Pharmacodynamic characterization of efflux and topoisomerase IV-mediated fluoroquinolone resistance in Streptococcus pneumoniae

Karl J. Madaras-Kelly1,2,*, Christopher Daniels2, Marissa Hegbloom1,2 and Michelle Thompson1,2

1 Veterans Affairs Medical Center, 500 W. Fort Street, Boise, ID 83702; 2 College of Pharmacy, Idaho State University, Pocatello, ID, USA

Received 6 August 2001; returned 4 March 2002; revised 15 April 2002; accepted 29 April 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Most in vitro investigations of fluoroquinolone resistance involving Streptococcus pneumoniae have described genotypic changes in quinolone resistance-determining regions (QRDRs) that occur as the result of exposure to fluoroquinolones obtained with static antimicrobial concentrations. The objectives of this study were to determine whether differences exist between moxifloxacin, sparfloxacin and levofloxacin antimicrobial effect (AME) and their ability to select out stepwise mutations with wild-type, efflux-expressing and parC-mediated fluoroquinolone resistance while simulating the in vivo dosing and pharmacokinetics of the respective agents.

Methods: A one-compartment pharmacodynamic model simulated fluoroquinolone dosing regimens. Duplicate 24 h experiments were carried out in Mueller–Hinton broth with 3% horse blood at 1 x 108 cfu/mL. Reserpine (10 mg/L) was added to select experiments conducted with efflux-expressing strains. AME was expressed as the area under the time–concentration kill curve (AUEC24). Strains expressing increased MIC post-time–concentration kill curve (TCKC) were evaluated for changes in QRDR.

Results: Moxifloxacin exhibited a greater AME against all isolates. Efflux was generally associated with partial loss of AME for all fluoroquinolones, and levofloxacin retained no AME against parC-expressing S. pneumoniae. Increased fluoroquinolone MIC post-TCKC was more common with efflux expression. The addition of reserpine was associated with enhanced AME for levofloxacin and moxifloxacin, but was not associated with altered resistance selection. Isolates recovered post-TCKC from experiments involving efflux- or parC mutation-containing isolates generally exhibited a more than four-fold increase in MIC, which was associated with commonly reported substitutions in both parC and gyrA.

Conclusion: The results of this study generally indicate that resistance selection under pharmacodynamic conditions is similar to results reported with static fluoroquinolone concentrations. While moxifloxacin AME was greater than levofloxacin and sparfloxacin, the overall selection of resistant isolates did not differ.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluoroquinolone resistance in Streptococcus pneumoniae is thought to involve, either individually or in combination, three possible mechanisms: (i) an efflux transporter mechanism; (ii) alterations in topoisomerase IV (parC or parE); and (iii) alterations in the DNA gyrase (gyrA or gyrB). Generally, the selection of high-level resistance involves a stepwise process involving a parC mutation first, followed by a gyrA mutation.1,2 Efflux may facilitate mutant selection by decreasing intracellular concentrations of fluoroquinolone.3 Most in vitro investigations of fluoroquinolone resistance in S. pneumoniae have described genotypic mutations that are manifest as changes in MIC. In these experiments, resistant isolates are selected by exposure to fluoroquinolones at low multiples of the MIC under static antimicrobial concentrations.16 Few studies have attempted to link fluoroquinolone pharmacokinetics to the resistance selection process.7,8 The objectives of this study were two-fold. First, to determine whether differences exist between moxifloxacin, sparfloxacin and levofloxacin in their ability to select out stepwise mutations in wild-type S. pneumoniae while simulating the in vivo dosing and pharmacokinetics of the respective agents. Secondly, to characterize the antimicrobial effect (AME) and the ability to select out further stepwise mutations in S. pneumoniae with known fluoroquinolone efflux and/or topoisomerase IV (parC) resistance mechanisms while simulating in vivo dosing and pharmacokinetics of the respective fluoroquinolones.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Three sets of time–concentration kill curve (TCKC) experiments that simulated fluoroquinolone dosage regimens were carried out, using four strains of S. pneumoniae. The first set of experiments compared fluoroquinolone AMEs, as well as the likelihood of selecting out mutants from a reference strain and a clinical isolate of S. pneumoniae with no known resistance mechanisms. A second set of TCKC experiments compared fluoroquinolone AME and the likelihood of selecting out resistant organisms from two isolates of S. pneumoniae with known resistance mechanisms. One isolate exhibited phenotypic expression of fluoroquinolone efflux, as characterized by resistance to norfloxacin and restoration of initial MIC with the addition of reserpine. This isolate did not possess topoisomerase or DNA gyrase mutations in the quinolone resistance-determining regions (QRDRs). A second isolate exhibited both a parC mutation and phenotypic expression of fluoroquinolone efflux, characterized in the same manner. In a third set of TCKC experiments, reserpine was added to the pharmacodynamic models along with the fluoroquinolone against the efflux-positive isolates. The purpose of these experiments was to allow comparison of fluoroquinolone activity on AME and resistance selection with and without fluoroquinolone efflux.

In vitro model

TCKC experiments were conducted as in a one-compartment in vitro pharmacodyanamic model described previously.9 The model consisted of a hollow glass sealed chamber (c. 500 mL) with inflow and outflow ports. Using a peristaltic pump, antibiotic-free, phosphate-buffered Mueller–Hinton broth supplemented with 3% lysed horse blood (SMHB) was forced into the system at a pre-determined rate so that an equal volume of medium was displaced, resulting in the simulation of a mono-exponential pharmacokinetic process.10 Before experiments were carried out, a pre-determined inoculum of S. pneumoniae was instilled into the system, followed by a single bolus addition of either moxifloxacin, levofloxacin or sparfloxacin. Each experiment was carried out in duplicate for 24 h at 37°C.

Antibiotics and pharmacokinetics

Stock solutions of moxifloxacin (Bayer, New Haven, CT, USA), sparfloxacin (Bertek Pharmaceuticals, Sugarland, TX, USA) and levofloxacin (Ortho-McNiel, Raritan, NJ, USA) were prepared using the appropriate amounts of sterile distilled water for injection and frozen in aliquots at –70°C until they were needed for individual experiments. Reserpine (Sigma, St Louis, MO, USA) was dissolved in 12 M glacial acetic acid and diluted to a concentration of 7 mg/mL in sterile distilled water for injection. Reserpine was dissolved immediately before each TCKC experiment, and was introduced into the pharmacodynamic model as a 1 mL bolus injection. The maximal peak concentration (Cmax) of reserpine introduced to the pharmacodynamic model was c. 10 mg/L. TCKC experiments were designed to simulate serum pharmacokinetic characteristics reflective of in vivo dosage regimens.1113 Target Cmaxs were 4.5, 1.3 and 6.0 mg/L for moxifloxacin, sparfloxacin and levofloxacin, respectively. Mono-exponential half-lives (t) were 12, 16 and 6 h for moxifloxacin, sparfloxacin and levofloxacin, respectively.

The fluoroquinolone concentrations simulated in the models were confirmed utilizing a microbiological assay.14 Briefly, plates were prepared from antibiotic media 2 (Difco, Detroit, MI, USA) with the pH adjusted to c. 7.7 at 25°C, and Escherichia coli ATCC 25922 was used as the study organism at a density equivalent to a 0.5 McFarland standard. Twenty-microlitre duplicate aliquots of standard solutions prepared in SMHB, as well as samples recovered from the 1 and 8 h TCKC timepoints, were incubated at 37°C for 24 h. The levofloxacin assay was linear from 0.3 to 10.0 mg/L, whereas the sparfloxacin and moxifloxacin assays were linear from 0.3 to 5 mg/L.

Bacteria and susceptibility testing

S. pneumoniae ATCC 49619 and a clinical isolate (SP 2136) were studied in the first set of experiments. These isolates lacked alterations in the QRDR and did not phenotypically express fluoroquinolone efflux. In the second set of TCKC experiments, two additional isolates of S. pneumoniae were studied. Strain 49619 EFX/C was a derivative of ATCC 49619 that expressed an efflux phenotype and had a mutation in parC (Ser-80->Tyr). This isolate was obtained by plating 104 cfu of ATCC 49619 on Trypticase soy agar (TSA) with 5% sheep blood supplemented with norfloxacin (4 x MIC). The other strain, 49619 EFX, was a mutant recovered at the 24 h time point during a TCKC experiment using levofloxacin and ATCC 49619. This strain expressed an efflux phenotype, but lacked alterations in the QRDR. Before undertaking TCKC experiments, isolates were subcultured three times consecutively in increasing volumes of SMHB to allow the organisms to attain exponential growth. The final culture (c. 1.5 L) was concentrated by centrifugation at 7000 rpm for 10 min. The pellet was resuspended in 20 mL of SMHB, and serial dilution was used to provide confirmation of the bacterial density. Depending on the isolate being studied, between 4 and 16 mL was injected into each model to provide a starting bacterial inoculum of c. 1 x 108 cfu/mL for each TCKC experiment. Growth controls (± reserpine) verified that each isolate could attain exponential growth. Susceptibility testing was performed on wild-type isolates and on all isolates recovered from the TCKC experiments after 24 h. Homogenates of a minimum of five to 10 colonies recovered from the 24 h sampling point of the TCKC were used to assess post-TCKC susceptibility. Susceptibility testing was performed using Etest according to the manufacturer’s specifications. Fluoroquinolone resistance post-TCKC was defined as at least a two-unit (mg/L) increase in MIC on the Etest strip as compared with the wild-type isolate.15

TCKC pharmacodynamics

At pre-determined timed intervals, samples of media were removed from the pharmacodynamic models for quantification of bacteria utilizing serial 10-fold dilution with aliquots plated on TSA supplemented with 5% sheep blood. After incubation for 18–24 h at 37°C, plates containing between 30 and 300 cfu were counted. The theoretical lower limit of bacterial counting accuracy was 3.0 x 102 cfu/mL. TCKCs were constructed by plotting log10 cfu/mL versus time.

Analysis of AME was expressed as the area under the time–concentration kill curve (AUEC24).16 The AUEC24 only incorporated time–concentration data points that were above the lower theoretical limit of bacterial counting. To compensate for differences in initial starting inocula, AUEC24 was standardized by dividing AUEC24 by the cfu/mL at time zero.17

Efflux determination

Phenotypic expression of efflux-mediated resistance was determined by an agar dilution method:18 1 x 104 cfu of each isolate was inoculated on to duplicate plates containing serially increasing concentrations of norfloxacin or norfloxacin plus reserpine (10 mg/L). Selected experiments were conducted with increased reserpine concentrations (50 and 100 mg/L) against isolates recovered post-TCKC that exhibited high-level fluoroquinolone resistance not explained by mutations in QRDR.

Amplification of S. pneumoniae QRDRs of parC, parE, gyrA and gyrB genes

All parent isolates and all post-TCKC isolates that exhibited an increased MIC underwent genotypic analysis by PCR for changes in their QRDR. Bacterial genomic DNA was extracted using the High Pure PCR Template kit (Boehringer-Mannheim, Indianapolis, IN, USA). The QRDRs of parC, parE, gyrA and gyrB were amplified via PCR using isolated chromosomal DNA as the template. PCR primers for the QRDRs and conditions for amplification were as described by Pan et al.2 All PCR primers were synthesized by the Idaho State University Molecular Research Core Facility. To identify mutations at the level of nucleotide sequence, PCR products were electrophoresed on 2% agarose gels, the appropriate band excised from the gel and PCR product purified using the Qiagen QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, CA, USA). Nucleotide sequences of PCR products were determined directly using an ABI Automated Fluorescence Sequencer and mutations were identified using the MegAlign program of DNAStar sequence analysis software.

Analysis

ANOVA and Dunn’s test were used to compare the AME for intra- and inter-species comparisons, and P < 0.0167 was considered statistically significant for intergroup comparisons. The AME for specific fluoroquinolones with and without reserpine was compared using Student’s t-test, and the {chi}2 or Fisher’s exact test was used to compare the likelihood of developing resistance for efflux versus non-efflux strains post-TCKC. P < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The initial susceptibility patterns and phenotypic expression of fluoroquinolone efflux of the four isolates of S. pneumoniae are summarized in Table 1. ATCC 49619 and SP 2136 were fully susceptible to all fluoroquinolones tested, and reserpine had no effect on the MIC of norfloxacin. The levofloxacin MIC for isolate 49619 EFX was two-fold higher than for ATCC 49619, but moxifloxacin and sparfloxacin MICs were the same. Relative to ATCC 49619, MICs of moxifloxacin, levofloxacin and sparfloxacin EFX/C were one- to two-fold higher. In contrast, norfloxacin MICs for 49619 EFX and 49619 EFX/C were eight-fold higher than the parent isolate, ATCC 49619. The addition of reserpine restored norfloxacin susceptibilities, suggesting the phenotypic expression of a fluoroquinolone efflux pump.


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Table 1.  Susceptibility data for pre-TCKC isolates
 
All three fluoroquinolones were effective at reducing the viable count of ATCC 49619 by at least 3 logs (Figure 1). Although not significantly different, a trend was evident favouring the moxifloxacin AME compared with sparfloxacin (P = 0.06) and levofloxacin (P = 0.11), although sparfloxacin and levofloxacin regimens did not appear to differ (P = 0.53). S. pneumoniae was recovered from four of the six TCKC experiments conducted with ATCC 49619. Analysis of post-TCKC strains revealed that only one isolate exhibited an increased MIC. This isolate was recovered from an experiment involving levofloxacin. The post-TCKC MIC of levofloxacin increased from the initial MIC of 0.75 to 1.5 mg/L. Analysis of the QRDR from this isolate did not reveal any changes; however, the isolate did express fluoroquinolone efflux, whereas the parent isolate did not (Table 1). This isolate was subsequently named 49619 EFX.



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Figure 1. TCKC profiles of moxifloxacin (diamonds), sparfloxacin (triangles) and levofloxacin (squares) versus ATCC 49619 (expressed as mean of duplicate experiments).

 
Against SP 2136, the AME was less pronounced for all three fluoroquinolones compared with ATCC 49619 (Figure 2). The AME of moxifloxacin was significantly greater than that of sparfloxacin (P = 0.003) or levofloxacin (P = 0.008). The sparfloxacin AME was greater than that of levofloxacin, but the difference was not significant (P = 0.08). S. pneumoniae was recovered from all six TCKCs conducted with SP 2136; however, none of the post-TCKC isolates exhibited an increased MIC.



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Figure 2. TCKC profiles of moxifloxacin (diamonds), sparfloxacin (triangles) and levofloxacin (squares) versus SP 2136 (expressed as mean of duplicate experiments).

 
The AME against 49619 EFX was significantly less (P < 0.001) for all three fluoroquinolones in comparison with ATCC 49619. However, there were no significant differences in AME for fluoroquinolones individually versus ATCC 49619, nor were there any statistical differences in the AME of individual fluoroquinolones against 49619 EFX (Figure 3). The addition of reserpine to TCKC was associated with enhanced AME for moxifloxacin (P < 0.03) and levofloxacin (P < 0.03) (Table 2). S. pneumoniae was recovered from 12 of the 13 TCKC experiments conducted with 49619 EFX. However, unlike non-efflux-producing strains ATCC 49619 or SP 2136, analysis of post-TCKC isolates revealed that seven of 13 strains exhibited an increased MIC (P = 0.03). One of four moxifloxacin, four of four sparfloxacin and two of five levofloxacin post-TCKC isolates developed resistance. A MIC increase occurred in three of seven TCKC experiments conducted without reserpine and four of six TCKC experiments conducted with reserpine (P = 0.26). The majority of isolates expressing resistance exhibited high-level resistance, with MICs >= 32 mg/L and corresponding mutations in both parC and gyrA (Table 3).



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Figure 3. TCKC profiles of moxifloxacin (diamonds), sparfloxacin (triangles) and levofloxacin (squares) versus 49619 EFX (expressed as mean of duplicate experiments).

 

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Table 2.  AME of moxifloxacin, levofloxacin and sparfloxacin ± reserpine against efflux- and non-efflux-positive S. pneumoniae
 

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Table 3.  Post-TCKC susceptibility and QRDR analysis data
 
Although not statistically different, the fluoroquinolone AME against 49619 EFX/C was less pronounced than the AME against 49619 EFX (P = 0.08) or ATCC 49619 (P = 0.07) (Figure 4). There was also a trend towards a greater AME for moxifloxacin (P = 0.04) as compared with levofloxacin against 49619 EFX/C. When the fluoroquinolone-specific AME against 49619 EFX/C was compared with the AME against ATCC 49619 or 49619 EFX, only the levofloxacin AME against 49619 EFX/C was signficantly less than against ATCC 49619 (P = 0.05). In fact, levofloxacin exhibited almost no AME against 49619 EFX/C, as the number of cfu/mL was above the dilutional capacity of 1 x 107 for many of the TCKC time points. The addition of reserpine to TCKC did not significantly change the AME for any of the three fluoroquinolones (Table 3). S. pneumoniae were recovered from all 12 TCKC experiments conducted with 49619 EFX/C. In a similar manner to 49619 EFX, analysis of post-TCKC-recovered isolates revealed that six of 12 strains exhibited an increase in MIC over baseline. Three of four moxifloxacin, two of four sparfloxacin and one of four levofloxacin post-TCKC isolates exhibited further increases in MIC. A MIC increase occurred in four of six TCKC experiments conducted without reserpine and two of six TCKCs with reserpine (P = 0.26). MICs varied in isolates expressing resistance, with the majority of isolates expressing high-level fluoroquinolone resistance. In two TCKCs conducted with 49619 EFX/C (one moxifloxacin, one levofloxacin) high-level resistance did not correlate with the presence of mutations in gyrA, gyrB or parE (Table 2). These two isolates were tested for hyperproduction of efflux by agar dilution with serial increases in norfloxacin concentrations with and without 50 and 100 mg/L of reserpine. The MIC of norfloxacin alone was 64 mg/L for both isolates. The MIC was reduced to 16 and 32 mg/L with 10 mg/L reserpine, and 8 mg/L with 50 mg/L reserpine. However, control experiments conducted with reserpine alone at 50 and 100 mg/L demonstrated inhibition of bacterial growth.



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Figure 4. TCKC profiles of moxifloxacin (diamonds), sparfloxacin (triangles) and levofloxacin (squares) versus 49619 EFX/C (expressed as mean of duplicate experiments). Levofloxacin cfu/mL was above the serial dilution limit for time points at 4–16 h.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The differences in pharmacokinetics and intrinsic antimicrobial potency, coupled with the frequency of mutation selection by particular fluoroquinolones, may play a significant role in determining whether fluoroquinolone resistance arises in S. pneumoniae. However, most in vitro investigations of fluoroquinolone resistance in S. pneumoniae have described genotypic changes that occur as the result of exposure to fluoroquinolones at low multiples of the MIC under static conditions. To our knowledge, our study is the first that has characterized the specific resistance mechanisms that occur by exposure to dynamic changes in drug concentrations with in vitro pharmacodynamic modelling.

Despite initial MICs suggesting that all organisms were susceptible to all three fluoroquinolones, there were large differences in AME between moxifloxacin, sparfloxacin and levofloxacin. All three fluoroquinolones exhibited pronounced AME against isolates without QRDR changes or the presence of efflux. However, moxifloxacin, and to a lesser extent sparfloxacin, retained activity against S. pneumoniae that exhibited phenotypic fluoroquinolone efflux. Despite an MIC of 1.5 mg/L, levofloxacin retained partial AME against the isolate possessing efflux only, and almost no AME against the isolate possessing both phenotypic efflux and a parC mutation. However, the exact degree of levofloxacin AME against 49619 EFX/C was not exemplified in these TCKCs, as the organism replicated above the serial dilution capacity of most time points. Moxifloxacin retained a relatively more pronounced AME against the isolate possessing a topoisomerase IV mutation in parC.

The presence of an energy-dependent efflux transport mechanism has been reported to be present in c. 45% of fluoroquinolone-resistant S. pneumoniae.18 Pharmacokinetic simulations against isolates without QRDR changes or the presence of efflux revealed the selection of only one isolate with an increase in MIC. This isolate did not contain alterations in QRDR, but was an efflux-producing isolate. In contrast, the majority of pharmacokinetic simulations against 49619 EFX resulted in the further selection of organisms with increases in MIC and corresponding changes in parC and gyrA. These results would support the findings of Beyer et al.,3 which suggest that the probability of avoiding efflux reduces the potential for selection of further resistance.

The results seen following addition of reserpine to our TCKC experiments conducted with isolates expressing efflux indicated enhanced AME for moxifloxacin and levofloxacin, but not sparfloxacin. Theoretically, moxifloxacin AME should not have been influenced by reserpine, as moxifloxacin has been reported to be relatively unaffected by pmrA-mediated efflux due to its C-8 methoxy substitution.3 However, at least one moxifloxacin-resistant isolate exhibiting a four-fold MIC increase attributed to efflux has been described.19

In contrast to static experiments conducted with reserpine, the addition of reserpine to TCKC experiments of efflux-expressing isolates did not prevent the development of further resistance.3,20 While reserpine is freely soluble in glacial acetic acid, we cannot exclude the possibility that reserpine precipitated during TCKC because the solubility of this drug is poor in aqueous solutions. Aeschlimann et al.,21 working with NorA-expressing Staphylococcus aureus, reported difficulty in maintaining the solubility of reserpine in TCKC.

Of interest, two post-TCKC isolates from experiments conducted with moxifloxacin and levofloxacin against 49619 EFX/C exhibited high-level resistance that was not fully explained by analysis of known QRDRs. This isolate possessed an alteration in parC and expressed efflux before TCKC. The MICs of moxifloxacin and levofloxacin were 0.25 and 1.5 mg/L, respectively. Post-TCKC analysis of recovered isolates indicated moxifloxacin and levofloxacin MICs of 4 and 32 mg/L, respectively. Subsequent agar dilution experiments conducted with 10, 50 and 100 mg/L of reserpine and serial concentrations of norfloxacin revealed a two-fold decrease in MIC with the addition of 10 mg/L reserpine and a three-fold decrease in MIC with the addition of 50 mg/L reserpine. However, 50 mg/L reserpine independently inhibited the growth of both isolates, whereas 10 mg/L did not. We did not directly measure the MIC post-TCKC of moxifloxacin or levofloxacin with reserpine, but chose to maintain consistency with the agar dilution method used to assess pre-TCKC isolates for efflux expression.

These experiments indicate that the high-level resistance post-TCKC might be due in part to hyperproduction of efflux; however, the full effect of inhibition of efflux with reserpine remains unknown. Hyperproduction of efflux without mutation in QRDR has rarely been shown to be responsible for high-level resistance to ofloxacin.8 An alternative possibility is that topoisomerase II mutations or other mutations outside of the sequenced QRDR resulted in phenotypic changes in MIC.22

Finally, the majority of mutations reported in our study involved Ser-80->Tyr for parC, and Ser-83->Phe, Ser-83->Tyr or Glu-87->Lys for gyrA, all of which have been reported as relatively frequent mutations in fluoroquinolone-resistant S. pneumoniae.6 The results of our investigation tend to suggest that selection of specific mutational changes recovered from clinical infections, as well as those selected under static fluoroquinolone concentrations, parallel those selected by dynamic conditions utilizing in vitro pharmacodynamic models.18


    Acknowledgements
 
This work was funded by an unrestricted educational grant from Bayer Pharmaceuticals.


    Footnotes
 
* Corresponding author. Tel: +1-208-422-1146; Fax: +1-208-422-1147; E-mail: kmk{at}otc.isu.edu Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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