1 Division of Microbiology, University of Leeds, Leeds LS2 9JT; 3 ICRF Mutation Detection Facility, St Jamess University Hospital, Leeds; 4 Division of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK; 2 Bayer, Global Strategic Marketing Anti Infective, D-42096 Wuppertal, Germany
Received 10 April 2002; returned 18 June 2002; revised 9 August 2002; accepted 12 September 2002
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Abstract |
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Keywords: DNA, topoisomerases, fluoroquinolones, mutation detection, denaturing high-performance liquid chromatography, DHPLC
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Introduction |
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Ideally, to allow the analysis of large numbers of sequences, mutation detection should be sensitive, non-hazardous, relatively inexpensive and fully or at least semi-automated to minimize time and labour cost.4 Recently, a new method that meets these criteriadenaturing high-performance liquid chromatography (DHPLC)was developed for comparative DNA sequencing.5,6 This is a high-capacity technique for detecting mutations, which has been used for screening DNA samples for single-nucleotide polymorphisms and inherited mutations in the human genome.4 The accuracy of mutation polymorphism detection by DHPLC has been acknowledged.4 To our knowledge, this is the first report to describe the use of this technique in relation to the detection of mutations in the bacterial genome.
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Materials and methods |
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In the DHPLC system, DNA fragments are carried by a liquid buffer current through the system thus forming a liquid (mobile) phase. Separation of DNA fragments by size is achieved by differential absorption or partition between the liquid phase and the matrix (stationary phase) in the system DNA Sep column (Transgenomic, Inc., Omaha, NE, USA). The liquid phase is driven by a binary pump from the buffer reservoirs, through the analytical DNA Sep column and detector, and subsequently into the waste reservoir. DNA fragment separation takes place in the analytical column. DNA fragments are detected by a UV detector and the analogue signal is converted into a digital value. The results are presented as chromatograms, i.e. a series of peaks corresponding to DNA fragments. The principle behind mutation detection by DHPLC is the differential retention of homo- and heteroduplex DNA on the column. DNA binding to the column is mediated by the ion pairing reagent triethylammonium acetate (TEAA) and disrupted by a linear gradient of acetonitrile, which results in the movement of the DNA from the stationary phase into the mobile phase and progress through the column to the detector. DHPLC identifies mutations and polymorphisms by detecting heteroduplexes that contain mismatched nucleotides, formed from two PCR products. Sequence variation creates a mixed population of homoduplexes and heteroduplexes when wild-type and mutant DNA are denatured and re-annealed. When the mixed population of DNA fragments is analysed by HPLC at partially denaturing temperatures, the heteroduplexes elute from the column in a linear acetonitrile gradient faster than homoduplex DNA because of their reduced melting temperature. Thus, when non-mutant (homoduplex) DNA is analysed, a single peak is observed in the chromatogram; in contrast, when mutant (heteroduplex) samples are assessed, two or more peaks will be present. Each test sample is assayed in the presence of a comparator sequence, in the present instance the non-mutant wild-type sequence, to ensure that heteroduplex DNA is formed and mutations will be detected. The analysis temperature is critical to the success of mutation detection by DHPLC, as differential retention of homo- and heteroduplex DNA is only observed under conditions of partial denaturation. To determine the appropriate temperature for analysis of an individual DNA fragment, retention times at different temperatures are determined and a melting curve is plotted (Figure 1a). The melting temperature (Tm) of the DNA fragment under analysis is determined from the melting curve and mutation detection is subsequently carried out at 1°C below Tm. In practice, it may be necessary to run samples at more than one temperature. Biphasic or multiple shoulder melting curves, as shown in Figure 1b, can indicate a need for analysis at multiple temperatures. The sensitivity of mutation detection by DHPLC in mammalian systems has been investigated extensively and is estimated at 97% or greater.7,8
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Three methicillin-sensitive and three methicillin-resistant, non-clonal (as defined by PFGE analysis) clinical S. aureus isolates, susceptible to ciprofloxacin (MIC 0.4 mg/L), were used to derive mutants resistant to ciprofloxacin, levofloxacin, sparfloxacin, trovafloxacin and moxifloxacin. Reference strain S. aureus NCTC 6751 was used as a control to show that there are no mutations in the target genes in the parent strains.
Selection of the in vitro generated mutants
In vitro mutants with reduced susceptibilities to ciprofloxacin, levofloxacin, sparfloxacin, trovafloxacin and moxifloxacin were recovered using a stepwise selection method. Initially each isolate was grown in antibiotic-free Iso-Sensitest broth overnight at 37°C. Iso-Sensitest agar containing the fluoroquinolones at 2 x, 2.5 x, 3 x and 4 x MIC for the parent strain were spread with 100 µL of the broth suspension of each strain and incubated at 37°C for 1824 h. First step mutant (FSM) colonies were picked from these plates. Second step mutants (SSM) were selected by growing FSM in Iso-Sensitest broth containing the concentration of fluoroquinolone at which each had been selected. Each broth culture was then plated out in the same way as described previously for the isolation of FSM. Colonies growing on plates containing concentrations of fluoroquinolones above the concentrations used for the isolation of FSM were picked as SSM. Third step mutants were sought in the same manner.
PCR amplification of the grlA, grlB, gyrA and gyrB genes
PCR amplifications of 225500 bp regions spanning the QRDRs of gyrA, grlA, gyrB and grlB genes were carried out as described previously,9,10 using genomic DNA from the parent strains and in vitro generated mutants with different levels of resistance to fluoroquinolones.
Nucleotide sequence analysis
All PCR-generated products from the clinical strains and 60 mutants with reduced susceptibilities to one or more fluoroquinolones were purified using a Qiagen purification kit (Qiagen, Germany) and then subjected to nucleotide sequence analysis using an ABI Prism 377 automated sequencer.
DHPLC analysis
In vitro mutants with defined mutations in grlA, grlB, gyrA or gyrB, as determined by nucleotide sequence analysis, were selected for examination using DHPLC. The PCR products of these in vitro mutants were then run as a blinded collection on an HPLC DNA Sep column under partial heat denaturation. For DHPLC analysis, 35 µL (100 ng of DNA) of PCR product generated from the in vitro mutant were mixed with an equal volume of PCR product from the respective parental strain, denatured at 95°C for 5 min and cooled to 65°C to allow formation of heteroduplexes. DHPLC was carried out using Transgenomic Wave HPLC equipment (Transgenomic) and a DNA Sep column (Transgenomic).7 Analysis was carried out at a flow rate of 0.9 mL/min. Elution of DNA from the column was detected by absorbance at 260 nm. DHPLC data analysis was based on subjective comparison of sample and reference chromatograms. Any sequence variation in PCR products results in reduced column retention time and changes in peak profiles.
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Results |
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A good melting curve was obtained from every sample; therefore, DHPLC analysis was only carried out at one temperature (1°C below Tm). Peak number was the most important criterion for assigning the presence of a mutation. A single peak in the wild-type sample was replaced by two, three or four peaks when mutations were present in the test sequence. Examples of chromatograms are given in Figure 2.
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Discussion |
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Quinolone resistance, unlike bacterial resistance to many other antimicrobials, relies upon mutations in endogenous genes rather than the acquisition of additional genetic information. This makes genetic characterization of quinolone resistance more demanding using current technology, because it is sometimes necessary to identify a single nucleotide change rather than detect the acquisition of substantial amounts of exogenous DNA. Development and application of DHPLC will simplify considerably the identification of mutations associated with quinolone resistance. Third generation fluorinated quinolones such as moxifloxacin and gatifloxacin are potent antimicrobial agents that are highly efficacious in the treatment of a variety of bacterial infections, especially those caused by Gram-positive organisms. The rate at which resistance emerges to fluoroquinolones in Gram-positive and -negative bacteria to these new agents is thought to be very low.1 Nevertheless, experience has shown that resistance to other quinolones does emerge as a result of stepwise mutations, which can result in very high levels of resistance to these compounds.13,14 Accordingly, the ability to detect early mutations would be of value in the continuing evaluation of resistance mechanisms for both established and newly developed antimicrobial compounds.
Quinolone resistance can be mediated by both single point mutation in a single gene or by multiple mutations in four or more targets.15 Thus, an examination of quinolone resistance allows the capabilities and the flexibility of the DHPLC system to be fully tested. In particular, it provides a paradigm for DHPLC identification of mutations conferring resistance to both single and multi-target drugs. The data presented here provide evidence that DHPLC detects all of the functional mutations in QRDR encoding regions of the four genes grlA, gyrA, grlB and gyrB. It is tempting to speculate that a rapid, multiplexed PCR for all the quinolone targets could be developed, so that resistance to quinolones, however mediated, may be detected in clinical isolates in a single screening event. There is also a potential for developing degenerate primers for cross-species detection of quinolone resistance. Instead of distinct peaks, such a system would give an output that would represent a specific resistance-genotype fingerprint.
Additionally, the use of degenerate PCR primers and multiplexing could allow a single reaction to detect numerous resistance alleles in a whole range of bacterial species. A rapid-PCR protocol, followed by high-throughput DHPLC potentially could eventually enable the clinician to be informed on sensitivity patterns in less than half a day (optimization and streamlining of protocol may allow results to be obtained in as short a time as 2 h). Primers directed at 16S RNA or insertion sequences could be used for rapid strain typing when following outbreaks, and provide a simple way of monitoring clonal spread for real-time and predictive epidemiology. Such a system would mean a several-fold reduction in laboratory turn-around time, and could result in a reduction in empirical antimicrobial therapy. We recommend this technique as a rapid (5 min/sample), high-capacity (96 samples per run) method for the detection of resistance alleles and for characterizing antibiotic resistance in bacteria.
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Acknowledgements |
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Footnotes |
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References |
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