An integron cassette encoding erythromycin esterase, ere(A), from Providencia stuartii

Isabelle Plante1,2, Daniela Centrón3 and Paul H. Roy1,2,*

1 Centre de Recherche en Infectiologie, Centre Hospitalier de l’Université Laval, 2705 Boul. Laurier; 2 Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Université Laval, Ste-Foy, Québec, Canada; 3 University of Buenos Aires, Faculty of Medicine, Department of Microbiology, Paraguay 2155, Piso 12, 1121 Capital Federal, Argentina

Received 21 October 2002; returned 13 December 2002; revised 17 January 2003; accepted 21 January 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We have mapped the variable region of the two class 1 integrons found in the multiresistant strain Providencia stuartii 1723. Integron 1 contains a new arrangement of gene cassettes, aacA4-aadB-aadA1, conferring resistance to all aminoglycosides used for clinical treatment. Integron 2 contains a variant of the gene cassette ere(A), coding for an erythromycin esterase, whose nucleotide sequence shares 93.7% DNA identity with ere(A) from Escherichia coli BM2195 plasmid pIP1100.

Keywords: resistance, aminoglycosides, sequencing


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The widespread use of antibiotics has rapidly increased the problem of dissemination of antibiotic resistance genes among bacterial strains responsible for infectious diseases. The transfer of antibiotic resistance genes is possible since these genes are often on plasmids, transposons and integrons. Integrons are genetic elements composed of a variable region containing one or more gene cassettes. Three classes have been found in clinical strains of multidrug-resistant bacteria, but class 1 integrons predominate.1 Class 1 integrons have identical or nearly identical integrase genes (intI1),1 which code for the site-specific recombinase2 responsible for cassette insertion.3 Class 1 integrons also include the attI1 site4 where the cassettes are integrated and a promoter, Pc, responsible for the transcription of the cassette-encoded genes.5 Gene cassettes include a gene followed by a 59-base element (or attC site), which is composed of imperfect inverted repeat sequences and which participates in recombination events. The unique ability of integrons to move individual gene cassettes has caused a rapid and flexible spread of resistance.6 More than 60 gene cassettes containing different resistance genes have been identified in Gram-negative bacilli,7 conferring resistance to very different families of antibiotics such as aminoglycosides, ß-lactams, chloramphenicol, trimethoprim and more recently to erythromycin8,9 and rifampicin.10


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In the present work, we analysed the two integrons found in the multiresistant strain Providencia stuartii 1723, a clinical strain that was isolated in a hospital in Argentina.

PCR mapping of the first integron was carried out on total DNA in a thermal cycler (Perkin-Elmer DNA Thermal Cycler 480). Oligonucleotides used for PCR mapping are listed in Table 1. All had an annealing temperature of 55°C. To amplify the DNA, we began with a 10 min denaturation step at 94°C. The 30 subsequent cycles were 1 min of denaturation at 94°C, 1 min of annealing at 55°C and 5 min of elongation at 72°C. Finally, we continued elongation for 10 min at 72°C. Amplification products were analysed on a 1% agarose gel stained with ethidium bromide.11


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Table 1.  Oligonucleotides used for PCR analysis of integrons
 
The first stage of PCR mapping was to amplify the variable region of integrons using primers specific for 5' and 3' conserved segments. The second stage was to map cassettes using one primer in the 5' or 3' conserved sequence and the second in a cassette.

The composition of the variable region of the second integron of P. stuartii 1723 was determined by direct sequencing of an amplicon of 1.6 kb made with primers 5'CS and 3'CS (Figure 1). Nucleotide sequence determination was carried out on an Applied Biosystems 373 DNA sequencer with XL UpGrade using ABI Prism Bigdye terminator cycle sequencing ready reaction kits with the AmpliTaq DNA polymerase FS (Fluorescent Sequencing) protocol as recommended by the manufacturer (Perkin-Elmer, Mississauga, Ontario, Canada). Sequencing primers were usually 20-mers selected from the last 60–80 nucleotides read from chromatograms and synthesized on an Applied Biosystems 394 DNA/RNA synthesizer (Foster City, CA, USA). Electropherograms were visualized using Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA). DNA sequence analysis was carried out using the Genetics Computer Group software package (Wisconsin Package Version 9.0; Genetics Computer Group, Madison, WI, USA).



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Figure 1. Map of the variable region of the two integrons of P. stuartii 1723. Conserved segments are designated by terms 5'CS and 3'CS. The PCR mapping technique is illustrated. The length of each amplicon (black line) is indicated, as well as the predicted size (in parentheses). A, primer 5'CS; B, primer 3'CS; C, primer aadB; D, primer aadB-COOH; E, primer aacA4-COOH; F, primer aadA-Tn21; G, primer aadA-COOH; H, primer aacA4.

 
PCR and sequencing results were confirmed by the hybridization of restriction fragments with the probes 5'CS and 3'CS. These fragments were made from the total DNA of the strain. The probes detected two fragments in each case (data not shown), showing that P. stuartii 1723 contains two integrons.

Conjugation experiments were carried out between P. stuartii 1723 and E. coli HB101 (supE44, hsdS20, recA13, ara-14, proA2, lacY1, galK2, rpsL20, xyl-5, mtl-1) to determine whether resistance was transferable. The transconjugants were selected on BHI agar with 1 g/L streptomycin and 100 mg/L erythromycin. A PCR analysis of the total DNA of one transconjugant confirmed the transfer of the two integrons from P. stuartii 1723 to E. coli HB101. Moreover, the transconjugant was a strain of E. coli, as confirmed by the result of an API-20E identification test.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The PCR mapping technique revealed the new gene cassette arrangement aacA4-aadB-aadA1 in the variable region of the first integron, which is shown in Figure 1. The aacA4 gene cassette explains the resistance of P. stuartii 1723 to amikacin (MIC 16 mg/L) and tobramycin (MIC 64 mg/L), whereas aadB confers resistance to gentamicin and tobramycin (MIC 64 mg/L of these two antibiotics), and aadA1 to streptomycin (MIC 128 mg/L) and spectinomycin (MIC > 128 mg/L).

Direct sequencing of the 1.6 kb amplicon revealed that the second integron of P. stuartii 1723 contains the gene cassette ere(A), which is very similar (93.7% DNA identity) to ere(A) from E. coli BM2195 plasmid pIP1100 (accession no. M11277)12 and to a variant ere(A) in GenBank (accession no. AF326209, >99% DNA identity).9 The ere(A) gene, which encodes an erythromycin esterase, could explain the resistance of P. stuartii 1723 to erythromycin (MIC 128 mg/L).

The complete nucleotide sequence of the gene cassette ere(A) of P. stuartii 1723 is 1376 bp long. The analysis of this nucleotide sequence revealed an open reading frame (ORF) that codes for a polypeptide of 408 amino acids. This gene is a cassette, since it contains a 59-base element including the conserved 7 bp core site GTTRRRY at the right-hand end. However, the left-hand end contains the core site RYRYAAC rather than the conserved RYYYAAC inverse core site.13 A 59-base element is also present downstream of the ere(A) gene of pIP1100, although the gene was not identified as being in a cassette.12 This could indicate that both genes are borne on a cassette. Moreover, the 59-base elements of the two genes are closely related. An ere(A) gene cassette has also been found in the variable region of an integron in a multiresistant strain of Klebsiella pneumoniae,8 but its sequence was not published.

The two integrons of P. stuartii 1723 are on a conjugative plasmid, since they were transferable to the strain Escherichia coli HB101 by conjugation. The MICs for P. stuartii 1723, E. coli HB101 and the transconjugant P. stuartii 1723 x E. coli HB101 were determined for amikacin, gentamicin, streptomycin and erythromycin by the broth dilution method. The results are shown in Table 2.


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Table 2.  MIC of some antibiotics for the strains P. stuartii 1723, E. coli HB101 and the transconjugant 1723 x HB101
 
Concerning the erythromycin family of antibiotics, the macrolides are relatively broad-spectrum antibiotics with more activity against Gram-positive than Gram-negative bacteria, because the antibacterial activities of macrolides against Gram-negative bacilli are influenced by pH, with increasing potency (lower MICs) as the pH rises to 8.5. Nevertheless, several macrolide resistance genes have been found in Gram-negative bacteria, including ere(A), ere(B), erm(B), mph(A) and mph(B).14 For Gram-positive bacteria, several more genes have been described.14

In conclusion, we report a P. stuartii isolate harbouring two class I integrons, one with a new arrangement of cassettes, aacA4-aadB-aadA1, conferring resistance to all aminoglycosides available for clinical use, and the other belonging to a new family of erythromycin resistance genes in Gram-negative bacilli.

Nucleotide sequence accession number

The nucleotide sequence of the ere(A) gene from P. stuartii 1723 appears in the GenBank database under accession no. AF099140.


    Acknowledgements
 
This work was supported by grant MT-13564 from the Canadian Institutes for Health Research (CIHR) to P.H.R.


    Footnotes
 
* Corresponding author. Tel: +1-418-654-2705; Fax: +1-418-654-2715; E-mail: paul.roy{at}crchul.ulaval.ca Back


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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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