Ceftazidime-resistant E. coli
and K. pneumoniae
strains were isolated from Barnes-Jewish Hospital in St. Louis. The number of presumed ESBL-producing isolates has been large, compared to data recorded from other single centers in the United States (3
), but the proportion of ESBL-producing isolates among all isolates remains <5%.
TEM-43 showed a hydrolytic profile similar to those of other TEM variants originating from the United States, such as TEM-10, TEM-12, and TEM-26 (3
). All these enzymes exhibit hydrolytic activity against ceftazidime as well as aztreonam. Like TEM-10, TEM-26, and TEM-28, cefotaxime was hydrolyzed by TEM-43 but at a lower level. TEM-43 had a significantly better affinity (lower Km
) for cephaloridine, cefotaxime, and aztreonam than the TEM-1 parent enzyme. However, the Km
of TEM-43 for cephaloridine was increased compared to those of the other related ESBLs. Imipenem and cefoxitin were poor substrates (Table ) for TEM-43, as expected for the TEM-derived ESBLs.
The substitution profile of TEM-43 is similar to that of TEM-6; both of these ESBLs have the same amino acid changes at positions 104 and 164 (14
). Both of these enzymes are members of the His164-substituted TEM variants, a growing family of ESBLs that is less numerous than extended-spectrum TEMs with Ser164 substitutions. When TEM-6 was evaluated for hydrolysis characteristics in our laboratory (5
), the hydrolytic profile was similar to that for TEM-43. However, aztreonam was hydrolyzed, with TEM-6 having a relative Vmax
that was approximately 15% of the observed hydrolysis rate for ceftazidime. This is in contrast to TEM-43, which hydrolyzes both aztreonam and ceftazidime with identical kcat
The unique ESBL substitution in TEM-43 is the threonine-for-methionine substitution at position 182. This change at position 182 has been reported for an inhibitor-resistant TEM-type β-lactamase, TEM-32, (7
) and for two ESBLs, TEM-20 and TEM-52 (23
). In a study with laboratory-derived TEM mutants (7
), substitution of only Thr for Met at position 182 gave a TEM enzyme with increased hydrolytic properties for penicillins and the early cephalosporins; no data on the hydrolysis of expanded-spectrum β-lactams were provided.
TEM-32 additionally has an isoleucine-for-methionine substitution at position 69 (this substitution has been shown to be the dominant factor for inhibitor resistance [12
]) but no changes from TEM-1 at positions 104 and 164. Assuming that the Ile69 substitution rather than the Thr182 substitution is fully responsible for inhibitor resistance, as shown by Farzaneh et al. (7
), it was expected that the TEM-43 β-lactamase would be susceptible to inhibition by the classic β-lactamase inhibitors. It was also reported that the hydrogen bond between the hydroxyl of Thr182 and the carbonyl of Glu64 was expected to be responsible for the increase in catalytic activity of TEM-32 (21
). Although the Met182Thr substitution at position 182 was identified for TEM-43, as it was in the inhibitor resistant TEM-32 β-lactamase, resistance to the inhibitors was not observed.
Substitution of the threonine at position 182 has recently been identified in the TEM β-lactamases as a global suppressor (9
). This substitution was shown to restore enzymatic activity to TEM variants with multiple mutations and provide additional protein stability. The Thr182 substitution alone may not convey a selective enzymatic advantage to the wild-type TEM-1 enzyme with respect either to the ability to be inhibited or to the ability to hydrolyze poor substrates. However, good substrates may be hydrolyzed somewhat more efficiently (7
). It is possible that the differences between the relative hydrolysis profiles for ceftazidime and aztreonam with TEM-6 and TEM-43 may be related to the Thr182 mutation. Huang and Palzkill (9
) hypothesized that the Thr182 mutation, considered to be a relatively “neutral” mutation, may allow more destabilizing mutations to occur to respond to antibiotic pressure without compromising the ability of the β-lactamase to function. It is quite likely that similar amino acid substitutions that appear to have no noticeable effect on enzymatic function will be identified. Because some of these neutral substitutions may not alter the pI or the phenotypic response to standard antibiotics, they will remain covert until a second mutation occurs. It is expected that these kinds of mutants already exist among the normal distribution of TEM-producing isolates. These may be identified only when additional ESBLs or inhibitor-resistant TEM variants are sequenced.