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 Riccio, M. L.
Right arrow Articles by Rossolini, G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Riccio, M. L.
Right arrow Articles by Rossolini, G. M.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, May 2003, p. 1746-1748, Vol. 47, No. 5
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.5.1746-1748.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Novel 3-N-Aminoglycoside Acetyltransferase Gene, aac(3)-Ic, from a Pseudomonas aeruginosa Integron

Maria Letizia Riccio,1 Jean-Denis Docquier,1 Emanuela Dell'Amico,1 Francesco Luzzaro,2 Gianfranco Amicosante,3 and Gian Maria Rossolini1*

Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, I-53100 Siena,1 Laboratorio di Microbiologia, Ospedale di Circolo di Varese, I-21100, Varese,2 Dipartimento di Scienze e Tecnologie Biomediche, Università di L'Aquila, I-67100, L'Aquila, Italy3

Received 23 October 2002/ Returned for modification 16 December 2002/ Accepted 27 January 2003


   ABSTRACT
Top
 Abstract
Text
 References
 
A novel gene, aac(3)-Ic, encoding an AAC(3)-I aminoglycoside 3-N-acetyltransferase, was identified on a gene cassette inserted into a Pseudomonas aeruginosa integron that also carries a blaVIM-2 and a cmlA7 gene cassette. The aac(3)-Ic gene product is 59 and 57% identical to AAC(3)-Ia and AAC(3)-Ib, respectively, and confers resistance to gentamicin and sisomicin.


   TEXT
Top
 Abstract
 Text
References
 
Acetylation of aminoglycosides by acetyltransferases is one of the major mechanisms of acquired resistance to these compounds (2, 11). Acetylation, by a large number of different enzymes, may occur at 1-, 3-, 6'-, and 2'-amino groups of aminoglycosides and can involve virtually all the medically useful compounds (11). The 3-N-aminoglycoside acetyltransferases [AAC(3) enzymes] and the 6'-N-aminoglycoside acetyltransferases [AAC(6') enzymes] are among the modifying enzymes most commonly encountered in clinical isolates (2, 10).

For the AAC(3) enzymes, several different proteins with different substrate specificities have been identified (18). The AAC(3)-I enzymes confer resistance to gentamicin, sisomicin, and fortimicin (astromicin) and are widespread among Enterobacteriaceae and nonfastidious gram-negative nonfermenters (9, 18). Two members of this group, AAC(3)-Ia and AAC(3)-Ib, divergent by approximately 30% of amino acid residues, have been identified (17, 21), with minor variants of each lineage (4, 7-9). The aac(3)-I alleles are found on mobile gene cassettes inserted into integrons (17, 18), a location that evidently facilitates their spread among different replicons and eventually among different strains and that likely accounts for the diffusion of these genes in the clinical setting.

In this work we report on the identification and characterization of a novel AAC(3)-I determinant, named aac(3)-Ic, that represents a third evolutionary lineage in this group of resistance genes.

Clinical isolate. Pseudomonas aeruginosa VA-182/00 is a multidrug-resistant strain isolated in the year 2000 from an inpatient at the Varese University Hospital in northern Italy. Two acquired ß-lactamases (the VIM-2 metalloenzyme and the PER-1 extended-spectrum serine enzyme) that confer an exceedingly broad profile of resistance to ß-lactams have already been characterized in this isolate (3). In vitro susceptibility to antimicrobial agents was determined by a macrodilution broth method (13) using cation-adjusted Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.). P. aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used for quality control of susceptibility testing. Aminoglycoside compounds were from Sigma Chemical Co. (St. Louis, Mo.) unless otherwise specified. Netilmicin was from Essex (Munich, Germany); isepamicin was from Schering-Plough (Kenilworth, N.J.). P. aeruginosa VA-182/00 exhibited resistance to most aminoglycosides (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. MICs of various aminoglycosides for P. aeruginosa VA-182/00 and E. coli DH5{alpha}(pMLR-36/02), carrying the cloned aac(3)-Ic genea

 
Characterization of the variable region of In182, containing a novel AAC(3) determinant. The blaVIM-2 metallo-ß-lactamase gene of VA-182/00 was shown to be carried on the chromosome (3), but the structure of the cognate integron has not been investigated. A PCR mapping and sequencing approach was adopted to investigate this point. PCR was carried out as described previously, as was sequencing of amplification products and of cloned fragments (16). Both strands were sequenced.

A blaVIM-2 specific primer (VIM1/2-for, 5'-TCTGGCTGAATGGCCCACCTC) was used in combination with the INT/3CS primer (16). This yielded a 2.5-kb amplification product which contained the right-hand moiety of the variable region of the blaVIM-2-containing integron, spanning the 3' end of the blaVIM-2 cassette, and two additional gene cassettes (Fig. 1). The left-hand moiety of this integron was then amplified using the INT/5CS primer (16) and a primer designed on the basis of the sequence of the second gene cassette (AAC-r [see below]). This resulted in a 2.1-kb amplification product, partially overlapping the previous one, spanning the blaVIM-2 cassette and most of the second cassette (Fig. 1). Assembly of sequence data revealed an original array of three gene cassettes inserted into the recombination site of a type 1 integron with a 3'-conserved segment (3'-CS) containing a qacE{Delta}1 allele; this integron was named In182 (Fig. 1).



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 1. Structure of the variable region of In182 from P. aeruginosa VA-182/00, which contains a blaVIM-2 cassette, the new aac(3)-Ic cassette, and a cmlA7 cassette. Hatched rectangles, 5'- and 3'-CS regions; filled arrows, cassette-borne resistance genes; circles, their attC recombination sites. The locations of primers INT/5CS, INT/3CS, VIM1/2-for, and AAC-r, used for PCR mapping of this region, are also shown (see text for more details).

 
The first cassette of In182 contains a blaVIM-2 gene which differs from other blaVIM-2 genes only by a silent G-to-T mutation at position 485 (the first nucleotide of the blaVIM-2 open reading frame [ORF] is taken as position 1) (15). The attC recombination site (59-base element) of this cassette is identical to that of blaVIM-2 cassettes found in other integrons including In56, In58, In59, In105, and In106 (14, 15, 22).

The second cassette contains an ORF encoding a protein that exhibits 56.4 to 59.6% identity to known AAC(3)-I enzymes and is notably divergent from both AAC(3)-Ia and AAC(3)-Ib (Fig. 2). This determinant, therefore, represents a third evolutionary lineage of aac(3)-I genes and was named aac(3)-Ic. The attC recombination site of the aac(3)-Ic cassette is quite different from those found in gene cassettes carrying aac(3)-Ia (57% nucleotide identity) and shows greater similarity (76% nucleotide identity) to that of the blaGES-1/IBC-1 cassettes (Fig. 3). Interestingly, this type of recombination site is also related to that found in the aadA1 cassette of In2, which is almost identical in the regions overlapping the internal 2L and 2R core sites and differs from the former by a large deletion (52 bp) in the central region (Fig. 3).



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 2. Comparison of the AAC(3)-Ic protein encoded by the second gene cassette of integron In182 with other AAC(3)-I proteins including AAC(3)-Ia from integron In4 (21), the AAC(3)-Ia variant from plasmid pUO901 (9), the AAC(3)-Ia variant from Acinetobacter baumannii AC-54/97 (8), the AAC(3)-Ia variant from integron InAB1 (7), AAC(3)-Ib from P. aeruginosa Stone 130 (17), and the AAC(3)-Ib variant from integron In60 (4). Amino acid residues conserved in at least two different lineages of AAC(3)-I enzymes are shaded. Residues that differ within each lineage are italicized.

 


View larger version (18K):
[in this window]
[in a new window]
  		FIG. 3. Nucleotide sequence comparison of the attC recombination site of the aac(3)-Ic cassette from integron In182 with other attC recombination sites including that of the blaGES-1 cassette of integron In60 (4) (identical to that of the blaIBC-1 cassette [6]), those of the aac(3)-Ia cassettes from integron In4 (21) [identical to that of the aac(3)-Ia cassette from pUO901 (9)], integron InAB1 (7), and an integron of Acinetobacter baumannii AC-54/97 (8), and that of the aadA1 cassette of In2 (20). The termination codon of each resistance gene is underlined. The locations of the 1L, 2L, and 2R core sites of the recombination elements (19) are indicated by arrows. Residues that are conserved in other sequences, in comparison with the attC recombination site of the aac(3)-Ic cassette, are shaded.

 
The third cassette is identical to that found in a plasmid-borne type 1 integron from an uncultured environmental bacterium (A. Schlueter, GenBank accession no. AY115475). It contains an allelic variant of the cmlA gene, known to be responsible for nonenzymatic resistance to chloramphenicol (5). The product of the cmlA allele of In182 is different from other known CmlA proteins and is most closely related to CmlA5 (12) and CmlA6 (1), from which it differs by single amino acid residues. Therefore, this allele was named cmlA7.

Cloning and expression of the aac(3)-Ic gene in Escherichia coli. The aac(3)-Ic gene was amplified from the genomic DNA of P. aeruginosa VA-182/00 by using primers AAC-f (5'-GATGATCTCTACTCAAACC) and AAC-r (5'-TTAGGCAGCAGGTTGAGG) [nucleotides corresponding to the start and stop codons of aac(3)-Ic are underlined]. The 472-bp amplification product was cloned in the plasmid vector pGEM-T-Easy by using the pGEM-T-Easy Vector System I (Promega Corp., Madison, Wis.). One of the recombinants (pMLR36/02), which contained the cloned aac(3)-Ic gene in the same orientation as the Plac promoter flanking the plasmid polylinker, and in which the authenticity of the cloned fragment had been confirmed by sequencing, was used for expression experiments with E. coli DH5{alpha}. In pMLR36/02 the aac(3)-Ic ORF was expected to be expressed, since the start codon overlaps the termination codon of the LacZ {alpha}-peptide-encoding sequence, creating an artificial operon. Compared to E. coli DH5{alpha}, DH5{alpha}(pMLR36/02) showed a notable reduction in susceptibilities to gentamicin and sisomicin and a slight reduction in susceptibilities to amikacin and tobramycin. Susceptibilities to streptomycin, kanamycin, neomycin, isepamycin, and netilmicin were unaffected (Table 1). These results confirmed that the aac(3)-Ic gene could contribute to aminoglycoside resistance with a pattern typical of AAC(3)-I enzymes (18). However, this enzyme was responsible only in part for the aminoglycoside resistance phenotype of VA-182/00.

Nucleotide sequence accession number. The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL sequence database and assigned accession no. AJ511268.


   ACKNOWLEDGMENTS
 
This work was supported in part by grant 2001068755_003 (PRIN 2001) from the Italian Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR) and by a grant from Essex Italia S.p.a.


   FOOTNOTES
 
* Corresponding author. Mailing address: Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, Policlinico Le Scotte, 53100 Siena, Italy. Phone: 39 0577 233327. Fax: 39 0577 233325. E-mail: rossolini{at}unisi.it. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Aubert, D., L. Poirel, J. Chevalier, S. Leotard, J. M. Pages, and P. Nordmann. 2001. Oxacillinase-mediated resistance to cefepime and susceptibility to ceftazidime in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 45:1615-1620.[Abstract/Free Full Text]
  2. Davies, J., and G. D. Wright. 1997. Bacterial resistance to aminoglycoside antibiotics. Trends Microbiol. 5:234-239.[CrossRef][Medline]
  3. Docquier, J. D., F. Luzzaro, G. Amicosante, A. Toniolo, and G. M. Rossolini. 2001. Multidrug-resistant Pseudomonas aeruginosa producing PER-1 extended-spectrum serine-ß-lactamase and VIM-2 metallo-ß-lactamase. Emerg. Infect. Dis. 7:910-911.[Medline]
  4. 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]
  5. George, A. M., and R. M. Hall. 2002. Efflux of chloramphenicol by the CmlA1 protein. FEMS Microbiol. Lett. 209:209-213.[CrossRef][Medline]
  6. 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]
  7. 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]
  8. Gombac, F., M. L. Riccio, G. M. Rossolini, C. Lagatolla, E. Tonin, C. Monti-Bragadin, A. Lavenia, and L. Dolzani. 2002. Molecular characterization of integrons in epidemiologically unrelated clinical isolates of Acinetobacter baumannii from Italian hospitals reveals a limited diversity of gene cassette arrays. Antimicrob. Agents Chemother. 46:3665-3668.[Abstract/Free Full Text]
  9. Javier Terán, F., M. Alvarez, J. E. Suárez, and M. C. Mendoza. 1991. Characterization of two aminoglycoside-(3)-N-acetyltransferase genes and assay as epidemiological probes. J. Antimicrob. Chemother. 28:333-346.[Abstract/Free Full Text]
  10. Miller, G. H., F. J. Sabatelli, R. S. Hare, Y. Glupczynski, P. Mackey, D. Shlaes, K. Shimizu, K. J. Shaw, and the Aminoglycoside Resistance Study Groups. 1997. The most frequent aminoglycoside resistance mechanisms—changes with time and geographic area: a reflection of aminoglycoside usage patterns? Clin. Infect. Dis. 24(Suppl. 1):S46-S62.
  11. Mingeot-Leclercq, M. P., Y. Glupczynski, and P. M. Tulkens. 1999. Aminoglycosides: activity and resistance. Antimicrob. Agents Chemother. 43:727-737.[Free Full Text]
  12. Naas, T., Y. Mikami, T. Imai, L. Poirel, and P. Nordmann. 2001. Characterization of In53, a class 1 plasmid- and composite transposon-located integron of Escherichia coli which carries an unusual array of gene cassettes. J. Bacteriol. 183:235-249.[Abstract/Free Full Text]
  13. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. Approved standard M7-A5. National Committee for Clinical Laboratory Standards, Wayne, Pa.
  14. Poirel, L., T. Lambert, S. Tuerkoglue, E. Ronco, J.-L. Gaillard, and P. Nordmann. 2001. Characterization of class 1 integrons from Pseudomonas aeruginosa that contain the blaVIM-2 carbapenem-hydrolyzing ß-lactamase gene and two novel aminoglycoside resistance genes. Antimicrob. Agents Chemother. 45:546-552.[Abstract/Free Full Text]
  15. Poirel, L., T. Naas, D. Nicholas, 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]
  16. Riccio, M. L., N. Franceschini, L. Boschi, B. Caravelli, G. Cornaglia, R. Fontana, G. Amicosante, and G. M. Rossolini. 2000. Characterization of the metallo-ß-lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of blaIMP allelic variants carried by gene cassettes of different phylogeny. Antimicrob. Agents Chemother. 44:1229-1235.[Abstract/Free Full Text]
  17. Schwocho, L. R., C. P. Schaffner, G. H. Miller, R. S. Hare, and K. J. Shaw. 1995. Cloning and characterization of a 3-N-aminoglycoside acetyltransferase gene, aac(3)-Ib, from Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 39:1790-1796.[Abstract]
  18. Shaw, K. J., P. N. Rather, R. S. Hare, and G. H. Miller. 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Rev. 57:138-163.[Abstract/Free Full Text]
  19. Stokes, H. W., D. B. O'Gorman, G. D. Recchia, M. Parsekhian, and R. M. Hall. 1997. Structure and function of 59-base element recombination sites associated with mobile gene cassettes. Mol. Microbiol. 26:731-745.[CrossRef][Medline]
  20. Stokes, H. W., and R. M. Hall. 1989. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol. Microbiol. 3:1669-1683.[Medline]
  21. Wohlleben, W., W. Arnold, L. Bissonnette, A. Pelletier, A. Tanguay, P. Roy, G. Gamboa, G. Barry, E. Aubert, and J. Davies. 1989. On the evolution of Tn21-like multiresistance transposons: sequence analysis of the gene (aacC1) for gentamicin acetyltransferase-3-I (AAC(3)-I), another member of the Tn21-based expression cassette. Mol. Gen. Genet. 217:202-208.[CrossRef][Medline]
  22. Yum, J. H., K. Yi, H. Lee, D. Yong, K. Lee. J. Kim, G. M. Rossolini, and Y. Chong. 2002. Molecular characterization of metallo-ß-lactamase-producing Acinetobacter baumannii and Acinetobacter genomospecies 3 from Korea: identification of two new integrons carrying the blaVIM-2 gene cassette. J. Antimicrob. Chemother. 49:837-840.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, May 2003, p. 1746-1748, Vol. 47, No. 5
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.5.1746-1748.2003
Copyright © 2003, 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 Riccio, M. L.
Right arrow Articles by Rossolini, G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Riccio, M. L.
Right arrow Articles by Rossolini, G. M.

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