1 Swedish Institute for Infectious Disease Control, Department of Bacteriology, S-171 82 Solna; 2 Karolinska Institute, Microbiology and Tumour Biology Center, S-171 77 Stockholm; 3 Karolinska Institute, Division of Clinical Bacteriology, Karolinska University Hospital Huddinge, S-141 86 Huddinge, Sweden
Received 27 August 2004; returned 4 October 2004; revised 15 October 2004; accepted 19 October 2004
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Abstract |
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Methods: Norfloxacin-resistant mutants were isolated and by DNA sequencing the mutations conferring resistance were identified. Mutant fitness was determined by measuring growth rates in vitro. Mutants with reduced growth rates were serially passaged to obtain growth-compensated mutants. The level of DNA supercoiling was determined by isolating plasmid DNA from the susceptible, resistant and compensated mutants and comparing the topoisomer distribution patterns by gel electrophoresis in the presence of chloroquine.
Results: Low-level resistance (448 mg/L) was caused by single mutations in gyrA or gyrB. Among these strains, three out of eight mutants showed lower fitness, whereas high-level resistant (>256 mg/L) mutants with double mutations in gyrA and parC, parE, nfxB or unknown genes all showed a reduced fitness. Slow-growing resistant mutants with a gyrA mutation had decreased DNA supercoiling. After serial passage in laboratory medium, mutant fitness was increased by compensatory mutation(s) that restored supercoiling to normal levels. The compensatory mutation(s) was not located in any of the genes (gyrAB, topA, parCE, hupB, fis, hupN, himAD or PA5348) that were expected to affect supercoiling.
Conclusions: Our results show that no cost and compensatory mutations are common in quinolone-resistant P. aeruginosa.
Keywords: antibiotic resistance , supercoiling , compensation , biological cost
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Introduction |
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Quinolones act by inhibiting the target enzymes DNA gyrase and topoisomerase IV, each comprised of two subunits: GyrA and GyrB, and ParC and ParE, respectively.6 Quinolones block replication and cell growth by trapping DNA gyrase on DNA as a DNAgyraseantibiotic complex, in which both DNA strands are broken.6 Resistance to quinolones is mediated by mutations in DNA gyrase, topoisomerase IV as well as by mutations in regulatory genes for different efflux systems.1,7 Furthermore, plasmid-borne resistance to quinolones has been reported in Klebsiella pneumoniae and Escherichia coli and in these cases the plasmids carry a quinolone-resistance gene (qnr) encoding a protein that blocks the inhibition of DNA gyrase caused by the drug.810 DNA gyrase is the primary target of quinolones in Gram-negative bacteria, and a majority of the resistance mutations found in gyrA and gyrB are clustered to the respective quinolone resistance-determining regions (QRDRs).11
Antibiotics target functions essential for bacterial survival, such as DNA gyrase, RNA polymerase, the ribosome and cell wall biosynthetic enzymes. Mutations in these targets that lead to resistance are commonly associated with a fitness cost, observed as a reduced growth rate and/or virulence in the absence of antibiotic.12,13 Bacteria can, in many cases, compensate for their loss of fitness by accumulating compensatory mutations that partly or fully restore the function impaired by the resistance mutation.12,13 The biological cost of antibiotic resistance in P. aeruginosa has been examined in mutants that overproduce multidrug resistance efflux pumps.14 These mutants showed a reduced fitness, which was observed as decreased survival in water, impaired production of phenazines and proteases, as well as lowered virulence. Since quinolones are commonly used for treatment of Pseudomonas infections, and the major mechanism of resistance is by alteration of topoisomerases, we wanted to study the fitness of such resistant mutants.
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Materials and methods |
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From several independent 1 mL Luria-Bertani (LB) cultures, norfloxacin-resistant mutants were isolated by plating 108109 P. aeruginosa PAO1 cells on Luria agar (LA) plates containing 2, 8 or 80 mg/L norfloxacin (Sigma). The plates were incubated at 37°C and one colony from each independent culture was collected. Resistant mutants were purified by two successive single-colony isolations on plates containing the same concentration of norfloxacin and subsequently frozen at 70°C. One mutant isolated on 8 mg/L norfloxacin was chosen for further selection on 80 mg/L norfloxacin (see below). Bacterial cells were grown in LB broth at 37°C. MICs were determined by using the Etest (AB Biodisk, Solna, Sweden).
Clinical isolates
Twenty quinolone-resistant P. aeruginosa isolates from Danish cystic fibrosis patients have been characterized and described previously and were kindly provided by Nils Høiby.4
Isolation of compensatory mutations
Two slow-growing norfloxacin-resistant gyrA mutants, DA6254 (mutation ACC83ATC) and DA6252 (mutation GAC87TAC), were chosen for selection of compensated mutants. Evolution experiments were performed by serial transfer of 106 bacteria to 1 mL of fresh LB medium and growth at 37°C to a final density of 109 cells/mL before the next transfer. Samples were taken daily from each culture and streaked on LA plates, and the colony size was compared with the susceptible parent and the resistant mutant. The cultures were cycled until the majority of the cells showed a faster growth phenotype (larger colonies). As a control experiment, several lineages of the susceptible parental strain PAO1 were cycled in parallel and during that time no changes were seen in the growth rate of these lineages.
Mutation rates
Independent cultures were inoculated with 103 cells and grown overnight. Undiluted cultures were plated on LA, norfloxacin plates at the indicated concentrations (2 and 8 mg/L) to determine the number of resistant mutants. Suitable dilutions were plated on LA plates to determine the number of cells present in the cultures. Mutation rates (µ) to fluoroquinolone resistance were calculated according to the equation: µ=lnP0/N where P0 designates the ratio of the number of cultures without mutants divided by the total number of cultures, and N designates the total number of bacteria plated.15 The compensatory mutation rate was calculated according to a previously described theory.16,17
Growth rate and biofilm measurements
The growth rates of the isolated mutants were determined using a Bioscreen C Analyzer System (Labsystems, Helsinki, Finland). 106 cells were inoculated into 400 µL of LB on a bioscreen plate and incubated with continuous shaking at 37°C. The absorbance was measured at 540 nm. Each strain was assayed in four independent cultures in two separate experiments. Relative growth rates were calculated as the ratio of the growth rate of the reference strain divided by the growth rate of the test strain. Growth rates of clinical isolates were related to the most-fit strain, which had a growth rate similar to P. aeruginosa PAO1. Measurement of biofilm formation was performed according to the method described by O'Toole et al18
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PCR amplification
Chromosomal DNA from the isolated P. aeruginosa mutants was prepared using a DNeasy kit (Qiagen Inc.) and subjected to PCR. PCR amplification of the QRDR of the gyrAB and parCE genes, as well as the complete coding sequence of the mexR and nfxB genes, was carried out with primers designed by Jalal & Wretlind.19,20 The oligonucleotide primers used for PCR amplification and DNA sequencing for the complete genes of gyrAB, parCE and the other sequenced genes (topA, hupN, fis, hupB, himAD and PA5348) are described in Table 1. The PCR reaction was run under the following conditions: denaturation for 5 min at 94°C, followed by 30 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 1 min, and a final extension step of 7 min at 72°C. The reactions were performed with AmpliTaq DNA polymerase (Applied Biosystems) in a DNA thermal cycler, GeneAmp PCR System 2400 (Perkin-Elmer). The PCR products were analysed by agarose gel electrophoresis, stained with ethidium bromide and visualized under UV light.
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PCR products were purified using GFX PCR DNA and a Gel Band Purification Kit (Amersham Pharmacia Biotech Inc.). Purified PCR products were processed for DNA sequencing using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Warrington, UK) in the ABI 3100 Genetic Analyzer (Perkin-Elmer).
Gel electrophoretic separation of topoisomers
Plasmid RSF1010 was transferred by electroporation to the parental susceptible strain PAO1, two resistant gyrA mutants, DA6254 (ACC83ATC) and DA6252 (GAC87TAC), and compensated mutants from these strains. Plasmid DNA was isolated using a Qiagen plasmid purification kit (Qiagen Inc.) and analysed by agarose gel electrophoresis in the presence of chloroquine, as previously described by Matsuo et al.21 with some modifications. Agarose gels (1%) were run for 22 h at 2 V/cm in 1 x TAE buffer containing 5 mg/L chloroquine and were washed for 3 h in distilled water before staining with ethidium bromide. At this concentration of chloroquine, topoisomers that were more supercoiled prior to electrophoresis migrated more rapidly through the gel.
RNA purification and synthesis of cDNA
Bacteria, grown in LB broth in the logarithmic phase, were harvested by centrifugation at 3000g for 10 min. The supernatants were discarded and the pellets re-suspended in 500 µL of 10 mM Tris, pH 8.0. Cell density was adjusted to 1.5 x 108 cells/mL, as determined by comparison with a McFarland standard no. 0.5. The cell suspensions were kept on ice during these procedures. The suspensions (200 µL;
0.3 x 108 cells) were used for isolation of total RNA by the protocol described in the High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany). The total elution volume of RNA was 50 µL at the final step. RNA was stored at 70°C until used in a cDNA preparation reaction. The concentration of RNA was determined spectrophotometrically at 260 nm. Total RNA (1 µg) was used as a template for reverse transcription (RT) in 20 µL volumes, as described in the 1st Strand cDNA Synthesis Kit for RT-PCR (Roche). In the RT reaction, we used random primers p(dN)6. The cDNA was stored at 20°C until used in real-time PCR in a LightCycler (LC; Roche).
Real-time PCR
The primers are described in Table 2. A master mix containing 4 mM MgCl2 and 2 µM each gene-specific primers was prepared on ice using a LightCycler-FastStart DNA Master SYBR Green I kit (Roche). Eighteen microlitres of master mix was combined with 2 µL cDNA in the capillaries, which were sealed and placed in the LightCycler. The following temperature profile was used for real-time PCR: after initial denaturation at 95°C for 600 s, amplification was carried out for 45 cycles with denaturation at 95°C for 15 s, annealing at 62°C for 5 s and extension at 72°C for 16 s. The temperature transition rate was 20°C/s. Melting curve analysis was conducted in one cycle with three segments: 95°C for 60 s, 67°C for 60 s and 95°C for 0 s. The temperature transition rate was 20°C/s for the first and second segments and 0.1°C for the third segment. mRNA for the ribosomal protein RpsL was used as a reference in the real-time PCR assay and the relative amount of mRNA for efflux pump proteins (not shown) was calculated, based on the standard curve and for DNA gyrase as well as topoisomerase IV, where 3.4 cycles caused a 10-fold increase in the PCR product. A strain was considered to hyperproduce mexB mRNA if the cDNA level was >3 x wild-type (MexB is produced constitutively). For the other proteins, the limit was >10 x wild-type.20 In control experiments without reverse transcriptase or RNase-treated samples, no PCR product was obtained.
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Results |
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We isolated norfloxacin-resistant mutants on three different concentrations of norfloxacin (2, 8 and 80 mg/L) to obtain a broad spectrum of mutants. Ten mutants from each selection condition were chosen for further investigation. We examined their resistance mechanism and determined the effect of these mutations on bacterial growth and biofilm formation. One mutant with each type of mutation is presented in Table 3. The mutation rate to norfloxacin resistance for mutants isolated on 2 and 8 mg/L was determined to be 1 x 109. Since these were spontaneous mutants they are likely to be single-point mutations. We identified the resistance mechanism by sequencing the gyrAB genes. The majority of the mutants (17/20) isolated on these concentrations of norfloxacin had a mutation in the QRDR of either gyrA or gyrB. Six of 20 mutants had a mutation in gyrA at codon 83 (ACCATC), which is the most reported target for quinolone resistance in several species.7
Resistance mutations were also identified at codon 87 in gyrA as well as at codon 323, 467 and 469 in gyrB. In addition, another rarely reported mutation was identified in gyrA at codon 83, ACC
GCC. Three mutants had no mutation in the QRDR of gyrA, gyrB, parC or parE or in the nfxB or mexR genes. Twelve of the 20 mutants isolated on 2 and 8 mg/L norfloxacin showed increased resistance to tetracycline (MIC equal or greater than 48 mg/L) in addition to norfloxacin resistance. Resistance to tetracycline and quinolones may be mediated by increased expression of efflux systems, such as MexAB-OprM and MexCD-OprJ, which are regulated by mexR and nfxB, respectively1
. We did not identify any mutations in these genes. However, we also analysed the mexB and mexD mRNA transcript levels using real-time PCR. Six of the 12 mutants with increased resistance to tetracycline showed a 5- to 1000-fold increased expression of MexD (data not shown). The remaining six tetracycline-resistant mutants could have alterations in other efflux systems.22
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Fitness effects of norfloxacin resistance
The fitness of quinolone-resistant mutants was measured as growth rate in LB medium and the ability of the strains to form biofilm in microtitre dishes. Among the resistant strains with a single mutation in gyrA or gyrB, only three out of eight mutation types showed fitness costs when measured as growth rate in vitro (Figure 1). One of the six mutants with a gyrA mutation at codon 83 (ACCATC), DA6254, showed a reduced fitness. In addition, another mutant with a single mutation in gyrA, DA6252, also showed reduced growth rate. Both DA6254 and DA6252 were subjected to compensatory evolution (see below). The third type of mutation that showed decreased fitness was the gyrB (GAG469GAC) mutant, DA6249. However, this mutant also showed increased tetracycline resistance and expression of mexD, indicating that it had an additional unidentified mutation. In strains with double mutations and high-level resistance, considerable reductions in growth rate were seen for all strains. In addition, we examined the fitness of several non-isogenic clinical isolates obtained from cystic fibrosis patients. These clinical isolates have been characterized previously and the majority of these strains have resistance mutations in several targets.4
In spite of the non-isogenicity of these strains, which might be expected to confound any correlation between resistance level and fitness, a weak negative correlation between fitness and level of resistance was still seen (Figure 2). Thus, the growth rate of strains with MIC values of 8 and 16 mg/L norfloxacin appeared lower than for strains that were inhibited at 2 mg/L (P<0.05, determined using one-sample t-test confirmed by MannWhitney U-test). We also measured biofilm formation in the resistant mutants, but no correlation between growth rates in LB medium or MIC values and the ability to form biofilm could be seen. Thus, the susceptible parent strain and the resistant mutants isolated in vitro showed similar levels of biofilm formation. The clinical isolates, on the other hand, formed little biofilm, as compared with strain PAO1 and its mutant derivatives (data not shown). We do not know the reason for the reduced biofilm formation, but it is unlikely to result from the reduced growth rate per se since the slow-growing mutant derivatives of PAO1 still showed normal biofilm formation.
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Two low-fitness resistant clones, DA6252 and DA6254, with single mutations in gyrA (GAC87TAC and ACC83ATC, respectively) were subjected to compensatory evolution by serial passage in LB medium. The ACC83ATC mutant chosen for compensatory evolution is likely to carry an additional unknown mutation since its growth rate was reduced as compared with other independently isolated ACC83ATC mutants. After only 2030 generations of growth, compensated mutants had appeared that showed a growth rate similar to the susceptible parent (Figure 1). In a control experiment, we serially passaged the susceptible parent strain. No changes in fitness were seen in this strain, indicating that the compensatory mutations found are specific suppressors of the resistance mutations and that they are not causing a general gain in fitness. Although the compensated strains retained the original resistance mutation, the MIC of norfloxacin decreased significantly from 64 mg/L (DA6252) and 48 mg/L (DA6254) to 12 mg/L. The fitness of the compensated strains was also investigated by measuring growth at 30°C and 42°C. All compensated mutants grew as well as wild-type at both 30°C and 42°C. However, biofilm formation at 37°C was slightly decreased (data not shown). From the kinetics of appearance of fast-growing mutants, the population sizes during serial passage and the fitness of the resistant and compensated mutants, we could calculate the mutation rate for compensatory mutations to 106/cell/generation.16,17
DNA supercoiling
We investigated alterations in supercoiling by electroporating the 8.7 kbp plasmid RSF1010 into the susceptible, resistant and compensated strains. Plasmid DNA was isolated and analysed by agarose gel electrophoresis in the presence of chloroquine. The topology of reporter plasmid DNA is thought to reflect the chromosomal topology. Chloroquine is an intercalating ligand that introduces supercoiling of closed circular DNA in the positive direction and without changing the linking number.26 The relative positions of the topoisomer with the average linking number gives an estimate of the mean degree of supercoiling of the DNA. At the chloroquine concentration used in this assay, 5 mg/L, negatively supercoiled forms show greater mobility than relaxed topoisomers. The majority of DNA from the susceptible parent strain was negatively supercoiled, illustrated by the highest mobility (Figure 3, lane 1, bottom band). Resistant strains were shifted to lower mobilities (Figure 3, lanes 2 and 5), indicating decreased supercoiling, whereas growth-compensated strains showed topoisomer distribution patterns similar to the susceptible parent (Figure 3, lanes 3, 4 and 6). These results show that the decreased fitness associated with these two gyrA resistance mutations is caused by decreased supercoiling and that the compensatory mutations act to restore supercoiling to normal levels.
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Since the fitness alterations in the resistant mutants and the compensated mutants were associated with changed supercoiling it is likely that the compensated mutants have acquired mutations that increase supercoiling to counteract the action of the gyrA mutation. The degree of supercoiling in DNA is determined by the balance between DNA-relaxing activity and DNA-supercoiling activity, regulated by the opposing actions of topoisomerase I and DNA gyrase, respectively.27 To try to identify the compensatory mutations, we sequenced the complete topA, gyrAB and parCE genes and their corresponding promoter regions from the resistant and compensated mutants, but no additional mutations were identified. Another possibility could be increased expression of the DNA gyrase or topoisomerase IV genes, but we could not detect any differences in the expression of these genes (data not shown). Other proteins thought to affect supercoiling are the histone-like proteins HU (encoded by hupB), integration host factor (encoded by himAD) and the DNA binding protein Fis (encoded by fis). In addition, P. aeruginosa has two probable DNA binding proteins similar to HU, annotated PA3940 (hupN) and PA5348. However, no mutations were detected in any of these genes or promoter regions.
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Discussion |
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The decreased fitness is associated with changed supercoiling. Thus, it was seen that two resistant mutants with reduced growth rate had decreased supercoiling (Figure 3). One of these mutants, DA6252, has a resistance mutation at codon 87 in gyrA, and this target has previously been shown to reduce supercoiling activity in E. coli.30,31 However, the second strain that showed decreased supercoiling, DA6254, has a mutation in gyrA at codon 83 and this target has not been associated with any changes in supercoiling activity in E. coli.30,31 Decreased supercoiling indicates the presence of a less efficient DNA gyrase, which could cause increased accumulation of positive supercoils in front of a replication fork or transcription complex.6 This could, in turn, slow replication/transcription and thereby reduce the growth rate. Resistant mutants with a reduced fitness could rapidly accumulate additional compensatory mutations that restored growth to the wild-type level. These mutations most likely act to restore supercoiling to the normal level (Figure 3). However, we have sequenced most of the target genes known or predicted to affect the level of supercoiling without identifying any compensatory mutations. In addition, we investigated the expression of the DNA gyrase or topoisomerase IV genes, but could not detect any changes in the levels of these transcripts. It is notable that the compensatory mutations arose at a high rate (about 106), which is much higher than that expected for a typical spontaneous point mutation (1011 to 109).32 One possibility is that compensation can occur by any type of loss-of-function (deletion, insertion, base pair substitution etc.) mutation in an unknown gene, which would be expected to result in a high mutation rate.32
The cost of quinolone resistance caused by topoisomerase mutations has been studied in E. coli,30 Salmonella typhimurium,33 Streptococcus pneumoniae34 and Staphylococcus aureus.35 The primary target of quinolones differs between Gram-negative and Gram-positive bacteria, being DNA gyrase and topoisomerase IV, respectively. The biological cost of quinolone resistance differs between different bacteria and depends on the level of resistance and the number of resistance mutations. For the five bacterial species examined, highly resistant mutants with multiple mutations show a significantly reduced fitness. However, for low-level resistant mutants with single mutations the cost depends on the bacterial species. Thus, no or low cost was seen in S. pneumoniae34 and S. typhimurium,33 whereas single, low-level resistance mutations conferred a variable cost in E. coli depending on strain background.30 Similar to our findings, compensatory mutations that reduce costs have been observed in some other species (S. typhimurium and S. aureus).33,35 For S. typhimurium the specific mechanism of compensation is not understood, but for S. aureus compensation appears to involve increased expression of the gyrAB and topB transcripts.35
In conclusion, the present study shows that no cost and compensatory mutations are common in quinolone-resistant P. aeruginosa. The clinical occurrence of these types of mutations is likely to contribute to the long-term persistence of resistant bacteria, underlining the importance of implementing efficient strategies to treat and prevent spread of resistant strains before they have become stably established in bacterial populations.13
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Acknowledgements |
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Footnotes |
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References |
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2 . Lambert, P. A. (2002). Mechanisms of antibiotic resistance in Pseudomonas aeruginosa. Journal of the Royal Society of Medicine 95, Suppl.41, 226.[ISI][Medline]
3
.
Doring, G., Conway, S. P., Heijerman, H. G. et al. (2000). Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus. European Respiratory Journal 16, 74967.
4
.
Jalal, S., Ciofu, O., Hoiby, N. et al. (2000). Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrobial Agents and Chemotherapy 44, 7102.
5
.
Pitt, T. L., Sparrow, M., Warner, M. et al. (2003). Survey of resistance of Pseudomonas aeruginosa from UK patients with cystic fibrosis to six commonly prescribed antimicrobial agents. Thorax 58, 7946.
6 . Drlica, K. & Zhao, X. (1997). DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiology and Molecular Biology Reviews 61, 37792.[Abstract]
7 . Piddock, L. J. (1999). Mechanisms of fluoroquinolone resistance: an update 1994-1998. Drugs 58, 1118.[ISI][Medline]
8 . Martinez-Martinez, L., Pascual, A. & Jacoby, G. A. (1998). Quinolone resistance from a transferable plasmid. Lancet 351, 7979.[CrossRef][ISI][Medline]
9
.
Wang, M., Tran, J. H., Jacoby, G. A. et al. (2003). Plasmid-mediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai, China. Antimicrobial Agents and Chemotherapy 47, 22428.
10
.
Tran, J. H. & Jacoby, G. A. (2002). Mechanism of plasmid-mediated quinolone resistance. Proceedings of the National Academy of Sciences, USA 99, 563842.
11 . Willmott, C. J. & Maxwell, A. (1993). A single point mutation in the DNA gyrase A protein greatly reduces binding of fluoroquinolones to the gyrase-DNA complex. Antimicrobial Agents and Chemotherapy 37, 1267.[Abstract]
12 . Andersson, D. I. & Levin, B. R. (1999). The biological cost of antibiotic resistance. Current Opinion in Microbiology 2, 48993.[CrossRef][ISI][Medline]
13 . Andersson, D. I. (2003). Persistence of antibiotic resistant bacteria. Current Opinion in Microbiology 6, 4526.[CrossRef][ISI][Medline]
14
.
Sanchez, P., Linares, J. F., Ruiz-Diez, B. et al. (2002). Fitness of in vitro selected Pseudomonas aeruginosa nalB and nfxB multidrug resistant mutants. Journal of Antimicrobial Chemotherapy 50, 65764.
15 . Lea, D. E. & Coulson, C. A. (1949). The distribution of the number of mutants in bacterial populations. Journal of Genetics 49, 26485.[ISI]
16 . Maisnier-Patin, S., Berg, O. G., Liljas, L. et al. (2002). Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium. Molecular Microbiology 46, 35566.[CrossRef][ISI][Medline]
17 . Nilsson, A. I., Kugelberg, E., Berg, O. G. et al. (2004). Experimental adaptation of Salmonella typhimurium to mice. Genetics 168, in press.
18 . O'Toole, G. A., Pratt, L. A., Watnick, P. I. et al. (1999). Genetic approaches to study of biofilms. Methods in Enzymology 310, 91109.[ISI][Medline]
19 . Jalal, S. & Wretlind, B. (1998). Mechanisms of quinolone resistance in clinical strains of Pseudomonas aeruginosa. Microbial Drug Resistance 4, 25761.[ISI][Medline]
20 . Oh, H., Stenhoff, J., Jalal, S. et al. (2003). Role of efflux pumps and mutations in genes for topoisomerase II and IV in fluoroquinolone-resistant Pseudomonas aeruginosa strains. Microbial Drug Resistance 9, 3238.[CrossRef][ISI][Medline]
21 . Matsuo, M., Ohtsuka, Y., Kataoka, K. et al. (1996). Transient relaxation of plasmid DNA in Escherichia coli by fluoroquinolones. Journal of Pharmacy and Pharmacology 48, 9857.[ISI][Medline]
22 . Poole, K. (2004). Efflux-mediated multiresistance in Gram-negative bacteria. Clinical Microbiology and Infection 10, 1226.
23
.
Komp Lindgren, P., Karlsson, A. & Hughes, D. (2003). Mutation rate and evolution of fluoroquinolone resistance in Escherichia coli isolates from patients with urinary tract infections. Antimicrobial Agents and Chemotherapy 47, 322232.
24
.
Mouneimne, H., Robert, J., Jarlier, V. et al. (1999). Type II topoisomerase mutations in ciprofloxacin-resistant strains of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 43, 626.
25 . Higgins, P. G., Fluit, A. C., Milatovic, D. et al. (2003). Mutations in GyrA, ParC, MexR and NfxB in clinical isolates of Pseudomonas aeruginosa. International Journal of Antimicrobial Agents 21, 40913.[CrossRef][ISI][Medline]
26 . Mizushima, T., Kataoka, K., Ogata, Y. et al. (1997). Increase in negative supercoiling of plasmid DNA in Escherichia coli exposed to cold shock. Molecular Microbiology 23, 3816.[CrossRef][ISI][Medline]
27 . Menzel, R. & Gellert, M. (1983). Regulation of the genes for E. coli DNA gyrase: homeostatic control of DNA supercoiling. Cell 34, 10513.[ISI][Medline]
28
.
Takenouchi, T., Sakagawa, E. & Sugawara, M. (1999). Detection of gyrA mutations among 335 Pseudomonas aeruginosa strains isolated in Japan and their susceptibilities to fluoroquinolones. Antimicrobial Agents and Chemotherapy 43, 4069.
29
.
Akasaka, T., Tanaka, M., Yamaguchi, A. et al. (2001). Type II topoisomerase mutations in fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa isolated in 1998 and 1999: role of target enzyme in mechanism of fluoroquinolone resistance. Antimicrobial Agents and Chemotherapy 45, 22638.
30
.
Bagel, S., Hullen, V., Wiedemann, B. et al. (1999). Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli. Antimicrobial Agents and Chemotherapy 43, 86875.
31
.
Barnard, F. M. & Maxwell, A. (2001). Interaction between DNA gyrase and quinolones: effects of alanine mutations at GyrA subunit residues Ser(83) and Asp(87). Antimicrobial Agents and Chemotherapy 45, 19942000.
32
.
Hudson, R. E., Bergthorsson, U., Roth, J. R. et al. (2002). Effect of chromosome location on bacterial mutation rates. Molecular Biology and Evolution 19, 8592.
33
.
Giraud, E., Cloeckaert, A., Baucheron, S. et al. (2003). Fitness cost of fluoroquinolone resistance in Salmonella enterica serovar Typhimurium. Journal of Medical Microbiology 52, 697703.
34 . Gillespie, S. H., Voelker, L. L. & Dickens, A. (2002). Evolutionary barriers to quinolone resistance in Streptococcus pneumoniae. Microbial Drug Resistance 8, 7984.[CrossRef][ISI][Medline]
35
.
Ince, D. & Hooper, D. C. (2003). Quinolone resistance due to reduced target enzyme expression. Journal of Bacteriology 185, 688392.