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Antimicrobial Agents and Chemotherapy, October 2006, p. 3485-3487, Vol. 50, No. 10
0066-4804/06/$08.00+0     doi:10.1128/AAC.00363-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SME-3, a Novel Member of the Serratia marcescens SME Family of Carbapenem-Hydrolyzing ß-Lactamases

Anne Marie Queenan,1* Wenchi Shang,1 Paul Schreckenberger,2 Karen Lolans,3 Karen Bush,1 and John Quinn3

Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Raritan, New Jersey,1 Loyola University Medical Center, Maywood, Illinois,2 CIDRI, John H. Stroger Hospital of Cook County, Chicago, Illinois3

Received 24 March 2006/ Returned for modification 12 May 2006/ Accepted 14 July 2006


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ABSTRACT
 
Imipenem-resistant Serratia marcescens isolates were cultured from a lung transplant patient given multiple antibiotics over several months. The strains expressed SME-3, a ß-lactamase of the rare SME carbapenem-hydrolyzing family. SME-3 differed from SME-1 by a single amino acid substitution of tyrosine for histidine at position 105, but the two ß-lactamases displayed similar hydrolytic profiles.


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TEXT
 
The spread of ß-lactamases among members of the family Enterobacteriaceae is an increasing trend throughout the world. While the vast majority of ß-lactamases cannot hydrolyze carbapenems, several types of carbapenem-hydrolyzing enzymes have been described. The most common are the molecular class B metalloenzymes that are found on mobile elements (14); however, several types of class A, or functional group 2f (2), carbapenemases have also been detected.

The first class A carbapenemase was identified in a Serratia marcescens strain isolated in 1982 (16). SME-1 has a broad hydrolysis spectrum that includes penicillins, cephalosporins, aztreonam, and carbapenems. Since the discovery of SME-1 in London, United Kingdom, SME-1- and SME-2-expressing S. marcescens isolates have been found in the United States in Massachusetts, California, and Illinois (9). The occurrence of SME enzymes has been infrequent, with the most recent clinical isolates of S. marcescens expressing the SME-1 ß-lactamase detected in Chicago, Ill. (5), and Texas (H. S. Sader, L. M. Deshpande, T. R. Fritsche, and R. N. Jones, Abstr. 45th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C2-104, 2005).

In this work, we identified and characterized a novel variant of the SME family that was found in an imipenem-resistant S. marcescens strain from a lung transplant patient in Chicago. The SME-3 ß-lactamase gene differed from the SME-1 gene by a single amino acid substitution of tyrosine for histidine at position 105. This substitution did not affect the overall hydrolytic spectrum; however, changes in the binding affinities of some substrates were observed (A. M. Queenan, P. Schreckenberger, K. Lolans, K. Bush, and J. P. Quinn, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-1664, 2004).

A 61-year-old lung transplant patient required 3 months of hospitalization and received multiple antimicrobial therapies, including trimethoprim-sulfamethoxazole, itraconazole, ceftriaxone, cefepime, and levofloxacin. No carbapenems were administered during this period. Imipenem-resistant S. marcescens was isolated from the sputum and, 2 weeks later, from pleural fluid. Concurrent with the detection of the imipenem-resistant isolates, an imipenem-susceptible strain of S. marcescens was also identified.

The ß-lactam MICs for the imipenem-resistant S. marcescens isolate (isolate OC7554) and the imipenem-susceptible S. marcescens isolate (isolate OC7555) were determined and compared with those for the original SME-1 strain, S. marcescens S6, by CLSI broth microdilution methods (4). The imipenem-resistant strain had meropenem and doripenem MICs of 8 and 4 µg/ml, respectively, and was resistant to cefoxitin but was susceptible to ceftazidime (Table 1). Isoelectric focusing (IEF) was performed with freeze-thaw lysates from the clinical strains (3) by using prepared Ampholine PAGplates (GE Healthcare), with ß-lactamase detection by nitrocefin. The imipenem-resistant strains had an enzyme with a pI of ~9.5 that migrated with the known SME-1 protein (data not shown). Plasmid isolation with a QIAGEN plasmid mini kit yielded no visible bands. The MIC pattern of an imipenem-resistant, ceftazidime-susceptible S. marcescens isolate and the IEF results strongly suggested that this ß-lactamase would be a member of the SME family.


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  		TABLE 1. S. marcescens strains and susceptibilities

Primers specific for SME sequences amplified ~1.2-kb products from whole-cell lysates of both of the imipenem-resistant isolates but not the susceptible isolate (data not shown). The entire blaSME-coding region was amplified with primers IRS5 and IRS6 by using Taq polymerase (Roche Diagnostics, Indianapolis, IN), as described previously (9). PCR products from three reactions per strain were sequenced by ACGT, Inc. (Wheeling, IL). Sequencing revealed identical SME genes, with the predicted amino acid sequence differing from the S. marcescens S6 sequence (GenBank accession number U60295) by a single substitution of tyrosine for histidine at Ambler position 105 (1). The GenBank accession number assigned to SME-3 is AY584237.

The two imipenem-resistant S. marcescens isolates and the susceptible strain isolated from the lung transplant patient, along with previously characterized reference strains (9), were subjected to pulsed-field gel electrophoresis (PFGE). Fifty micromolar thiourea was added to the running buffer to reduce background smearing (11). The degree of relatedness between strains was evaluated according to the guidelines of Tenover et al. (13), in which strains with seven or more band differences are considered unrelated. The imipenem-resistant S. marcescens isolates isolated 2 weeks apart were identical by PFGE and not related (>16 band differences) to the imipenem-susceptible strain from the same patient (Fig. 1). The S. marcescens strains with SME-3 had at least 11 band differences from SME-expressing strains from London and the United States.


Figure 1
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  		FIG. 1. PFGE of SpeI-digested DNA from S. marcescens isolates. Lanes 1 to 4, imipenem-resistant strains with characterized SME ß-lactamases (9); lane 1, S6; lane 2, OC4124; lane 3, OC4126; lane 4, OC4176; lanes 5 and 6, independently isolated imipenem-resistant strains from lung transplant patient; lane 7, imipenem-susceptible isolate from the same patient.

SME-3 was subcloned into the NcoI and SalI sites of pET24d, expressed in Escherichia coli BL21(DE3) (EMD Biosciences, San Diego, CA), and purified by fast-performance liquid chromatography (9). Protein purity, as determined with colloidal blue-stained NuPAGE gels (Invitrogen, Carlsbad, CA), was 98%. Protein quantitation was done with a MicroBCA system (Pierce, Rockford, IL). Kinetic parameters were determined spectrophotometrically, with hydrolysis measured as described previously (12). Initial rates were used to calculate Km and Vmax values from a Hanes plot.

SME-3 hydrolyzed a broad range of substrates, similar to those reported for SME-1. The highest kcat values obtained were for cephaloridine, followed by those for ampicillin, imipenem, and aztreonam (Table 2). Meropenem and doripenem had kcat values 100-fold lower than those of imipenem. Ceftazidime and cefoxitin were poorly hydrolyzed, with kcat values ≤0.3 s–1, while cefotaxime, cefepime, and ceftobiprole were hydrolyzed with kcat values of 1 to 7 s–1. SME-3 was inhibited by clavulanic acid and tazobactam, with Ki values of 0.24 µM and 0.14 µM, respectively. When the kinetic parameters for SME-3 were compared to those for SME-1 (9), a few differences in the Km values were observed. For cephaloridine, the SME-3 Km values were lower (400 µM compared to 770 µM for SME-1), while for aztreonam, the Km value was higher (769 µM for SME-3 compared to 488 µM for SME-1). As a result of changes in binding affinities, the hydrolytic efficiency of SME-3, expressed as kcat/Km, was increased for cephaloridine and decreased for aztreonam.


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  		TABLE 2. Kinetic parameters of the SME-3 ß-lactamase

The amino acid change in SME-3 is a tyrosine substitution for the histidine at amino acid position 105. This histidine is conserved in the other group 2f carbapenemases NMC and IMI and was speculated to be important for carbapenem hydrolysis in early studies (8, 10). However, the class A KPC carbapenemases have a tryptophan at amino acid position 105 (15). When random mutations of SME-1 at position 105 were selected for in vivo imipenem resistance, the aromatic amino acids tryptophan and tyrosine were the most common substitutions (6). While the histidine at amino acid position 105 is clearly not required for enzyme function, its presence at the entrance to the active site appears to play a subtle role in the binding affinities of some substrates.

Carbapenem resistance in S. marcescens due to the SME ß-lactamases continues to be an infrequent, sporadic problem in hospitals, and treatment with a carbapenem is not a prerequisite for their emergence. The source of these strains remains unknown. The SME genes are chromosomal and have not been associated with any mobile elements (7). This information and the fact that these S. marcescens strains remain susceptible to extended-spectrum cephalosporins are the likely reasons that these strains have not, fortunately, become a widespread problem.


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FOOTNOTES
 
* Corresponding author. Mailing address: Antimicrobial Agents Research Team, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 1000 Route 202 South, OMP Research B210, Raritan, NJ 08869. Phone: (908) 704-5515. Fax: (908) 707-3501. E-mail: aqueenan{at}prdus.jnj.com. Back


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Antimicrobial Agents and Chemotherapy, October 2006, p. 3485-3487, Vol. 50, No. 10
0066-4804/06/$08.00+0     doi:10.1128/AAC.00363-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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  • Queenan, A. M., Bush, K. (2007). Carbapenemases: the Versatile {beta}-Lactamases. Clin. Microbiol. Rev. 20: 440-458 [Abstract] [Full Text]  
  • Poirel, L., Wenger, A., Bille, J., Bernabeu, S., Naas, T., Nordmann, P. (2007). SME-2-Producing Serratia marcescens Isolate from Switzerland. Antimicrob. Agents Chemother. 51: 2282-2283 [Full Text]  

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