Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates

María del Mar Tavíoa,*, Jordi Vilab, Joaquím Ruizb, José Ruiza, Antonio Manuel Martín-Sáncheza and María Teresa Jiménez de Antab

a Microbiology, Department of Clinical Sciences, School of Medicine, University of Las Palmas de Gran Canaria, Dr. Pasteur s/n, 35080 Las Palmas; b Department of Microbiology, Hospital Clinic, School of Medicine, University of Barcelona, Villarroel 170, 08036 Barcelona, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Eighteen quinolone-resistant isolates of Escherichia coli were selected by exposing ten clinical isolates to increasing concentrations of norfloxacin and lomefloxacin. The mutant isolates showed a multiple-antibiotic-resistance phenotype. All of them contained single mutations in gyrA consisting of the substitution of Ser-83->Leu (n = 14), Val (n = 1) or Ala (n = 1) and the substitution of Asp-87->Asn (n = 2). Only one concomitant mutation in parC (Ser-80->Arg) was detected. Four parent isolates exhibited a single mutation in gyrA which required <=12 mg/L of norfloxacin to be inhibited. Fluoroquinolone resistance, in the 18 quinolone-resistant mutants, was a result of mutations affecting DNA gyrase plus decreased fluoroquinolone uptake. This latter mechanism of resistance was a combined effect of an absence of OmpF and an increase in active efflux in eight isolates, or an increased active efflux alone in the remaining ten selected mutants.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The quinolones are a group of synthetic antimicrobial agents that includes nalidixic acid and the fluorinated quinolones. Different mechanisms are involved in the development of resistance to fluoroquinolones. Some are the consequence of mutations involving the genes that encode DNA gyrase1,2,3,4,5 and topoisomerase IV.1,4,6,7 Other mutations affect the accumulation of quinolones; they include those affecting either the expression of porins1,4,8 or lipopolysaccharide,9,10 or the active efflux of quinolones from the bacterial cell.1,4,11,12 In this context, previous studies have described the involvement of a decreased accumulation of fluoroquinolones in mutants of Escherichia coli with a multiple-antibiotic-resistance phenotype (Mar mutants). This phenotype is due to the reduced expression of OmpF and the overexpression of active efflux systems.12,13 In this study, the roles of the mechanisms currently described in the acquisition of resistance to quinolones were analysed. Eighteen E. coli mutants were selected by exposure to increasing concentrations of norfloxacin and lomefloxacin. Differences and similarities between each mutant and its parent isolate were characterized to elucidate the respective roles of changes of targets, permeability and active efflux in the development of resistance to fluoroquinolones.


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

Ten clinical isolates of E. coli obtained from the Microbiology Service of the Insular Hospital of Gran Canaria (Spain) that were susceptible to fluoroquinolones, were used as parent isolates (the PS isolates). Spontaneous resistant mutants were selected by exposing these isolates to increasing concentrations of norfloxacin (the NorE isolates) and lomefloxacin (the LmfE isolates) until the MIC of the selective agent was >=8 mg/L. E. coli JF699 (OmpA deficient isolate—J. Foulds isolate), E. coli JF703 (OmpF deficient isolate—J. Foulds isolate), E. coli KL16 isolate (K. B. Low isolate) (donated by Dr Mary K. B. Berlyn, E. coli Genetic Stock Center, New Haven, CT, USA)14 and E. coli ATCC 25922 (donated by Dr Federico Uruburu, Spanish Type Culture Collection, Valencia, Spain) were also used.

Antibiotics and determination of susceptibility

MICs were determined by an agar dilution method according to the NCCLS guidelines.15 Antimicrobial agents were supplied by their manufacturers: ciprofloxacin (Bayer, Barcelona, Spain), lomefloxacin (G. D. Searle & Co, Madrid, Spain), ofloxacin (Roussel, Madrid, Spain), temafloxacin (Abbott, Madrid, Spain), sparfloxacin (Rhône– Poulenc–Rorer, Madrid, Spain) and cephalothin (Lilly, Madrid, Spain). Tetracycline, nalidixic acid and norfloxacin were obtained from Sigma Chemical Co. (Madrid, Spain).

Amplification and DNA sequencing of the quinolone resistance-determining region of the gyrA, gyrB, parC and parE genes

The different gene sequences (gyrA, gyrB, parC and parE) were amplified by the polymerase chain reaction (PCR) using the method and primers previously described.5,6,16 The purified PCR product was processed for DNA sequencing and analysed in an automatic DNA sequencer.

Preparation and analysis of outer membrane proteins (OMP) and lipopolysaccharide (LPS)

Outer membrane extracts were prepared using the method described by Sawai et al.17 Each membrane suspension (10 µg) in loading buffer was electrophoresed in a 6 M urea–10% SDS (sodium dodecyl sulphate) polyacrylamide gel. The LPS extracts from intact cells were obtained18 and analysed using 13.5% polyacrylamide gel electrophoresis (PAGE) as described previously.18 The LPS bands were visualized by silver staining.19

Norfloxacin accumulation determination

Uptake measurements were repeated at least three times by the method described by Hirai et al.9,20 Intracellular accumulation of norfloxacin was always measured at the same time in each parent isolate and the quinolone-resistant mutants derived from it, with and without the presence of 50 and 100 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP). Bacterial cells were grown to mid-log phase at 37°C in antibiotic medium 3 (Difco, Madrid, Spain), and each bacterial cell suspension was prepared in sodium phosphate buffer (50 mM, pH 7.0) to an optical density of 1.5 at 520 nm wavelength. The total bacterial suspension of each isolate was divided into three at the beginning of each assay, to measure in parallel norfloxacin uptake with and without 50 and 100 µM CCCP. CCCP was added to two parts of the bacterial suspension 10 min before norfloxacin was added.21 Each bacterial suspension (8 mL) was collected and chilled at 5, 15 and 30 min after the addition of norfloxacin 10 mg/L. The cells of each sample were sedimented by centrifugation and washed once in 2 ml of sodium phosphate buffer (50 mM, pH 7.0). The cells were then suspended in 200 µL of the same buffer. The suspension was immersed in boiling water for 7 min and then centrifuged.9,20 The concentrations of norfloxacin in the intracellular extracts were measured by bioassay using Klebsiella pneumoniae ATCC 100319,20 (donated by Dr Federico Uruburu, Spanish Type Culture Collection, Valencia, Spain). The norfloxacin concentration in each intracellular extract was measured by bioassay at least six times. The accepted standard deviation for all the uptake results was always <=5% with respect to each mean value of several measurements taken at 5, 15 and 30 min, with or without CCCP.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutant isolation and selection

Three consecutive selective steps were needed for the development of mutants, for which the selective agent MIC was >=8 mg/L, except for the NorE1, LmfE1, NorE5, LmfE5 and LmfE7 isolates, which were obtained after two consecutive selective steps. The mutation frequencies of the different selective steps with both norfloxacin or lomefloxacin ranged between 10–9 and 10–4. Only the most resistant mutants selected (norfloxacin or lomefloxacin MIC >= 8 mg/L) were studied (18 isolates).

Determination of MICs

The susceptibilities to various antibiotics of the mutants and their parent isolates are shown in Table I. Many of the selected mutants showed an increased level of resistance to tetracycline and cephalothin as well as to quinolones (Table I).


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Table I. Susceptibilities of wild-type isolates, parent isolates and mutants
 
Analysis of mutations in DNA gyrase and topoisomerase IV

To understand the role of mutations of DNA gyrase and topoisomerase IV in the development of resistance to quinolones in the mutants, the sequences of the quinolone resistance-determining region of the gyrA, gyrB, parC and parE genes were studied (Table II). This analysis demonstrated that the PS1, PS5, PS7 isolates and 14 of the quinolone-resistant mutants exhibited a mutation in the gyrA gene (Ser-83->Leu) that was generated by a C->T transversion in the codon TCG. For the NorE6 and LmfE6 isolates the substitution was Asp-87->Asn, which was generated from G->A transversion in the codon GAC. In addition, the T->G transversion in the codon TCG resulted in a change of Ser-83->Ala in the GyrA subunit in the PS10 and NorE10 isolates. For the LmfE10 isolate the substitution of Ala-83->Val, which was generated from the codon GCG by a CRT transversion, was demonstrated. No double mutations in the gyrA gene were detected. Only one isolate (NorE5) was also shown to contain a second mutation at codon 80 (Ser), which was generated by a T->A transversion from codon AGT in the parC gene, leading to Ser-80->Arg substitution. No mutations were detected in the amplified fragment of the gyrB or parE genes.


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Table II. Substitutions in the GyrA and ParC subunits in parent isolates and mutants
 
Electrophoretic profiles of outer membrane proteins and lipopolysaccharide

All the parent isolates expressed the C and F porins. The NorE1, NorE2, LmfE2, NorE3, LmfE3, LmfE4, NorE5, LmfE5, NorE8, LmfE9 and NorE10 isolates (Figure) showed decreased expression of an electrophoretic band migrating at the OmpF position. The NorE1 and LmfE9 isolates did not express a band with an electrophoretic mobility in urea–SDS polyacrylamide gels consistent with OmpC (Figure 1). Examination of the outer membrane LPS revealed that all the parent isolates (except the PS5 and PS6 isolates) and most of the quinolone-resistant mutants exhibited smooth LPS, i.e. high molecular mass polymers were expressed. They were not detected in the PS5, NorE5, PS6, PS8 and NorE10 isolates' LPS profiles, resulting in the expression of rough LPS. Similarly, the comparison of LPS profiles from selected mutants with those of their respective parent isolates demonstrated a slightly decreased expression of the latter polymers in the NorE1, LmfE1, LmfE7 and LmfE10 isolates. The LmfE5, LmfE7 and LmfE9 isolates showed a decreased expression of low molecular mass polymers.



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Figure. Electrophoretic profiles of outer membrane protein extracts in a 6 M urea–10% polyacrylamide gel. (a) Lanes: 1, E. coli KL16; 2, E. coli JF703; 3, E. coli JF699; 4, E. coli ATCC 25922; 5, PS1; 6, NorE1; 7, LmfE1; 8, PS7; 9, LmfE7. (b) Lanes: 1, E. coli KL16; 2, E. coli ATCC 25922; 3, PS2; 4, NorE2; 5, LmfE2. (c) Lanes: 1, E. coli KL16; 2, E. coli ATCC 25922; 3, PS3; 4, NorE3; 5, LmfE3; 6, PS4; 7, NorE4; 8, LmfE4. (d) Lanes: 1, E. coli KL16; 2, E. coli ATCC 25922; 3, PS5; 4, NorE5; 5, LmfE5; 6, PS9; 7, NorE9; 8, LmfE9. (e) Lanes: 1, E. coli KL16; 2, E. coli ATCC 25922; 3, PS6; 4, NorE6; 5, LmfE6; 6, PS8; 7, NorE8; 8, PS10; 9, NorE10; 10; LmfE10.

 
Norfloxacin accumulation

Eleven mutants (the NorE1, NorE2, LmfE2, LmfE4, NorE5, LmfE5, NorE6, LmfE6, NorE8, NorE9 and LmfE9 isolates) showed a lower accumulation of norfloxacin than their PS isolates; their norfloxacin uptake range was 16 to 77% in comparison with their parent isolates. In the remaining seven mutants the difference in the norfloxacin accumulation was lower than or equal to the standard deviation (5%), except in the NorE4 and LmfE7 isolates, which accumulated 50% and 26% more norfloxacin than their parent isolates, respectively. Furthermore, three parent isolates (the PS5, PS6 and PS7 isolates) accumulated 68–75% and the remaining seven PS isolates accumulated <50% than the KL16 isolate (Table III). The difference between the effect of 50 and 100 µM CCCP in the intracellular accumulation of norfloxacin both in the quinolone-resistant mutants and in the parent isolates was lower than the maximum accepted standard deviation for uptake, 5% (Table III). The addition of both 50 and 100 µM CCCP resulted in increases in intracellular norfloxacin concentrations that were higher than 20% in all the parent isolates, with the exceptions of the PS2, PS3 and PS4 isolates (Table III). The effect of CCCP was more noticeable in 12 of the mutants, in which the presence of CCCP resulted in a two- to six-fold increment in the norfloxacin uptake (Table III).


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Table III. Classification of quinolone-resistant mutants according to OmpF expression and norfloxacin uptake
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Development of multiple-antibiotic-resistance phenotypes in the mutants studied

The highest frequency of selection of mutations was obtained with the parent isolates that exhibited a higher level of resistance to nalidixic acid.12 All the quinolone-resistant mutants showed a multiple-resistance phenotype, which has been described in E. coli isolates after selection with fluoroquinolones20 and other agents.12

The involvement of substitutions in DNA gyrase and topoisomerase IV subunits in the quinolone resistance

Analysis of amino acid changes in the subunits of topoisomerases revealed that the substitution of Ser-83->Leu in the GyrA subunit of DNA gyrase was the most frequently identified change, as has been described by others.1,4,5 The 14 quinolone-resistant isolates and the PS1, PS5 and PS7 isolates in which this mutation was present exhibited a high level of resistance to nalidixic acid (MIC >= 128 mg/L) and ciprofloxacin and norfloxacin MICs >= 0.25 mg/L. the Ser-83->Ala substitution in DNA gyrase in the PS10 isolate was associated with both lower nalidixic acid and fluoroquinolone MICs than the substitution Ser-83->Leu, although the PS10 isolate was more resistant to quinolones than the other six parent isolates, which had no mutation of topoisomerases.2 The appearance of a second change in the amino acid sequence of the ParC subunit in the NorE5 isolate (Ser-80->Arg) was not associated with higher fluoroquinolone MICs with respect to the LmfE5 isolate. It is likely that the lower fluoroquinolone uptake exhibited by the LmfE5 isolate in comparison with the NorE5 isolate was important in determining the greater resistance to quinolone compared with the NorE5 isolate, despite the parC gene mutation in this latter isolate. Similarly, the NorE6 and LmfE6 isolates showed the same change (Asp-87->Asn) in the GyrA subunit as the N-113 E. coli isolate described by Oram & Fisher,3 but MICs of quinolones were higher in the LmFE6 and NorE6 isolates than in the N-113 isolate. Finally, our results as in previous studies show that mutations in the gyrB and parE genes do not play a major role in the acquisition of quinolone resistance.5,16

Changes in outer membrane proteins and lipopolysaccharide

The role of OMP and LPS in the development of resistance to quinolones was studied by comparing the different selected mutants with their parent isolates. SDS–PAGE analysis of outer membranes revealed that the increased resistance to quinolones was associated with the lack of OmpF in 11 quinolone-resistant mutants, this porin being expressed in the ten parent isolates. In this context, it has been shown that several mutations (marA, soxQ1, cfxB1, nfxC1, etc.) reduce ompF expression after transcription and that this reduction is related to the expression of the micF locus (an antisense RNA complementary to ompF mRNA).12,14 In this study, the lack of OmpF was associated with a decreased norfloxacin uptake in only eight of the mutants (the NorE1, NorE2, LmfE2, LmfE4, NorE5, LmfE5, NorE8 and LmfE9 isolates). The lack of OmpF in the NorE3, LmfE3 and NorE10 isolates was not associated with a significant diminution of norfloxacin uptake, i.e. higher than 5% in the steady state, with respect to their parent isolates. It should be noted that for the PS3 and PS10 isolates, a lower level of norfloxacin uptake was characteristic in comparison with KL16 isolate wild-type. This feature could contribute to increased resistance to quinolones in the NorE3 and LmfE3 isolates when low uptake is combined with mutations in the gyrA gene.1,4 The influence of the lack of expression of high molecular mass polymers of LPS on the quinolone MICs in the PS5, NorE5, PS6, PS8 and NorE10 isolates cannot be excluded as previous work has proposed.1,9,10 In this context, the NorE5 and NorE10 isolates, as the NorC mutants,20 exhibited rough LPS, lack of OmpF expression and accumulated <50% of the norfloxacin concentration compared with the amount accumulated by the KL16 isolate.1,20

Norfloxacin uptake and its role in parent isolates and mutants

The study of intracellular accumulation of norfloxacin demonstrated that all the parent isolates accumulated less norfloxacin than the KL16 wild-type isolate, despite the fact they did not lose the expression of OmpF. In this context, seven of the parent isolates accumulated less than 50% of norfloxacin compared with the KL16 isolate, although 11 mutants accumulated even less norfloxacin than their parent isolates (Table III). The addition of 50 or 100 µM CCCP resulted in increased norfloxacin accumulation in all the parent isolates and mutants with the exceptions of the PS2, PS3, PS4, NorE3, LmfE3 and NorE4 isolates, showing that proton-dependent active efflux influenced the intracellular level of norfloxacin in 22 of the isolates studied. Nevertheless, the involvement of a non-proton-dependent active efflux could explain the lower norfloxacin uptake, with respect to the KL16 wild-type isolate, in the NorE3, LmfE3 and NorE4 isolates, as well as in the PS2, PS3 and PS4 isolates.1,11 Furthermore, the presence of active efflux when added to the effect of change (Asp-87->Asn) in the GyrA subunit in the NorE6 and LmfE6 isolates, could explain the higher fluoroquinolone MICs in the NorE6 and LmfE6 isolates when compared with the N-113 isolate.3 In this context, the influence of an enhanced proton-dependent active efflux on norfloxacin accumulation in the NorE9 and NorE10 isolates was seen. The increased norfloxacin MICs (four- to 32-fold) in the LmfE1, LmfE7 and LmfE10 isolates with respect to the PS1, PS7 and the PS10 isolates were caused by decreased fluoroquinolone uptake. The addition of 50 or 100 µM CCCP resulted in an increase in norfloxacin uptake from 62% to 80% in the LmfE1 and LmfE10 isolates, and 17% in the LmfE7 isolate. These results are in accordance with the higher norfloxacin MICs in the LmfE1 and LmfE10 isolates than in the LmfE7 isolate when compared with their parent isolates. These latter mutants showed an increased resistance to tetracycline and/or cephalothin in association with a rise in the MIC of nalidixic acid. These antimicrobial agents have been described as substrates of the AcrAB efflux system in E. coli.13,22 The overexpression of the AcrAB system could explain the multiresistance shown by the LmfE1, LmfE7 and LmfE10 isolates. Further studies are in progress to reveal the involvement of this efflux system in these mutants.

The diversity of behaviour with respect to the OmpF expression and proton-dependent active efflux in the 18 mutants studied suggests a classification into five different groups (Table III):

In summary, DNA gyrase mutations were the principal determining factors in the acquisition of a higher level of resistance to quinolones in the isolates in this study. A decreased permeability, caused by changes in OMPs, and an increased active efflux were complementary in determining the MICs of the quinolones. These latter two mechanisms could facilitate the development of multiple-antibiotic-resistance phenotypes and, in turn, contribute to the selection of quinolone resistance in the course of clinical use.


    Acknowledgments
 
We thank the Scientific and Technical Service of the University of Barcelona for their advice and materials concerning DNA sequencing. This work was supported by a grant from The Spanish Society of Chemotherapy and by a subsidy (93/167) from The Education, Culture and Sports Council, Canary Autonomous Government and by SAF 97/0091 from DGICyT Spain.


    Notes
 
* Corresponding author. Tel:+34-28-451459; Fax: +34-28-451416; E-mail: mtavio{at}infovia.ulpgc.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 9 February 1999; returned 3 June 1999; accepted 26 July 1999