1 Centre de Recherche en Infectiologie, Centre Hospitalier de lUniversité 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: resistance, aminoglycosides, sequencing
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
|
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 6080 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).
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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 |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Martinez, E. & de la Cruz, F. (1990). Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes. EMBO Journal 9, 127581.[Abstract]
3 . Collis, C. M., Grammaticopoulos, G., Briton, J., Stokes, H. W. & Hall, R. M. (1993). Site-specific insertion of gene cassettes into integrons. Molecular Microbiology 9, 4152.[ISI][Medline]
4
.
Partridge, S. R., Recchia, G. D., Scaramuzzi, C., Collis, C. M., Stokes, H. W. & Hall, R. M. (2000). Definition of the attI1 site of class1 integrons. Microbiology 146, 285564.
5 . Collis, C. M. & Hall, R. M. (1995). Expression of antibiotic resistance genes in the integrated cassettes of integrons. Antimicrobial Agents and Chemotherapy 39, 15562.[Abstract]
6 . Roy, P. H. (1997). Dissémination de la résistance aux antibiotiques: le génie génétique à loeuvre chez les bactéries. Médecine/Sciences 13, 92733.
7 . Hall, R. M. & Collis, C. M. (1998). Antibiotic resistance in gram negative bacteria: the role of gene cassettes and integrons. Drug Resistance Updates 1, 10919.[ISI]
8 . Jones, M. E., Peters, E., Weersink, A. M., Fluit, A. C. & Verhoef, J. (1997). Widespread occurrence of integrons causing multiple antibiotic resistance in bacteria. Lancet 349, 17423.[ISI][Medline]
9
.
Peters, E. D. J., Leverstein-van Hall, M. A., Box, A. T. A., Verhoef, J. & Fluit, A. C. (2001). Novel gene cassettes and integrons. Antimicrobial Agents and Chemotherapy 45, 29614.
10
.
Tribuddharat, C. & Fennewald, M. (1999). Integron-mediated rifampin resistance in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 43, 9602.
11 . Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
12 . Ounissi, H. & Courvalin, P. (1985). Nucleotide sequence of the gene ereA encoding the erythromycin esterase in Escherichia coli. Gene 35, 2718.[CrossRef][ISI][Medline]
13 . Stokes, H. W., OGorman, D. B., Recchia, G. D., Parsekhian, M. & Hall, R. M. (1997). Structure and function of 59-base element recombination sites associated with mobile gene cassettes. Molecular Microbiology 26, 73145.[ISI][Medline]
14
.
Roberts, M. C., Sutcliffe, J., Courvalin, P., Jensen, L. B., Rood, J. & Seppala, H. (1999). Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants. Antimicrobial Agents and Chemotherapy 43, 282330.