JAC Advance Access originally published online on March 9, 2007
Journal of Antimicrobial Chemotherapy 2007 59(4):742-745; doi:10.1093/jac/dkl538
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Characterization of sulphonamide resistance genes and class 1 integron gene cassettes in Enterobacteriaceae, Central African Republic (CAR)
1 Institut Pasteur de Bangui, Bangui, Central African Republic 2 Université Pierre et Marie Curie, Paris 6, EA 2392, Laboratoire de Bactériologie, F-75005, Paris, France 3 Service de Bactériologie-Hygiène, Hôpital Tenon, AP-HP, Paris, France
* Corresponding author. Service de Bactériologie-Hygiène, Hôpital Tenon, AP-HP, rue de la Chine, 75970 Paris cedex 20, France. Tel: +33 1 56 01 70 1; Fax: +33 1 56 01 61 08; E-mail: guillaume.arlet{at}tnn.aphp.fr
Received 11 July 2006; returned 8 August 2006; revised 11 December 2006; accepted 12 December 2006
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Abstract |
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Objectives: The aim of this study was to characterize genes encoding sulphonamide resistance and gene cassettes associated with class 1 integrons in trimethoprim-sulphamethoxazole resistant Enterobacteriaceae recovered from Bangui, Central African Republic (CAR).
Methods: We studied 78 clinical Enterobacteriaceae isolates, including 16 extended-spectrum ß-lactamases producers, 10 Salmonella and 9 Shigella, resistant to trimethoprim-sulphamethoxazole as assessed by the disc diffusion method. PCR was used to test for sul1 and sul2 genes. Class 1 integron resistance gene cassettes were characterized by directly sequencing PCR products obtained with primers recognising 5' and 3' conserved regions.
Results: The sul1 gene was found in 67 isolates, the sul2 gene in 72 isolates and both genes in 62 isolates, while the int1 gene was found in 74 isolates. The most prevalent dfr genes were dfrA7 (49%), dfrA1 (17%) and dfrA2d (13%).
Conclusion: These results illustrate the wide distribution of sulphonamide and trimethoprim resistance genes among Enterobacteriaceae in Bangui (CAR).
Keywords: co-trimoxazole resistance, gene cassettes, antimicrobial agents
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Introduction |
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Sulphonamides and trimethoprim are inexpensive antibiotics that have a synergistic effect.1 Consequently, they have been used in combination (co-trimoxazole) since 1968 for a wide range of clinical indications including uncomplicated urinary tract infections, enteric bacterial diseases and respiratory tract infections.1 Plasmid-mediated resistance to sulphonamides and trimethoprim is normally due to the acquisition of novel target enzymes that are naturally resistant: dihydropteroate synthases for sulphonamides and dihydrofolate reductases for trimethoprim.2 Three resistance genes, sul1, sul2 and sul3 encoding dihydropteroate synthases and more than 20 dihydrofolate reductase (dfr) genes have been described. Both groups of genes are associated with class 1 integrons residing in plasmids and/or the bacterial chromosome.2–4
Our knowledge of resistance to sulphonamides and trimethoprim in developing countries is not extensive. Some reports indicate that the prevalence of resistance in enterobacterial pathogens isolated in these countries is very high (33–96%) compared to isolates from developed countries (7–24%).3,5–7
Recent studies in the Central African Republic (CAR) showed that more than 76% of Enterobacteriaceae recovered from urinary tract infections in 2000–2002 and from bloodstream infections in 1999 were resistant to co-trimoxazole.8,9
Here, we report the molecular characterization of sulphonamide resistance genes and gene cassettes associated with class I integrons in various Enterobacteriaceae including extended spectrum ß-lactamase (ESBL) producers and enteric pathogens such as Salmonella and Shigella recovered at the Pasteur Institute, Bangui (CAR).
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Materials and methods |
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Bacterial isolates and antimicrobial susceptibility testing
Sixty-one ESBL-negative-Enterobacteriaceae isolated between October 2004 and June 2005 and 17 ESBL-producing Enterobacteriaceae isolated between January 2003 and October 2005, were recovered at Pasteur Institute in Bangui (CAR) from clinical samples sent by four public health centres, one paediatric hospital and ambulatory medical visits; all these centres are located in Bangui, the capital of Central African Republic. The isolates (one isolate/patient) were associated with urinary tract infections (n = 47), pneumonia in an AIDS patient (n = 1), wound infections (n = 5), vaginal colonizations (n = 5), ear infection (n = 1), meningitis infection (n = 1), bacteraemia (n = 2) and diarrhoeal diseases (n = 16). These pathogen isolates included several bacterial species: Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Shigella spp. and Salmonella spp (Table 1).
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All isolates were tested for their susceptibility to antimicrobial agents including trimethoprim-sulphamethoxazole (1.25/23.75 µg) using a standard disc diffusion method on Mueller–Hinton agar (Bio-Rad, Marnes-La-Coquette, France) and interpreted according to the recommendations of the Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM at www.sfm.asso.fr). The isolates were scored as resistant and were selected if their inhibition zone diameter for co-trimoxazole was <10 mm. E. coli ATCC 25922 was used as control.
During this period, 73% of the ESBL-negative Enterobacteriaceae recovered from urinary tract infections were resistant to co-trimoxazole.
The ESBL-negative isolates included: E. coli, K. pneumoniae, Enterobacter cloacae and Enterobacter aerogenes (Table 1). The ESBLs were previously characterized (except one) as CTX-M-3, CTX-M-15, SHV-12 and SHV-2a.10
PCR screening of sul genes, class 1 integrase gene and characterization of class 1 integron resistance gene cassettes
All the PCR assays were carried out in a total volume of 50 µL mixture containing the following reagents: DNA (100 ng), Primers (1 µM), dNTP (200 µM), Tris-HCl (10 mM; pH 8.3), KCl (50 mM), MgCl2 (1.5 mM) and 1 U of Taq DNA polymerase.
The strains were screened for sul1 and sul2 by a multiplex PCR using specific oligonucleotide primers (Table 2) as previously described.11 PCR using specific primers (Table 2) used to detect the sul3 gene involved an annealing temperature at 53°C. Three E. coli clinical isolates (strains 02-57295, 02-58161 and 03-709) obtained in December 2002 and January 2003, in Tenon Hospital (France) and harbouring the intI1 gene and the sul1 gene, the sul2 gene and the sul3 gene, respectively were used as positive controls (A. Doloy, G. Arlet, personal data). E. coli DH10B was used as negative control for PCR assays.
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The integrase gene (int1) was detected by PCR using specific primers that amplified an amplicon of 899 bp (Table 2). The PCR conditions were as follows: initial denaturation (94°C for 5 min) followed by 35 cycles (94°C for 30 s, 60°C for 40 s and 72°C for 1 min). A final extension was performed at 72°C for 7 min.
The variable region of class 1 integrons was amplified by PCR using primers (Table 2) specific for the 5' conserved segment (5'CS) and 3' conserved segment (3'CS). Thermal cycler conditions were 94°C for 5 min followed by 35 cycles (94°C for 30 s, 60°C for 1 min, and 72°C for 2 min) and a final extension at 72°C for 7 min.
PCR products were subjected to DNA sequencing using PCR primers with an ABI PRISM 3100 Genetic Analyser sequencing (Applied Biosystems). Additional sequencing primers (Table 2) were designed using Oligo4 software and used for DNA sequencing.
Sequences obtained were analysed by comparison with the sequences in databases by BLASTN (www.ncbi.nlm.nih.gov) and Clustal W (http://www.ebi.ac.uk/).
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Results and discussion |
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The PCR analysis indicated that 72 isolates (92%) carried the sul1 gene, 67 (86%), the sul2 gene and 62 (80%) carried both sul1 and sul2 genes while sul3 was not found (Table 1). The frequencies of sul1 and sul2 genes found among our isolates are in conformity with the data previously reported.3,11 However, both sul1 and sul2 gene frequencies were higher in the current isolates than has been previously published from European countries.11–14
Seventy-four isolates (95%) were positive for the class 1 integrase gene while nine isolates (11%) were negative for sul1 but positive for sul2 as has previously described in Portuguese Salmonella and London E. coli strains.12,14 Three other E. coli isolates were sul1 positive and int1 negative. Previously, one plasmid has been described carrying a class 1 integron and a truncated int1 gene which could have given similar results.15
Six dfr genes encoding dihydrofolate reductase were identified in 76 isolates (97%) with dfrA7 frequencies being the most common (38 isolates; 48%) (Table 1). This correlates with a previous study of Senegalese isolates.16 The dfrA1 gene was the second most common, in 13 strains (17% of isolates) (Table 1) and has previously been associated with blood culture isolates in European hospitals.17 dfrA2d was the third most common gene, in 10 strains (13% of isolates) (Table 1) and in the CAR isolates is associated with ESBLs. Two E. coli isolates carried dfrA5 gene.
We detected three linkages between streptomycin resistance and dfr genes and one linkage with erythromycin esterase [ere(A)] and dfr genes (Table 1). All four of these linkages have previously been described.18–22
In CAR, the combinations of trimethoprim-sulfamethoxazole and pyrimethamine-sulfadoxine (Fansidar®) has been extensively used for bacterial treatment and antimalarial prophylaxis, respectively.8,23 Thus, it is not surprising that 73% of urinary pathogen Enterobacteriaceae were resistant to co-trimoxazole and that 90% of the isolates examined carried sul and int1 genes and > 95% carried dfr genes, similar results have been found in other studies of African isolates but this carriage rate is higher than reported in Europe or America.16, 18–22,24
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None to declare.
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Acknowledgements |
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T. Frank receives a fellowship from the Institut Pasteur de Paris and the French Government. This work was financed by grants from the French Government (Project 2001-168, FSP-C1 ‘Antibiorésistance’) and by the Faculté de Médecine Pierre et Marie Curie, Université Pierre et Marie Curie (Paris VI).
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References |
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