TABLE 4

Clones selected on IPM, 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
−16170CTXATMCAZIPMETPFOXPENCTXATMCAZIPMFOX
GES-1 (wild type)CAG0.750.25120.250.00643.4 ± 0.61.0 ± 0.2<0.1<0.1<0.1<0.17.7 ± 0.5
Round 1
    GES-I1S0.1250.0941.50.50.064127.7 ± 1.2<0.1<0.1<0.10.15 ± 0.00060.16 ± 0.0486 ± 24
Round 2
    GES-I2TS0.190.12520.750.0942412.4 ± 3.2<0.1<0.1<0.10.18 ± 0.020.27 ± 0.06ND
Round 3
    GES-I3TTS10.191220.25>25637.8 ± 2.1<0.1<0.1<0.10.64 ± 0.040.97 ± 0.13150 ± 12
Constructs
    GES-I4T41.51280.380.0166NDNDNDNDNDNDND
    GES-I5TS0.50.1940.750.1948NDNDNDNDNDNDND
  • a The antibiotic concentrations used for selection were the following: round 1, 0.1 μg/ml; round 2, 0.125 μg/ml; and round 3, 0.25 μg/ml.

  • b The amino acid positions were assigned according to Ambler, except for the change at −1, which corresponds to a single nucleotide change at position −1 with respect to the ATG start codon, in which the nucleotide number relative to it is indicated. 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.