Prevalence and types of class 1 integrons in aminoglycoside-resistant Enterobacteriaceae from several Chilean hospitals

Angélica Reyes1, Helia Bello1, Mariana Domínguez1, S. Mella1, R. Zemelman2 and G. González1,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Members of the family Enterobacteriaceae are responsible for a variety of nosocomial infections, treatment of which is limited due to their increasing resistance to antibiotics. Some bacterial genes encoding antibiotic resistance comprise the major part of gene cassettes, most of which are associated with integrons. In this work, the carriage of class 1, 2 and 3 integrons was investigated in 191 Enterobacteriaceae isolates from clinical specimens of hospitalized patients. Class 1 integrons were found to be the most common, whereas no class 3 integrons were detected. The variable regions of 13 class 1 integrons were characterized and four types were found. Type 1 harbours only ant(3'')I, type 2 harbours ant(2'')I and ant(3'')I, type 3 harbours aac(6')Ib and ant(3'')I and type 4 lacks inserted gene cassettes.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Members of the family Enterobacteriaceae, such as Escherichia coli, Klebsiella pneumoniae subsp. pneumoniae, Klebsiella oxytoca, Serratia marcescens, Proteus spp., Enterobacter spp., Salmonella spp., Shigella spp., Yersinia spp. and Providencia spp., are frequently identified as aetiological agents of nosocomial and community-acquired infections.1 In hospital, immunosuppressed patients, especially those admitted to intensive care units, are susceptible to infections produced by one or other of these bacteria.2 The frequent use of antibiotics has contributed greatly to the selection of antibiotic-resistant clones of these microbes,3 and this resistance is frequently mediated by genes localized on extrachromosomal genetic elements, such as plasmids, transposons and gene cassettes inserted into integrons. This location favours dissemination of resistance genes among nosocomial bacteria.4

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 (qacE{Delta}1), 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

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 1998–2000, 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 Tris–HCl, 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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Table 1.  Sequences of oligonucleotides used as primers for PCR
 
The identities of gene cassettes inserted in the variable regions were determined by PCR analysis, using specific primer pairs shown in Table 1, and the same PCR programme was used to detect integrons.

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 qacE{Delta}1 genes, respectively.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Presence of integrons

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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Table 2.  General features of isolates characterized for class 1 integrons
 
The ant(3'')I gene was also detected in all strains, and always in a class 1 integron adjacent to the 3'CS. When primers sulpro3, which targets the 5'CS, and aadA.R, which targets ant(3'')I, were employed, three PCR products of sizes 980, 1500 and 1800 bp were recovered, indicating the presence of different sequences between ant(3'')I and the 5'CS. Thus, amplicons of 980 bp identified integrons carrying only the ant(3'')I gene cassette, whereas amplicons of 1500 and 1800 bp indicated carriage of the aac(6')Ib or ant(2'')I gene cassette between the 5'CS and ant(3'')I, respectively.

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 qacE{Delta}1 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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hall & Stokes10 initially described integrons as genetic elements that behave as gene expression vectors and that express resistance to different antibiotics, a general description later confirmed by other authors.4,1315 Our results provide additional evidence to support this view. The prevalence of class 1 integrons among isolates of Enterobacteriaceae investigated in this work agrees with that reported by others.16,17 Whereas gene cassettes encoding the aminoglycoside-modifying enzymes AAC(6')Ib, ANT(2'')I and ANT(3'')I were detected in the study, those for AAC(3)I and AAC(6')II were not (where AAC and ANT stand for aminoglycoside acetyltransferase and nucleotidyltransferase). Whereas the aac(6')Ib gene was found in all 13 isolates investigated, only in the E. coli isolates was it class 1 integron associated. The selective pressure exerted by aminoglycosides, mainly amikacin, in Chilean hospitals might account for this finding. Del Solar et al.18 have previously reported that AAC(6')Ib is commonly produced by nosocomial Gram-negative isolates in Chilean hospitals.

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.


    Acknowledgements
 
We gratefully acknowledge the Fondo Nacional de Ciencia y Tecnologia (FONDECYT) of Chile for supporting this investigation (Grant 1000352), and also the Laboratories of Microbiology of the different hospitals included in the study for their kind collaboration in the selection of clinical isolates.


    Footnotes
 
* Corresponding author. Tel: +56-41-20-32-37; Fax: +56-41-24-59-75; E-mail: ggonzal{at}udec.cl Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Holt, J., Krieg, N., Sneath, P., Staley, J. & Williams, S. (1994). Bergey’s Manual of Determinative Bacteriology, 9th edn, pp. 175–87. Williams & Wilkins, Baltimore, MD, USA.

2 . Archibald, L., Phillips, L., Monnet, D., McGowan J. E., Tenover, F. C. & Gaynes, R. P. (1997). Antimicrobial resistance in isolates from inpatients and outpatients in the United States: increasing importance of the intensive care unit. Clinical Infectious Diseases 24, 211–5.[ISI][Medline]

3 . Amyes, S. & Gemmell, C. (1992). Antibiotic resistance in bacteria. Journal of Medical Microbiology 36, 4–29.[ISI][Medline]

4 . Rechia, G. & Hall, R. (1995) Gene cassettes: a new class of mobile element. Microbiology 141, 3015–27.[ISI][Medline]

5 . Bennet, P. M. (1999). Integrons and gene cassettes: a genetic construction kit for bacteria. Journal of Antimicrobial Chemotherapy 43, 1–4.[Free Full Text]

6 . Collis, C. & Hall, R. (1995). Expression of antibiotic resistance in the integrated cassettes of integrons. Antimicrobial Agents and Chemotherapy 39, 155–62.[Abstract]

7 . Gravel, A., Messier, N. & Roy, P. H. (1998). Point mutations in the integron integrase IntI1 that affect recombination and/or substrate recognition. Journal of Bacteriology 180, 5437–42.[Abstract/Free Full Text]

8 . Fluit, A. C. & Schmitz, F. J. (1999). Class 1 integrons, gene cassettes, mobility, and epidemiology. European Journal of Clinical Microbiology and Infectious Diseases 18, 761–70.[CrossRef][ISI][Medline]

9 . Jones, M. E., Peters, E., Weersink, A.-M., Fluit, A. & Verhoef, J. (1997). Widespread occurrence of integrons causing multiple antibiotic resistance in bacteria. Lancet 349, 1742–3.[ISI][Medline]

10 . Hall, R. & Stokes, H. (1993). Integrons: novel DNA elements which capture genes by site-specific recombination. Genetica 90, 115–32.[ISI][Medline]

11 . Rosser, S. & Young, H.-K. (1999). Identification and characterization of class 1 integrons in bacteria from an aquatic enviroment. Journal of Antimicrobial Chemotherapy 44, 11–8.[Abstract/Free Full Text]

12 . Senda, K., Arakawa, Y., Ichiyama, S., Nakashima, K., Ito, H., Oshuka, S. et al. (1996). PCR detection of metallo-ß-lactamase gene (blaIMP) in Gram-negative rods resistant to broad-spectrum ß-lactams. Journal of Clinical Microbiology 34, 2909–13.[Abstract]

13 . Lee, Y., Han, H., Seong, C., Lee, H. & Jung, J. (1998). Distribution of genes coding for aminoglycoside acetyltranferases in gentamicin resistant bacteria isolated from aquatic environment. Journal of Microbiology 36, 249–55.[ISI]

14 . Martinez-Freijo, P., Fluit, A., Schmitz, F.-J., Grek, V., Verhoef, J. & Jones, M. (1998). Class I integrons in Gram-negative isolates from different European hospitals and association with decreased susceptibilitiy to multiple antibiotic compounds. Journal of Antimicrobial Chemotherapy 42, 689–96.[Abstract]

15 . Ploy, M., Lambert, T., Gassama, A. & Denis, F. (2000). The role of integrons in dissemination of antibiotic resistance. Annales Biologie Clinique 58, 439–44.

16 . Chang, C., Chang, L., Chang, Y., Lee, T. & Chang, S. (2000). Characterisation of drug resistance gene cassettes associated with class 1 integrons in clinical isolates of Escherichia coli from Taiwan, ROC. Journal of Medical Microbiology 49, 1097–102.[Abstract/Free Full Text]

17 . White, P., McIver, C. & Rawlinson, W. (2001). Integrons and gene cassettes in the Enterobacteriaceae. Antimicrobial Agents and Chemotherapy 45, 2658–61.[Abstract/Free Full Text]

18 . Del Solar, E., García, A., Bello, H., Domínguez, M., González, G. & Zemelman, R. (1995). Mecanismos enzimáticos de resistencia a antibióticos aminoglicósidos en bacilos Gram negativos de hospitales chilenos. Revista Médica de Chile 123, 293–7.[ISI][Medline]

19 . Seward, R., Lambert, T. & Towner, K. (1998). Molecular epidemiology of aminoglycoside resistance in Acinetobacer spp. Journal of Medical Microbiology 47, 455–62.[Abstract]

20 . Lévesque, C., Piché, L., Larose, C. & Roy, P. (1995). PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrobial Agents and Chemotherapy 39, 185–91.[Abstract]

21 . Gravel, A., Fournier, B. & Roy, P. H. (1998). DNA complexes obtained with the integron integrase IntI1 at the att1 site. Nucleic Acids Research 26, 4347–55.[Abstract/Free Full Text]

22 . Hannecart-Pokorni, E., Depuydt, F., De Wit, L., Van Bossuyt, E., Content, J. & Vanhoof, R. (1997). Characterization of the 6'-N-aminoglycoside acetyltransferase gene aac(6')-Il associated with a sul1-type integron. Antimicrobial Agents and Chemotherapy 41, 314–8.[Abstract]