JAC Advance Access originally published online on August 5, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):848-852; doi:10.1093/jac/dkl315
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Mutators among CTX-M ß-lactamase-producing Escherichia coli and risk for the emergence of fosfomycin resistance
1 Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency 61 Colindale Avenue, London NW9 5EQ, UK 2 Laboratory of Health Care Associated Infection, Centre for Infections, Health Protection Agency London, UK 3 Barts and The London School of Medicine and Dentistry London, UK
*Corresponding author. Tel +44-208-8327-7236; Fax +44-208-8327-6264; E-mail: matthew.ellington{at}hpa.org.uk
Received 10 May 2006; returned 30 May 2006; revised 7 July 2006; accepted 12 July 2006
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Abstract |
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Objectives: Fosfomycin is a possible oral treatment for lower urinary tract infections caused by Escherichia coli with CTX-M extended-spectrum ß-lactamases but is vulnerable to mutational resistance. Hypermutability among natural E. coli populations might facilitate the emergence of resistance to fosfomycin. We therefore examined the prevalence of mutators amongst urinary isolates of E. coli producing CTX-M ß-lactamases.
Methods: Urinary E. coli isolates with CTX-M ß-lactamases (n = 220) were screened for resistance to both rifampicin and fosfomycin, as well as a mutator phenotype, by rifampicin and fosfomycin disc assays. Mutation frequencies for 10 isolates, identified as mutators by the initial disc screen, were determined in triplicate on agar with rifampicin or fosfomycin at 4x MIC and with fosfomycin or nitrofurantoin at 256 mg/L.
Results: The disc screen identified 10 likely mutators and quantitative tests indicated that 9 of these had mutation frequencies of 8.0 x 10–6–1.5 x 10–4 for fosfomycin and 0.1–2.3 x 10–6 for rifampicin. These mutators were diverse in terms of PFGE type and 4 of the 10 were confirmed as strong mutators with rifampicin and fosfomycin. Only the strongest mutator isolate and hypermutable MutS– control strain consistently gave single-step mutants resistant to 256 mg/L fosfomycin. No nitrofurantoin-resistant mutants were selected from any isolate, although they could be selected from the hypermutable MutS– control strain.
Conclusions: Mutator phenotypes were found among E. coli expressing CTX-M ß-lactamases and were independent of strain type. These had an increased propensity to fosfomycin resistance.
Keywords: hypermutators , nitrofurantoin , UTIs
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Introduction |
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Escherichia coli with CTX-M ß-lactamases have emerged rapidly in Europe and Asia, widely supplanting TEM and SHV types as the major extended-spectrum ß-lactamases (ESBLs).1 Uropathogenic E. coli with CTX-M ESBLs are often resistant to multiple antibiotics.1 Carbapenems are the preferred treatment for serious infections, but fosfomycin and nitrofurantoin are oral options for urinary tract infections (UTIs), although only the latter is widely used in the UK. The molecular mechanism(s) of nitrofurantoin resistance are poorly defined, but may involve mutations affecting NADH reductases and nitro-reductases,2 and resistance seems to occur relatively rarely.3 Most fosfomycin resistance arises through mutations affecting uptake.4 These emerge at higher frequencies (up to 1 in 104).4 From a 2004 survey of cephalosporin-resistant Enterobacteriaceae our laboratory has determined that fosfomycin and nitrofurantoin are highly active against most E. coli isolates producing CTX-M ESBLs (75% of 230 isolates for nitrofurantoin and 98% of 230 for fosfomycin) (R. Hope, personal communication), highlighting the potential utility of these antibiotics.
Clinical isolates with rifampicin resistance frequencies between 4 x 10–8 and 4 x 10–7 (weak mutators) have been detected in 
25% of E. coli.5 However, the true extent of any elevation for a mutation frequency of 4 x 10–8 could be questioned. Strong mutators (rifampicin mutation frequencies >4 x 10–7) have been found in up to 7.5% of E. coli6 and to be more prevalent in ESBL-producing than non-ESBL-producing E. coli.7 Hypermutable phenotypes in clinical isolates of E. coli occur most often via a defective methyl-directed DNA mismatch repair (MMR) pathway.8 If frequent among isolates with CTX-M enzymes, hypermutability could increase the risk of resistance to fosfomycin or nitrofurantoin emerging; however, no published data exist assessing such a risk. In the present work, we determined the prevalence of mutators amongst multiresistant E. coli expressing CTX-M enzymes from UTIs and the risk they pose for the emergence of resistance.
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Materials and methods |
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Bacterial isolates, strains and growth
A total of 220 E. coli with CTX-M enzymes, all from UTIs, referred during 2003/4 and characterized previously,1 were retained for the study. These comprised 172 sporadic isolates and 48 representatives of five major PFGE-defined clones (A–E) that are prevalent in the UK.1 All the isolates were susceptible to both fosfomycin and nitrofurantoin according to BSAC breakpoints9 and did not show frank resistance to a 30 µg rifampicin disc. A hypermutable MutS-negative E. coli control strain (1413) and its non-hypermutable parent (1411)10 (Table 1) were supplied by R. Lloyd (Nottingham, UK).
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Bacteria were cultured aerobically with brain heart infusion broth, Columbia-blood agar or Iso-Sensitest agar (Oxoid, Basingstoke, UK) at 37°C. Antibiotics and discs were obtained from Sigma-Aldrich (Poole, UK) and Oxoid (Basingstoke, UK), respectively.
Screening for mutator phenotypes
Bacteria were screened for a mutator phenotype using a previously described rifampicin and fosfomycin disc test method.11 This was carried out for three independent broth cultures with a starting inoculum of 103 cfu in order to minimize the probability of a pre-existing mutant being present. Isolates yielding <30 and <10 colonies, around 50 µg fosfomycin and 30 µg rifampicin discs, respectively, were designated as non-mutators. Those yielding >70 and >10, respectively, were considered to be hypermutators. Those yielding 30–70 and >10, or >70 and <10, around fosfomycin and rifampicin discs were presumed to be weak mutators.
Susceptibility testing and 4x MIC mutation frequency determination
MICs of rifampicin and fosfomycin were determined by BSAC methodology.9 Mutation frequencies were determined10 for the emergence of resistance to 4x MIC and 256 mg/L fosfomycin (both tested in the presence of 100 mg/L glucose-6-phosphate), 4x MIC rifampicin, and 256 mg/L nitrofurantoin in triple determinations from three independent broth cultures (9 data points in total) started from 103 cfu. For tests with 256 mg/L nitrofurantoin or fosfomycin, 50 mL Luria–Bertani broth cultures were inoculated with 50 µL of a 10–3 dilution of fully grown culture (to give an inoculum of 103–104 cfu), incubated overnight to stationary phase, were harvested, resuspended in 600 µL of broth and 200 µL aliquots spread on to selective agar plates. Selective plates were incubated at 37°C for 24 h and the number of colonies counted and expressed as a fraction of the number of viable cells as determined on drug-free agar.
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Results |
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Initial screen for mutators
In triplicated experiments the MutS– strain and 10/220 (4.5%) CTX-M E. coli isolates yielded >70 colonies around 50 µg fosfomycin discs and >10 colonies around 30 µg rifampicin discs, indicating them to be likely mutators (Figure 1a and b). These 10 clinical isolates were of diverse PFGE types and included sporadic isolates, along with representatives of strains B, D and E; none of the 18 strain type A and C isolates tested was deemed to be a potential mutator. A further 98 isolates either inconsistently gave >70 and >10 colonies around fosfomycin and rifampicin discs in the triplicated experiments, or gave either >70 colonies around fosfomycin discs or >10 around rifampicin discs, but not to both, suggesting them to be, at best, weak mutators or to contain a sub-population with decreased susceptibility to either fosfomycin or rifampicin; a further 32 isolates that gave 30–70 colonies around 50 µg fosfomycin discs but fewer than 10 around 30 µg rifampicin discs were also counted as possible weak mutators; E. coli 1411 (parent of the MutS– strain) and the remaining 80 clinical isolates gave <30 colonies around 50 µg fosfomycin discs and <10 colonies around 30 µg rifampicin discs, indicating non-mutator status (Figure 1a and b).
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Mutation frequencies
Sixteen clinical isolates were selected on the basis of the initial disc test screen for mutation frequency studies with fosfomycin and rifampicin at 4x MIC. These comprised 10 mutator candidates and six non-mutator isolates. E. coli 1411 and 1413 (MutS–) control strains were also tested.
The non-mutator control (1411) and the six putative non-mutators had mutation frequencies of 3.0–6.7 x 10–6 for fosfomycin and 1.0–3.9 x 10–8 for rifampicin, confirming non-mutator status5,6 (Figure 1c and d). Mutation frequencies for the MutS– control were 2.7 ± 0.9 x 10–4 for fosfomycin and 2.9 ± 0.5 x 10–6 for rifampicin (Figure 1c and d), representing 50-fold increases compared with its parent. Of 10 likely mutators identified in the initial screen, five were confirmed as weak mutators (frequencies of 0.8–2.6 x 10–5 for fosfomycin and 1.0–3.0 x 10–7 for rifampicin) and four were confirmed as strong or hypermutators (frequencies of 0.15–1.5 x 10–4 for fosfomycin and 0.6–2.3 x 10–6 for rifampicin) (Figure 1c and d). The remaining isolate (Mtr8) did not have elevated mutations frequencies in these tests. Thus, 9/220 (4.1%) isolates studied were confirmed as mutators (strong and weak) and 4/220 (1.8%) were confirmed strong or hypermutators. Five of the nine mutators were individual isolates amongst the 30 representatives of strains B, D and E tested; the remaining 4 were individual isolates amongst the 172 sporadic isolates tested. Thus, the occurrence of a mutator phenotype was not dependent on strain type.
To determine the risk that mutators pose for the rapid development of high-level resistance to fosfomycin and nitrofurantoin in a single-step, we determined the frequency at which mutants resistant to 256 mg/L fosfomycin or nitrofurantoin (higher than the BSAC breakpoints for resistance, >128 and >32 mg/L, respectively9) occurred, via a modified protocol with an inoculum of 2 x 109 cfu per plate. Only the strongest mutator amongst the clinical isolates (mutation frequency of 2.3 ± 1.1 x 10–6 for rifampicin and 1.5 ± 1.0 x 10–4 for fosfomycin at 4x MIC) consistently gave single-step mutants at 256 mg/L fosfomycin (Table 1). Nitrofurantoin-resistant mutants were not selected from any of the clinical isolates although single mutants were raised in one of the three experiments with the hypermutable MutS– control strain, (mutation frequency = 6.7 x 10–9) (Table 1).
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Discussion |
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Fosfomycin and nitrofurantoin are potentially useful antibiotics for treating uncomplicated UTIs caused by CTX-M-producing E. coli. In this study we have, for the first time, examined the clonality and prevalence of mutators amongst UTI-associated E. coli from the UK and the risk that they pose for the emergence of resistance to these antibiotics.
Of 220 multiresistant urinary isolates of E. coli expressing CTX-M enzymes, 10 were suggested to be mutators by the disc screen method and 9 of these 10 were confirmed to have elevated mutation frequencies to fosfomycin and rifampicin. The disc screen first described by Galan et al.11 thus seems usefully predictive for detecting mutator isolates of E. coli. Moreover, both the disc test and mutation frequency determination tests use low inocula and thus exclude the selection of pre-existing mutants resistant to both agents tested, minimizing the risk of misidentifying mutators.
Prevalences of hypermutators amongst UTI isolates of E. coli as high as 7.5% have been reported;6 this compares with 1.8% in the present study, and 4.1% for confirmed weak and strong mutators combined. In addition, a recent study reported up to 43% of ESBL-producing E. coli in Spain to be weak mutators—defined as having rifampicin mutation frequencies of 4 x 10–8–4 x 10–7. The lower limit of this range may have contributed to this being the highest prevalence of mutators ever reported,7 a point discussed elsewhere.12 To date, the most common lesions found in naturally occurring E. coli mutators have been in the mutHLS genes of the DNA methyl-directed MMR pathway.8 Future studies will focus on determining the sequence of these and other candidate mutator genes from the mutator isolates identified in this initial study. E. coli producing CTX-M ESBLs are increasingly prevalent worldwide and have spread widely in the UK. Five major strain types A–E are prevalent,1 along with many sporadic producers, amongst the CTX-M-ß-lactamase-producing E. coli present in the UK. Amongst the isolates tested, mutator status did not correlate with strain type; rather, individual isolates of strains B, D and E and sporadic isolates were mutators and the absence of mutator isolates of strains A and C among 18 representatives tested does not preclude the possibility that they do occur.
Our results show an enhanced risk for the emergence of fosfomycin resistance amongst the mutator isolates at 4x MIC and that the strongest of these mutators yielded variants resistant to fosfomycin at 256 mg/L in vitro, suggesting that resistance might arise rapidly with wide clinical use. However, resistance to fosfomycin has not emerged rapidly when fosfomycin has been used therapeutically,3 and it has even been suggested that the fitness costs associated with resistance4,13 might limit the emergence of resistance to fosfomycin in vivo,4 unless compensatory mutations occur. It remains to be seen whether hypermutators predispose towards the selection of more successful uropathogenic fosfomycin-resistant strains.
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None to declare.
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Acknowledgements |
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We would like to thank Russell Hope (Antibiotic Resistance Monitoring and Reference Laboratory) for nitrofurantoin and fosfomycin susceptibility data. Funding was provided by the Department of Health, UK, through grant no. 91 of the Resistance to Antibiotics and other Antimicrobial Agents Research Programme. Parts of this work were presented at the Sixteenth European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Nice, France, 1–4 April 2006 (Ellington MJ, Livermore DM, Pitt TL et al. Mutators among CTX-M ß-lactamase-producing E. coli pose a risk for the emergence of fosfomycin resistance. Clin Microbiol Infect 2006; 12 Suppl 4: Abstract P1236).
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References |
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1 Woodford N, Ward ME, Kaufmann ME, et al. (2004) Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum ß-lactamases in the UK. J Antimicrob Chemother 54:735–43.
2
Breeze AS and Obaseiki-Ebor EE. (1983) Nitrofuran reductase activity in nitrofurantoin-resistant strains of E. coli K12: some with chromosomally determined resistance and others carrying R-plasmids. J Antimicrob Chemother 12:543–7.
3 Guay DR. (2001) An update on the role of nitrofurans in the management of urinary tract infections. Drugs 61:353–64.[CrossRef][Web of Science][Medline]
4
Nilsson AI, Berg OG, Aspevall O, et al. (2003) Biological costs and mechanisms of fosfomycin resistance in E. coli. Antimicrob Agents Chemother 47:2850–8.
5
Baquero MR, Nilsson AI, Turrientes MC, et al. (2004) Polymorphic mutation frequencies in E. coli: emergence of weak mutators in clinical isolates. J Bacteriol 186:5538–42.
6
Denamur E, Bonacorsi S, Giraud A, et al. (2002) High frequency of mutator strains among human uropathogenic E. coli isolates. J Bacteriol 184:605–9.
7
Baquero MR, Galan JC, del Carmen TM, et al. (2005) Increased mutation frequencies in E. coli isolates harboring extended-spectrum ß-lactamases. Antimicrob Agents Chemother 49:4754–6.
8 Chopra I, O'Neill AJ, Miller K. (2003) The role of mutators in the emergence of antibiotic-resistant bacteria. Drug Resist Updat 6:137–45.[CrossRef][Web of Science][Medline]
9 BSAC. BSAC Methods for Antimicrobial Susceptibility Testing, Version 5, January 2006. http://www.bsac.org.uk/_db/_documents/version_5_.pdf (14 April 2006, date last accessed).
10
Miller K, O'Neill AJ, Chopra I. (2002) Response of E. coli hypermutators to selection pressure with antimicrobial agents from different classes. J Antimicrob Chemother 49:925–34.
11
Galan JC, Tato M, Baquero MR, et al. (2004) Fosfomycin and rifampin disk diffusion tests for detection of E. coli mutator strains. J Clin Microbiol 42:4310–2.
12 Hall LMC and Henderson-Begg SK. (2006) Hypermutable bacteria isolated from humans—a critical analysis. Microbiol in press.
13 Marchese A, Gualco L, Debbia EA, et al. (2003) In vitro activity of fosfomycin against Gram-negative urinary pathogens and the biological cost of fosfomycin resistance. Int J Antimicrob Agents 22:53–9.
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