JAC Advance Access originally published online on September 19, 2006
Journal of Antimicrobial Chemotherapy 2006 58(5):1054-1057; doi:10.1093/jac/dkl361
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In vivo selection during ofloxacin therapy of Escherichia coli with combined topoisomerase mutations that confer high resistance to ofloxacin but susceptibility to nalidixic acid
1 Service de Bactériologie-Virologie-Hygiène, AP-HP CHU Henri Mondor Université Paris XII, 51 Avenue du Maréchal de Lattre de Tassigny, 94010 Créteil Cedex, France 2 Unité de Contrôle Épidémiologique et Prévention de l'Infection, AP-HP CHU Henri Mondor Créteil, France 3 Inserm U722 and University Paris 7 Denis Diderot Site Xavier Bichat, France
*Corresponding author. Tel: +33-1-49-81-28-31; Fax: +33-1-49-81-28-39; E-mail: emmanuelle.cambau{at}hmn.aphp.fr
Received 26 May 2006; returned 2 July 2006; revised 25 July 2006; accepted 10 August 2006
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Objectives: To investigate quinolone resistance mechanisms in an Escherichia coli clinical isolate (Ar2) resistant to ofloxacin but susceptible to nalidixic acid selected after 10 days of ofloxacin therapy in a patient with prostatitis.
Methods: Molecular typing (ERIC-PCR and RAPD), antibiotic susceptibility and gyrA, gyrB, parC and parE QRDR sequences were compared for E. coli Ar2 and a wild-type E. coli (Ar1) isolated 2 months earlier in the same patient. Ofloxacin-resistant mutants were selected in vitro in order to reproduce the mutations observed and the original phenotype.
Results: The two strains were similar with regard to antibiotic susceptibility except quinolones and for ERIC-PCR and RAPD patterns, suggesting a clonal relationship and acquisition of quinolone resistance by chromosomal mutation. Quinolone MICs were 3, 0.12, 0.05 and 0.02 mg/L of nalidixic acid, ofloxacin, levofloxacin and ciprofloxacin, respectively, for E. coli Ar1 and 6, 32, 8 and 1 mg/L, respectively, for E. coli Ar2. The strain Ar2 harboured two substitutions, Gly-81
Asp in GyrA and Ser-80Arg in ParC. Introduction into E. coli Ar2 of the wild-type gyrA fully complemented fluoroquinolone resistance. Although the strain was not a hypermutator, ofloxacin first-step resistant mutants with gyrA mutations were easily obtained from E. coli Ar1 and 25% of them were at codon 81. In vitro stepwise combination of Gly-81Asp in GyrA and Ser-80Arg in ParC reproduced the original phenotype in E. coli KL16.
Conclusions: A double topoisomerase mutant was selected in vivo by 10 days ofloxacin. The mutations were originally combined for a result of ofloxacin resistance but nalidixic acid susceptibility.
Keywords: fluoroquinolones , parC , DNA gyrase A , QRDRs , gyrA , prostatitis
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Introduction |
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Quinolone resistance mechanisms in Escherichia coli are active efflux, reduced uptake, topoisomerase protection due to plasmid-mediated qnr genes, acetylation, and point mutations in the quinolone resistance-determining regions (QRDRs) of the DNA gyrase (gyrA and gyrB) and the topoisomerase IV (parC and parE) genes.1,2 Substitutions at Ser-83 or Asp-87 in GyrA and of Ser-80 and Glu-84 in ParC are the most common in E. coli.1–4 Substitutions at other positions are rare in clinical isolates.3 Although a single mutation in gyrA confers resistance to nalidixic acid, additional mutations in gyrA and/or in parC genes, occurring in a stepwise manner, are required to obtain high-level fluoroquinolone resistance.5,6 Mutations in the gyrB and parE genes confer a low-level resistance and are less frequently observed.1–3
We routinely test nalidixic acid susceptibility in addition to fluoroquinolone susceptibility, and we observed an E. coli clinical isolate that was highly resistant to ofloxacin, and susceptible to nalidixic acid. Investigation of the mutations in the type II topoisomerase genes in comparison with a fluoroquinolone susceptible strain previously isolated in the same patient showed that this clinical isolate acquired a rare gyrA mutation combined with a parC mutation. Isogenic mutants showed that such a combination of mutations results in an original susceptibility phenotype. This study is the second report of a clinical isolate harbouring a Gly-81Asp GyrA substitution,5 and the first report of such a double mutant selected in vivo.
Case report
On 8 December 2004, the E. coli clinical strain Ar2, resistant to ofloxacin and susceptible to nalidixic acid, was recovered from two blood cultures of a 78-year-old patient with a catheter-related bacteraemia. Two months earlier (2 October 2004), this patient was initially admitted in the ICU ward for septic shock with acute prostatitis. At that time, an E. coli isolate (strain Ar1), susceptible to all quinolones, was obtained from blood and urinary tract specimens. For this episode, he received a 4 week antibiotic course: amoxicillin for 72 h, cefotaxime for 15 days, followed by ofloxacin for 10 days. He was then transferred to the nephrology unit because of acute renal failure and pancreatitis, and needed long-term parenteral nutrition. During this prolonged hospitalization, he presented skin ulceration on the right knee where the strain Ar2 was first recovered (15 November 2004). Skin ulceration preceded cutaneous colonization of the parenteral nutrition-catheter that caused bacteraemia. Catheter-related bacteraemia was successfully treated with removal of the catheter and cefotaxime for 14 days.
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Materials and methods |
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Antimicrobial susceptibility was determined by the disc diffusion method (www.sfm.asso.fr/) and MICs of quinolones by the Etest (AB Biodisk, Solna, Sweden).
Amplification of the QRDRs in gyrA (521 bp fragment), gyrB (287 bp fragment), parC (230 bp fragment) and parE (201 bp fragment) genes used the primers gyrAfwd, 5'-CTGCGCGGGCTGTGTTATAATT-3'; gyrArv, 5'-CCGTGCCGTCATAGTTATCAA-3'; gyrBfwd, 5'-CTGCCGGGCAAACTGGCAGA-3'; gyrBrv, 5'-TCGACGTCCGCATCGGTCAT-3'; parCfwd, 5'-GTATGCGATGTCTGAACT-3'; parCrv, 5'-TTCGGTGTAACGCATTGC-3'; parEfwd, 5'-GCCTGGCAAACTGGCTGATGTA-3'; parErv, 5'-ATATCGTGCACTTCCTGCGAAGC-3', in a 50 µL reaction mixture containing 1 pmol of primer, 0.2 mM dNTPs, 1x reaction buffer, 1.5 mM MgCl2, 1.25 U of Taq polymerase and 150 ng of DNA extracted using the QIAmp DNA Mini Kit (Qiagen, Courtaboeuf, France). Thirty-five cycles of the following temperature profile were run: 30 s at 94°C, 30 s at 40°C (for gyrB), 50°C (for parC) or 57°C (for gyrA and parE) and 30 s at 72°C. PCR fragments were directly sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit and the ABI Prism 3100 automated sequence analyser after purification with MontageTM PCR Centrifugal Filter Devices (Millipore, Molsheim, France).
E. coli Ar2 was transformed with the plasmid pRM386 (AMP–R) containing the wild-type gyrA gene of E. coli.5
Molecular typing was achieved by ERIC-PCR (primer ERIC2, 5'-AAGTAAGTGACTGGGGTGAGCGC-3') and RAPD (primer 1254, 5'-CCGCAGCCAA-3').
Ofloxacin-resistant mutants were selected by plating 109 cfu onto Mueller-Hinton agar containing from 0.25 to 4 mg/L of ofloxacin (Sanofi-Aventis France) as described previously.6 To search for a hypermutable character, the mutation frequencies of the strains E. coli Ar1 and E. coli Ar2 were estimated by monitoring the strains capacities to generate mutations conferring resistance to rifampicin in at least six independent cultures.7
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Clonal relatedness between E. coli Ar1 and E. coli Ar2
The two strains were resistant to sulphonamides and trimethoprim, and susceptible to ß-lactams and aminoglycosides. E. coli Ar1 was susceptible to quinolones whereas E. coli Ar2 was categorized intermediate to ciprofloxacin, resistant to ofloxacin and susceptible to nalidixic acid (Table 1). The highest MIC increase between the two strains was observed for ofloxacin and levofloxacin (128-fold).
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ERIC-PCR and RAPD patterns were similar for the two strains (data not shown) suggesting a clonal relationship and a mechanism of acquired fluoroquinolone resistance by chromosomal mutation.
Topoisomerase mutations
Complementation of E. coli Ar2 with the wild-type gyrA gene resulted in a 32- to 128-fold decrease in the MICs of ofloxacin and ciprofloxacin whereas nalidixic acid MIC was nearly the same (Table 1). E. coli Ar1 was devoid of mutation in gyrA, gyrB, parC and parE QRDRs. E. coli Ar2 harboured two mutations, one in gyrA and one in parC, leading to the substitutions Gly-81Asp in GyrA and Ser-80Arg in ParC, respectively. There was no mutation in gyrB or in parE QRDRs, confirming the result of complementation tests.
In vitro selection of mutants
In order to determine whether the E. coli isolates had the ability to acquire uncommon mutations, we sought a hypermutable character. The two strains Ar1 and Ar2 showed mutation frequencies to rifampicin resistance of 6 x 10–9 and 5 x 10–9, respectively, while mutator and non-mutator control strains presented mutation frequencies of 6 x 10–7 and 9.9 x 10–9, respectively. We concluded that the strains Ar1 and Ar2 were not hypermutators.
Single-step mutants were selected in vitro by ofloxacin from E. coli Ar1 at a frequency of 10–8, as usually observed.6 Out of the 12 mutants studied, 3 (25%) showed an uncommon gyrA mutation at codon 81, but leading to the Gly-81Cys substitution, and none had the particular quinolone resistance phenotype of E. coli Ar2 (Table 2). Since we did not obtain the exact Gly-81Asp substitution by selection from E. coli Ar1, we tried to obtain isogenic mutants by conducting a second step in vitro selection by ofloxacin from E. coli SSc1, which is a mutant of E. coli KL16 previously obtained in our laboratory (data not shown) and that harbours the Gly-81Asp substitution in GyrA. Three of the ofloxacin-resistant mutants of E. coli SSc1 harboured an additional parC mutation. One (SSc1-1) harboured the exact combination of Gly-81Asp in GyrA and Ser-80Arg in ParC as observed in E. coli Ar2, and its quinolone resistance pattern was similar to that of E. coli Ar2. Although the MIC of ofloxacin for E. coli SSc1-1 was 2-fold lower than that for E. coli Ar2, ciprofloxacin and nalidixic acid MICs were the same (Table 2). Small intrinsic differences in the parental strains, i.e. E. coli Ar1 and E. coli KL16, might explain the lower ofloxacin MIC observed in the E. coli KL16 mutant.
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Discussion |
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We report the first case of clinical selection by ofloxacin of an E. coli strain (E. coli Ar2) with the combination of the very rare substitution Gly-81
Asp in GyrA and the common Ser-80Arg in ParC that confer high resistance to ofloxacin and retain susceptibility to nalidixic acid.
In E. coli, quinolone resistance due to topoisomerase gene mutations is acquired through a single-step gyrA mutation and results in high-level nalidixic resistance and low-level resistance to fluoroquinolones.1–3 High-level fluoroquinolone resistance is obtained by an additional mutation in gyrA and/or parC in a second- or a third-step model.4 In E. coli, GyrA substitutions are more frequently at positions 83 (Ser to Leu, Trp, Ala or Val) and 87 (Asp to Asn, Gly, Tyr, His, or Val).2,3 Although substitutions have been described at other codons (67, 82, 84 and 106), they occur less frequently and many of them have only been detected in in vitro mutants.2,3 Substitutions at position 81 in GyrA (Gly-81Cys, Gly-81Ser or Gly-81His) are very uncommon and have been described in Salmonella enterica, Serratia marcescens and Acinetobacter baumannii.2,8,9 In E. coli, they have only been described in one in vitro mutant (Gly-81Cys),2 and in one clinical isolate (Gly-81Asp).5 The strains harbouring GyrA substitution at position 81 usually show low-level resistance to fluoroquinolones (MICs of ciprofloxacin from 0.1 to 1 mg/L), but only those harbouring the GyrA Gly-81Asp substitution were as susceptible to nalidixic acid as wild-type strains and thus showed a ‘dissociated phenotype’ of quinolone resistance.5 In the context of the structure of E. coli gyrase, Gly-81Asp substitution in GyrA results in the replacement of a neutral amino acid with a negatively charged amino acid and in the juxtaposition of two residues (Asp-81 and Asp-82) in the vicinity of the 
-helix 4 that constitutes the actual proposed quinolone-binding site with the two major amino acids, Ser-83 and Asp-87.10 It has been shown that when the second Asp-82 residue is substituted with Gly, it abolishes the nalidixic acid susceptibility.11 In the mycobacterial model of gyrase, position 81 was thought to be important for quinolone orientation towards gyrase, particularly through the binding of the C-7 ring of fluoroquinolones.12 The Gly-81Asp modification in GyrA may produce steric hindrance responsible for a decreased binding of molecules with a bulky group at position C-7.5,12 Contrary to fluoroquinolones, nalidixic acid lacks a C-7 ring and its binding might not be hampered. Because of the O-ring between N-1 and N-8, ofloxacin and levofloxacin may be specifically hampered by the mutation at codon 81.
This is the first description of the combination of the substitution Gly-81Asp in GyrA with another substitution in DNA gyrase or topoisomerase IV. The Ser-80Arg substitution in ParC is common in addition to gyrase substitutions at positions 83 or 87 and has been shown to increase the level of resistance to all quinolones, including nalidixic acid.2,4 In our isogenic second-step mutant harbouring Ser-80Arg in ParC, MICs of ofloxacin and ciprofloxacin increased but not the MIC of nalidixic acid. This may be because DNA gyrase is the primary target in E. coli and the susceptible character of the primary target (herein Gly-81Asp GyrA is responsible for a nalidixic acid susceptibility character) is dominant over the resistant character conferred by the secondary target (herein Ser-80Arg in ParC).4 Full complementation with the wild-type gyrA gene showed that gyrA and parC mutations were responsible for the quinolone resistance acquired by E. coli Ar2, although other additional mutations outside of QRDRs are possible.
We tried to reproduce in vitro the combination of the double mutation with isogenic mutants. Since results of the in vitro selection by ofloxacin from E. coli Ar1, which was supposed to be the parental strain of E. coli Ar2 (similar DNA fingerprinting, and similar susceptibility to other antibiotics), showed a higher occurrence of gyrA mutations than in other studies,1–4,10,13 we investigated the strains E. coli Ar1 and Ar2 for a hypermutable character.7 Since they were not mutators, we hypothesized that the high occurrence of gyrA mutations may be due to ofloxacin. To our knowledge, this molecule has been rarely used for in vitro selection. Whereas most of the gyrA mutations obtained by selection with other quinolones were reported at codons 83 and 87,1–3 we observed mutations at codon 81 for 25% of the mutants selected by ofloxacin. Although the resulting substitution was not Gly-81Asp as observed in E. coli Ar2, we may attribute the frequency of mutation at codon 81 to ofloxacin. Since out of the twelve mutants selected in vitro by ofloxacin, a second uncommon gyrA mutation at codon 119 was observed, we may suppose that E. coli Ar1 harbours unknown mutations that facilitate the occurrence of mutations in the topoisomerase type II genes. Mutation at codon 119 has been rarely described and quinolone MICs for such mutants are similar to those for our mutant.3,13
Although in theory a bacterial population of >1014 cells would be needed to select for a double (gyrA and parC) mutant, E. coli Ar2 was probably selected in vivo during the 10 days of ofloxacin exposure, when the E. coli inoculum should have been greatly reduced after the 2 week course of ß-lactams. Selection may have been facilitated by the prostate site of infection or skin colonization, which are difficult to eradicate. The fact that resistance to ofloxacin, ciprofloxacin and nalidixic acid was dissociated shows that microbiologists may not categorize quinolone susceptibility on the basis of testing a single quinolone.
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None to declare.
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Acknowledgements |
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A preliminary report was presented at the Forty-fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2005 (abstract C1-1040).
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Yu X, Susa M, Knabbe C, et al. (2004) Development and validation of a diagnostic DNA microarray to detect quinolone-resistant Escherichia coli among clinical isolates. J Clin Microbiol 42:4083–91.
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Cambau E, Bordon F, Collatz E, et al. (1993) Novel gyrA point mutation in a strain of Escherichia coli resistant to fluoroquinolones but not to nalidixic acid. Antimicrob Agents Chemother 37:1247–52.
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Gilbert DN, Kolhlepp SJ, Slama KA, et al. (2001) Phenotypic resistance of Staphylococcus aureus, selected Enterobacteriaceae, and Pseudomonas aeruginosa after single and multiple in vitro exposures to ciprofloxacin, levofloxacin and trovafloxacin. Antimicrob Agents Chemother 45:883–92.
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Denamur E, Bonacorsi S, Giraud A, et al. (2002) High frequency of mutator strains among human uropathogenic Escherichia coli isolates. J Bacteriol 184:605–9.
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Weigel LM, Steward CD, Tenover FC. (1998) gyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae. Antimicrob Agents Chemother 42:2661–7.
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