JAC Advance Access originally published online on March 6, 2008
Journal of Antimicrobial Chemotherapy 2008 61(5):1007-1015; doi:10.1093/jac/dkn077
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Original research |
Low selection of topoisomerase mutants from strains of Escherichia coli harbouring plasmid-borne qnr genes
1 Université Paris12, IFR10, Créteil, France; 2 AP-HP, CHU Henri Mondor, Service de Bactériologie-Virologie-Hygiène, Créteil, France 3 Hôpital d'Instruction des Armées Bégin Laboratoire de Biologie, Saint-Mandé, France 4 Ecole du Val de Grâce, Paris, France
* Correspondence address. Laboratoire de Bactériologie-Virologie-Hygiène, CHU Henri Mondor, 51 rue du maréchal de Lattre de Tassigny, 94010 CRETEIL Cedex, France. Tel: +33-1-49812831; Fax: +33-1-49812839; E-mail: emmanuelle.cambau{at}hmn.aphp.fr
Received 16 August 2007; returned 24 October 2007; revised 16 January 2008; accepted 5 February 2008
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Abstract |
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Objectives: To investigate mutations in the type II topoisomerase genes in quinolone-resistant mutants selected from bacteria harbouring plasmid-borne qnr genes.
Methods: Mutants were selected by nalidixic acid, ciprofloxacin and moxifloxacin from two Escherichia coli reference strains and corresponding transconjugants harbouring qnrA1, qnrA3, qnrB2 or qnrS1 genes.
Results: The proportion of resistant mutants selected by the three quinolones was, respectively, in the same range for qnr-positive transconjugants and reference strains. Only 20% (65/329) of the mutants selected from the transconjugants showed a gyrase mutation, whereas 79% (94/119) of those from the reference strains without a qnr gene did (P < 0.0001). At four times the MIC of the selector quinolone, gyrA mutants represented 49% and 95% of the mutants selected with nalidixic acid, 4% and 94% with ciprofloxacin and 0% and 54% with moxifloxacin for qnr-positive transconjugants and reference strains, respectively. Mutations within gyrA were distributed at codon 87 (D87G, H, N or Y) and at codon 83 (S83L) with three novel mutations (gyrA Ser83stop, gyrA Asp82Asn and gyrB insertion of Glu at 465) and three rare mutations (gyrA Gly81Asp, gyrA Asp82Gly and gyrA Ser431Pro), mainly obtained from reference strains after moxifloxacin selection. Strikingly, none of the mutants selected by moxifloxacin from qnr-positive transconjugants harboured a mutation in the topoisomerase genes.
Conclusions: Topoisomerase mutants are rarely selected by ciprofloxacin and moxifloxacin from strains harbouring qnr. This suggests that the quinolone resistance-determining region domains are protected from quinolones by the Qnr protein and consequently other mechanisms are developed to acquire a further step of fluoroquinolone resistance.
Keywords: quinolones , ciprofloxacin , gyrase , moxifloxacin , QRDR
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Introduction |
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Qnr is a new mechanism of quinolone resistance mediated by topoisomerase protection.1 The original qnr gene, known now as the ‘qnrA1’ allele, was the first plasmid-borne quinolone resistance gene to be described.2 Two other transferable quinolone resistance genes have been described subsequently although they act via distinct mechanisms: aac6'-Ib-cr mediating acetylation of norfloxacin and ciprofloxacin,3 and qepA mediating efflux.4
Classical mechanisms of quinolone resistance are due to chromosomal mutations in the genes encoding the quinolone targets or in regulatory genes affecting permeability or efflux.5,6 Bacterial targets of quinolones are the type II topoisomerases: DNA gyrase (GyrA2GyrB2) and topoisomerase IV (ParC2ParE2). Mutations in the DNA gyrase genes (gyrA and gyrB) and in the topoisomerase IV genes (parC and parE) mainly occur in the quinolone resistance-determining regions (QRDRs) of each subunit at codons 83 and 87 (numbering system in Escherichia coli) in gyrA and codons 426, 447 and 464 in gyrB.6–11 Since gyrase is the primary target in E. coli, mutations in parC or parE are only observed in second- or third-step mutants and in association with gyrase mutations.7,12–15
The qnr genes reported so far are qnrA,16 qnrB17 and qnrS18 sharing ca 50% identity.19 Various alleles of qnrA, qnrB and qnrS genes have also been described and share 84% to 99% identity with the original gene.20 qnr genes encode 215–226 amino acid proteins, belonging to the pentapeptide repeat family.21–23 In vitro assays conducted with QnrA1 and QnrB1 showed that they protect DNA gyrase and topoisomerase IV from the inhibitory activity of quinolones in a proportional manner.16,18,24,25 Qnr may thus interfere with the binding of quinolones to DNA gyrase and topoisomerase IV. Our hypothesis was that the interaction site between Qnr and DNA gyrase may overlap that of quinolones and DNA gyrase, which corresponds to the QRDRs. Consequently, mutations conferring quinolone resistance in strains producing Qnr might not be located within the conventional QRDRs.
qnr genes are transferred by conjugation of multidrug-resistant plasmids between enterobacterial species2 and confer a similar low level of quinolone resistance in E. coli recipients.17,18,26 Other mechanisms of resistance, either conferred by other plasmid-borne genes or chromosomal-borne genes of the recipient strains, are additive to Qnr26 and their combination results in a high level of quinolone resistance, usually observed in clinical strains.27,28 In these clinical qnr-positive strains, we previously described classical topoisomerase mutations at codon 83 or 87 in GyrA and at codon 80 or 84 in ParC.27,29 However, it is not clear if these topoisomerase mutations occurred in strains already harbouring qnr genes, in order to bring a further step of quinolone resistance, or if the qnr gene was gained by quinolone-resistant strains with topoisomerase mutations.
Our objective was to investigate whether strains harbouring qnr genes develop similar topoisomerase mutations as qnr-negative strains. We conducted selection experiments with quinolones from: (i) E. coli transconjugants harbouring a qnr gene, either qnrA (two different alleles), qnrB or qnrS; and (ii) reference strains of E. coli as a control. Type II topoisomerase mutations were sought in randomly selected mutants from different experiments. We observed that although the proportions of mutants were similar, gyrase mutations were significantly rarer after quinolone selection from qnr-positive strains than from E. coli strains lacking qnr.
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Parental strains used in the selection experiments
Parental strains were transconjugants obtained after conjugation between clinical strains harbouring a qnr gene and E. coli J53 resistant to sodium azide (gift of W. Sougakoff). Donors were Enterobacter cloacae Hm477 harbouring qnrA1,30 Klebsiella pneumoniae He96 harbouring qnrA3,30 E. cloacae Ps0429 harbouring qnrB2 (this study; gift of J. Robert) and E. cloacae Hm-05184 harbouring qnrS1 (this study). The transconjugants E. coli Tc(qnrA1), E. coli Tc(qnrA3), E. coli Tc(qnrB2) and E. coli Tc(qnrS1) were obtained after 40 min of mating in Mueller–Hinton (MH) broth as previously described for E. coli Tc(qnrA1).30 E. coli J53 and E. coli KL16 were reference strains used as comparators for in vitro selection of quinolone-resistant mutants.
Selection of resistant mutants by quinolones
Strains were grown at 37°C overnight in antibiotic-free MH broth. Hundred millilitre cultures were centrifuged and the pellet was suspended in 5 mL of sterile broth to give an inoculum of 
109–1010 cfu/mL. Agar plates containing nalidixic acid, ciprofloxacin and moxifloxacin at concentration of MIC (MIC value referring to the parental strains) x1, MIC x2, MIC x4, MIC x8 and MIC x16 were inoculated with 100 µL of cell suspension and incubated at 37°C. One to five colonies were randomly taken from each selecting plate. The stability of the qnr gene in the quinolone-resistant mutants was assessed by PCR detection.27,31 Selection experiments were performed twice on the same day and the whole first-step experiment was repeated four times for E. coli Tc(qnrA1) and E. coli J53, and two times for E. coli Tc(qnrA3), E. coli Tc(qnrB2), E. coli Tc(qnrS1) and E. coli KL16, respectively.
A second-step selection by ciprofloxacin and moxifloxacin was performed for E. coli Tc(qnrA1). Mutants of E. coli Tc(qnrA1) obtained from the first-step and the second-step of selection were studied for MICs of nalidixic acid, ciprofloxacin, moxifloxacin (agar dilution method) and susceptibility to other antibiotics (agar diffusion method with discs from Bio-Rad, Marnes la Coquette, France).
PCR and DNA sequencing of DNA gyrase and topoisomerase IV genes
QRDRs in gyrA, gyrB, parC and parE were amplified from total DNA, as described previously, in order to obtain a 479 bp fragment from nucleotide position –42 to position 436 of gyrA, a 259 bp fragment from position 1227 to 1485 of gyrB, a 194 bp fragment from position 155 to 348 in parC and a 155 bp fragment from position 1214 to 1368 in parE. 27 Reactions were performed in an iCycler (Bio-Rad) with 0.25 mM of mixed deoxynucleotide triphosphate (dNTPs), 2.5 U of Taq DNA polymerase (QBiogene), 5 µL of buffer, 0.5 µM of each primer (Proligo) and 5 µL of DNA in a final volume of 50 µL. The whole gyrA and gyrB genes were amplified with primers 5'-CTGCGCGGCTGTGTTATAATT-3' and 5'-GCCCAGACTTTGCAGCCTGG-3' for gyrA generating a 2723 bp fragment, and with primers 5'-TTCGAAGATGTTTACCGTG-3' and 5'-GGCCTGATAAGCGTAGCGC-3' for gyrB generating a 2480 bp fragment. PCR was performed with 0.35 mM dNTPs, 1 U of DNA polymerase (Kit Expand Long Template PCR, Roche), 5 µL of the buffer 3, 0.35 µM of each primer and 5 µL of DNA in a final volume of 50 µL. The hybridization temperatures were 57°C for amplification of gyrA and parE QRDRs, 50°C for amplification of parC QRDR, 40°C for amplification of gyrB QRDR and 50°C for the whole gyrA and gyrB genes.
PCR amplified fragments were purified using the Kit Montage PCR (Millipore) and were sequenced using the Kit ‘BigDye terminator v3.1 Cycle Sequencing’ and the sequencer ABI PRISM 3100 (Applied Biosystems). Sequences were analysed with Chromas and BioEdit softwares. For the whole gyrA and gyrB genes, sequencing of the amplification fragments was done by chromosome walking with the following primers: 5'-CTGCGCGGCTGTGTTATAATT-3', 5'-CGCCGTAGGTATGGCAACC-3', 5'-CGTCACCGCCGTGAAGTGG-3', 5'-GGTCGTGACGCTCTCTCAC-3', 5'-CCGTAACCGTTTTGCGTTG-3', 5'-GCCCAGACTTTGCAGCCTGG-3' for gyrA, and 5'-TTCGAAGATGTTTACCGTG-3', 5'-ACCAATGTGACCGAGTTCG-3', 5'-TCGACGTCCGCATCGGGTCAT-3', 5'-CTGGTATCTGAGTACAACG-3', 5'-GGCCTGATAAGCGTAGCGC-3' for gyrB.
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Proportion of quinolone-resistant mutants from E. coli harbouring a qnr gene
Quinolone-resistant mutants were selected from the four transconjugants and the two reference E. coli strains by plating on MH plates containing concentrations determined as MIC folds as described earlier. MICs are presented in Table 1, as well as the concentration of quinolones at which no mutant was selected, determined for the highest inocula (109–1010 cfu), which are close to mutant prevention concentration (MPC).32 Although MPCs of ciprofloxacin and moxifloxacin for the transconjugants were 10-fold higher than those for the E. coli strains lacking qnr, the MPC/MIC ratios were similar in the two groups, between 4 and 16 for ciprofloxacin and moxifloxacin. Mutants were systematically checked for the presence of qnr. The stability of the qnr gene (proportion of qnr-positive mutants among mutants selected) was 95% for qnrA1, 77% for qnrA3, 80% for qnrB1 and 100% for qnrS. Only mutants harbouring qnr were studied further.
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Proportions of resistant mutants, determined from selection experiments with transconjugants and reference strains, were compiled and presented in Figure 1. For the three quinolones, the data did not significantly differ between qnr-positive E. coli transconjugants and E. coli recipients lacking qnr selected at two times and four times the MIC.
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Occurrence of gyrase mutations in strains harbouring a qnr gene
Mutants were collected from distinct experiments (independently grown cultures) according to the quinolone used for the selection, the parental strain and the concentration at which mutants were selected. This concentration was higher for qnr-positive transconjugants than for strains lacking qnr: 0.25, 0.5 and 1 mg/L versus 0.03 and 0.06 mg/L for ciprofloxacin; and 1 and 4 mg/L versus 0.12 and 0.25 mg/L for moxifloxacin. Clones were replicated separately on antibiotic-free MH agar plates and were also tested for growth on antibiotic-containing MH. Overall, 448 first-step mutants were examined for QRDR sequences in gyrA, gyrB, parC and parE. The results are summarized in Table 2 in comparison with the qnr gene. No mutations were observed in parC and in parE, which is not surprising for first-step mutants of E. coli. Overall, 36% (159/448) of the mutants harboured a point mutation in the gyrase genes, mostly in gyrA (154/159, 97%). Mutation was observed in gyrB only for five (5/448, 1.1%) mutants. The proportion of gyrase mutants, and especially of gyrA mutants, was compared between mutants selected from quinolone susceptible and reference E. coli strains and those from transconjugants harbouring a qnr gene (Table 2). Data for qnrA1 and qnrA3 were compiled since they led to similar results. The proportion of gyrase mutants was only 20% for mutants selected from qnr-positive transconjugants and differed significantly from the proportion of 79% measured for the qnr-negative E. coli strains (P < 0.0001). All qnr genes led to a similar low proportion of gyrase mutants. Among gyrase mutations, gyrA mutations were the most frequent whatever the parental strain: 97% for reference strains, 98% for E. coli Tc(qnrA), 100% for E. coli Tc(qnrB) and 83% (5 out of 6) for E. coli Tc(qnrS). From qnr-positive transconjugants, gyrA mutants were more often selected by nalidixic acid than by ciprofloxacin [61/124 (49%) versus 4/100 (4%), P < 0.001], although they were equally selected [37/39 (95%) versus 32/34 (94%)] from the qnr-negative reference strains. It is of interest that overall moxifloxacin selected few gyrase mutants [only 25 out of the 156 mutants (16%)] and that these mutants were obtained from the qnr-negative strains. From reference strains, the proportion of gyrase mutants was lower with moxifloxacin (54%) than with ciprofloxacin (94%) and with nalidixic acid (95%). We checked that moxifloxacin-resistant mutants still harboured the qnr allele, showed a significant increase in quinolone MICs and were devoid of topoisomerase mutations outside the QRDRs. Further analysis of the mutants from E. coli Tc(qnrA1) was carried out as appears below.
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Distribution and type of gyrase mutations at the QRDR codons
Substitutions in GyrA and in GyrB were analysed with regard to the parental strain and the quinolone used for selection (Table 3). Most of the substitutions were at positions 83 and 87. Although they were equally distributed at these two positions in mutants obtained from qnr-negative strains, substitution of Ser83 was more frequently observed than that of Asp87 in mutants obtained from qnr-positive transconjugants (34% and 39% versus 13% and 6%, respectively, P = 0.02). The Ser83Leu substitution was preponderant [83/159 (52%)]. Various substitutions of Asp87 were observed resulting in Gly, Tyr, His, Asn or Val. Four substitutions were at position 81 or 82: Gly81Cys, Gly81Asp, Asp82Gly and Asp82Asn. They are either new (Asp82Asn) or uncommon. The corresponding mutants were selected exclusively from the reference strains and three of them (Gly81Cys, Gly81Asp and Asp82Gly) by moxifloxacin. Strikingly, we found in a mutant selected from E. coli Tc (qnrA1), the substitution of the serine codon (TCG) at position 83 into a Stop codon of the Amber-type (TAG). This has never been described previously in a topoisomerase gene.
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gyrB mutations were rare and were observed in mutants selected from the reference strains for three of them, one from E. coli Tc(qnrA3) and one from E. coli Tc(qnrS1). They concerned various codons (431, 447 and 464). An insertion of a Glu codon between the codons 465 and 466 was observed, which has never been described previously.
Two-step selection from the qnrA1-positive transconjugant
In order to reproduce the successive steps of selection observed in vivo, we performed two steps of selection from E. coli Tc(qnrA1), taken as a representative qnr-positive transconjugant. First-step selection was as described earlier. Second-step mutants were selected by ciprofloxacin (MH plates containing 0.25, 0.5, 1, 2 and 4 mg/L) and moxifloxacin (MH plates containing 0.5, 1, 2, 4, 8 and 16 mg/L) from: (i) the mutant HmN11, which was selected by nalidixic acid and harboured a gyrA mutation (Ser83Leu); (ii) the mutant HmC7, selected by ciprofloxacin and lacking QRDR mutation; and (iii) the mutant HmM5, selected by moxifloxacin and lacking QRDR mutation.
In the second-step mutants obtained from the gyrA mutant (HmN11), either selected by ciprofloxacin or by moxifloxacin, we observed an additional parC mutation at codon 80 (Ser80Ile), at codon 84 (Glu84Lys) or at codon 78 (Gly78Asp). However, most (17/27, 63%) of the mutants did not harbour additional mutation in either the gyrase or the topoisomerase IV genes. For these mutants, the mutation gyrA Ser83Leu was indeed conserved. Out of the second-step mutants selected from a first-step mutant without gyrase mutation (HmC7 or HmM5), only two (2/24, 8%) gained a gyrA mutation (Ser83Leu). The remaining 22 mutants were sequenced for the whole gyrA and gyrB and did not show mutation outside the QRDRs.
Quinolone resistance levels of topoisomerase mutants of E. coli Tc(qnrA1)
MICs of quinolones for first-step (n = 128) and second-step (n = 113) mutants obtained from E. coli Tc(qnrA1) distributed into four groups with regard to the MICs and the QRDR mutation (Table 4 and Figure 2). The first group (I) includes clones for which geometric mean MICs were close to those for the parental strain, and thus were not considered as true quinolone-resistant mutants. The second group (II) includes clones with a moderate increase in the MIC of the three quinolones and without QRDR mutation. Associated resistance to tetracycline, chloramphenicol, trimethoprim and β-lactams led us to hypothesize that they were potential permeability or efflux mutants.5
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The third group (III) includes clones with at least one topoisomerase mutation and was divided into three subgroups: first-step mutants with one gyrA mutation (subgroup a), second-step mutants with single gyrA and parC mutation (subgroup b) and second-step mutants with only one gyrA mutation (subgroup c). From the MIC results for group IIIa, we observed that one gyrA mutation occurring in a strain harbouring qnrA1 led to a 16- to 32-fold increase in nalidixic acid MIC (final MIC of 512 mg/L), and only a 2- to 4-fold increase in ciprofloxacin MIC (final MIC of 0.25 mg/L) and moxifloxacin, respectively. These MICs were similar values to MICs determined against first-step mutants with one gyrA mutation obtained from reference strains lacking qnr: geometric mean MIC values being 311 mg/L for nalidixic acid, 0.3 mg/L for ciprofloxacin and 0.7 mg/L for moxifloxacin. Additional mutation in parC (group IIIb) resulted in a further increase in ciprofloxacin and in moxifloxacin MICs (final MICs of 4 and 8 mg/L). It should be pointed out that nalidixic acid MIC decreased 4-fold (from 512 to 128 mg/L) for the mutants harbouring Asp78Gly alteration in ParC in addition to Ser83Leu alteration in GyrA. The mutants of the group IIIc showed lower MICs than those of the group IIIb and might have acquired a mechanism of efflux or reduced permeability in addition to the gyrase mutation, since ciprofloxacin MICs increased more than moxifloxacin and nalidixic acid MICs.
The fourth group (IV) includes second-step mutants for which MICs of the three quinolones rose 20-fold and were without mutation in the topoisomerase genes. Geometric mean MICs of nalidixic acid and ciprofloxacin were lower than those of clones of group III, but moxifloxacin MICs were up to 16 mg/L, i.e. 64-fold the MIC for the parental strain (Figure 2). For the clones of this group, we re-sequenced the qnrA gene and its environment and no additional mutations were found.
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Qnr proteins, involved in quinolone resistance, have been shown to interact with DNA gyrase and topoisomerase IV and to protect them from quinolone inhibition.18,24,25 In order to gain information on the interaction site between Qnr and DNA gyrase, we aimed to identify the QRDR, i.e. the interaction site between quinolones and DNA gyrase, for a DNA gyrase bound by Qnr proteins. Identification of the QRDR in the presence of Qnr was achieved through in vitro selection of quinolone-resistant mutants of E. coli harbouring a qnr gene. Different qnr genes (qnrA, qnrB and qnrS) and alleles (qnrA1 and qnrA3) were tested and gave similar results. The results were compared with those determined in the absence of Qnr, i.e. in reference qnr-negative strains.
Although the frequency of selection of quinolone-resistant mutants was reported to be 10–100 times higher in the presence of Qnr at critical concentrations,2,33 we did not observe a significant difference between the proportions of resistant mutants determined with transconjugants and with reference strains, the latter proportions being close to that previously observed by others.34,35 However, proportions slightly varied according to the selection experiment and the parental strain, even if the conditions of the experiment were the same (quinolone concentration measured as MIC folds, isogenic parental strains). This is commonly reported in quinolone selection experiments.29,34 MPC values observed for qnr-positive transconjugants were similar to those observed for strains with a first step of quinolone resistance such as Salmonella enterica strains with a gyrA mutation,29 and MPC/MIC ratios were similar to those observed for quinolone selection in E. coli.36 We noticed that quinolone MIC values were not significantly higher in mutants selected in the presence of qnr than in the absence of qnr. This could be explained by the fact that MIC reflects more the sensitivity of the unmutated target, i.e. topoisomerase IV, which remains the same if Qnr is bound to gyrase or if gyrase is mutated.
Although our hypothesis was that when Qnr is bound to gyrase, it hampers the access of quinolones to QRDRs, we were surprised by the low occurrence of mutation in the QRDRs after a first-step selection with the transconjugants. Such a low proportion of gyrase mutants has never been described before for E. coli. In previous studies, most of the E. coli mutants selected in vitro by nalidixic acid, norfloxacin or ciprofloxacin were reported to be gyrase mutants,6,7,10,12,37,38 which is concordant with data we obtained for the control reference strains. Topoisomerase mutation outside the QRDRs was not observed. Mechanisms other than topoisomerase mutation have occurred to bring the significant increase in quinolone MICs observed in this study. These mechanisms are not likely to have been enhanced efflux or decrease in permeability since the MICs of other antibiotics were not increased. One can suggest that overproduction of Qnr may result in high-level quinolone resistance, but mutation in the qnr gene itself or in its environment was not found in the mutants. Further study of these mutants will be necessary in order to design possible new mechanisms of resistance.
The important result of this study is that topoisomerase mutation rarely occurs in qnr-positive strains. This suggests that the interaction of the quinolones with the topoisomerase QRDR domains may be impeded by the Qnr protein. The impossibility to produce topoisomerase mutants in the presence of Qnr is particularly obvious with ciprofloxacin and moxifloxacin, since only four gyrase mutants (representing 4% of the mutants) were selected by ciprofloxacin and none (0/110) by moxifloxacin.
DNA gyrase mutations selected in the presence of Qnr were mainly at positions 83 and 87 in GyrA, which are the classic hot spots of mutations observed in clinical quinolone-resistant strains.39 gyrA mutations occur rarely at positions other than 83 and 87, such as 51, 67, 81, 84, 106 and 119.6,7,39 Although three novel mutations (Ser83stop in GyrA, Asp82Asn in GyrA and Glu insertion at 465 in GyrB) and three rare mutations (Gly81Asp in GyrA,40 Asp82Gly in GyrA41 and Ser431Pro in GyrB42) were observed in our selection study, most of them were selected from the reference strains. The occurrence of rare mutations may be explained by the specific characters of the host cell E. coli J53 used for conjugation and as a control strain. We had doubts at first about the Ser83stop mutation in GyrA since gyrase genes are essential genes43 and the mutant grew normally. A probable explanation would be the presence in the cell of specific RNAt suppressors that are able to transform Amber stop codons into amino acid residues such as Trp or Leu, which are known to confer quinolone resistance when located at position 83 in GyrA.44 Such RNAt suppressors have been described in some of the E. coli K-12 derivatives and may occur in strains of its lineage such as E. coli J53.45
In mutants obtained from qnr-positive transconjugants as well as in those obtained from reference strains, gyrA mutation occurred at the first-step selection, and parC mutation was observed only in second-step mutants already harbouring a gyrA mutation. This is in agreement with DNA gyrase being the primary target of quinolones in E. coli and topoisomerase IV being the secondary target.6,7,12,13 These results showed that the presence of Qnr did not modify the respective role of the two type II topoisomerases as primary and secondary targets of quinolones. From the two-step selection of qnrA-positive transconjugants, again, no topoisomerase mutant was observed after selection by moxifloxacin, and few were observed after selection by ciprofloxacin. Substitutions in topoisomerase IV were Ser80Ile and Gly84Lys in ParC, which are common, and Gly78Asp, which is rare.13 A corresponding substitution in GyrA (Gly81Asp) was previously shown to confer an original pattern of quinolone resistance with resistance to ciprofloxacin and susceptibility to nalidixic acid.40 We confirmed the specific effect of the substitution Gly78Asp in ParC since a decrease in nalidixic acid MIC was observed in this mutant.
Results concerning moxifloxacin differed from those obtained with nalidixic acid and ciprofloxacin. First, topoisomerase mutations occurred less frequently than with nalidixic acid or ciprofloxacin even after selection from reference strains of E. coli and second, when observed, these mutations were uncommon (positions 81 and 82 in GyrA, insertion in GyrB and Gly79Asp in ParC). The occurrence of gyrase mutants after selection by moxifloxacin has not been extensively studied in E. coli since this new fluoroquinolone targets Gram-positive bacteria such as pneumococci and staphylococci.46 In S. pneumoniae, mutations are classically observed at positions corresponding to 83 and 87 in GyrA (E. coli numbering).6 This suggests that the interaction of moxifloxacin with E. coli topoisomerases may be different.
It has been hypothesized that qnr genes were transferred from bacterial progenitors, such as Shewanella algae and Vibrionaceae,47 and that qnr emerged among clinical strains because of the quinolone selective pressure.1,2,19,47 However, the low level of resistance conferred by qnr26,27,48 seems not high enough to lead to clinical selection, even in non-urinary infections,49 and the addition of another mechanism of resistance is necessary to pass over the critical breakpoints.26 qnr-positive clinical strains reported in recent years were indeed highly resistant to ciprofloxacin28,30,50 and harboured multiple mechanisms such as mutations in gyrA and in parC,27 or decreased permeability.51 Although some qnr-positive clinical isolates are ‘pure’ qnr-positive strains and thus remain ciprofloxacin susceptible,30,52 it is not clear whether the first step of resistance is the acquisition of the qnr gene or the occurrence of a topoisomerase mutation. From the in vitro selection experiments conducted in this study, we showed that qnr-positive strains very rarely developed topoisomerase mutations using ciprofloxacin as a selector, which is one of the most prescribed quinolones, even in the two-step selection experiment, which reproduces the clinical selection of ciprofloxacin-resistant E. coli.10,27,29,30,36,53 Therefore, it is probable that most often the qnr gene is acquired by strains having already developed topoisomerase mutations. The advantage conferred may be more due to other genes present on the multiresistance plasmid, such as those encoded extended-spectrum β-lactamases,54 than to the additional quinolone resistance.
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This work was supported by a grant from the Chancellerie de l'Université de Paris.
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None to declare.
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