JAC Advance Access originally published online on March 5, 2008
Journal of Antimicrobial Chemotherapy 2008 61(5):1016-1019; doi:10.1093/jac/dkn078
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Original research |
Development of Escherichia coli rifaximin-resistant mutants: frequency of selection and stability
1 Centre de Recerca en Salut Internacional de Barcelona (CRESIB), Hospital Clinic/IDIBAPS, C/Villarroel 170, 08036 Barcelona, Spain 2 Servicio de Microbiologia, Hospital Clinic/IDIBAPS, C/Villarroel 170, 08036 Barcelona, Spain
* Corresponding author. Tel: +34-932275400, ext. 3388; Fax: +34-932279853; E-mail: joruiz{at}clinic.ub.es
Received 8 September 2007; returned 30 October 2007; revised 17 January 2008; accepted 31 January 2008
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Abstract |
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Objectives: To select rifaximin-resistant mutants of Escherichia coli and to establish the frequency of mutation, cross-resistance with other antimicrobial agents and the stability of the mutants obtained.
Methods: Four E. coli isolates [two enteroaggregative E. coli (EAEC) and two enterotoxigenic E. coli (ETEC)] were used to obtain rifaximin-resistant mutants. The frequency of mutation in the presence of rifaximin, rifampicin and ciprofloxacin was established by growth on plates containing serial dilutions of antibiotic above the bacterial MIC. To determine the stability of rifaximin resistance, 28 selected resistant mutants were grown for 20 consecutive cultures on antibiotic-free plates. Every 10 days, the MICs of rifaximin, chloramphenicol, nalidixic acid and ciprofloxacin were established.
Results: The frequency of mutation in the presence of rifaximin ranged between 5.7 x 10–7 and 1.6 x 10–6 in the case of the ETEC isolates, and between 2.0 x 10–8 and 9.3 x 10–8 in the case of the EAEC isolates; the frequency of mutation in the presence of rifampicin was in the order of 10–8 and no mutant in the presence of ciprofloxacin was obtained. Twenty-six out of 28 selected mutants exhibited resistance levels around or higher than 256 mg/L. In all cases, the resistance was stable, and no reversion towards the original parental MIC was observed. In no case was the MIC of chloramphenicol, nalidixic acid or ciprofloxacin affected.
Conclusions: Rifaximin has a low level of resistance selection, although it may select stable highly resistant mutants in a single step. Periodical surveillance of the levels of rifaximin resistance is required to detect the possible appearance of rifaximin-resistant clinical isolates. Further studies to characterize in-depth the mechanisms of stable resistance to rifaximin are necessary.
Keywords: frequency of mutation , diarrhoea , non-absorbable antibiotics
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Introduction |
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Rifaximin is a non-absorbable rifamycin derivative agent. Although this characteristic has classically been considered as a limitation, it has been proposed as an alternative to the current therapeutic schedules for some digestive infections, such as the traveller's diarrhoea (TD).1 In fact, due to this characteristic, rifaximin has no-systemic involvement, does not alter the microbiota population (other than those based in the intestinal area) and presents high concentrations in the intestinal lumen, higher than the usual MICs described in vitro for the main enteropathogens.2 Another proposed goal associated with the use of this agent is the possibility of reserving other antimicrobial agents, which are able to be used to treat systemic infections, such as the fluoroquinolones that are currently used as first-line drugs in the treatment of TD, diminishing their pressure on the microorganisms, thereby minimizing their ecological impact and favouring a lower selection of quinolone-resistant strains. However, no study has been performed regarding the potential of rifaximin to select resistant mutants, and its possible effect on the activity of other antimicrobial agents has not been established. Resistance to antimicrobial agents is one of the main problems related to the current use of antibiotics. Thus, the ability of antimicrobial agents to lead to the development of resistant mutants, their effect on the susceptibility levels of other antimicrobial agents and the stability of the mutants are relevant factors that must be established to determine the therapeutic value of novel antibacterial agents.
The aim of this study was to obtain rifaximin-resistant mutants in vitro and establish the frequency of mutation, the possible cross-resistance with other agents and the stability of the acquired resistance.
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Materials and methods |
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Microorganisms
Four diarrhoeic Escherichia coli clinical isolates [two enterotoxigenic E. coli (ETEC) and two enteroaggregative E. coli (EAEC)] recovered as a cause of TD were included in this study. The isolates were identified by conventional biochemical methods, while the diarrhoeic character (presence of the gene encoding the LT toxin, as well as presence of the pAA plasmid) was determined by PCR as described previously.2
Antibacterial susceptibility levels
The MICs of chloramphenicol, nalidixic acid, ciprofloxacin (Sigma, St Louis, MO, USA) and rifaximin (Bama-Geve, Barcelona, Spain) were determined by agar dilution methodology.2,3 E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 were used as quality controls.
Determination of spontaneous single-step resistance rates
To determine the spontaneous single-step resistance rates, one colony of the selected strains was grown overnight on brain heart infusion (Becton–Dickinson, Le Pont de Claix, France). Aliquots of 100 µL were directly spread onto Mueller–Hinton (Becton Dickinson) plates containing rifaximin at 8, 16, 32, 64 and 128 mg/L (equivalent to 1x, 2x, 4x, 8x and 16x MIC). Simultaneously, serial dilutions designed to calculate the size of the original inoculum was performed. The plates were read at 24 h. The initial inocula as well as the frequency of spontaneous resistance selected with rifaximin were calculated as described previously.4 The experiments were repeated four times, and the frequency of mutation considered was the mean of the experiments. Additionally, the frequency of mutation in the presence of rifampicin and ciprofloxacin was established following the same methodology.
Stability of the selected rifaximin-resistant mutants
Twenty-eight resistant E. coli mutants were selected from the plates used to determine the mutation frequency in the presence of rifaximin. These strains were grown in Mueller–Hinton medium in the absence of antibiotic pressure for 20 consecutive cultures. Every 10 days, the MICs of all the antibacterial agents included in this study were determined.
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Results |
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The frequency of selection of rifaximin-resistant mutants ranged between 5.7 x 10–7 and 1.6 x 10–6 in the case of the ETEC strains, and between 2.0 x 10–8 and 9.3 x 10–8 in the case of the EAEC isolates (Table 1). The frequency of mutation of each strain was independent of the antibiotic concentration in the plates (Table 1). No in vitro ciprofloxacin-resistant mutants were obtained, while the frequency of mutation in the presence of rifampicin was in the order of 10–8 (Tables 2 and 3).
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Twenty-eight resistant mutant strains were selected to analyse the levels and stability of acquired rifaximin resistance, as well as the possible presence of cross-resistance with another agents. Only two of the selected mutants (strains 11 and 14) presented a low/moderate stable resistance level of 32–64 mg/L. The remaining mutants consistently presented rifaximin MIC levels
256 mg/L. After 20 consecutive cultures on plates without antibiotic pressure, reversion of rifaximin resistance towards the parental MICs was not observed (Table 4).
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No alteration was observed in the MICs of nalidixic acid, ciprofloxacin and chloramphenicol at any time. Thus, in all cases, the MIC of ciprofloxacin was lower than 0.007 mg/L, whereas that of nalidixic acid and chloramphenicol ranged between 0.5 and 2 mg/L, and between 0.5 mg/L and 4 mg/L, respectively. No differences higher than 4x between parental strains and their respective obtained mutants were observed.
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Discussion |
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The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. In our case, the frequency of mutation ranged between 10–6 and 10–8. In the case of EAEC, the frequency of mutation in the presence of rifaximin was similar to that established in the present study for rifampicin, whereas in the case of ETEC, the frequency of mutation in the presence of rifaximin was slightly higher than those frequencies described for rifampicin both in the present study and in the literature.5 In both EAEC and ETEC, the frequency of mutation in the presence of rifaximin was slightly higher than that established for ciprofloxacin.5
In all cases, ciprofloxacin selects mutants at a lower frequency than rifaximin and rifampicin. However, it is worth mentioning that all the isolates presented an MIC of ciprofloxacin lower than 0.007 mg/L. Thus, it is possible that the selection plates presented excessive ciprofloxacin concentrations in comparison with the other agents analysed.
The rifaximin-resistant mutants obtained were stable and did not revert when the selection pressure disappeared, suggesting the presence of chromosomal mutations, probably in the rpoB gene, as has been described for rifampicin and other derivatives of rifamycin.6 However, other possibilities should be considered, such as the stable deregulation of efflux systems due to different factors such as the presence of mutations or stable alterations (i.e. insertion of an IS or transposons) in their promoter regions.7 Obviously, mutations in the rpoB gene or deregulations of efflux pumps, as well as other possible alterations, may be present together (studies in development).
The fact that 26 out of 28 analysed mutants presented a stable rifaximin MIC of around 256 mg/L or higher, irrespective of the rifaximin concentration present in the plate on which they were selected, together with the absence of differences in the selection rates among the different selective plates, suggests that these high resistance levels may be related to a single mutation step. Thus, despite the high intestinal concentrations of rifaximin, of up to 8000 µg/g in faeces,1 rifaximin should be used with caution, because of the possibility of developing rifaximin resistance during treatment, which may result in both therapeutic failure and the spread of stable rifaximin-resistant strains. In order to evaluate the real risk in clinical practice, the availability of rifaximin in the intestinal mucosal surfaces should be determined.
Only 2 out of 28 mutant strains (strains 11 and 14) exhibited a slight but stable increase in the rifaximin MIC. In the case of other antibacterial agents, such as quinolones, it has been described that different specific substitutions in their targets may result in different levels of resistance.8 The differences observed in the present study may be the result of a similar phenomenon. Furthermore, as mentioned previously, the presence of a stable deregulation of an efflux pump or the presence of stable alterations in the outer membrane cannot be ruled out.
The results obtained show that neither stable nor unstable quinolone-resistant or quinolone-intermediate mutants were obtained. This is of special interest, since the presence of diminished susceptibility to quinolones is a risk factor for developing full resistance that may be related to therapeutic failure.9 Additionally, the lack of effect on the MICs of quinolones and chloramphenicol suggests that rifaximin does not act by diminishing the expression level of the OmpF porin or overexpressing the AcrAB-TolC efflux pump. Thus, at least in the mutants analysed, rifaximin does not act on marRAB or other similar multiresistant regulation systems.10
In summary, this study describes the low levels of resistance selection by rifaximin, but the possibility of selecting highly resistant mutants in a single step. Although rifaximin is a promising alternative in the treatment of diarrhoeal infections, periodic surveillance is required to detect the possible appearance of clinical isolates exhibiting resistance. Further studies designed to establish the bioavailability of rifaximin in the mucosal surfaces as well as to characterize the mechanisms of stable resistance to rifaximin in-depth are necessary, since selection of rifaximin-resistant mutants during normal rifaximin treatment may result in therapeutic failure.
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Funding |
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J. R. is the recipient of grant CP05/0130 from Fondo de Investigaciones Sanitarias. The research of J. V. is supported by grant 2005 SGR00444 from the Department d'Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya, Spain. This study was also funded by Laboratorios Bama-Geve.
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Transparency declarations |
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The sponsor of the study (Bama-Geve) had no role in study design, data collection, data interpretation or writing the report. J. R. had full access to all the data in the study and had final responsibility for the decision to submit for publication. We declare that we have no conflict of interest.
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References |
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1 Jiang ZD, Ke S, Palazzini E, et al. In vitro activity and fecal concentration of rifaximin after oral administration. Antimicrob Agents Chemother (2000) 44:2205–6.
2 Ruiz J, Mensa L, O'Callaghan C, et al. In vitro antimicrobial activity of rifaximin against enteropathogens causing traveler's diarrhea. Diagn Microbiol Infect Dis (2007) 59:473–5.[CrossRef][Web of Science][Medline]
3 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Sixteenth Informational Supplement M100-S16 (2006) Wayne, PA, USA: CLSI.
4
Ruiz J, Jurado A, Garcia-Méndez E, et al. Frequency of selection of fluoroquinolone resistant mutants of Neisseria gonorrhoeae exposed to gemifloxacin and four other quinolones. J Antimicrob Chemother (2001) 48:545–8.
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Miller K, O'Neill AJ, Chopra I. Response of Escherichia coli hypermutators to selection pressure with antimicrobial agents from different classes. J Antimicrob Chemother (2002) 49:925–34.
6 Xu M, Zhou YN, Goldstein BP, et al. Cross-resistance of Escherichia coli RNA polymerase conferring rifampin resistance to different antibiotics. J Bacteriol (2005) 1887:2763–92.
7 Piddock LJV. Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin Microbiol Infect (2006) 19:382–402.
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Ruiz J. Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA Gyrase protection. J Antimicrob Chemother (2003) 51:1109–17.
9 Ruiz J, Gómez J, Navia MM, et al. High prevalence of nalidixic acid resistant, ciprofloxacin susceptible phenotype among clinical isolates of Escherichia coli and other Enterobacteriaceae. Diagn Microbiol Infect Dis (2002) 42:257–61.[CrossRef][Web of Science][Medline]
10 Randall LP, Woodward MJ. The multiple antibiotic resistance (mar) locus and its significance. Res Vet Sci (2002) 72:87–93.[CrossRef][Web of Science][Medline]
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