1 Service de Microbiologie, Hôpital Saint Antoine, Assistance Publique-Hôpitaux de Paris, Paris; 2 Laboratoire de Bactériologie, EA 2392, UFR Saint Antoine, Université Paris 6, Paris; 3 Service de Microbiologie, Hôpital Louis Mourier, Assistance Publique-Hôpitaux de Paris, Colombes; 4 CNRS, Unité FRE 2125, Quimper; 5 Service de Bactériologie, Hôpital Tenon, Assistance Publique-Hôpitaux de Paris, 4 rue de la Chine, 75970 Paris cedex 20, France
Received 4 October 2004; returned 20 October 2004; revised 17 December 2004; accepted 20 December 2004
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Abstract |
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Methods: S. marcescens SMSA was isolated from an intra-abdominal wound of a patient previously treated with ceftazidime. A susceptible strain, SLS73, was used as a control. Susceptibility testing, PCR, DNA sequencing, molecular cloning, site-directed mutagenesis and determination of kinetic parameters were carried out to investigate the mechanism of resistance to ceftazidime.
Results: MICs of ceftazidime were 64 and 0.2 mg/L for SMSA and SLS73, respectively. Sequencing of the ampC gene of SMSA was carried out. When compared with the closest AmpC enzyme, the S. marcescens S3 ß-lactamase, the novel protein showed E57Q, Q129K and S220Y substitutions. The S220Y substitution is located in the omega loop. Introduced by mutagenesis in the ampC gene of SLS73, this substitution conferred the same level of resistance to ceftazidime. The catalytic efficiency (kcat/Km) of the mutated enzyme toward ceftazidime was increased by about 100-fold.
Conclusions: We present another example of in vivo selection of broad-spectrum resistance by amino acid substitution in the omega loop of chromosomal AmpC ß-lactamase in S. marcescens.
Keywords: extended-spectrum activities , mutagenesis , amino acid substitutions
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Introduction |
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The aim of this study was to characterize the mechanism of ceftazidime resistance in a clinical isolate of S. marcescens.
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Materials and methods |
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ß-Lactamase induction was studied by the disc method described by Mahlen et al.7 The production of extended-spectrum ß-lactamase was tested by the double-disc synergy test.
MICs were determined by the agar dilution method with MuellerHinton agar (Bio-Rad, Marnes-la-Coquette, France). MIC results (Table 1) were interpreted according to the guidelines of the CA-SFM, the French Society for Microbiology (www.sfm.asso.fr).
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The genes coding for the following transferable ß-lactamases (TEM, SHV, CTX-M type enzymes) were detected by PCR amplification from total DNA as previously described.9 The clustal W program (http://www.infobiogen.fr) was used to align multiple protein sequences.
By site-directed mutagenesis,10
the substitution S220Y was introduced in AmpC of the wild-type isolate of S. marcescens, SLS73, in order to analyse its effect. The ampC gene was amplified using oligonucleotide primers SM Upper Sac (5'-CGAGCTCGGCAGCCGTAAAGGAATGACA-3') (position 14821501 of AJ271368) and SM Lower Hind (5'-CCAAGCTTGGGCAGCATCGGTGGTAGGG-3') (position 27652747 of AJ271368), which contained SacI and HindIII restriction sites at their 5' ends, respectively (underlined). The PCR product was cloned into the pBK-CMV cloning vector (Stratagene, La Jolla, CA, USA). The recombinant plasmid obtained, termed pBK-SerS, was used to transform competent Escherichia coli JM101 [supE thi (lac-proAB) F(traD36 proAB+ lacIq lacZ-
M15)]. A site-specific mutagenesis using a mutagenic primer in PCR was used as described previously.10
The primers required for PCR mutagenesis consisted of a forward mutagenic primer Ser Sca Upper and the previously described primers, SM Upper Sac and SM Lower Hind. Ser Sca Upper (5'-GAGTACTACGGCATCAAGTCCA-3') (position 22882309 of AJ271368) contained a double-base mismatch and introduced the S220Y substitution and the ScaI restriction site. The recombinant plasmid obtained was named pBK-SerR. It was used to transform competent E. coli JM101 and was digested with ScaI to control the mutagenesis. Moreover, the insert was sequenced to verify the created mutation using SM Upper Sac and SM Lower Hind primers.
The purification of ß-lactamases from E. coli pBK-SerR and E. coli pBK-SerS and studies of their kinetic constants for substrates were determined (Table 2) by computerized microacidimetric assay as described by Labia et al.11
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Results and discussion |
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SLS73 had a typical susceptibility pattern of wild-type S. marcescens.1 For SMSA, the MICs of ticarcillin and ceftazidime were at least 128 times higher than those for SLS73. The MICs of cefotaxime, cefepime and cefpirome were eight times higher for SMSA than those for SLS73. TEM, SHV or CTX-M type extended-spectrum ß-lactamase genes could not be detected in the SMSA strain.
The susceptibility pattern of SMSA was peculiar: a higher resistance level to ceftazidime than to cefotaxime (four-fold) and the inducibility of ß-lactamase suggested modifications of the structure and activity of its enzyme.46 Moreover, MICs of piperacillin (Table 1) were in accordance with a low level of AmpC expression in the two S. marcescens strains.1
The sequence of the PCR product of the ampC gene of SMSA (1062 bp) (Figure 1) showed closest similarity to S. marcescens S3 ß-lactamase.4 The deduced sequence showed three amino acid differences. Two of these differences represent amino acids not previously seen at these positions: substitution S220Y, located in the omega loop, and substitution Q129K, located in a peripheral position. The omega loop is a region known to be critical for hydrolysis of oxyimino-cephalosporins in class A and class C ß-lactamases.12 Matsumura et al.4 demonstrated that an E219K amino acid substitution in AmpC of S. marcescens results in an enzyme with extended-substrate specificity to oxyimino-cephalosporins.
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The E. coli pBK-SerR enzyme exhibited an increased catalytic efficiency (kcat/Km) toward ceftazidime (>100-fold), and toward cefepime and cefpirome (>30-fold). This was mostly due to an increase in the kcat value despite greatly reduced affinities, in comparison with that of E. coli pBK-SerS. The kinetic parameters observed here were in agreement with those of SRT-1,3,4 for which an increased relative Vmax toward ceftazidime has been observed ( > 20-fold). The kcat value increased by about 400-fold for cefotaxime in the E. coli pBK-SerR enzyme, but this was nearly completely counterbalanced by a strong increase in Km; the catalytic efficiency remained about the same in the E. coli pBK-SerR enzyme.
Comparing the crystal structures of three AmpC ß-lactamases, of C. freundii, E. cloacae and E. coli, similar results were obtained concerning the terminal nitrogen (NZ) of Lys-67. This nitrogen is located at hydrogen bond-distance with the backbone carbonyl of Ala-220, the side-chain amide of Asn-152 and the hydroxyl of Ser-64. NZ is also in close proximity to the phenolic hydroxyl of the highly conserved Tyr-150. A model was built for the 3-D structures of the two S. marcescens ß-lactamases using Swiss Model.1315 In the model of the E. coli pBK-SerR enzyme, the position of NZ of Lys-67 was altered by about 1 Å, enlarging the enzymic cavity (Figure 2). The same conclusions are also possible for SRT-1 with substitution at position 2193,4 and 520R with substitution at position 705 which are both close to position 67.
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Only one clinical isolate of S. marcescens resistant to ceftazidime, as well as to cefepime and cefpirome, with mutations in AmpC was reported.6 This strain (HD) had a 4-amino-acid deletion located in the H-10 helix of the ß-lactamase. In our strain SMSA, which produces a naturally inducible AmpC, if the S220Y substitution was combined with overproduced production of ß-lactamase, we suggest that none of the ß-lactam antibiotics except imipenem would be active.
The EMBL accession number for the nucleotide sequence reported in this paper is AJ698133.
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Acknowledgements |
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