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JAC Advance Access originally published online on October 13, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1274-1278; doi:10.1093/jac/dkl404
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Analysis of the mechanisms of fluoroquinolone resistance in urinary tract pathogens

Hafizah Y. Chenia1,2, Balakrishna Pillay1 and Dorsamy Pillay1,3,*

1 Department of Microbiology, School of Microbiology and Biochemistry University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa 2 Department of Microbiology University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa 3 Department of Biotechnology Durban University of Technology, PO Box 1334, Durban 4000, South Africa

*Correspondence address. Centre for Research Management and Development, Durban University of Technology, PO Box 1334, Durban 4000, South Africa. Tel: +27-31-2042576; Fax: +27-31-2042946; E-mail: gansen{at}dut.ac.za

Received 25 May 2005; returned 30 March 2006; revised 30 August 2006; accepted 7 September 2006


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Objectives: To characterize the mechanisms of fluoroquinolone resistance in urinary tract pathogens exhibiting a multiple antibiotic resistance phenotype as well as a high-level resistance to fluoroquinolones.

Methods: Nineteen Escherichia coli urinary tract infection pathogens exhibiting high-level resistance to fluoroquinolones were characterized in this study. Alterations in outer membrane proteins (OMPs) and lipopolysaccharide (LPS) were analysed by PAGE. Changes to the quinolone resistance-determining regions (QRDRs) of GyrA and ParC were determined by PCR and DNA sequencing. The presence of the qnrA gene was determined by PCR amplification. Ciprofloxacin uptake was determined spectrophotometrically using the quinolone accumulation assay.

Results: OMP analysis showed decreased expression, the absence of certain proteins or the presence of proteins with altered molecular weights when compared with wild-type strains. Most isolates possessed a smooth LPS phenotype. Isolates had double mutations in GyrA codons 83 and 87, in addition to a ParC alteration at Ser-80/Glu-84. Isolates accumulated varying levels of ciprofloxacin, and upon the addition of carbonyl cyanide m-chlorophenylhydrazone, increased accumulation was observed in all instances. E. coli isolates with a rough LPS phenotype appeared to accumulate higher levels of ciprofloxacin compared with those with a smooth LPS phenotype expressing OmpF normally, or even those not possessing OmpF. All E. coli isolates tested demonstrated active efflux of ciprofloxacin. Plasmid-mediated quinolone resistance (presence of the qnrA gene) was observed in 36.8% of isolates.

Conclusions: A combination of target gene alterations, altered OM permeability, presence of the qnrA gene and active efflux appear to act together to produce a high-level, multiresistance phenotype.

Keywords: fluoroquinolone resistance , OMP , LPS , GyrA and ParC alterations , energy-dependent efflux systems


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An evolution in the aetiology and antimicrobial susceptibilities of urinary tract infection (UTI) organisms is being continually observed among outpatients.1 Most UTI cases in the hospital and community setting are initially treated empirically based on the frequency of potential pathogens, local antimicrobial resistance rates and illness severity.1 Due to previous fluoroquinolone usage, under-dosing and mono-therapy against moderately susceptible pathogens, fluoroquinolone resistance has developed among these UTI pathogens.

Resistance to fluoroquinolones is a result of a combination of mechanisms acting either singly or in combination to produce the resistance phenotype. Point mutations within DNA gyrase (gyrA and gyrB genes) cause a reduction in the affinity of the enzyme for fluoroquinolones and decrease the susceptibility of the organisms to fluoroquinolones.2,3 Topoisomerase IV (parC gene) is the secondary target for fluoroquinolone action in the absence of a susceptible DNA gyrase.3 Other mechanisms involve mutations affecting the intracellular accumulation of fluoroquinolones in the cell envelope, i.e. those affecting the increased/decreased expression of outer membrane proteins (OMPs),2–4 alterations in the lipopolysaccharide (LPS) component, and/or efflux of fluoroquinolones from bacterial cells.5 Mutations involving the bacterial cell envelope tend to confer a multiple drug resistance phenotype to several chemically unrelated but clinically important drugs. The plasmid-encoded, integron-associated qnr gene has been sporadically found in isolates of Shigella spp., Klebsiella pneumoniae and Escherichia coli. It is responsible for low-level fluoroquinolone resistance, especially in strains with normal OMP, and may supplement resistance due to other mechanisms such as DNA gyrase mutations, efflux pump activation or deficiencies in OMP.6

It has been suggested that quinolone resistance is higher in developing countries than in developed countries. Use of less active quinolones such as nalidixic acid, and/or the use of low dosages of the more potent fluoroquinolones such as ciprofloxacin, results in the selection of mutants.1 This study characterizes fluoroquinolone resistance mechanisms involving the cell envelope in clinically resistant E. coli UTI microorganisms and alterations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC, and evaluates the role of ciprofloxacin efflux in the resistance phenotype of clinical E. coli isolates.


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Bacterial strains

Nineteen E. coli UTI pathogens (indicated in Table 1) displaying resistance to ciprofloxacin, grepafloxacin, lomefloxacin, norfloxacin and sparfloxacin were selected for study from a private medical pathology laboratory collection in Durban, South Africa. Additionally, isolates displayed resistance to ampicillin, co-trimoxazole, piperacillin and nalidixic acid by disc diffusion testing according to NCCLS guidelines.7 Ciprofloxacin MICs were determined using the Etest according to the manufacturer's instructions (AB Biodisk, Solna, Sweden).


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  		Table 1. Characterization of quinolone resistance mechanisms found in fluoroquinolone-resistant UTI isolates

 
Outer membrane protein profile and lipopolysaccharide analysis

OMPs were prepared with N-lauryl-sarcosine using a modification of the method of Sawai et al.8 Samples were subjected to denaturing-PAGE using the discontinuous Tris–glycine buffer system in 12% polyacrylamide gels. LPS extracts were prepared by boiling bacterial OMP preparations for 10 min in sample buffer. After cooling, 50 µg of proteinase K was added and samples were incubated overnight at 55°C and boiled at 100°C for 10 min.9 LPS profiles were obtained by electrophoresis of the protease-resistant LPS preparations in 12% polyacrylamide gels and visualized by silver-staining using the Quick-Silver staining kit (Amersham, UK). E. coli strain HB 101 was used as a fluoroquinolone-susceptible control for OMP and LPS profile analysis.

gyrA and parC QRDR amplification and DNA sequencing

The gyrA and parC gene fragments of six E. coli isolates (F5, F8, F9, F13, F14 and F18) were amplified using previously described primers.10,11 PCR amplimers were purified and processed for automated sequencing. Alignments of DNA sequences to reference gyrA and parC sequences, obtained from GenBank, and protein translations were performed using DNAMAN version 5.0 (Lynnon BioSoft, Canada).

Quinolone accumulation assay

Ciprofloxacin uptake was assayed for 6 of the 19 isolates by a modified method of Mortimer and Piddock.4 Ciprofloxacin (Bayer AG, Germany) was added to a final concentration of 10 mg/L, and accumulation measured with and without the addition of 100 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP), an electron transport chain uncoupler that inhibits energy-dependent efflux. Following addition of ciprofloxacin, 0.5 mL samples were removed at 15 s intervals for 1 min, at 30 s intervals for up to 5 min, then at 10, 15, 20, 30, 45, 60, 90 and 120 min intervals. The fluorescence of the supernatant was determined with a Luminescence Spectrometer LS-50B (Perkin Elmer, USA) at excitation and emission wavelengths of 275 and 440 nm, respectively. The concentration of ciprofloxacin in the supernatant was calculated by comparison with a standard curve for ciprofloxacin (0.01–0.250 mg/L) in 0.1 M glycine hydrochloride (pH 3). Accumulation of ciprofloxacin (ng/mg cells) was calculated by subtracting the absorbed ciprofloxacin at time zero from the amount of ciprofloxacin eluted at various time intervals. The experiments were performed in duplicate on two separate occasions in order to ensure reproducibility. The accepted standard deviation for all accumulation results was ≤10% with respect to the mean values taken at the respective sampling times.2

Amplification of the qnrA gene

Genomic and plasmid DNA from all isolates were screened for the presence of the qnrA gene using previously described primers6 and amplification conditions.10


   Results and discussion
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Up to 80% of UTI pathogens isolated from outpatient urine samples in Durban, South Africa are E. coli. About 5 years ago, ~4% of these E. coli isolates were resistant to fluoroquinolones. This has currently increased dramatically to around 11% (A. K. Peer, personal communication). The isolates under study represent the initial fluoroquinolone-resistant strains isolated in this geographical area.

Most E. coli isolates (73.7%) expressed both the OmpC (38–40 kDa) and OmpF (36 kDa) proteins identical to that of the fluoroquinolone-susceptible control strain, E. coli HB 101 (Table 1). Two isolates (F17 and F20) showed OmpF expression with decreased OmpC expression. Five isolates expressed the OmpC protein only. Only isolates F9 and F18 expressed rough (R) LPS profiles, while the remainder displayed the smooth (S) LPS phenotype.

The loss of OmpC or OmpF observed in some isolates has been described previously in fluoroquinolone-resistant E. coli isolates.2,5 Decreased expression or absence of OmpF has been implicated in increased permeability of the cell envelope to hydrophilic agents, including ciprofloxacin. Fluoroquinolone exposure is not always implicated in LPS alteration of resistant E. coli isolates.12 This was also evident in the present study where 17/19 isolates showed no significant LPS alterations (Table 1).

The presence of multiple mutations in gyrA and/or parC is required for high-level fluoroquinolone resistance in E. coli.3 The most common mutation observed in clinical isolates is in GyrA at codon 83, followed by that at codon 87. In ParC, the common substitutions appear to be at codons 80 and/or 84.3 All six E. coli isolates assayed possessed two mutations in gyrA and a single mutation in parC resulting in alterations at codons Ser-83 and Asp-87 in GyrA and codons 80 or 84 in ParC, respectively (Table 1). The majority of isolates displayed Ser-83->Leu and Asp-87->Asn alterations in GyrA and a Ser-80->Ile alteration in ParC. GyrA and ParC alterations could not be correlated with ciprofloxacin Etest MICs which ranged from 2 to >32 mg/L (Table 1).

Decreased accumulation is associated with an increase in bacterial permeability to antibacterial agents or the overexpression of efflux pumps.2–5 Ciprofloxacin accumulation assay data showed an increase in the steady-state concentrations in all isolates tested and accumulation appeared to double following the addition of 100 µM CCCP (Figure 1a). Thus, it is possible to suggest that the efflux mechanism described previously in E. coli is present and active in these isolates.2 The six E. coli isolates assayed for ciprofloxacin accumulation showed varying accumulation levels (Figure 1b). Elution at 20 min showed that these isolates accumulated between 6.5 and 20 ng ciprofloxacin/mg cells, with an average of 11.4 ng/mg cells. After CCCP addition, these values increased to 30–82 ng ciprofloxacin/mg cells, averaging 46.7 ng/mg. The difference in accumulation ranged from 10.0 to 75.5 ng/mg, with the latter being demonstrated by isolate F18. The concentrations of ciprofloxacin accumulated for E. coli isolates at 120 min were 2.5–28.5 ng ciprofloxacin/mg cells, with an average of 13.8 ng/mg. At 120 min, post-CCCP addition, the concentrations of ciprofloxacin accumulated were in the range of 37–89 ng ciprofloxacin/mg cells, with an average of 53.8 ng/mg. The difference in accumulation was calculated to range from 86.5 to 115.5 ng ciprofloxacin/mg cells. The greatest level of accumulation at this time was for isolate F18.


Figure 1
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  		Figure 1. (a) Time-dependent accumulation of ciprofloxacin by six E. coli UTI isolates without CCCP and following CCCP addition at 5 min. Results for accumulation by isolates F5, F8, F9, F13, F14 and F18 without CCCP are depicted by filled circles, inverted triangles, squares, diamonds, triangles and ovals, respectively. Results for accumulation by isolates following CCCP addition are depicted by open circles, triangles squares, diamonds, inverted triangles and ovals, respectively. (b) Time-dependent differential accumulation of ciprofloxacin by six E. coli UTI isolates. The difference in ciprofloxacin accumulation by isolates F5, F8, F9, F13, F14 and F18 are depicted by filled circles, open circles, filled inverted triangles, open triangles, filled squares and open squares, respectively. Difference in ciprofloxacin accumulation = (accumulation in presence of CCCP)–(accumulation without CCCP).

 
Everett et al.2 found that strains with rough LPS accumulated <50% of the concentration of ciprofloxacin compared with wild-type strains. However, in this study, isolates (F9 and F18) with rough LPS appeared to accumulate ciprofloxacin to a greater degree than isolates with a smooth LPS phenotype expressing OmpC and/or OmpF (Figure 1). Decreases in the amount of OmpF were found to be associated with decreased accumulation of fluoroquinolones in E. coli.13 In the present study, of the isolates assayed for ciprofloxacin accumulation, only isolates F14 and F18 demonstrated loss of OmpF, however, their LPS profiles were different and this may account for the difference in ciprofloxacin accumulation. Active efflux either singly or in tandem with OMP alterations would be responsible for the low accumulation of ciprofloxacin seen in isolates assayed (14–25 ng ciprofloxacin/mg cells) in the absence of CCCP.

Although plasmid-mediated quinolone resistance is reported to occur rarely and shows poor persistence,6 the 543 bp qnrA gene fragment was amplified from plasmid and genomic DNA of 36.8% (7/19) of UTI study isolates (Table 1). The presence of this gene might play a role in initiating the quinolone resistance phenotype or play a supplementary role, enhancing the effect of target genes and efflux activity by 4- to 128-fold.

Traditionally, the multiple antibiotic resistance (Mar) phenotype has been associated with reduced expression of OmpF and overexpression of the efflux systems.5 However, a diversity of phenotypes was identified in the E. coli UTI isolates, including: (i) an absence of OmpF and increased efflux (isolates F14 and F18); (ii) no OM alterations but reduced accumulation due only to increased active efflux (isolates F5, F8 and F9); and (iii) decreased OmpF expression coupled with active efflux (isolate F13). The differential accumulation of ciprofloxacin by isolates F14 and F18 might be attributed to differences in LPS and/or potentially in MarR, which would affect the expression of the AcrAB efflux pump.14

It is apparent that in the UTI isolates examined, GyrA and ParC alterations, decreased permeability due to alterations in OMPs and LPS, in conjunction with active efflux play an important role in the fluoroquinolone resistance phenotypes observed. It would be short-sighted to underestimate the effect of efflux-mediated resistance to ciprofloxacin and the other quinolones, even though it generally results in low-level resistance. Activation and/or increased expression of efflux pumps as a result of mutations within the global regulator operons, such as marRAB, would play a critical role in determining the success of fluoroquinolone-resistant clinical isolates. In conjunction with multiple mutations in gyrA and parC, altered OMPs and LPS, increased efflux and the presence of the qnr gene would favour the survival of DNA gyrase mutants as opposed to susceptible strains. This would facilitate the development of high-level resistance mutants in the clinical setting. Microorganisms are constantly evolving and their acquisition of a combination of beneficial mutations within target genes, in concert with a variety of OM modifications and efflux systems, would allow these versatile organisms to survive and reproduce in the face of antagonistic therapeutic agents.


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None to declare.


   Acknowledgements
 
This work was supported by a research grant from the University of Durban-Westville and the URDP of the National Research Foundation of South Africa to D. P. Dr A. K. Peer and Ms N. Pillay are acknowledged for providing bacterial cultures.


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1 Gales AC, Jones RN, Gordon KA, et al. (2000) Activity and spectrum of 22 antimicrobial agents tested against urinary tract infection pathogens in hospitalized patients in Latin America: report from the second year of the SENTRY Antimicrobial Surveillance Program (1998). J Antimicrob Chemother 45:295–303.[Abstract/Free Full Text]

2 Everett MJ, Jin YF, Ricci V, et al. (1996) Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrob Agents Chemother 40:2380–6.[Abstract]

3 Ruiz J. (2003) Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA gyrase protection. J Antimicrob Chemother 51:1109–17.[Abstract/Free Full Text]

4 Mortimer PGS and Piddock LJV. (1991) A comparison of methods used for measuring the accumulation of quinolones by Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus. J Antimicrob Chemother 28:639–53.[Abstract/Free Full Text]

5 Cohen SP, McMurry LM, Hooper DC, et al. (1989) Cross-resistance to fluoroquinolones in multiple-antibiotic-resistant (Mar) Escherichia coli selected by tetracycline or chloramphenicol: decreased drug accumulation associated with membrane changes in addition to OmpF reduction. Antimicrob Agents Chemother 33:1318–25.[Abstract/Free Full Text]

6 Rodriguez-Martinez JM, Pascual A, Garcia I, et al. (2003) Detection of the plasmid-mediated quinolone resistance determinant qnr among clinical isolates of Klebsiella pneumoniae producing AmpC-type ß-lactamase. J Antimicrob Chemother 52:703–6.[Abstract/Free Full Text]

7 National Committee for Clinical Laboratory Standards. (2000) Methods for Diffusion Disk Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically: Approved Standard M7-A5(Wayne, PA, USA, NCCLS).

8 Sawai T, Hiruma R, Kawana N, et al. (1982) Outer membrane permeation of ß-lactam antibiotics in Escherichia coli, Proteus mirabilis and Enterobacter cloacae. Antimicrob Agents Chemother 22:585–92.[Abstract/Free Full Text]

9 Carlone GM, Thomas ML, Rumschlag HS, et al. (1986) Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species. J Clin Microbiol 24:330–2.[Abstract/Free Full Text]

10 Vila J, Ruiz J, Goni P, et al. (1995) Mutation in the gyrA gene of quinolone-resistant clinical isolates of Acinetobacter baumannii. Antimicrob Agents Chemother 39:1201–3.[Abstract]

11 Vila J, Ruiz J, Goni P, et al. (1996) Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli. Antimicrob Agents Chemother 40:491–3.[Abstract]

12 Piddock LJV, Hall MC, Walters RN. (1991) Phenotypic characterization of quinolone-resistant mutants of Enterobacteriaceae selected from wild type, gyrA type and multiply resistant (marA) type strains. J Antimicrob Chemother 28:185–98.[Abstract/Free Full Text]

13 Ma D, Cook DN, Alberti DM, et al. (1993) Molecular cloning and characterization of acrA and acrE genes of Escherichia coli. J Bacteriol 175:6299–313.[Abstract/Free Full Text]

14 Randall LP and Woodward MJ. (2002) The multiple antibiotic resistance (mar) locus and its sigificance. Res Vet Sci 72:87–93.[CrossRef][Web of Science][Medline]


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