JAC Advance Access published online on August 15, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn331
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Original research |
Prevalence and diversity of integrons and associated resistance genes in faecal Escherichia coli isolates of healthy humans in Spain
1 Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain 2 CIBIR, Unidad de Microbiología Molecular, Logroño, Spain 3 Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain
* Correspondence address. Área de Bioquímica y Biología Molecular, Departamento de Agricultura y Alimentación, Universidad de La Rioja, Madre de Dios 51, 26006 Logroño, Spain. Tel: +34-941-299750; Fax: +34-941-299721; E-mail: carmen.torres{at}unirioja.es
Received 18 April 2008; returned 22 May 2008; revised 21 July 2008; accepted 23 July 2008
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Abstract |
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Objectives: To analyse the prevalence and diversity of integrons in faecal Escherichia coli isolates from healthy humans in Spain.
Methods: One hundred E. coli isolates were obtained in Levine agar plates from faecal samples of 100 healthy humans during March to October 2007. Susceptibility to 16 antimicrobial agents was determined by the disc diffusion method. The presence and characterization of class 1, 2 and 3 integrons, as well as the presence of other antimicrobial resistance genes, were performed by PCR and DNA sequencing.
Results: Integrases associated with class 1 and/or class 2 integrons were identified in 29 E. coli isolates (intI1 gene in 26 isolates, intI2 in 1 isolate and intI1 + intI2 in 2 isolates), the remaining 71 isolates being free of these integrons. Seven different gene cassette arrangements were demonstrated in 27 of the 28 intI1-positive isolates and were as follows (number of isolates): dfrA1 + aadA1 (12), aadA (8), dfrA17 + aadA5 (3), dfrA7 (1), dfrA5 (1), dfrA1 (1) and dfrA12 + orfF + aadA2 (1). Four isolates presented defective class 1 integrons lacking the 3'-conserved region. The three isolates containing class 2 integrons harboured the dfrA1 + sat + aadA1 gene cassette array in their variable region. Integron-positive isolates showed higher percentages of resistance to streptomycin, ampicillin, tetracycline, trimethoprim, sulfamethoxazole, chloramphenicol and nalidixic acid than integron-negative isolates. Sixty-five percent of the integron-positive isolates belonged to phylogenetic groups A or D.
Conclusions: A high prevalence of integrons was detected in faecal E. coli of healthy humans. Individuals in the community could be a reservoir of integron-containing E. coli isolates.
Key Words: E. coli , antimicrobial resistance , gene cassettes
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Introduction |
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Integrons play an important role in the antimicrobial resistance of clinical Escherichia coli strains because they are able to capture, integrate and express gene cassettes encoding proteins associated with antimicrobial resistance. Furthermore, the capture of genes is particularly important when these integrons are mobilized by broad-host-range conjugative plasmids or transposons. The presence of integrons in clinical multiresistant E. coli isolates recovered from the hospital environment is frequently reported. However, there are relatively few reports about the presence of integrons in healthy individuals.1–3 The objective of our study was to determine the prevalence and diversity of class 1, 2 and 3 integrons in faecal E. coli isolates of healthy humans and to analyse the associated resistance genes and the phylogenetic groups of the integron-positive isolates.
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Materials and methods |
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One hundred faecal samples were recovered between March and October 2007 from healthy humans living in two regions of Spain, Madrid (23 samples) and La Rioja (77 samples), located in the centre and in the north of the country, respectively. Eighty-nine percent of the individuals lived in urban areas. The age of healthy humans ranged from 3 to 85 years (<10 years, 5%; 20–40 years, 50%; 40–60 years, 29% and >60 years, 16%), and none of them were exposed to antimicrobial agents or to a hospital environment in the 3 months prior to sample recovery. All individuals (or their parents in the case of children) gave informed consent for participation in this study.
Faecal samples were seeded on Levine agar plates and were incubated at 37°C for 24 h. One colony per sample with a typical E. coli morphology (black and metallic green colonies) was recovered and identified by classical biochemical methods (Gram, TSI, indole, Methyl-Red-Voges-Proskauer and urease) and by PCR amplification of the uidA gene, which encodes a β-glucuronidase protein specific for E. coli.
Susceptibility to 16 antimicrobial agents (ampicillin, amoxicillin/clavulanic acid, cefoxitin, cefotaxime, ceftazidime, imipenem, aztreonam, gentamicin, streptomycin, kanamycin, nalidixic acid, ciprofloxacin, trimethoprim/sulfamethoxazole, sulphonamides, tetracycline and chloramphenicol) was determined for all isolates by the disc diffusion method.4 E. coli ATCC 25922 was used as a control strain.
The presence of class 1, 2 and 3 integrons was analysed in all E. coli isolates obtained in this study. PCR amplification was used to detect the intI1, intI2 and intI3 genes, as well as the 3'-conserved region (3' CS) of class 1 integrons (qac
E+sul1 genes).5 The characterization of the variable region of class 1 and 2 integrons was performed by PCR and subsequent DNA sequencing.5 The presence of genes associated with ampicillin (blaTEM, blaSHV and blaOXA-1), tetracycline [tet(A)–tet(E), tet(G) and tet(M)], streptomycin (aadA), sulphonamide (sul1, sul2 and sul3), kanamycin [aph(3')-Ia and aph(3')-IIa] and chloramphenicol resistance (cmlA and floR) was also analysed by PCR.5 Chloramphenicol–acetyltransferase activity was studied, as described previously.5 The identification of the major phylogenetic groups of the integron-positive isolates was carried out by PCR.6 Positive and negative controls from our E. coli strain collection of the University of La Rioja were included in all PCRs.
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Results and discussion |
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One hundred E. coli isolates were obtained from the studied faecal samples (one isolate per sample), and the presence of integrons was demonstrated in 29 of them (29%). The intI1 gene was identified in 26 of these isolates, the intI2 gene in 1 isolate and both the intI1 and intI2 genes in 2 additional isolates (Table 1). The intI3 gene was not identified in our bacterial collection, and similar results were also previously reported by others.1,3
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Seven different gene cassette arrangements were demonstrated in 27 of the 28 intI1-positive isolates and were as follows (number of isolates): dfrA1 + aadA1 (12), aadA (8), dfrA17 + aadA5 (3), dfrA7 (1), dfrA5 (1), dfrA1 (1) and dfrA12 + orfF + aadA2 (1) (Table 1). It is interesting that the qac
E + sul1 3' CS fragment was missing in 4 of the 28 intI1-positive isolates (14.3%). The genetic composition of the variable region of these four isolates was studied in detail by PCR amplification using the primer walking strategy; dfrA1, dfrA5 and dfrA12 + orfF + aadA2 gene cassette arrangements were found in three of these isolates, but all the PCRs performed were negative for the remaining isolate (E. coli Pn240). The presence of these 3' CS-lacking integrons has also been reported at low frequencies in E. coli recovered from healthy humans and animals.2,5 The three isolates of our study containing class 2 integrons presented the same gene cassette array in their variable region, i.e. dfrA1 + sat + aadA1, also being frequent in other studies.1,3,5 Most of the gene cassettes found within the variable region of class 1 integrons in our E. coli isolates corresponded to different variants of dfrA and aadA genes (43% and 54%, respectively), and similar results were previously obtained in commensal E. coli isolates from healthy subjects (33% and 55%, respectively).1 These genes are associated with trimethoprim and streptomycin resistance, respectively, and dfrA1 + aadA1 was the combination most frequently detected not only in our study but also in E. coli isolates recovered from healthy and sick humans, animals and foods in other studies.1–3,5,7–9 The reason for the wide distribution of some successful integrons with a specific arrangement is not known, although the possible inclusion of these integrons in transposons and/or plasmids could explain their wide dissemination in different environments.
The antimicrobial resistance phenotypes were studied in all E. coli isolates of our study, and the percentages of resistance detected were as follows (% among integron-positive/% among integron-negative isolates): sulphonamides (97/17), trimethoprim/sulfamethoxazole (79/7), streptomycin (79/17), tetracycline (86/8), ampicillin (83/16), amoxicillin/clavulanic acid (7/3), chloramphenicol (21/1), nalidixic acid (14/6), kanamycin (3/3) and gentamicin (0/3). All isolates were susceptible to cefoxitin, ceftazidime, cefotaxime, imipenem, aztreonam and ciprofloxacin. All 29 integron-positive isolates showed resistance to at least three antimicrobial agents, as found in other studies,1,8,9 but only 10 of our 71 integron-negative isolates presented this characteristic (14%). Higher percentages of resistance to some antimicrobial agents (streptomycin, ampicillin, tetracycline, trimethoprim, sulfamethoxazole, chloramphenicol and nalidixic acid) were observed among integron-positive isolates with respect to integron-negative isolates. This fact could be explained by the presence of resistance genes in the conserved or variable region of integrons (as is the case for genes associated with sulfamethoxazole, trimethoprim and streptomycin resistance) or by the inclusion of resistance genes in the same mobile elements that carry integrons.
Table 2 shows the antimicrobial resistance genes detected in our isolates. A blaTEM gene was detected in 17 of the 35 ampicillin-resistant isolates and the blaSHV-1 gene in 1 additional isolate. Regarding tetracycline resistance, a diversity of tet genes was found in the resistant integron-positive isolates [tet(A), tet(B), tet(C) and tet(D)], the tet(A) gene being the predominant one. It has been previously suggested that the tet(A) gene and class 1 integrons are often located on the same conjugative plasmid.10
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The following genes were identified among the 40 sulphonamide-resistant isolates (integron-positive/integron-negative isolates): sul1 (15/0), sul2 (3/9), sul3 (1/0) and sul1+sul2 (9/1). The prevalence of sul1 and sul2 genes in our integron-positive isolates (86% and 43%, respectively) was similar to previously reported prevalences (67% and 56%, respectively) in class 1 integron-containing E. coli isolates from healthy children.2 The aph(3')-Ia gene was found in two of the three kanamycin-resistant E. coli isolates. Among the six chloramphenicol-resistant integron-positive isolates, the cmlA gene was found in one isolate and chloramphenicol–acetyltransferase activity was demonstrated in four other isolates.
The phylogenetic groups D and A were the most prevalent ones among our integron-containing isolates (10 and 9 isolates, respectively; 65.5%), and groups B1 and B2 were each identified in five isolates. Similar to our data, class 1 integrons were detected more frequently among E. coli isolates of the phylogenetic group A, and some authors suggest that B2 E. coli isolates could be less resistant to antimicrobials than non-B2 isolates.1,3,7
In conclusion, a high prevalence of integrons was detected in faecal E. coli isolates of healthy humans (29%), the dfrA1 + aadA1 gene cassette combination being most frequently found in their variable region, and this fact suggests that individuals in the community could be a reservoir of integron-containing E. coli isolates.
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This work was partially supported by the Project SAF2006- 14207-C02 from the Ministry of Education and Science of Spain. L. V. has a fellowship from the Spanish Ministry of Education and Science (SAF2006-14207-C02-01), and S. S. has a fellowship from the Gobierno de La Rioja, Spain (Colabora 2007/15).
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Transparency declarations |
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None to declare.
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References |
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1 . Cocchi S, Grasselli E, Gutacker M, et al. Distribution and characterization of integrons in Escherichia coli strains of animal and human origin. FEMS Immunol Med Microbiol (2007) 50:126–32.[CrossRef][Web of Science][Medline]
2 . Infante B, Grape M, Larsson M, et al. Acquired sulphonamide resistance genes in faecal Escherichia coli from healthy children in Bolivia and Peru. Int J Antimicrob Agents (2005) 25:308–12.[CrossRef][Web of Science][Medline]
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Skurnik D, Le Menac'h A, Zurakowski D, et al. Integron-associated antimicrobial resistance and phylogenetic grouping of Escherichia coli isolates from healthy subjects free of recent antibiotic exposure. Antimicrob Agents Chemother (2005) 49:3062–5.
4 . Clinical and Laboratory Standards Institute. Performance Standards For Antimicrobial Susceptibility Testing: Seventeenth Informational Supplement Approved Standard M100-S17 (2007) Wayne, PA, USA: CLSI.
5 . Sáenz Y, Briñas L, Domínguez E, et al. Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal, and food origins. Antimicrob Agents Chemother (2004) 48:3995–4001.
6
.
Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol (2000) 66:4555–8.
7
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Machado E, Ferreira J, Novais A, et al. Preservation of integron types among Enterobacteriaceae producing extended-spectrum β-lactamases in a Spanish hospital over a 15-year period (1988 to 2003). Antimicrob Agents Chemother (2007) 51:2201–4.
8
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Sunde M. Prevalence and characterization of class 1 and class 2 integrons in Escherichia coli isolated from meat and meat products of Norwegian origin. J Antimicrob Chemother (2005) 56:1019–24.
9
.
Kang HY, Jeong YS, Oh JY, et al. Characterization of antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea. J Antimicrob Chemother (2005) 55:639–44.
10
.
Sunde M, Norström M. The prevalence of, associations between and conjugal transfer of antibiotic resistance genes in Escherichia coli isolated from Norwegian meat and meat products. J Antimicrob Chemother (2006) 58:741–7.
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