 ± 0.6<0.1<0.10.6 ± 0.07
Round 4
    GES-A5TKAA12>256>2560.250.064623.6 ± 2.512.2 ± 1.238.0 ± 2.510.9 ± 0.7<0.1<0.10.5 ± 0.04
Constructs
    GES-A6A0.381.5480.250.0123NDNDNDNDNDNDND
    GES-A7KA0.7532>2560.250.0124NDNDNDNDNDNDND
    GES-A8AA112>2560.380.0254NDNDNDNDNDNDND
  • a The antibiotic concentrations used for selection were the following: round 1, 1 μg/ml; round 2, 16 μg/ml; round 3, 128 μg/ml; and round 4, 256 μg/ml.

  • b The amino acid positions were assigned according to Ambler. The amino acid changes compared to the wild-type GES-1 are indicated.

  • c All the GES alleles are expressed in the highly susceptible E. coli TOP10 cells.

  • d Specific activities were measured by UV spectrophotometry from crude extracts of E. coli TOP10 producing the indicated variant for each of the indicated antibiotics (PEN, CTX, CAZ, ATM, FOX, and IPM). The mean and standard deviation (SD) are indicated. ND, not determined.

  • e The IC50s of clavulanic acid were measured with PEN as a substrate. The mean and the SD are indicated.