Abstract
We applied in vitro evolution to an Escherichia coli strain containing blaCTX-M-2 and obtained 10 independent mutant blaCTX-M-2 alleles that confer elevated resistance to ceftazidime (MIC ≥ 32 μg/ml) but lost the ability to confer resistance to cefepime. All alleles had a Pro-to-Ser substitution at position 167.
The CTX-M β-lactamases are plasmid-mediated, Ambler class A extended-spectrum β-lactamases that confer resistance to penicillins and most oxyimino-cephalosporins with the usual exception of ceftazidime (12, 14). Unique to the β-lactamases in the CTX-M family is their ability to provide a high level of resistance to cefepime, with MICs often exceeding 8 μg/ml (15). In comparison, many TEM β-lactamases can confer high levels of resistance to ceftazidime but do not efficiently hydrolyze cefepime (6). Four CTX-M enzymes (CTX-M-15, CTX-M-16, CTX-M-27, and CTX-M-19) that confer increased levels of resistance to ceftazidime have been identified (5). In some cases, this increase in ceftazidime resistance has appeared in association with a reduction in the level of cefepime resistance conferred by these enzymes (5).
The in vitro evolution method developed by Barlow and Hall has been used to determine the evolutionary potential of the β-lactamase genes blaTEM-1 and blaCMY-2 (2, 4). Barlow and Hall showed that the blaTEM-1 and blaCMY-2 determinants could give rise to alleles that conferred increased resistance to cefepime (2, 3). However, blaTEM and ampC alleles conferring resistance to cefepime have not yet been detected in clinical isolates, whereas blaCTX-Ms have been identified in many cefepime-resistant isolates.
The gene blaCTX-M-2 is a particularly important blaCTX-M allele because it confers resistance to cefepime, is prevalent in diverse geographic regions, such as Argentina (13) and Japan (9), and is found in many species of the Enterobacteriaceae (13). To our knowledge, no descendants of blaCTX-M-2 are resistant to ceftazidime. Thus, we wanted to determine if this phenotype was within the evolutionary potential of blaCTX-M-2.
Ten independent libraries of mutant blaCTX-M-2 alleles were generated in Escherichia coli DH5αE and selected for ceftazidime resistance by using a previously described in vitro evolution method (4). Briefly, blaCTX-M-2 was mutagenized by using Mutazyme DNA polymerase (Stratagene, La Jolla, Calif.). Mutagenized genes were digested with enzymes BspHI and SacI, cloned into the BspHI and SacI sites of pACSE3 (4), and transformed into E. coli DH5αE with selection for tetracycline resistance (15 μg/ml). A previously described freeze-thaw procedure was used to prepare cell lysates for isoelectric focusing (IEF) (7). IEF was performed by the method of Matthew and Harris (8). The IEF gels were stained with a 0.05% (0.96 mM) solution of nitrocefin (BD Biosciences, San Jose, Calif.) to visualize the β-lactamases. The isoelectric points of the evolved CTX-M-2 alleles were estimated by comparison with TEM-1 (pI 5.4), SHV-5 (pI 8.2), TEM-3 (pI 6.3), and MIR-1 (pI 8.4) β-lactamases. MICs were determined by broth microdilution according to NCCLS guidelines (10). Quality control organisms included E. coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853.
Ten mutant libraries were subcultured independently in L-Broth (Becton Dickinson Microbiology Systems, Sparks, Md.) containing twofold serial dilutions of ceftazidime (8 to 32 μg/ml) and incubated overnight at 37°C. Only one cycle of mutagenesis and selection was required to obtain cells that were able to grow in ceftazidime at a concentration of 32 μg/ml. The cultures that grew at 32 μg/ml were selected and used as inocula for cultures containing cefepime (4 to 32 μg/ml); however, 4 μg/ml was the highest concentration of cefepime at which growth occurred. After overnight incubation in cefepime (4 μg/ml), the cultures were passaged two more times in L-Broth containing ceftazidime (32 to 512 μg/ml). After selection, plasmid DNA was prepared from each library by using the highest concentration of ceftazidime that still supported growth. The plasmid DNA was transformed into susceptible E. coli DH5αE cells to eliminate any host selection that may have occurred during the selection with ceftazidime (3). A single colony from each transformation was used for further phenotypic analysis and DNA sequence analysis.
The MICs of several antimicrobial agents tested against individual clones from the 10 independent populations of mutant blaCTX-M-2 alleles are shown in Table 1. All of the mutant blaCTX-M-2 alleles mediated ceftazidime resistance, with MICs ranging from 32 to 128 μg/ml compared to a ceftazidime MIC of 4 μg/ml for the parent blaCTX-M-2 allele. The cefepime MICs mediated by the mutant blaCTX-M-2 alleles were 4 to 5 doubling dilutions lower than those of the parent allele. Based on these data, it appears that there is a trade-off between cefepime resistance and ceftazidime resistance for blaCTX-M-2, which may explain why no descendants of blaCTX-M-2 capable of conferring ceftazidime resistance have been detected in clinical settings.
TABLE 1.
MICs for clones expressing CTX-M-2 and evolved allelesa
| β-Lactam | MICs for CTX-M-2 | MICs for clone no. indicated
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | ||
| AMP | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 |
| AMC | 16 | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
| PIP | >128 | >128 | >128 | >128 | >128 | >128 | >128 | >128 | >128 | >128 | >128 |
| TZP | ≤1 | ≤1 | ≤1 | ≤1 | ≤1 | ≤1 | ≤1 | ≤1 | ≤1 | ≤1 | ≤1 |
| TIM | 128 | 64 | 64 | 64 | 16 | 16 | 32 | 32 | 16 | 32 | 32 |
| ATM | 32 | 8 | 8 | 8 | 2 | 8 | 16 | 4 | 8 | 16 | 4 |
| ATM + CLA | 0.12 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.12 | 0.06 | 0.06 | 0.06 | 0.06 |
| CFZ | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 |
| CTT | ≤2 | ≤2 | ≤2 | ≤2 | ≤2 | ≤2 | ≤2 | ≤2 | ≤2 | ≤2 | ≤2 |
| FOX | 4 | 4 | 4 | 8 | 4 | 4 | 4 | 4 | 4 | 8 | 4 |
| CTX | >64 | 16 | 16 | 16 | 8 | 8 | 16 | 16 | 16 | 32 | 16 |
| CTX + CLA | 0.12 | 0.25 | 0.12 | 0.25 | 0.06 | 0.25 | 0.25 | 0.12 | 0.25 | 0.12 | 0.12 |
| CPD | >128 | >128 | >128 | >128 | >128 | >128 | >128 | >128 | >128 | >128 | >128 |
| CPD + CLA | 1 | 4 | 4 | 8 | 1 | 4 | 8 | 1 | 2 | 1 | 1 |
| CAZ | 4 | 128 | 128 | 128 | 32 | 64 | 128 | 64 | 128 | 128 | 128 |
| CAZ + CLA | 0.5 | 2 | 2 | 1 | 0.5 | 4 | 4 | 1 | 4 | 2 | 1 |
| ZOX | 4 | 1 | 1 | 2 | 0.5 | 1 | 4 | 0.5 | 0.5 | 1 | 1 |
| CRO | >64 | 64 | 64 | 32 | 16 | 16 | 32 | 32 | 32 | 64 | 32 |
| CRO + CLA | 0.12 | 0.25 | 0.25 | 0.12 | ≤0.06 | 0.25 | 0.5 | 0.12 | 0.25 | 0.25 | 0.12 |
| FEP | >32 | 2 | 2 | 2 | 1 | 1 | 2 | 1 | 1 | 1 | 1 |
| FEP + CLA | 0.06 | 0.12 | 0.06 | 0.06 | 0.06 | ≤0.03 | 0.12 | ≤0.03 | ≤0.03 | ≤0.03 | 0.5 |
| IPM | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.25 | 0.5 | 0.5 | 0.25 |
| MEM | ≤0.25 | ≤0.25 | ≤0.25 | ≤0.25 | ≤0.25 | ≤0.25 | ≤0.25 | ≤0.25 | ≤0.25 | ≤0.25 | ≤0.25 |
AMP, ampicillin; AMC, amoxicillin-clavulanic acid; PIP, piperacillin; TZP, piperacillin-tazobactam; TIM, ticarcillin-clavulanic acid; ATM, aztreonam; CLA, clavulanic acid; CFZ, cefazolin; CTT, cefotetan; FOX, cefoxitin; CTX, cefotaxime; CPD, cefpodoxime; CAZ, ceftazidime; ZOX, ceftizoxime; CRO, ceftriaxone; FEP, cefepime; IPM, imipenem; MEM, meropenem. MICs are expressed in micrograms per milliliter.
The DNA sequences of the mutant blaCTX-M-2 alleles are shown in Table 2. An IEF analysis of CTX-M-2 demonstrated two bands, with pIs of 7.95 and 8.0, while one of the mutant β-lactamases with a single mutation (Clone 3) showed two bands with pIs of 7.90 and 7.95 (data not shown). All 10 blaCTX-M-2 alleles have a Pro-to-Ser mutation at position 167 by the numbering system of Ambler et al. (1), which indicates that this mutation is important for resistance to ceftazidime. This mutation occurs naturally in blaCTX-M-19, a member of the blaCTX-M-9 group (12) that hydrolyzes ceftazidime (MIC = 128 μg/ml) but has decreased ability to hydrolyze cefepime (MIC = 4 μg/ml) with respect to its ancestor blaCTX-M-18 (MIC = 16 μg/ml).
TABLE 2.
Mutations recovered from the evolved CTX-M-2 alleles
| DNA site | Mutation | Amino acid substitution for clone no. indicatedb
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | ||
| 23 | G→A | R4H | |||||||||
| 25 | T→C | S5P | |||||||||
| 36 | G→A | S | |||||||||
| 38 | T→C | V9A | |||||||||
| 59 | C→T | S | |||||||||
| 67 | A→T | S19C | |||||||||
| 96 | C→T | S | |||||||||
| 208 | G→C | A67P | |||||||||
| 246 | G→A | S | |||||||||
| 267 | C→T | S | |||||||||
| 301 | C→A | S | |||||||||
| 369 | T→G | S | |||||||||
| 376 | G→A | G123S | |||||||||
| 421 | C→T | S | |||||||||
| 453 | G→T | S | |||||||||
| 503 | C→T | T165I | |||||||||
| 508 | C→T | P167S | P167S | P167S | P167S | P167S | P167S | P167S | P167S | P167S | P167S |
| 527 | T→C | I173T | |||||||||
| 604 | A→C | K197N | |||||||||
| 606 | G→A | S | |||||||||
| 769 | C→G | N254K | |||||||||
The position in the coding sequence is given.
S, silent mutation.
As shown by our data and those of others (11, 12), the identification of a β-lactamase as a CTX-M type cannot be made solely on the susceptibility pattern of cefotaxime and ceftazidime, since ceftazidime resistance has developed as a result of mutations in several blaCTX-M determinants, including blaCTX-M-15, blaCTX-M-16, and blaCTX-M-27 (5, 11). We anticipate the continued spread of these β-lactamase determinants among the Enterobacteriaceae.
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