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Antimicrobial Agents and Chemotherapy, October 2008, p. 3792-3794, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00387-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Molecular and Biochemical Characterization of SHV-56, a Novel Inhibitor-Resistant β-Lactamase from Klebsiella pneumoniae{triangledown}

Véronique Dubois,1* Laurent Poirel,2 François Demarthe,1 Corinne Arpin,1 Laure Coulange,1 Luciene A. R. Minarini,2 Marie-Christine Bezian,3 Patrice Nordmann,2 and Claudine Quentin1

UMR 5234, CNRS, Université de Bordeaux 2, Bordeaux,1 Service de Bactériologie-Virologie, INSERM U914: Emerging Resistance to Antibiotics, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine et Université Paris-Sud, K.-Bicêtre,2 Laboratoire de Bactériologie, Hôpital Saint-André, Bordeaux, France3

Received 21 March 2008/ Returned for modification 22 May 2008/ Accepted 23 July 2008


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ABSTRACT
 
A clinical strain of Klebsiella pneumoniae was found to possess the chromosomal gene blaSHV-56, encoding a new inhibitor-resistant β-lactamase with a pI of 7.6. SHV-56 is derived from SHV-11 by the single substitution K234R. This mutation therefore evidences a new critical site for inhibitor resistance among SHV enzymes.


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TEXT
 
Klebsiella pneumoniae is responsible for a wide range of community- and hospital-acquired opportunistic infections, such as urinary tract infections, pneumonia, or septicemia (13). This species is intrinsically susceptible to all antibiotics active against gram-negative bacilli, except to amino- and carboxypenicillins. This low-level resistance is due to the production of a chromosomally mediated broad-spectrum β-lactamase (SHV-1, mainly, or the LEN or OKP type) that is readily inhibited by β-lactamase inhibitors. Some isolates exhibit a high level of resistance to aminopenicillins and reduced susceptibility to amoxicillin-clavulanate and narrow-spectrum cephalosporins due to the acquisition of plasmid-mediated broad-spectrum β-lactamase or overproduction of the chromosomal enzyme (9). Inhibitor-resistant (IR) TEM-type enzymes conferring amoxicillin-clavulanate resistance and cephalothin susceptibility (2, 3, 8, 10) and expanded-spectrum β-lactamases conferring expanded-spectrum cephalosporin resistance (5) have been described in K. pneumoniae and other Enterobacteriaceae. SHV mutant enzymes are mainly expanded-spectrum β-lactamases (21); however, rare IR-SHV-type β-lactamases (SHV-10 and SHV-49) have been identified (6, 16). We report here SHV-56, the third variant of the SHV family to exhibit resistance to inhibitors, from a K. pneumoniae clinical strain.

K. pneumoniae Kp2792 was isolated from the urine of a 68-year-old obese woman hospitalized in 2003 in the infectious diseases ward at the Saint-André hospital, Bordeaux, France. This patient, admitted for leg erysipelas, was first treated by pristinamycin alone. After a febrile episode, amoxicillin-clavulanate was added at 6 g/day intravenously for one and one-half months. By the disk diffusion method, strain Kp2792 exhibited an IR phenotype without coresistances. The MICs, determined by the agar dilution method (http://www.sfm.asso.fr), confirmed that Kp2792 was highly resistant to the penicillins tested (MICs of 512 to >4,096 µg/ml) and susceptible to cephalothin (MIC of 8 µg/ml) (Table 1). The MICs of penicillins were reduced only slightly by clavulanate and more efficiently by sulbactam, while tazobactam totally restored the piperacillin susceptibility.


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  		TABLE 1. MICs of β-lactams for the clinical isolate Kp2792 and for E. coli JM109 strains expressing or not expressing SHV-11 or SHV-56 β-lactamase

Analysis of the β-lactamase content by isoelectric focusing (11) showed the presence of a single β-lactamase cofocusing with SHV-1 (pI 7.6). The transfer of β-lactam resistance by conjugation to a nalidixic acid-resistant mutant of Escherichia coli K12 by the filter mating technique failed to yield any transconjugant. Plasmid DNA analysis of strain Kp2792 by using a JET STAR plasmid DNA midi kit (Genomed, Löhne, Germany) did not reveal any plasmid. The selection of transformants on agar plates containing ampicillin (100 µg/ml) after electroporation of putative plasmid DNA extract into E. coli JM109 or TOP10 remained unsuccessful. Total DNA of Kp2792 was extracted as previously described (7), and PCR amplification using primers specific for genes encoding IR β-lactamases (blaTEM, blaSHV, blaOXA-1, blaOXA-2, blaOXA-10, and blaCARB) were performed under standard PCR conditions (18). The amplification did not yield any products except for the blaSHV gene expected for K. pneumoniae. Southern blot analysis followed by DNA-DNA hybridization using Kp2792 total and plasmid DNA preparations, either unrestricted or digested by EcoRI and/or BamHI enzyme; a blaSHV-specific probe; and a digoxigenin labeling and detection kit (Roche Applied Science, Meylan, France) confirmed the presence of a single chromosomally located blaSHV gene (data not shown). Subsequently, Kp2792 whole-cell DNA was totally restricted by EcoRI and ligated into the EcoRI site of the pBK-CMV cloning vector (Stratagene, La Jolla, CA). E. coli JM109 bacteria containing recombinant plasmids were selected on agar plates containing ampicillin (100 µg/ml) and kanamycin (50 µg/ml). The recombinant plasmid pC1 containing an insert of ca. 9.5 kb was obtained and subsequently subcloned, using BamHI and EcoRI digestion, into the same vector, giving pSC1 with a ca.-3.5-kb insert. The results of the disk diffusion method and MIC determination demonstrated that E. coli JM109 containing pSC1 exhibited the same IR phenotype as strain Kp2792 (Table 1), produced a β-lactamase with a pI of 7.6, and harbored a blaSHV gene.

The purified (MicrospinTM Sephacryl S-400; Amersham) blaSHV amplicons obtained from strain Kp2792 and recombinant plasmid pSC1 were sequenced on both strands by using a DYEnamic ET dye terminator kit (Amersham Biosciences, Orsay, France) and an ABI 310 automatic sequencer (Perkin-Elmer, Courtaboeuf, France). The results of sequence analysis revealed in both cases an identical blaSHV-type gene encoding a new β-lactamase variant, named SHV-56. The upstream promoter area showed a C at the second position of the –10 region, thus allowing basal SHV expression (17). blaSHV-56 differed from blaSHV-1 (4) by seven nucleotide substitutions that lead to two amino acid substitutions, Leu35->Gln and Lys234->Arg, according to the Ambler nomenclature (1). The Leu35->Gln substitution is frequently encountered and characteristic of the SHV-11 enzyme, which is a broad-spectrum β-lactamase inhibited with a low Ki by clavulanate (6, 12). The Lys234->Arg substitution has not been reported so far, and its role was investigated, especially with regard to β-lactamase inhibitor susceptibility. Thus, site-directed mutagenesis was performed using recombinant plasmid pE1 that expresses the SHV-11 β-lactamase (6), primers MD2792F (5'-GGTTTATCGCCGATAGGACCGGAGCTGG-3') and MD2792R (5'-CCAGCTCCGGTCCTATCGGCGATAAACC-3'), and a QuickChange site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands) as indicated by the manufacturer. This technique allowed the lysine at position 234 of SHV-11 to be replaced by an arginine, thus leading to SHV-56. The resulting mutant (pE1MD), whose sequence was double-checked, exhibited an IR phenotype (Table 1).

To further characterize the activity of SHV-56 and compare its hydrolytic properties to those of SHV-11, the enzymes were extracted as described previously (6) from E. coli bacteria containing the plasmids pE1 and pE1MD. The extract was loaded successively onto two Q-Sepharose columns with 20 mM diethanolamine (pH 9.2) and 20 mM Bis-Tris (pH 7) buffer, respectively (15). SHV-56 showed a penicillinase activity similar to that of SHV-11, whereas no hydrolysis was detected against expanded-spectrum cephalosporins (Table 2). The 50% inhibitory concentrations (IC50) of clavulanate, sulbactam, and tazobactam were determined by the rate of benzylpenicillin (100 µM) hydrolysis by purified proteins after 3 min of preincubation at 30°C. The IC50 were considerably higher for SHV-56 (2.5 µM and 0.75 µM, respectively) than for SHV-11 (0.12 µM and 0.13 µM, respectively), in contrast with the IC50 of sulbactam (0.3 µM for SHV-56 versus 2.4 µM for SHV-11), as confirmed by the MIC results. SHV-56 might display a different turnover number for sulbactam than for clavulanate and tazobactam. These data confirmed that SHV-56 was an IR β-lactamase owing to the Lys234->Arg substitution. Interestingly, position 234 is located in the β3-strand of SHV-56, very close to the Ser130 residue, and it takes part in the catalytic site. The amino acid Ser130 plays an important role in substrate turnover and catalysis (proton transfer), and mutations at this site have been previously implicated in site-directed-mutagenetic and natural IR enzymes (3, 19, 20, 22). It could be hypothesized that Arg234, possessing a longer side chain than a lysine at the same position, might change the Ser130 side chain position and thus its interaction with β-lactamase inhibitors. Moreover, regarding the sulbactam data, the Arg244 position might also be speculated to have a role in the inhibitor resistance.


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  		TABLE 2. Kinetic parameters of the purified β-lactamase SHV-56a

In conclusion, SHV-56 is a new IR β-lactamase belonging to the SHV family that has been identified in a clinical strain of K. pneumoniae. The enzyme was a direct derivative of the naturally occurring SHV β-lactamase in strain Kp2792, and it might have been selected in vivo since the patient had received a long course of treatment with amoxicillin-clavulanate. SHV-56 is derived from SHV-11 by the single substitution Lys234->Arg, which is reported here for the first time in an SHV-type enzyme and has never been observed in natural and artificial IR variants of the TEM family.

Nucleotide sequence accession number. The nucleotide sequence of the blaSHV-56 gene reported in this work is available in the GenBank nucleotide database under the accession number EU586041.


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ACKNOWLEDGMENTS
 
We thank Michel Laguerre and Hedi Mammeri for valuable advice on the β-lactamase structure.

This work was supported by grants from the Ministère de l'Education Nationale et de la Recherche from France (EA-525 from Université de Bordeaux 2, Bordeaux, France, and EA3539, Université Paris XI, Paris, France) and by a grant from the European Community (6th PCRD, LSHM-CT-2005-018705). L.A.R.M. received a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES (Brazil).


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Microbiologie, UMR-CNRS 5234, Université de Bordeaux 2, 146 Rue Léo Saignat, 33076 Bordeaux Cedex, France. Phone: 33 5 57 57 10 75. Fax: 33 5 56 90 90 72. E-mail: veronique.dubois{at}bacterio.u-bordeaux2.fr Back

{triangledown} Published ahead of print on 28 July 2008. Back


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REFERENCES
 
    1
  1. Ambler, R. P., A. F. Coulson, J. M. Frere, J. M. Ghuysen, B. Joris, M. Forsman, R. C. Levesque, G. Tiraby, and S. G. Waley. 1991. A standard numbering scheme for the class A β-lactamases. Biochem. J. 276:269-270.[Medline]
    2. 2
  2. Bermudes, H., F. Jude, C. Arpin, C. Quentin, A. Morand, R. Labia, J. Lemozy, D. Sirot, C. Chanal, C. Huc, H. Dabernat, and J. Sirot. 1997. Characterization of an inhibitor-resistant TEM (IRT) β-lactamase in a novel strain of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 41:222.[Free Full Text]
    3. 3
  3. Bermudes, H., F. Jude, E. B. Chaibi, C. Arpin, C. Bebear, R. Labia, C. Quentin, D. Sirot, P. Pouedras, C. Chanal-Claris, C. Cerceau, J. Sirot, A. Morand, J. Lemozy, C. Chanal, C. Huc, and H. Dabernat. 1999. Molecular characterization of TEM-59 (IRT-17), a novel inhibitor-resistant TEM-derived β-lactamase in a clinical isolate of Klebsiella oxytoca. Antimicrob. Agents Chemother. 43:1657-1661.[Abstract/Free Full Text]
    4. 4
  4. Bradford, P. A. 1999. Automated thermal cycling is superior to traditional methods for nucleotide sequencing of blaSHV genes. Antimicrob. Agents Chemother. 43:2960-2963.[Abstract/Free Full Text]
    5. 5
  5. Canton, R., A. Novais, A. Valverde, E. Machado, L. Peixe, F. Baquero, and T. M. Coque. 2008. Prevalence and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in Europe. Clin. Microbiol. Infect. 14:144-153.[CrossRef][Medline]
    6. 6
  6. Dubois, V., L. Poirel, C. Arpin, L. Coulange, C. Bebear, P. Nordmann, and C. Quentin. 2004. SHV-49, a novel inhibitor-resistant β-lactamase in a clinical isolate of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 48:4466-4469.[Abstract/Free Full Text]
    7. 7
  7. Dubois, V., L. Poirel, C. Marie, C. Arpin, P. Nordmann, and C. Quentin. 2002. Molecular characterization of a novel class 1 integron containing blaGES-1 and a fused product of aac3-Ib/aac6'-Ib' gene cassettes in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 46:638-645.[Abstract/Free Full Text]
    8. 8
  8. Girlich, D., A. Karim, L. Poirel, M. H. Cavin, C. Verny, and P. Nordmann. 2000. Molecular epidemiology of an outbreak due to IRT-2 β-lactamase-producing strains of Klebsiella pneumoniae in a geriatric department. J. Antimicrob. Chemother. 45:467-473.[Abstract/Free Full Text]
    9. 9
  9. Jarlier, V., and P. Nordmann. 2000. Sensibilité aux antibiotiques: entérobactéries et β-lactamines, p. 649-665. In J. Freney, F. Renaud, W. Hansen, and C. Bollet (ed.), Précis de bactériologie clinique. ESKA, Paris, France.
    10. 10
  10. Lemozy, J., D. Sirot, C. Chanal, C. Huc, R. Labia, H. Dabernat, and J. Sirot. 1995. First characterization of inhibitor-resistant TEM (IRT) β-lactamases in Klebsiella pneumoniae strains. Antimicrob. Agents Chemother. 39:2580-2582.[Abstract/Free Full Text]
    11. 11
  11. Mathew, M., A. M. Harris, M. J. Marshall, and G. W. Ross. 1975. The use of analytical isoelectric focusing for detection and identification of β-lactamases. J. Gen. Microbiol. 88:169-178.[Medline]
    12. 12
  12. Nuesch-Inderbinen, M. T., F. H. Kayser, and H. Hachler. 1997. Survey and molecular genetics of SHV β-lactamases in Enterobacteriaceae in Switzerland: two novel enzymes, SHV-11 and SHV-12. Antimicrob. Agents Chemother. 41:943-949[Abstract/Free Full Text]
    13. 13
  13. Podschun, R., and U. Ullmann. 1998. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin. Microbiol. Rev. 11:589-603.[Abstract/Free Full Text]
    14. 14
  14. Poirel, L., M. Guibert, D. Girlich, T. Naas, and P. Nordmann. 1999. Cloning, sequence analyses, expression, and distribution of ampC-ampR from Morganella morganii clinical isolates. Antimicrob. Agents Chemother. 43:769-776.[Abstract/Free Full Text]
    15. 15
  15. Poirel, L., C. Heritier, I. Podglajen, W. Sougakoff, L. Gutmann, and P. Nordmann. 2003. Emergence in Klebsiella pneumoniae of a chromosome-encoded SHV β-lactamase that compromises the efficacy of imipenem. Antimicrob. Agents Chemother. 47:755-758.[Abstract/Free Full Text]
    16. 16
  16. Prinarakis, E. E., V. Miriagou, E. Tzelepi, M. Gazouli, and L. S. Tzouvelekis. 1997. Emergence of an inhibitor-resistant β-lactamase (SHV-10) derived from an SHV-5 variant. Antimicrob. Agents Chemother. 41:838-840.[Abstract/Free Full Text]
    17. 17
  17. Rice, L. B., L. L. Carias, A. M. Hujer, M. Bonafede, R. Hutton, C. Hoyen, and R. A. Bonomo. 2000. High-level expression of chromosomally encoded SHV-1 β-lactamase and an outer membrane protein change confer resistance to ceftazidime and piperacillin-tazobactam in a clinical isolate of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 44:362-367.[Abstract/Free Full Text]
    18. 18
  18. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    19. 19
  19. Sulton, D., D. Pagan-Rodriguez, X. Zhou, Y. Liu, A. M. Hujer, C. R. Bethel, M. S. Helfand, J. M. Thomson, V. E. Anderson, J. D. Buynak, L. M. Ng, and R. A. Bonomo. 2005. Clavulanic acid inactivation of SHV-1 and the inhibitor-resistant S130G SHV-1 β-lactamase. Insights into the mechanism of inhibition. J. Biol. Chem. 280:35528-35536.[Abstract/Free Full Text]
    20. 20
  20. Sun, T., C. R. Bethel, R. A. Bonomo, and J. R. Knox. 2004. Inhibitor-resistant class A β-lactamases: consequences of the Ser130-to-Gly mutation seen in Apo and tazobactam structures of the SHV-1 variant. Biochemistry 43:14111-14117.[CrossRef][Medline]
    21. 21
  21. Tzouvelekis, L. S., and R. A. Bonomo. 1999. SHV-type β-lactamases. Curr. Pharm. Des. 5:847-864.[Medline]
    22. 22
  22. Wang, X., G. Minasov, and B. K. Shoichet. 2002. The structural bases of antibiotic resistance in the clinically derived mutant β-lactamases TEM-30, TEM-32, and TEM-34. J. Biol. Chem. 277:32149-32156.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, October 2008, p. 3792-3794, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00387-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Drawz, S. M., Bonomo, R. A. (2010). Three Decades of {beta}-Lactamase Inhibitors. Clin. Microbiol. Rev. 23: 160-201 [Abstract] [Full Text]  

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