Resistance among Escherichia coli to sulphonamides and other antimicrobials now little used in man

David C. Bean1,*, David M. Livermore2, Iro Papa1 and Lucinda M. C. Hall1

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


* Corresponding author. Tel: +44-20-7882-2325; Fax: +44-20-7882-2181; E-mail: d.c.bean{at}qmul.ac.uk

Received 10 June 2005; returned 25 July 2005; revised 15 August 2005; accepted 19 August 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: We investigated whether sulphonamide resistance in Escherichia coli remained prevalent in 2004, 9 years since the formal introduction of a UK prescribing restriction on co-trimoxazole. Resistance to other agents no longer in common use was also examined.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is widely supposed that reduction or cessation in the use of particular antibiotics will diminish the prevalence of resistance. There is evidence that this may be the case in some instances: a restriction in macrolide prescribing in Finland, for example, was followed by a reduction in erythromycin resistance among group A streptococci.1 In contrast, however, we demonstrated the persistence of sulphonamide resistance in Escherichia coli in the UK in 1999 despite a formal prescribing restriction implemented in 1995 and a 97% reduction in usage since 1991.2 Even more surprisingly, one of the genetic elements responsible for this sulphonamide resistance, sul2, had actually increased in prevalence among human E. coli despite the limited direct selection pressure.

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.


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

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 {chi}2 analysis and {chi}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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 391 urinary E. coli isolates were included; Table 1 shows the prevalence of resistance to each antibiotic tested compared with previous years. Resistance to sulphonamides stayed relatively constant from 1991 to 2004, fluctuating between 39.7% and 46%. Resistances to ampicillin, streptomycin, tetracycline and trimethoprim also fluctuated over the three time intervals with no consistent trend. In contrast, there were consecutive decreases in resistance to chloramphenicol, gentamicin and kanamycin from 1991 to 1999 to 2004, although not all were statistically significant.


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Table 1. Proportions (%) of resistant isolates from each collection (data for 1991 and 1999 taken from Enne et al.2)

 
Isolates that were resistant to sulphonamides in 2004 also tended to be resistant to other antibiotics (Table 2). Specifically, resistances to ampicillin, chloramphenicol, streptomycin, tetracycline and trimethoprim were significantly (P ≤ 0.01) associated with sulphonamide resistance. These associations were the same as those reported for 1991 and 1999,2 except that resistance to kanamycin, which was rare in 2004, was no longer associated with sulphonamide resistance.


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Table 2. Association of sulphonamide resistance with resistance to other antibiotics

 
Each of the 178 sulphonamide-resistant isolates from 2004 was tested by PCR for the presence of the sul1, sul2 and sul3 genes. In all but one isolate, resistance could be attributed to the presence of sul1 (19%), sul2 (53%) or both (28%), whereas none of the isolates was found to contain the recently described sul3 gene.4 The prevalence of the sul1 gene had increased to 21.2% in 2004 from 17.5% in 1999, while the prevalence of sul2, at 36.3%, had essentially remained unchanged from 1999 (Table 3). The trend of increasing numbers of isolates containing both the sul1 and sul2 genes continued in 2004, rising from 4.7% in 1991 to 10.3% in 1999, and 12.5% in 2004, although the latter increase was not statistically significant.


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Table 3. Proportion (%) of isolates from each collection with particular sul genes (data for 1991 and 1999 taken from Enne et al.2)

 
All the sulphonamide-resistant isolates from 2004 were also tested for the presence of the intI gene by PCR. The sul1 gene is an integral part of type 1 integrons and, as expected, the intI gene was present in all but two (97.6%) of the sul1-positive isolates. Interestingly, however, 33 isolates were PCR-positive for both the sul2 and intI genes, but negative for the sul1 gene.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The prevalence of sulphonamide resistance in E. coli from UTIs remained as high in 2004 as it was 5 years previously, despite an almost complete withdrawal of the antibiotic from human medical use in the UK. [Co-trimoxazole prescriptions have remained below 70 000 annually from 2000 to 2004 (personal communication, Peter Stephens, IMS Health) compared with over 4 million prescriptions annually in the late 1980s.2] The reason for this persistence remains unclear, although several hypotheses have been proprosed.2 One explanation, that insufficient time had elapsed to allow a decline in sulphonamide resistance to occur, prompted this study. Collection of bacterial isolates was exactly 5 years since the last survey, and 9 years after the prescribing restrictions were introduced. Although it remains possible that resistance will eventually decrease, it now seems unlikely that it will occur within any useful time frame.

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.


    Acknowledgements
 
We are grateful to the staff of the Microbiology Laboratory at the Royal London Hospital for provision of E. coli isolates. We thank Vincent Perreten for supplying sul3 plasmid pUVP4401 and Peter Stephens of IMS Health for provision of antibiotic prescribing data. This study was funded by the Department of Health (Resistance to Antibiotics and Other Antimicrobial Agents Programme, Grant No. 91).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1. Seppala H, Klaukka T, Vuopio-Varkila J et al. The effect of changes in the consumption of macrolide antibiotics on erythromycin resistance in group A streptococci in Finland. New Engl J Med 1997; 337: 441–6.[Abstract/Free Full Text]

2. Enne VI, Livermore DM, Stephens P et al. Persistence of sulphonamide resistance in Escherichia coli in the UK despite national prescribing restriction. Lancet 2001; 357: 1325–8.[CrossRef][ISI][Medline]

3. Andrews JM. BSAC standardized disc susceptibility testing method (version 4). J Antimicrob Chemother 2005; 56: 60–76.[Free Full Text]

4. Perreten V, Boerlin P. A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland. Antimicrob Agents Chemother 2003; 47: 1169–72.[Abstract/Free Full Text]

5. Enne VI, Bennett PM, Livermore DM et al. Enhancement of host fitness by the sul2-coding plasmid p9123 in the absence of selective pressure. J Antimicrob Chemother 2004; 53: 958–63.[Abstract/Free Full Text]

6. Radstrom P, Swedberg G. RSF1010 and a conjugative plasmid contain sulII, one of two known genes for plasmid-borne sulfonamide resistance dihydropteroate synthase. Antimicrob Agents Chemother 1988; 32: 1684–92.[ISI][Medline]

7. Sunde M, Norstrom M. The genetic background for streptomycin resistance in Escherichia coli influences the distribution of MICs. J Antimicrob Chemother 2005; 56: 87–90.[Abstract/Free Full Text]

8. Stokes HW, Hall RM. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol 1989; 3: 1669–83.[ISI][Medline]

9. Veterinary Medicines Directorate. Sales of Antimicrobial Products Authorised for Use as Veterinary Medicines, Antiprotozoals, Antifungals, Growth Promoters and Coccidiostats, in the UK. http://www.vmd.gov.uk/general/publications/AM-Sales-rpt-2003-v09.pdf (23 May 2005, date last accessed).

10. Bischoff KM, White DG, Hume ME et al. The chloramphenicol resistance gene cmlA is disseminated on transferable plasmids that confer multiple-drug resistance in swine Escherichia coli. FEMS Microbiol Lett 2005; 243: 285–91.[CrossRef][ISI][Medline]





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