TABLE 3.

Characterization of new β-lactamases

Data set requiredSpecific testing criteria
MICs using standardized
    methodologySelect antibiotics that most closely define the enzymatic properties. Strains to be tested should include (i) original clinical isolate, (ii) transformant or transconjugant with no other enzyme present, and (iii) host strain without any β-lactamase.
Full nucleotide and amino
    acid sequencesStandard methodology
Approved nameContact the curators of the website http://www.lahey.org/Studies/
Protein purificationAt least 90% purity on electrophoresis with high protein concn. No unrelated β-lactamase activity in the extract.
Isoelectric focusingIsoelectric point for all β-lactamases in original clinical isolate and for purified protein
Hydrolysis profile
    (kcat and Km values)Standard substrates are as follows: cephaloridinea (or cephalothin), benzylpenicillin, cloxacillin or oxacillin, cefotaxime, ceftazidime, cefoxitin, imipenem, and aztreonam. Additional substrates should be tested for enzymes with specific substrate profiles, e.g., doripenem, ertapenem, and/or meropenem for carbapenemases. Native (nontagged) enzymes should be studied.
Inhibition profile (IC50sb
    or inhibition constants)Standard inhibitors, including clavulanic acid, tazobactam, and EDTAc
  • a Cephaloridine is preferred (contact K. Bush for availability), but cephalothin can be substituted.

  • b Determined after 5 min of preincubation of enzyme and inhibitor. Substrate concentration and Km should be reported.

  • c Other metal ion-chelating agents, such as 1,10-o-phenanthroline or dipicolinic acid, can also be tested if an MBL is suspected.