AAC
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poirel, L.
Right arrow Articles by Nordmann, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poirel, L.
Right arrow Articles by Nordmann, P.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, April 2004, p. 1406-1409, Vol. 48, No. 4
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.4.1406-1409.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Molecular Analysis of Metallo-ß-Lactamase Gene blaSPM-1-Surrounding Sequences from Disseminated Pseudomonas aeruginosa Isolates in Recife, Brazil

Laurent Poirel,1 Marcelo Magalhaes,2 Miguel Lopes,2 and Patrice Nordmann1*

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France,1 Department of Tropical Medicine, Federal University of Pernambuco, Recife, Brazil2

Received 17 July 2003/ Returned for modification 8 October 2003/ Accepted 22 December 2003


   ABSTRACT
Top
 Abstract
Text
 References
 
The spread of clonally related carbapenem-resistant Pseudomonas aeruginosa producing the metallo-ß-lactamase SPM-1 was found in Recife, Brazil. Upstream of blaSPM-1, a novel common region (CR4) was identified, comprising an open reading frame, orf495, whose product shares significant identity with putative recombinases, such as Orf513. CR4 may be responsible for mobilization and expression of blaSPM-1.


   TEXT
Top
 Abstract
 Text
References
 
During the last decade, several acquired carbapenem-hydrolyzing enzymes have been identified in Pseudomonas aeruginosa, beginning with IMP-1 and derivatives, which are widespread in Japan (24, 25) and China (4) and are emerging in Europe and Canada (8, 13). Then, VIM-1 and VIM-2 enzymes were characterized, first in Italy and France (11, 22), respectively, but these enzymes are also highly prevalent in Korea and Greece (7, 14, 29). Recently a third member of acquired Ambler class B ß-lactamases, SPM-1, has been identified from a P. aeruginosa isolate in Sao Paulo, Brazil (28).

The aim of this study was to analyze carbapenem-resistant P. aeruginosa isolates recovered from different hospitals in Recife, Brazil, from November 2002 to January 2003. Nineteen P. aeruginosa strains resistant to ceftazidime and imipenem were isolated at the Marcelo Magalhaes clinical laboratory in Recife. Ten isolates were from patients of neurological wards and intensive care units of the Hospital Portugues, Recife, whereas nine strains were from patients hospitalized in similar units from several hospitals in Recife. Eleven imipenem-resistant isolates were identified as metallo-ß-lactamase producers by using the double disk-synergy test with EDTA (30), and these were retained for further genetical analysis. They were resistant to all ß-lactams except aztreonam. Isoelectric focusing analysis performed as described previously (20) showed that the 11 isolates expressed 3 ß-lactamases, with pI values of 6.5, 7.5, and 8.7, the last likely corresponding to the naturally occurring AmpC enzyme of P. aeruginosa. Under standard PCR conditions (20), a series of primers was used for detection of genes encoding Ambler class B ß-lactamases of the VIM, IMP, or SPM type as described previously (21). Positive results were obtained using the blaSPM-1-specific primers (SPM-1A, 5'-CTGCTTGGATTCATGGGCGC-3'; SPM-1B, 5'-CCTTTTCCGCGACCTTGATC-3'). The 11 isolates had the same blaSPM-1 gene previously identified in P. aeruginosa (28).

A genotypic comparison of the blaSPM-1-positive P. aeruginosa isolates performed by pulsed-field gel electrophoresis, using restriction enzyme SpeI, revealed that these isolates were clonally related (data not shown) (18, 27). Electroporation of putative plasmid DNA extracts from blaSPM-1-positive P. aeruginosa isolates performed as described previously (19) failed. Using pulsed-field gel electrophoresis and I-CeuI digestion followed by hybridization with PCR-generated probes as previously reported (9), a strong signal (located at ca. 400 kb) was obtained with the blaSPM-1-specific probe for SPM-1-producing P. aeruginosa that did not correspond to one of the bands that hybridized with the 16S ribosomal DNA-specific probe (data not shown). Thus, the blaSPM-1 gene was likely plasmid located. In addition, Southern hybridization experiments with BamHI-restricted DNA of SPM-1-producing P. aeruginosa isolates gave a unique signal (data not shown), indicating that the blaSPM-1 gene was present as a single copy in the P. aeruginosa genome.

To characterize the genetic structures surrounding the blaSPM-1 gene in P. aeruginosa, cloning experiments were performed using whole-cell DNA of a blaSPM-1-positive P. aeruginosa isolate and pBK-CMV as a cloning vector, as described previously (17). Selection of recombinant clones was performed using trypticase soy agar plates containing 100 µg of amoxicillin and 30 µg of kanamycin per ml, and two phenotypes were obtained.

A recombinant clone containing plasmid pMM-1 expressed a ß-lactamase with a pI value of 7.5 and possessed the blaSPM-1 gene, which conferred resistance to aminopenicillins, ceftazidime, and cefoxitin and reduced suceptibility to imipenem. Another recombinant clone, containing plasmid pMM-2, expressed a restricted-spectrum oxacillinase with a pI value of 6.5, not inhibited by clavulanic acid. Sequencing revealed a novel class D ß-lactamase gene, blaOXA-56, that was part of a blaOXA-10-like gene cassette structure. OXA-56 had three amino acid substitutions compared to OXA-35 (2) and OXA-7 (23).

Sequencing of the 3,312-bp insert of pMM-1 revealed an open reading frame upstream of blaSPM-1 encoding 495 amino acids (Fig. 1). The product of orf495 had 81% amino acid identity with Orf2, identified in the multidrug resistance region of an SXT-type conjugative transposon from Vibrio cholerae (10) and considered a recombinase of the so-called CR2 element (16), 58% identity with OrfA, identified in an Escherichia coli strain (5) and part of the so-called CR3 (16), and 54% identity with Orf513, identified upstream of several antibiotic resistance genes, such as blaCMY-9, blaCTX-M-2, blaCTX-M-9, catA2, dfrA10, and part of the so-called CR1 (1, 3, 6, 15). The definition of these conserved regions (CRs) included right-hand ends exhibiting a degree of identity between the CRs (16). Analysis of the nucleotide sequences located between orf495 and blaSPM-1 revealed a similar 33-bp right-hand boundary, sharing 31, 24, and 24 bp with those of CR3, CR2, and CR1, respectively (Fig. 1). Thus, orf495 may be part of a novel transposable structure called CR4. The putative recombinases of the CRs shared an overall important degree of identity (Fig. 2), whereas their functionality remain to be determined. Partridge and Hall (16) showed that CR1 is able to move by itself by homologous recombination events and that the right-hand end of this structure may be active by incorporating adjacent sequences. Upstream of orf495, the sequence encoding the C-terminal extremity of a putative GroEL chaperonin protein (called GroEL1) was present, with its product sharing 89 and 87% amino acid identity with GroEL proteins from Desulfitobacterium hafniense and Stenotrophomonas maltophilia, respectively, with two hairpin structures likely playing a role in transcription ending (Fig. 1).



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 1. Nucleotide sequence of a 3,775-bp fragment containing the blaSPM-1 gene and made of the 3,312-bp BamHI insert of recombinant plasmid pMM-1 completed by PCR sequencing. The deduced amino acid sequence is designated in single-letter code below the nucleotide sequence. The transcription orientations of the open reading frames are indicated by horizontal arrows. The right-hand boundary of CR4 is shaded in grey. The putative hybrid promoter sequences are underlined, consisting in the -35 region brought by the CR4 element and the -10 region belonging to the blaSPM-1 gene. The orf495 transposase gene of CR4 is indicated, as well as the two partial sequences of the groEL gene. The stars indicates stop codons, and a vertical arrow indicates the crossover point of the CR4-mediated putative recombination event. The hairpin structures are boxed and marked with head-to-head arrows. Another vertical arrow (position 3312) indicates the extremity of the cloned fragment in pMM-1.

 


View larger version (67K):
[in this window]
[in a new window]
  		FIG. 2. Comparison of the amino acid sequences of the different recombinases, consisting in Orf495 from CR4, Orf2 from CR3, Orf513 from CR1, and OrfA from CR2 (15). Stars indicate residues that are identical for the four proteins.

 
Downstream of blaSPM-1, sequence encoding an N-terminally truncated part of a similar protein (called GroEL2) was present (Fig. 1). PCR experiments using primers designed from the sequence reported by Toleman et al. (28) in combination with SPM-1A followed by sequencing revealed perfect identity with sequences identified in the blaSPM-1-positive P. aeruginosa strain 48-1997A, starting with a 16-bp-long hairpin structure located just after the putative recombination crossover point (Fig. 1). The C-terminal extremity of the GroEL2-encoding sequence present downstream of blaSPM-1 was very similar (94% nucleotide identity) to that of GroEL1 (Fig. 1). In addition, the noncoding sequences located downstream of both groEL genes were identical. Surprisingly, the 5' end of orf495 was identified in the 3'-end extremity of the groEL2 gene in our sequence, as well as in that reported by Toleman et al. (28). Thus, at least part of orf495 is also present on both sides of blaSPM-1 (Fig. 1).

These data indicated a possible duplication of part of the target genetic structures subsequent to a mobilization process that could result, as suggested (16), from a one-ended mobilization event enhancing the spread of blaSPM-1.

The genetic structures involved in this mobilization process may also enhance expression of the adjacent antibiotic resistance genes, as demonstrated for insertion sequence elements (12). Indeed, analysis of the 220 bp separating orf495 from blaSPM-1 revealed putative promoter sequences made of a -35 motif (TTGAAT) provided by the CR4 boundary and a putative original -10 motif (TACAAT) of blaSPM-1, constituting a hybrid promoter (Fig. 1).

Finally, the origin of this novel element, CR4, has to be determined, since orf495 is not present at least in the P. aeruginosa PAO1 genome (26). Although blaSPM-1 has a GC content of 48%, that of orf495 is 70%, consistent with a Pseudomonaceae origin.

This study describes a novel genetic element associated with a metallo-ß-lactamase gene that may constitute a tool for its dissemination.

Nucleotide sequence accession numbers. The sequences determined in this study have been assigned GenBank sequence accession no. AY341249 (for blaSPM-1) and AY445080 (for blaOXA-56).


   ACKNOWLEDGMENTS
 
This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, Paris, France.

We thank Marisa Castro for technical assistance. L.P. is a researcher from the INSERM.


   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.ap-hop-paris.fr. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Arduino, S. M., P. H. Roy, G. A. Jacoby, B. E. Orman, S. A. Pineiro, and D. Centron. 2002. blaCTX-M-2 is located in an unusual class 1 integron (In35) which includes Orf513. Antimicrob. Agents Chemother. 46:2303-2306.[Abstract/Free Full Text]
  2. Aubert, D., L. Poirel, A. Ben Ali, F. W. Goldstein, and P. Nordmann. 2001. OXA-35 is an OXA-10-related ß-lactamase from Pseudomonas aeruginosa. J. Antimicrob. Chemother. 48:717-721.[Abstract/Free Full Text]
  3. Boyd, D., G. A. Peters, A. Cloeckaert, K. S. Boumedine, E. Chaslus-Dancla, H. Imberechts, and M. R. Mulvey. 2002. Complete nucleotide sequence of a 43-kilobase genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104 and its identification in phage type DT120 and serovar Agona. J. Bacteriol. 183:5725-5732.
  4. Chu, Y. W., M. Afzal-Shah, E. T. Houang, M. I. Palepou, D. J. Lyon, N. Woodford, and D. M. Livermore. 2001. IMP-4, a novel metallo-ß-lactamase from nosocomial Acinetobacter spp. collected in Hong Kong between 1994 and 1998. Antimicrob. Agents Chemother. 45:710-714.[Abstract/Free Full Text]
  5. Cloeckaert, A., S. Baucheron, G. Flaujac, S. Schwarz, C. Kehrenberg, J. L. Martel, and E. Chaslus-Dancla. 2000. Plasmid-mediated florfenicol resistance encoded by the floR gene in Escherichia coli isolated from cattle. Antimicrob. Agents Chemother. 44:2858-2860.[Abstract/Free Full Text]
  6. Di Conza, J., J. A. Ayala, P. Power, M. Mollerach, and G. Gutkind. 2002. Novel class 1 integron (InS21) carrying blaCTX-M-2 in Salmonella enterica serovar Infantis. Antimicrob. Agents Chemother. 46:2257-2261.[Abstract/Free Full Text]
  7. Giakkoupi, P., G. Petrikkos, L. S. Tzouvelekis, S. Tsonas, N. J. Legakis, A. C. Vatopoulos, and the WHONET Greece Study Group. 2003. Spread of integron-associated VIM-type metallo-ß-lactamase genes among imipenem-nonsusceptible Pseudomonas aeruginosa strains in Greek hospitals. J. Clin. Microbiol. 41:822-825.[Abstract/Free Full Text]
  8. Gibb, A. P., C. Tribuddharat, R. A. Moore, T. J. Louie, W. Krulicki, D. M. Livermore, M. F. Palepou, and N. Woodford. 2002. Nosocomial outbreak of carbapenem-resistant Pseudomonas aeruginosa with a new blaIMP allele, blaIMP-7. Antimicrob. Agents Chemother. 46:255-258.[Abstract/Free Full Text]
  9. Héritier, C., L. Poirel, D. Aubert, and P. Nordmann. 2003. Genetic and functional analysis of the chromosome-encoded carbapenem-hydrolyzing oxacillinase OXA-40 of Acinetobacter baumannii. Antimicrob. Agents Chemother. 47:268-273.[Abstract/Free Full Text]
  10. Hochhut, B., Y. Lotfi, D. Mazel, S. M. Faruque, R. Woodgate, and M. K. Waldor. 2001. Molecular analysis of antibiotic resistance gene clusters in Vibrio cholerae O139 and O1 SXT constins. Antimicrob. Agents Chemother. 45:2991-3000.[Abstract/Free Full Text]
  11. Lauretti, L., M. L. Riccio, A. Mazzariol, G. Cornaglia, G. Amicosante, R. Fontana, and G. M. Rossolini. 1999. Cloning and characterization of blaVIM, a new integron-borne metallo-ß-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob. Agents Chemother. 43:1584-1590.[Abstract/Free Full Text]
  12. Mahillon, J., and M. Chandler. 1998. Insertion sequences. Microbiol. Mol. Biol. Rev. 62:725-774.[Abstract/Free Full Text]
  13. Nordmann, P., and L. Poirel. 2002. Emerging carbapenemases in Gram-negative aerobes. Clin. Microbiol. Infect. 8:321-331.[CrossRef][Medline]
  14. Oh, E. J., S. Lee, Y. J. Park, J. J. Park, K. Park, S. I. Kim, M. W. Kang, and B. K. Kim. 2003. Prevalence of metallo-ß-lactamase among Pseudomonas aeruginosa and Acinetobacter baumannii in a Korean University hospital and comparison of screening methods for detecting metallo-ß-lactamase. J. Microbiol. Methods 54:411-418.[CrossRef][Medline]
  15. Parsons, Y., R. M. Hall, and H. W. Stokes. 1991. A new trimethoprim resistance gene, dhfrX, in the In7 integron of plasmid pDG0100. Antimicrob. Agents Chemother. 35:2436-2439.[Abstract/Free Full Text]
  16. Partridge, S. R., and R. M. Hall. 2003. In34, a complex In5 family class 1 integron containing orf513 and dfrA10. Antimicrob. Agents Chemother. 47:342-349.[Abstract/Free Full Text]
  17. Poirel, L., D. Girlich, T. Naas, and P. Nordmann. 2001. OXA-28, an extended-spectrum variant of OXA-10 ß-lactamase from Pseudomonas aeruginosa and its plasmid- and integron-located gene. Antimicrob. Agents Chemother. 45:447-453.[Abstract/Free Full Text]
  18. 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]
  19. 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]
  20. 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]
  21. Poirel, L., and P. Nordmann. 2002. Acquired carbapenem-hydrolyzing ß-lactamases and their genetic support. Curr. Pharm. Biotechnol. 3:117-127.[CrossRef][Medline]
  22. Poirel, L., T. Naas, D. Nicolas, L. Collet, S. Bellais, J. D. Cavallo, and P. Nordmann. 2000. Characterization of VIM-2, a carbapenem-hydrolyzing metallo-ß-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob. Agents Chemother. 44:891-897.[Abstract/Free Full Text]
  23. Scoulica, E., A. Aransay, and Y. Tselentis. 1995. Molecular characterization of the OXA-7 ß-lactamase gene. Antimicrob. Agents Chemother. 39:1379-1382.[Abstract]
  24. Senda, K., Y. Arakawa, S. Ichiyama, K. Nakashima, H. Ito, S. Ohsuka, K. Shimokata, N. Kato, and M. Ohta. 1996. PCR detection of metallo-ß-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum ß-lactams. J. Clin. Microbiol. 34:2909-2913.[Abstract]
  25. Senda, K., Y. Arakawa, K. Nakashima, H. Ito, S. Ichiyama, K. Shimokata, N. Kato, and M. Ohta. 1996. Multifocal outbreaks of metallo-ß-lactamase-producing Pseudomonas aeruginosa resistant to broad-spectrum ß-lactams, including carbapenems. Antimicrob. Agents Chemother. 40:349-353.[Abstract]
  26. Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, I. T. Paulsen, J. Reizer, M. H. Saier, R. E. Hancock, S. Lory, M. V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959-964.[CrossRef][Medline]
  27. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.[Medline]
  28. Toleman, M. A., A. M. Simm, T. A. Murphy, A. C. Gales, D. J. Biedenbach, R. N. Jones, and T. R. Walsh. 2002. Molecular characterization of SPM-1, a novel metallo-ß-lactamase isolated in Latin America: report from the SENTRY antimicrobial surveillance programme. J. Antimicrob. Chemother. 50:673-679.[Abstract/Free Full Text]
  29. Tsakris, A., S. Pournaras, N. Woodford, M. F. Palepou, G. S. Babini, J. Douboyas, and D. M. Livermore. 2000. Outbreak of infections caused by Pseudomonas aeruginosa producing VIM-1 carbapenemase in Greece. J. Clin. Microbiol. 38:1290-1292.[Abstract/Free Full Text]
  30. Yong, D., K. Lee, J. H. Yum, H. B. Shin, G. M. Rossolini, and Y. Chong. 2002. Imipenem-EDTA disk method for differentiation of metallo-ß-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J. Clin. Microbiol. 40:3798-3801.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, April 2004, p. 1406-1409, Vol. 48, No. 4
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.4.1406-1409.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poirel, L.
Right arrow Articles by Nordmann, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poirel, L.
Right arrow Articles by Nordmann, P.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Clin. Vaccine Immunol. Clin. Microbiol. Rev.
J. Clin. Microbiol. ALL ASM JOURNALS