1 Departamento de Microbiología, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C., Concepción; 2 Facultad de Medicina, Universidad San Sebastián, Concepción, Chile
Received 31 January 2002; returned 15 July 2002; revised 2 September 2002; accepted 6 October 2002
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Abstract |
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Introduction |
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Integrons encode site-specific recombination systems that capture and express gene cassettes, thus becoming natural expression vectors of these genes.5,6 Based on the nature of the integrase,7 three classes of integrons, with clinical and epidemiological relevance for antibiotic resistance, have been described. Class 1, the best characterized integrons, have been reported in clinical and environmental isolates of several Gram-negative bacilli.8,9 Integrons of this class comprise two conserved segments flanking another, of variable length, within which are found antibiotic resistance gene cassettes.4,8 The 5' conserved end (5'CS) encodes a DNA integrase (IntI1) that mobilizes and inserts gene cassettes through a site-specific recombinational mechanism at a specific site (attI) adjacent to the IntI gene.10 Thus, this end of the integron behaves as a receptor for gene cassettes. The 5'CS also contains a promoter sequence, Pant, needed for the expression of most of the genes carried on cassettes.6 The 3' conserved end (3'CS) of class 1 integrons includes a truncated antiseptic resistance gene (qacE1), a sulphonamide resistance gene (sul1) and an open reading frame (orf5) of unknown function.4,5,10
In this study, the prevalence of integrons of different classes in 191 clinical isolates of enterobacteria from various Chilean hospitals was investigated. The variable regions of 13 class 1 integrons were genetically characterized.
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Materials and methods |
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One hundred and ninety-one clinical isolates of Enterobacteriaceae, identified as E. coli (128 strains), K. pneumoniae (26 strains), Proteus mirabilis (30 strains) and Shigella spp. (seven strains), isolated from various Chilean hospitals during 19982000, were included in the study. Thirteen strains (eight E. coli, two K. pneumoniae, three P. mirabilis) were selected for further study on the basis of the presence of class 1 integrons and aminoglycoside resistance. Isolates were from the following hospitals: Hospital Base, Lota; Hospital San Jose, Coronel; Hospital Base, San Carlos; Hospital Dr Guillermo Grant B. and Hospital Sanatorio Aleman, Concepción; Hospital Dr Gustavo Fricke, Viña del Mar; Hospital Clínico Pontificia Universidad Catolica de Chile and Hospital San Borja Arriarán, Santiago.
PCR
PCR was performed using 2.5 µL of 10x dNTPs mix (1.25 mM each of dATP, dCTP, dGTP and dTTP), 2.5 µL of each primer (0.5 µM), 2.5 µL of 10x PCR buffer, 1.25 µL of MgCl2 (50 mM), 3.6 µL of sterile distilled water (SDW), 0.15 µL of Taq DNA polymerase (5 U/µL) and 10 µL of the DNA template. Template was prepared by mixing 300 µL of an overnight bacterial culture and 700 µL of SDW, boiling the mixture for 15 min and then centrifuging at 14 000 rpm for 5 min. The supernatant was used directly as the source of template.
DNA was amplified by PCR using the following cycle conditions: 96°C for 30 s, 55°C for 1 min, 70°C for 3 min (one cycle), followed by 96°C for 15 s, 55°C for 30 s, 70°C for 3 min (25 cycles) and a final extension of 70°C for 5 min. The primers used to amplify the intI1 gene (class 1) were those described previously11 (intA, 5'-ATCATCGTCGTAGAGACGTCGG-3'; intB, 5'-GTCAAGGTTCTGGACCAGTTGC-3'). The intI2 gene (class 2) was amplified with the primers described by Daniela Centron (Universidad de Buenos Aires, Argentina, personal communication) (Inti2F, 5'-GCAAATGAAGTGCAACGC-3'; Inti2R, 5'-ACACGCTTGCTAACGATG-3') and the intI3 gene (class 3) with primers reported by Senda et al.12 (IntI3-200, 5'-GCAGGGTGTGGACGAATACG-3'; Int3-940, 5'-ACAGACCGAGAA-GGCTTATG-3').
The expected sizes of PCR products were 892 bp for intI1, 467 bp for intI2 and 760 bp for intI3. PCR products were confirmed to be intI1 amplicons by restriction with SphI, which results in two fragments of 393 and 499 bp. The intI2 gene PCR products were restricted with HaeIII, which results in two fragments of 197 and 270 bp.
Electrophoresis of amplification products
PCR products were concentrated by electrophoresis in 1% agarose gels at 100 V in 0.5x TAE buffer (0.4 M TrisHCl, 0.02 M Na2EDTA·2H2O, 0.2 M sodium acetate, 1.02 M acetic acid), and visualized by ultraviolet illumination after staining the gels with ethidium bromide (0.5 mg/L).
Characterization of class 1 integrons
The variable regions of integrons were amplified using primers sulpro3/CASS2 (Table 1). The PCR conditions used were those recommended by H. K. Young (University of Dundee, Scotland, personal communication): 94°C for 2 min, followed by 94°C for 10 s, 55°C for 30 s and 68°C for 5 min (10 cycles), and then 20 similar cycles, but with the annealing step increased by 20 s, plus a final extension at 65°C for 7 min.
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Characterization of the 3'CS of the class 1 integrons
Specific primer pairs Sul1/Sul1.rev and orf4/Sul1.rev (Table 1) were used to detect sul1 and qacE1 genes, respectively.
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Results |
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Among isolates of E. coli and K. pneumoniae, class 1 integrons were common (52 and 10 strains, respectively), whereas class 2 integrons were found in only 27 strains of E. coli, and of these 17 also carried a class 1 integron. None of the K. pneumoniae isolates harboured class 2 integrons. Among Shigella spp., class 2 integrons were found in five strains. One of these isolates also contained a class 1 integron. In P. mirabilis, class 1 and 2 integrons were more commonly found together (17 of 30 strains tested), followed by those strains that contained only class 2 integrons (four strains). Class 3 integrons were not detected in the isolates of Enterobacteriaceae investigated in this study.
Sizes of the variable regions of class 1 integrons
The variable regions of class 1 integrons investigated have sizes between 1400 and 2000 bp. Amplicons with sizes of 1400 bp were found in six strains, an indication that these isolates harbour integrons with only one gene cassette, given that an integron with no gene cassette yields a PCR product of
600 bp. Seven isolates yielded amplicons of
2000 bp, suggesting carriage of two gene cassettes. Eight strains of E. coli yielded a second amplicon of
580 bp, indicating the likely presence of a second integron lacking inserted gene cassettes.
Characterization of the variable regions of class 1 integrons
From PCR analysis, the following gene cassettes were identified: aac(6')Ib, ant(2'')I and ant(3'')I (Table 2). All isolates harboured aac(6')Ib, but only in the E. coli isolates was this gene located in the variable region of class 1 integrons.
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The 3'CS of class 1 integrons was detected in all isolates, PCR products obtained with primer pairs Sul1/Sul1.rev (408 bp) and orf4/Sul1.rev (872 bp), revealing the presence of sul1 and qacE1 genes, respectively.
From the gene cassettes found in this study and their assortments in the variable regions of the class 1 integron structure, four types of integrons were detected: type 1 carries ant(3'')I, type 2 carries ant(2'')I and ant(3'')I, type 3 carries aac(6')Ib and ant(3'')I, and type 4 lacks gene cassettes.
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Discussion |
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Since the sul1 gene is part of the conserved segment (3'CS) of class 1 integrons, and sulphonamide is commonly used in Chile, this is likely to exert pressure for the selection of sulphonamide-resistant Gram-negative bacteria that may also be resistant to other antibacterial agents encoded by gene cassettes inserted in integrons.
The types of class 1 integrons described in this work have been found in fermenting and non-fermenting Gram-negative bacilli, not only from clinical sources,16,19 but also in bacteria from aquatic environments,11 suggesting that these gene cassettes are maintained inside the integrons in the absence of overt antibiotic selective pressure. Type 2 integrons, also found in this study, have been reported in nosocomial isolates of Acinetobacter baumannii,19 indicating mobilization of these elements between bacteria of different genera. The type 3 class 1 integrons in the subset of isolates (Table 2) are likely to be responsible, in part, for the dissemination of the aac(6')Ib gene that was found in all the strains.
The results of this study demonstrate that carriage of integrons can, in part, explain the presence of resistance genes in bacteria obtained from environments in which the particular antibiotics are commonly present. Also, the gene cassettes located in integrons, as found in this study, can explain the broad resistance of these bacteria to several aminoglycosides.
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Acknowledgements |
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Footnotes |
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References |
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