1 Centre for Infectious Disease, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, 4 Newark Street, London E1 2AT, UK; 2 Antibiotic Resistance Monitoring & Reference Laboratory, Health Protection Agency Centre for Infections, London NW9 5HT, UK
Received 10 June 2005; returned 25 July 2005; revised 15 August 2005; accepted 19 August 2005
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Abstract |
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Methods: Consecutive urinary E. coli isolates were obtained at the diagnostic microbiology laboratory of the Royal London Hospital from January to March 2004. The presence of the sulphonamide resistance genes, sul1, sul2 and sul3, and the class I integrase gene, int1, were determined by PCR.
Results: Of the 391 E. coli isolates recovered in 2004, 45.5% were sulphonamide-resistant compared with 46.0% in 1999 and 39.7% in 1991. The sul2 gene remained the most prevalent sulphonamide resistance determinant, present in 81% of resistant isolates in 2004 compared with 79% and 67% in 1999 and 1991, respectively; 28% of resistant isolates carried both sul1 and sul2 genes; sul3 was not found. Resistance to streptomycin also remained common, whereas resistance to chloramphenicol and kanamycin had decreased since 1999.
Conclusion: Sulphonamide resistance in E. coli persists undiminished despite the prolonged withdrawal of this antibiotic in the UK; resistance to streptomycin also seems stable whilst that to chloramphenicol and kanamycin is declining.
Keywords: streptomycin , persistence , integrons
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Introduction |
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We suggested2 that a number of factors might explain the continued persistence of resistance: that insufficient time had elapsed; lack of fitness cost associated with carrying resistance genes; non-human use of sulphonamides; and linkage of sulphonamide resistance to determinants of resistance to antimicrobials that remain in common use. To address the first of these hypotheses, we sought to examine whether high rates of sulphonamide resistance still persisted in the East London community in 2004, 5 years after our earlier investigation. We also investigated trends in resistance to other antimicrobial agents that are no longer commonly used.
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Materials and methods |
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E. coli isolates were obtained from the diagnostic microbiology laboratory of the Royal London Hospital. All consecutive isolates from urinary specimens from 20 January through to 19 March 2004 were included. Isolates were identified as E. coli by the clinical laboratory (using ß-glucuronidase agar), and confirmed by appropriate growth characteristics on chromogenic UTI medium (Oxoid, Basingstoke, UK). Lactose-negative isolates were confirmed as E. coli using the indole test.
Antimicrobial susceptibility testing
Antibiotic susceptibility was determined by disc diffusion assay in accordance with British Society for Antimicrobial Chemotherapy (BSAC) guidelines,3 with the following amendments: kanamycin zones were interpreted based on criteria for gentamicin and a biological zone breakpoint for tetracycline was taken as <19 mm susceptible, 20 mm resistant, so as to divide the two clusters forming a bimodal distribution. Antibiotic discs were obtained from Oxoid (Basingstoke, UK).
PCR amplification
Amplification of the sul1 and intI genes was performed with the primers and conditions described previously;2 sul2 was amplified with sul2F 5'-TCG TCA ACA TAA CCT CGG ACA G-3' and sul2R 5'-GTT GCG TTT GAT ACC GGC AC-3'; primers for sul3 were as described by Perreten and Boerlin.4 PCR was performed using ReddyMix (Abgene, Epsom, UK) mastermix. Template DNA was prepared by suspending a bacterial colony in 100 µL of distilled water, incubating at 100°C for 10 min then removing cell debris by centrifugation.
Statistical methods
Tests of significance were made using 2 analysis and
2 for trend. Strength of association was demonstrated by the odds ratio. Statistical analysis was performed using MedCalc for Windows, version 8.0.2.0 (MedCalc Software, Mariakerke, Belgium).
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Results |
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Discussion |
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As previously noted, linkage of sulphonamide resistance to resistance to agents that remain in common use is likely to result in co-selection of sulphonamide resistance determinants. In 2004, sulphonamide resistance continued to be strongly associated with resistance to ampicillin and trimethoprim (odds ratios of 20.0 and 16.7, respectively). There also continued to be a strong association with resistance to streptomycin (odds ratio of 44.3), which is perhaps to be expected given that the sul2 gene is often found adjacent to the resistance genes strAB,5,6 while sul1 is often associated with aadA gene cassettes encoding aminoglycoside adenyltransferases in type 1 integrons.7 But streptomycin, like the sulphonamides, has had limited clinical use for many years and it is not obvious why resistance to either class of drug should have persisted. In contrast to the persistence of sulphonamide and streptomycin resistance, chloramphenicol resistance has steadily declined over all three time points, as have both gentamicin and kanamycin resistances.
Why should resistance to one antibiotic decline while others persist when the agents are withdrawn? Possible explanations for the persistence of sulphonamide (and streptomycin) resistance include the properties of the mobile elements on which the determinants are carried, and the potential selection pressures other than in human medical use. We have already demonstrated that a small sul2-carrying plasmid, p9123 (similar to other widely distributed plasmids), appears to confer a fitness advantage on its host, although two larger sul2 plasmids had a fitness cost.5 Furthermore, sul genes are distributed on a wide range of mobile genetic elements. If a sul gene can mobilize itself to new hosts at a rate similar to, or faster than it is lost due to the lack of selection, it will persist. The sul1 gene is an integral part of class 1 integrons, essentially forming the 3' conserved segment of these elements,8 while the sul2 gene is prolific on small non-conjugative plasmids,5 as well as some large conjugative plasmids.6 The efficient dissemination of the sul genes is likely to eventually produce new combinations of these with other genes. This view is supported by the increasing number of isolates carrying both sul1 and sul2, and by the large number of isolates carrying a class I integrase gene and sul2 but not sul1.
Non-human use of antimicrobial agents in agriculture may also be important. According to data from the UK Veterinary Medical Directorate,9 veterinary use of sulphonamides remains high, with 74 tonnes sold in 2003. Streptomycin is also used extensively in veterinary medicine, with 7 tonnes sold in the UK during 2003. It is also used to treat food plants in some countries. In contrast, chloramphenicol is banned for use in food animals in the UK; nevertheless, a recent study in the USA demonstrated that chloramphenicol resistance was high in E. coli from swine despite an apparent lack of selection.10
This study provides further evidence to challenge the assumption that withdrawal of an antibiotic will necessarily result in a reduction in resistance. In the case of sulphonamide resistance in E. coli, 9 years has not proved sufficient to detect any reduction in prevalence.
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Acknowledgements |
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References |
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