Construction of E. coli ΔampC.
An ampC gene isogenic mutant of E. coli DH5α was constructed to eliminate phenotypic variations which may result from AmpC production during in vitro evolution. The ampC gene was replaced by kanamycin resistance cassette in E. coli ΔampC::Kanr. The cassette was then eliminated by using the helper plasmid pCP20. Each step of the construction was checked by PCR (Fig. ). The resulting strain was designated E. coli ΔampC.
Collection of mutants.
After the first round of mutagenesis, 106 clones were able to grow in the presence of 4 μg/ml ceftazidime. Phenotype 1, which exhibited similar inhibition diameters for ceftazidime and cefotaxime, possessed 81 clones. Phenotype 2, which exhibited a smaller inhibition diameter for ceftazidime than for cefotaxime, possessed 25 clones. The clones belonging to phenotype 2 presented a great diversity of inhibition diameters for the other β-lactams, unlike the clones belonging to phenotype 1, which formed a homogenous group. A second round of mutagenesis was performed from six candidate mutants (one from phenotype 1 and five from phenotype 2), and 123 clones were obtained on agar containing 8 μg/ml ceftazidime; 22 clones belonged to phenotype 1, and 101 clones belonged to phenotype 2. The last round of mutagenesis was performed from four candidate mutants (two from phenotype 1 and two from phenotype 2); 60 clones had resistance phenotype 1, and 39 others had resistance phenotype 2.
Deduced amino acid sequences revealed 38 different substitutions and one to six substitutions per enzyme (Table ). One to three substitutions per CTX-M mutant were acquired after each cycle of mutagenesis.
Clones, their phenotypes, and the corresponding enzymes obtained from amino acid substitution in the CTX-M-9 enzyme during three cycles of mutagenesis
All CTX-M-9 mutants associated with resistance phenotype 1 harbored the Asp240Gly substitution, which is located in the
β3 strand (Fig. ). The CTX-M mutants harbored the Asp240Gly substitution alone (M-A1) or associated with substitution Val29Ala (M-A2) or Ile173Thr (M-A3) after the first round of mutagenesis. After the second round, the Asp240Gly substitution was associated with one (Ala219Asp in M-A1B2) or two (Gln87Leu and Arg276His in M-A1B1) additional substitutions. After the last round, three other substitutions appeared in mutants M-A1B2C1 (Gly289Trp, His197Arg, and Leu169Met) and M-A1B1C1 (His112Tyr, Thr230Ile, and Ala231Val).
FIG. 2. Crystallographic structure of the CTX-M-9 β-lactamase. The omega loop is in dark blue, the β3 strand is in red, the H11 α-helix is in brown, the N-terminal extremity of the H2 α-helix is in light blue, and the loop between (more ...)
The mutants involved in resistance phenotype 2 harbored a larger diversity of substitutions than those implicated in resistance phenotype 1. Twenty-four additional substitutions were observed (Table ). Substitutions at positions 167, 169, 179, and/or 164, which are located in the omega loop, appeared from the first round of mutagenesis (Fig. ).
The Pro167Ser substitution appeared alone in mutant M-A6 or in association with substitution Asn106Ser in mutant M-A5. Their derivatives M-A5B1 and M-A6B1 harbored the additional substitutions Thr159Ser and Ala109Thr, respectively, and were at the origins of 8 out of 10 mutants obtained during the third round of mutagenesis (M-A5B1C1, M-A5B1C2, and M-A6B1C1 to M-A6B1C6). These mutants harbored a complex combination of substitutions, which comprised substitutions Ala77Val, Gly146Arg, Glu166Val, Thr171Ser, Ala172Val, Glu201Asp, Thr209Ser, Thr227Ala, Ala231Val, Gln254Pro, and Pro268Ala (Table ).
The Leu169Gln substitution was observed after the first round of mutagenesis alone in mutant M-A4. After the second round of mutagenesis, this substitution was associated with substitution Asp240Gly in its derivative M-A4B1. During the third round of mutagenesis, a second type of substitution, Leu169Met, was observed in mutant M-A1B2C1. This enzyme, which derived from mutant M-A1B2, additionally harbored substitutions Asp240Gly, His197Arg, Ala219Asp, and Gly289Trp.
The Asp179Gly substitution was associated with substitutions Asn106Ser and Thr86Ala in mutant M-A7. During the second round of mutagenesis, the M-A7-encoding gene was at
the origins of the three mutants M-A7B1, M-A7B2, and M-A7B3, which harbored the additional substitutions Thr165Ile, Ala231Val, and/or Arg276His.
The Arg164His substitution was obtained in mutant M-A8 after the first round of mutagenesis in association with substitutions Ala231Val and Arg276Ser. This enzyme was at the origin of four additional mutants (M-A8B1 to M-A8B4) after a second round of mutagenesis. These mutants harbored the substitution at position 164 in combination with other substitutions of the omega loop: Pro167Ser, Pro167His, Asp179Asn, and Asp179Tyr.
MICs of β-lactams.
MICs of β-lactams were determined for the 30 clones collected for blaCTX-M sequencing (Table ). Overall, ceftazidime MICs increased from 1 μg/ml for CTX-M-9-producing E. coli ΔampC to 128 μg/ml for the mutant-producing clones (A6B1C1). MICs of ceftazidime ranged from 8 to 16, 8 to 64, and 16 to 128 μg/ml after the first, second, and third rounds of mutagenesis, respectively. Among the 30 CTX-M-producing clones, 16 had a high level of resistance to ceftazidime (MIC of ≥32 μg/ml). Of these, eight were obtained after the second round of mutagenesis, and the other eight were obtained after the third round. Clone A6B1C1, which had the highest ceftazidime MIC (128 μg/ml), was obtained after three cycles of mutagenesis.
β-Lactam MICs for CTX-M-producing mutants of E. coli ΔampC
In contrast to CTX-M-9-producing E. coli ΔampC, which exhibited higher MICs of cefotaxime than of ceftazidime (16 versus 1 μg/ml), mutant-producing E. coli ΔampC clones belonging to resistance phenotype 1 (A1 to A3, A1B1, A1B2, and A1B1C1) exhibited similar MICs of cefotaxime and ceftazidime (8 to 64 versus 8 to 64 μg/ml). MICs of ceftazidime were 8- to 64-fold higher for mutant-producing E. coli ΔampC than for the CTX-M-9-producing E. coli ΔampC. In comparison with CTX-M-9-producing E. coli ΔampC, mutant-producing E. coli ΔampC clones of resistance phenotype 1 exhibited no major modification of MICs of penicillins (64 to >2,048 versus 256 to >2,048 μg/ml), cephalothin (256 to 1,024 versus 1,024 μg/ml), cefuroxime (256 to 2,048 versus 1,024 μg/ml), and cefotaxime (8 to 64 versus 16 μg/ml).
The clones belonging to resistance phenotype 2 exhibited higher MICs of ceftazidime than of cefotaxime, unlike CTX-M-9-producing E. coli ΔampC. The MICs of cefotaxime for mutant-producing E. coli ΔampC defined two groups. Among the clones with cefotaxime MICs of 8 to 16 μg/ml (similar to those of CTX-M-9-producing E. coli DH5α-ΔampC), the MICs of amoxicillin were identical to those of CTX-M-9-producing E. coli ΔampC for clones A8B3, A5B1C2, A6B1C1, A6B1C4, and A6B1C6 and were lower (<1,024 μg/ml) for clones A4B1, A1B2C1, A7B1, and A7B2. The others clones exhibited cefotaxime MICs (0.25 to 2 μg/ml) lower than those for CTX-M-9-producing E. coli ΔampC. This decrease in cefotaxime MICs was not associated with major modifications of MICs of amoxicillin for clones A8, A5B1, A6B1, A8B1, A5B1C1, and A6B1C3 (1,024 to >2,048 μg/ml), but the MICs of amoxicillin were lower for clones A4, A5, A6, A7, A7B3, A8B2, A8B4, A6B1C2, and A6B1C5 (8 to 256 μg/ml).
For the 30 clones, the MICs of inhibitor-penicillin combinations, aztreonam, cefepime, cefpirome, and imipenem did not increase more than fourfold in comparison with those for CTX-M-9-producing E. coli ΔampC.
The mutants designated M-A1B1C1, M-A6B1C1, and M-A8B3, were overexpressed in E. coli BL21(DE3) from pET9a-derived plasmids and were purified by liquid chromatography. One to 3 milligrams of β-lactamase per liter of culture medium was obtained, and the purity was estimated to be >98%.
The kinetic parameters for these strains are shown in Table . M-A1B1C1 exhibited typical enzymatic features of CTX-M mutants. Lower Km values were obtained for penicillins (Km, 14 to 35 μM) than for cephalosporins (140 to 450 μM). Cephalothin was the best substrate (kcat, 3,800 s−1), and a higher kcat was observed for cefotaxime (550 s−1) than for ceftazidime (35 s−1). However, the kcat value against ceftazidime was 17-fold higher for M-A1B1C1 than for CTX-M-9. In addition, the ceftazidime Km value of M-A1B1C1 was lower than that of CTX-M-9 (450 μM versus 600 μM). Conversely, the kinetic constants of M-A6B1C1 and M-A8B3 were different from those of typical CTX-M enzymes, such as CTX-M-9. kcat values against penicillins and cephalothin were 7- to 25-fold lower for the two mutants than for CTX-M-9. kcat values of M-A6B1C1 were also significantly lower than those of CTX-M-9 for oxyimino cephalosporins (cefotaxime, 17 versus 450 s−1; ceftazidime, 0.1 versus 2 s−1). kcat values of M-A8B3 against these substrates were still about 10-fold lower than those of M-A6B1C1. Although the Km values for cephalothin and cefotaxime were similar for M-A6B1C1 (250 and 200 μM) and CTX-M-9 (150 and 120 μM), the Km value against ceftazidime was considerably lower for M-A6B1C1 than for CTX-M-9 (8 versus 600 μM). Km values of M-A8B3 were impressively low for all β-lactams (2 to 13 μM), in particular for oxyimino cephalosporins (2 to 4 μM).
Kinetic parameters of CTX-M-9 and derivative mutants M-A1B1C1, M-A6B1C1, and M-A8B3