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Journal of Clinical Microbiology, August 2006, p. 3008-3011, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.02576-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Pyrosequencing as a Rapid Tool for Identification of GES-Type Extended-Spectrum ß-Lactamases

Laurent Poirel, Thierry Naas, and Patrice Nordmann*

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, 94275 K.-Bicêtre, France

Received 12 December 2005/ Returned for modification 15 May 2006/ Accepted 5 June 2006


   ABSTRACT
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A pyrosequencing technique was used for identification of extended-spectrum ß-lactamases (ESBLs) of GES type. These ß-lactamases are isolated increasingly emerging in gram-negative bacteria worldwide. This rapid and reliable identification method is interesting, since GES variants, including not only expanded-spectrum cephalosporins but also carbapenems, cephamycins, and monobactams, are the only ESBLs that possess different hydrolysis profiles.


   TEXT
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Extended-spectrum ß-lactamases (ESBLs) of the GES type (also named IBC) have been reported increasingly in gram-negative rods, including Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae (5, 9, 11, 12, 14, 22). These GES variants have been identified in France, Greece, Portugal, South Africa, French Guyana, Argentina, and Brazil (1-3, 10, 13, 17-19). The current GES nomenclature is defined within the Lahey Clinic website (http://www.lahey.org/studies/webt.html). ß-Lactamase GES-1 was from France (12), GES-2 was from South Africa (14), GES-3, GES-4, GES-7, and GES-8 were from Greece (5, 6, 17), and GES-5 and GES-6 were from Japan (18, 19). In addition, GES-3 has also been reported recently in Korea (7).

ß-Lactamase GES-1 possesses a hydrolysis profile similar to those of classical clavulanic acid-inhibited Ambler class A ESBLs, including penicillins and expanded-spectrum cephalosporins but not cephamycins and carbapenems, inhibited also by clavulanic acid and tazobactam (12). Unlike most ESBLs, GES-1 did not possess hydrolytic activity toward aztreonam. ß-Lactamase GES-2 also hydrolyzes carbapenems and is less susceptible to inhibitors, due to a 2-bp substitution (nucleotide positions 493 and 494) (Fig. 1), leading to a single Gly170Asn change inside the omega loop of the catalytic site (14). A single nucleotide substitution at position 493 (Fig. 1) leading to a Gly170Ser change was identified in GES-3, GES-5, and GES-6 (Table 1). These GES variants hydrolyze carbapenems at a level as low as that for GES-2, which also hydrolyzes cephamycins. In addition, these variants are weakly susceptible to Ambler class A ß-lactamase inhibitors. Recently, GES-9, which differs from GES-1 by a single nucleotide substitution at position 709, leading to a Gly243Ser change, was studied (Table 1; Fig. 1) (11). GES-9 does not hydrolyze carbapenems, whereas its activity is broadened toward monobactams. Taking these different hydrolytic activities into account, including in several cases those for carbapenems, it is useful to have a rapid identification technique for these techniques.


Figure 1
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  		FIG. 1. Alignment of the nucleotide sequences of genes encoding ß-lactamases GES-1, GES-2, GES-3, and GES-9. Names correspond to the updated GES nomenclature (see the text). PCR amplification primers (GES-F, GES-R1bio, and GES-R2bio) are boxed, whereas sequencing primers (S1 and S2) are boxed and shaded. The vertical boxes indicate the locations of interest for critical amino acid residues. Nucleotide numbering of the blaGES genes are indicated above the nucleotide sequences.

 

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  		TABLE 1. Key amino acid substitutions of GES variants in relation to their hydrolysis spectra

 
A method of detection of blaGES-1/blaGES-2 genes by using a real-time PCR technique has been proposed (21). A competitive peptide nucleic acid-based multiplex PCR assay was also developed to recognize a blaGES-2 allele compared to other blaGES genes (20). With that technique, it was not possible to identify any non-blaGES-2 variant without further sequencing.

We report here PCR detection of blaGES genes coupled with a pyrosequencing-based methodology that provides the ability to detect rapidly the substitutions at the origin of differences in hydrolysis spectrum. Pyrosequencing is a reliable technique that allows fast identification of short DNA sequences. It has already been used as a rapid and convenient approach for detection of antibiotic resistances, such as ciprofloxacin resistance in Neisseria gonorrhoeae, rifampin resistance in Mycobacterium tuberculosis, macrolide resistance in Streptococcus pneumoniae, Streptococcus pyogenes, Mycobacterium avium, Campylobacter jejuni, and Haemophilus influenzae, and linezolid resistance in enterococci (4, 6, 15, 16, 23).

Four GES variants, as representatives of the four different hydrolysis spectra of GES enzymes, were retained. The blaGES-1 gene was amplified from K. pneumoniae ORI-1 (12), blaGES-2 from P. aeruginosa GW-1 (14), blaGES-3 from plasmid pRSB113 (gift from A. Schlueter), and blaGES-9 from P. aeruginosa DEJ (11). In addition, a collection of twenty-eight ESBL-positive enterobacterial isolates producing TEM-, SHV-, CTX-M-, PER-, and VEB-type ß-lactamases recovered from clinical specimens in 2002 in the Paris area were included in the study (8). Whole-cell DNAs were extracted by a boiling extract procedure, using one colony of each bacterial strain resuspended in 100 µl of distilled water. After a 5-min heating at 100°C, suspensions were centrifuged (5 min, 10,000 x g) and 2 µl of supernatant was used as the template. PCR experiments were performed, with 30 cycles consisting of 30-s denaturation at 94°C, 30-s annealing at 52°C, and 30-s extension at 72°C. PCRs specific for the detection of blaTEM, blaSHV, blaCTX-M, blaVEB, and blaPER genes were performed as described previously (8). Specific primers (GES-F as forward primer and GES-R1bio and GES-R2bio as reverse primers [Table 1; Fig. 1]) for all the blaGES genes studied were designed in order to amplify two fragments of 378 bp and 230 bp, respectively. The shortest product allowed analysis of codon 170 only by use of probe S1 whereas the longest PCR product encompassed also codon 243 that may be sequenced using probe S2 (Table 2; Fig. 1). Reverse primers were biotin labeled at their 5' end. All the primers and probes for pyrosequencing were purified by high-performance liquid chromatography (Sigma Proligo, Saint-Quentin Fallavier, France).


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  		TABLE 2. Primers used for blaGES-like genes amplification and sequencing primers used in this study

 
Pyrosequencing was performed by using a PSQ 96 sample preparation kit and a PSQ 96MA analyzer (AB Biotage, Uppsala, Sweden) by following the manufacturer's instructions. PCR products were obtained with each couple of primers using DNA of the four blaGES-positive isolates as templates. Those PCR products were of high intensity, consistent with a direct use for pyrosequencing (Fig. 2). However, no PCR product was obtained with the blaTEM-like-, blaSHV-like-, blaCTX-M-like-, blaPER-1-, or blaVEB-1-positive isolates tested, underlining the specificity of the PCR.


Figure 2
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  		FIG. 2. Agarose gel analysis of PCR products recovered from the GES producers tested, including strains harboring the blaGES-1 gene (lanes 1 and 5), the blaGES-2 gene (lanes 2 and 6), the blaGES-3 gene (lanes 3 and 7), and the blaGES-9 gene (lanes 4 and 8). Wells 1 to 4 correspond to PCR with GES-R1bio reverse primer (expected size, 378 bp), and wells 5 to 8 correspond to PCR with GES-R2bio reverse primer (expected size, 230 bp). M corresponds to the molecular weight marker (1-kb ladder; Invitrogen).

 
Unpurified amplified products recovered after PCR were captured and separated by using streptavidin-Sepharose beads, and the resulting single-stranded DNA was used as the template for pyrosequencing with appropriate pyrosequencing probes (Fig. 1). The overall experiment is based on a protocol detailed within the AB Biotage website (www.pyrosequencing.com). The different pyrograms obtained with the four variants tested are shown in Fig. 3. The amount of light released at each extension step is directly proportional to the amount of nucleotide added, the relative numbers of a given nucleotide being consequently appreciated by the relative peak heights on data traces. In a few minutes, pyrosequencing allowed the determination of the three key nucleotides, consequently identifying ß-lactamases GES-1, GES-2, GES-3, and GES-9 variants. If pyrosequencing is used in a prospective approach, the critical amino acid residues might be identified, even though the whole sequence of the GES enzyme would not be determined by using this technique.


Figure 3
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  		FIG. 3. Detection by pyrosequencing of the G170N or G170S substitutions of GES-2 and GES-3 (A), respectively, and the G243S substitution of GES-9 (B). The wild-type blaGES-1 sequence (a) has been tested together with those from the GES-2 (b), GES-3 (c), and GES-9 (d) producers. The deduced sequence is indicated above each peak for the blaGES-1 gene and above each peak that differs from blaGES-1 for others.

 
Conclusion. This pyrosequencing technique is a rapid tool for the identification of GES-type ESBLs. Pyrosequencing allows identification in a few minutes of critical amino acid residues which are related to clinically relevant substrate profiles. Consequently, this technique is convenient to prevent usage of possibly inefficient ß-lactam-containing treatments leading to therapeutic failures. Thus, in specific geographical areas, such as in France, Greece, South Africa, and Japan where different GES variants may coexist, it might be useful to be able rapidly to distinguish a classical GES-1-type ESBL from a nonclassical enzyme that may possess extended capability to hydrolyze carbapenems, cephamycins, and monobactams, or that may be less efficiently inhibited by inhibitors. Indeed, in those areas where GES enzymes coexist, this technique would be suitable to avoid sequencing delays. In addition, this pyrosequencing technique may also provide a rapid epidemiological tool for a preliminary survey on plasmid-mediated ESBLs. From extraction to sequencing results, the technique takes three hours, a length of time that may be reduced further by using a faster PCR-based technique, such as a Light Cycler-based technique.


   ACKNOWLEDGMENTS
 
This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, France, and by the European Community (6th PCRD, LSHM-CT-2003-503-335). L.P. is a researcher for the INSERM, Paris, France.

We are grateful to S. Marin, J. Hogg, and R. England from Biotage for their support of this work.


   FOOTNOTES
 
* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice{at}bct.aphp.fr. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Castanheira, M., R. E. Mendes, T. R. Walsh, A. C. Gales, and R. N. Jones. 2004. Emergence of the extended-spectrum ß-lactamase GES-1 in a Pseudomonas aeruginosa strain from Brazil: report from the SENTRY antimicrobial surveillance program. Antimicrob. Agents Chemother. 48:2344-2345.[Free Full Text]
  2. Duarte, A., F. Boavida, F. Grosso, M. Correia, L. M. Lito, J. M. Cristino, and M. J. Salgado. 2003. Outbreak of GES-1 ß-lactamase-producing multidrug-resistant Klebsiella pneumoniae in a university hospital in Lisbon, Portugal. Antimicrob. Agents Chemother. 47:1481-1482.[Free Full Text]
  3. 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]
  4. Gharizadeh, B., M. Akhras, M. Unemo, B. Wretlind, P. Nyren, and N. Pourmand. 2005. Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing. Int. J. Antimicrob. Agents 26:486-490.[CrossRef][Medline]
  5. Giakkoupi, P., L. S. Tzouvelekis, A. Tsakris, V. Loukova, D. Sofianou, and E. Tzelepi. 2000. IBC-1, a novel integron-associated class A ß-lactamase with extended-spectrum properties produced by an Enterobacter cloacae clinical strain. Antimicrob. Agents Chemother. 44:2247-2253.[Abstract/Free Full Text]
  6. Haanpera, M., P. Huovinen, and J. Jalava. 2005. Detection and quantification of macrolide resistance mutations at positions 2058 and 2059 of the 23S rRNA gene by pyrosequencing. Antimicrob. Agents Chemother. 49:457-460.[Abstract/Free Full Text]
  7. Jeong, S. H., I. K. Bae, D. Kim, S. G. Hong, J. S. Song, J. H. Lee, and S. H. Lee. 2005. First outbreak of Klebsiella pneumoniae clinical isolates producing GES-3 and SHV-12 extended-spectrum ß-lactamases in Korea. Antimicrob. Agents Chemother. 49:4809-4810.[Free Full Text]
  8. Lartigue, M. F., N. Fortineau, and P. Nordmann. 2005. Spread of novel expanded-spectrum ß-lactamases in Enterobacteriaceae in a university hospital in the Paris area, France. Clin. Microbiol. Infect. 11:588-591.[CrossRef][Medline]
  9. Mavroidi, A., E. Tzelepi, A. Tsakris, V. Miriagou, D. Sofianou, and L. S. Tzouvelekis. 2001. An integron-associated ß-lactamase (IBC-2) from Pseudomonas aeruginosa is a variant of the extended-spectrum ß-lactamase IBC-1. J. Antimicrob. Chemother. 48:627-630.[Abstract/Free Full Text]
  10. Pasteran, F., D. Faccone, A. Petroni, M. Rapoport, M. Galas, M. Vazquez, and A. Procopio. 2005. Novel variant blaVIM-11 of the metallo-ß-lactamase blaVIM family in a GES-1 extended-spectrum ß-lactamase-producing Pseudomonas aeruginosa clinical isolate in Argentina. Antimicrob. Agents Chemother. 49:474-475.[Free Full Text]
  11. Poirel, L., L. Brinas, N. Fortineau, and P. Nordmann. 2005. Integron-encoded GES-type extended-spectrum ß-lactamase with increased activity toward aztreonam in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 49:3593-3597.[Abstract/Free Full Text]
  12. Poirel, L., I. Le Thomas, T. Naas, A. Karim, and P. Nordmann. 2000. Biochemical sequence analyses of GES-1, a novel class A extended-spectrum ß-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. Antimicrob. Agents Chemother. 44:622-632.[Abstract/Free Full Text]
  13. Poirel, L., G. F. Weldhagen, C. De Champs, and P. Nordmann. 2002. A nosocomial outbreak of Pseudomonas aeruginosa isolates expressing the extended-spectrum ß-lactamase GES-2 in South Africa. J. Antimicrob. Chemother. 49:561-565.[Abstract/Free Full Text]
  14. Poirel, L., G. F. Weldhagen, T. Naas, C. De Champs, M. G. Dove, and P. Nordmann. 2001. GES-2, a class A ß-lactamase from Pseudomonas aeruginosa with increased hydrolysis of imipenem. Antimicrob. Agents Chemother. 45:2598-2603.[Abstract/Free Full Text]
  15. Ryoo, N. H., E. C. Kim, S. G. Hong, Y. J. Park, K. Lee, I. K. Bae, E. H. Song, and S. H. Jeong. 2005. Dissemination of SHV-12 and CTX-M-type extended-spectrum ß-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae and emergence of GES-3 in Korea. J. Antimicrob. Chemother. 56:698-702.[Abstract/Free Full Text]
  16. Sinclair, A., C. Arnold, and N. Woodford. 2003. Rapid detection and estimation by pyrosequencing of 23S rRNA genes with a single nucleotide polymorphism conferring linezolid resistance to enterococci. Antimicrob. Agents Chemother. 47:3620-3622.[Abstract/Free Full Text]
  17. Vourli, S., P. Giakkoupi, V. Miriagou, E. Tzelepi, A. C. Vatopoulos, and L. S. Tzouvelekis. 2004. Novel GES/IBC extended-spectrum ß-lactamase variants with carbapenemase activity in clinical enterobacteria. FEMS Microbiol. Lett. 234:209-213.[Medline]
  18. Wachino, J., Y. Doi, K. Yamane, N. Shibata, T. Yagi, T. Kubota, H. Ito, and Y. Arakawa. 2004. Nosocomial spread of ceftazidime-resistant Klebsiella pneumoniae strains producing a novel class A ß-lactamase, GES-3, in a neonatal intensive care unit in Japan. Antimicrob. Agents Chemother. 48:1960-1967.[Abstract/Free Full Text]
  19. Wachino, J., Y. Doi, K. Yamane, N. Shibata, T. Yagi, T. Kubota, and Y. Arakawa. 2004. Molecular characterization of a cephamycin-hydrolyzing and inhibitor-resistant class A ß-lactamase, GES-4, possessing a single G170S substitution in the omega-loop. Antimicrob. Agents Chemother. 48:2905-2910.[Abstract/Free Full Text]
  20. Weldhagen, G. F. 2004. Sequence-selective recognition of extended-spectrum ß-lactamase GES-2 by a competitive, peptide nucleic acid-based multiplex PCR assay. Antimicrob. Agents Chemother. 48:3402-3406.[Abstract/Free Full Text]
  21. Weldhagen, G. F. 2004. Rapid detection and sequence-specific differentiation of extended-spectrum ß-lactamase GES-2 from Pseudomonas aeruginosa by use of a real-time PCR assay. Antimicrob. Agents Chemother. 48:4059-4062.[Abstract/Free Full Text]
  22. Weldhagen, G. F., L. Poirel, and P. Nordmann. 2003. Ambler class A extended-spectrum ß-lactamases in Pseudomonas aeruginosa: novel developments and clinical impact. Antimicrob. Agents Chemother. 47:2385-2392.[Free Full Text]
  23. Zhao, J. R., Y. J. Bai, Q. H. Zhang, Y. Wang, M. Luo, and X. J. Yan. 2005. Pyrosequencing-based approach for rapid detection of rifampin-resistant Mycobacterium tuberculosis. Diagn. Microbiol. Infect. Dis. 51:135-137.[CrossRef][Medline]

Journal of Clinical Microbiology, August 2006, p. 3008-3011, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.02576-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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