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JAC Advance Access originally published online on February 18, 2005
Journal of Antimicrobial Chemotherapy 2005 55(4):496-499; doi:10.1093/jac/dki025
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Published by Oxford University Press 2005

Resistance to ceftazidime is associated with a S220Y substitution in the omega loop of the AmpC ß-lactamase of a Serratia marcescens clinical isolate

Nadia Hidri1,2, Guilène Barnaud3, Dominique Decré1,2, Claude Cerceau4, Valérie Lalande1, Jean Claude Petit1,2, Roger Labia4 and Guillaume Arlet2,5,*

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

* Corresponding author. Tel: +33-1-56-01-70-18; Fax: +33-1-56-01-61-08; Email: guillaume.arlet{at}tnn.ap-hop-paris.fr

Received 4 October 2004; returned 20 October 2004; revised 17 December 2004; accepted 20 December 2004


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: The aim of this study was to characterize the ampC ß-lactamase gene of a clinical isolate of Serratia marcescens resistant to ceftazidime.

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


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 References
 
Serratia marcescens produces an inducible chromosomally-encoded class C ß-lactamase.1,2 In case of overproduced AmpC cephalosporinase, the level of resistance to oxyimino-cephalosporins is high except for ceftazidime, cefepime and cefpirome.1,2 Until now, only a few strains of S. marcescens have been reported with an altered chromosomal AmpC that hydrolyses oxyimino-cephalosporins efficiently. Substitutions in AmpC were located in the omega loop in the clinical strain SRT-1 (from Glu to Lys at position 219),3,4 and near the serine active site in the laboratory mutant 520R (from Thr to Ile at position 70).5 Recently, a 12 nt deletion leading to a 4-amino-acid deletion located in the H-10 helix of AmpC was described in the clinical strain of S. marcescens HD resistant to ceftazidime, cefepime and cefpirome.6

The aim of this study was to characterize the mechanism of ceftazidime resistance in a clinical isolate of S. marcescens.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 References
 
S. marcescens SMSA was isolated at the Saint-Antoine Hospital (Paris, France) in 2000 from an intra-abdominal wound of a patient previously treated with ceftazidime. S. marcescens SLS73 was isolated in 1998 at the Saint-Louis Hospital (Paris, France). SLS73 produces an inducible ß-lactamase. Its ampR and ampC genes were cloned and sequenced previously (GenBank accession number AJ271368).

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 Mueller–Hinton 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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  		Table 1.. MICs (mg/L) of several ß-lactams for clinical strains of E. coli JM101, S. marcescens SLS73 and SMSA, and E. coli JM101 harbouring pBK-SerS or pBK-SerR

 
The ampC gene of SMSA was amplified and sequenced8 using oligonucleotide primers based on consensus sequences from the ampC genes of S. marcescens, named ‘Smar upper’ (5'-GCAGCCGTAAAGGAATGACA-3') (position 1482–1501 of AJ271368) and ‘Smar lower’ (5'-TGSAYGATGYGGTAAGCCGC-3') (S = G or C; Y = C or T) (position 2703–2684 of AJ271368).

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 1482–1501 of AJ271368) and ‘SM Lower Hind’ (5'-CCAAGCTTGGGCAGCATCGGTGGTAGGG-3') (position 2765–2747 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 {Delta}(lac-proAB) F’(traD36 proAB+ lacIq lacZ-{Delta}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 2288–2309 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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  		Table 2.. Kinetic parameters of ß-lactamases produced by E. coli pBK-SerR and E. coli pBK-SerS for various substrates

 

   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
References
 
Positive induction was observed for both strains with a characteristic flattening cefotaxime inhibition zone in front of the disc of cefoxitin, suggesting that both strains produced an inducible ß-lactamase (data not shown).

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.4–6 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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  		Figure 1.. Multiple amino acid sequence alignment of AmpC ß-lactamases from S. marcescens S3, 520R, SRT-1, HD, SMSA and SLS73 (AJ271368) strains. Dashes indicate identical amino acids. Asterisks indicate the 4-amino-acid deletion in AmpC of S. marcescens strain HD. The amino acid substitutions involved in the extension of the spectrum of the ß-lactamase are in bold. The omega loop is in bold and underlined. Conserved residues of class C ß-lactamases are underlined. Amino acids are numbered in accordance with the conventional numbering of E. cloacae P99 with the active-site serine at position 64 in bold. SMSA sequence after ‘/’ was not determined.

 
The MICs of ß-lactam antibiotics for the two transformants, E. coli pBK-SerS and E. coli pBK-SerR, are listed in Table 1. Ticarcillin and ceftazidime MICs for the transformant E. coli pBK-SerR were much higher than those for the strain E. coli pBK-SerS. The MICs of cefepime and cefpirome were increased by at least a factor of eight. Surprisingly, the MICs of cefotaxime were not significantly modified in the variant strain. These results were fully compatible with those obtained for the clinical isolates SMSA and SLS73.

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’.13–15 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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  		Figure 2.. Effect of Tyr-220 on the position of NZ of Lys-67, in native enzyme (right-hand NZ) and mutant enzyme (left-hand NZ). The arrow shows the alteration of this nitrogen position.

 
Another hot spot for mutations in AmpC of Enterobacteriaceae conferring resistance not only to ceftazidime but also to cefepime and cefpirome is the H-10 {alpha}-helix.6,16,17 The 4-amino-acid deletion located in the H-10 {alpha}-helix of the ß-lactamase of HD results in a marked decrease in the Km values.6 Similar kinetic parameter modifications were observed in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes with enzymes modified in the H-10 {alpha}-helix.16,17

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.


   Acknowledgements
 
This work was supported by a grant from UFR Saint-Antoine, Université Paris VI and by a grant from the European Community (contract LSHM-CT 2003–503335).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Livermore, D. M. (1995). ß-Lactamases in laboratory and clinical resistance. Clinical Microbiology Reviews 8, 557–84.[Abstract]

2 . Sanders, C. C. & Sanders, W. E. (1986). Type I ß-lactamases of gram-negative bacteria: interactions with ß-lactam antibiotics. Journal of Infectious Diseases 154, 792–800.[Web of Science][Medline]

3 . Matsumura, N. & Mitsuhashi, S. (1995). A ß-lactamase from Serratia marcescens hydrolysing the 2-carboxypenam T-5575. Antimicrobial Agents and Chemotherapy 39, 2132–4.[Abstract]

4 . Matsumura, N., Minami, S. & Mitsuhashi, S. (1998). Sequences of homologous ß-lactamases from clinical isolates of Serratia marcescens with different substrate specificities. Antimicrobial Agents and Chemotherapy 42, 176–9.[Abstract/Free Full Text]

5 . Raimondi, A., Sisto, F. & Nikaido, H. (2001). Mutation in Serratia marcescens AmpC ß-lactamase producing high-level resistance to ceftazidime and cefpirome. Antimicrobial Agents and Chemotherapy 45, 2331–9.[Abstract/Free Full Text]

6 . Mammeri, H., Poirel, L., Bemer, P. et al. (2004). Resistance to cefepime and cefpirome due to a 4-amino-acid deletion in the chromosome-encoded AmpC ß-lactamase of a Serratia marcescens clinical isolate. Antimicrobial Agents and Chemotherapy 48, 716–20.[Abstract/Free Full Text]

7 . Mahlen, S. D., Morrow, S. S., Abdalhamid, B. et al. (2003). Analyses of ampC gene expression in Serratia marcescens reveal new regulatory properties. Journal of Antimicrobial Chemotherapy 51, 791–802.[Abstract/Free Full Text]

8 . Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, USA 74, 5463–7.[Abstract/Free Full Text]

9 . Eckert, C., Gautier, V., Saladin-Allard, M. et al. (2004). Dissemination of CTX-M-type ß-lactamases among clinical isolates of Enterobacteriaceae in Paris, France. Antimicrobial Agents and Chemotherapy 48, 1249–55.[Abstract/Free Full Text]

10 . Nelson, R. M. & Long, G. L. (1989). A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Analytical Biochemistry 180, 147–51.[CrossRef][Web of Science][Medline]

11 . Labia, R., Andrillon, J. & Le Goffic, F. (1973). Computerized microacidimetric determination of ß-lactamase Michaelis–Menten constants. FEBS Letters 33, 42–4.[CrossRef][Web of Science][Medline]

12 . Knox, J. R., Moews, P. C. & Frère, J. M. (1996). Molecular evolution of bacterial ß-lactam resistance. Chemistry and Biology 3, 937–47.[CrossRef][Web of Science][Medline]

13 . Guex, N. & Peitsch, M. C. (1997). SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–23.[CrossRef][Web of Science][Medline]

14 . Peitsch, M. C. (1995). Protein modeling by e-mail. Bio/technology 13, 658–60.[CrossRef]

15 . Peitsch, M. C. (1996). ProMod and Swiss-Model: internet-based tools for automated comparative protein modelling. Biochemical Society Transactions 24, 274–9.[Web of Science][Medline]

16 . Barnaud, G., Benzerara, Y., Gravisse, J. et al. (2004). Selection during cefepime treatment of a new cephalosporinase variant with extended-spectrum resistance to cefepime in an Enterobacter aerogenes clinical isolate. Antimicrobial Agents and Chemotherapy 48, 1040–2.[Abstract/Free Full Text]

17 . Barnaud, G., Labia, R., Raskine, L. et al. (2001). Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate. FEMS Microbiology Letters 195, 185–90.[CrossRef][Web of Science][Medline]


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