Results of analytical IEF and of molecular characterization of β-lactamase determinants carried by 70 putative ESBL producersd

No. of isolatesIEF band(s)aResult of colony hybridizationEnzyme(s)bMICc (μg/ml) of:
blaTEM probeblaPER probeCTXCAZFEPATM
52pI 5.2e + pI 5.6++TEM-2 + PER-116->6416-3232-644-32
18pI 5.9+TEM-524-162-82-162
  • a In the bioassay, the pI 5.2 and 5.9 bands showed activity against cefotaxime, ceftazidime, cefepime, and aztreonam, while the pI 5.6 band did not show activity against any of the above compounds.

  • b According to the sequence of the determinant encoding the mature enzyme.

  • c CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; and ATM, aztreonam. The MICs of ESBL producers were determined by standard broth macrodilution test (14).

  • d In vitro susceptibility to expanded-spectrum cephalosporins and aztreonam is also shown.

  • e The pI 5.2 enzyme was produced at a lower level in seven of these isolates, as shown by a lower intensity of the band consistently observed in replicated experiments after loading the same amount of crude extracts. Assay of cefotaximase activity in the extracts of three isolates, including two of those producing a higher amount of the enzyme and one of those producing a lower amount (the same isolates selected for sequence analysis, see text), revealed a higher activity in the former isolates (specific activities, 310 ± 35 and 335 ± 40 nmol/min/mg of protein) than in the latter isolate (specific activity, 195 ± 20 nmol/min/mg of protein). No consistent differences, however, were observed between the MICs of CTX, CAZ, FEP, and ATM for isolates producing the enzyme at a higher level and those for isolates producing the enzyme at a lower level.