1 Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, 5th Floor, Basic Medical Sciences Building, 730 William Avenue, Winnipeg, Manitoba, R3E 0W3, Canada; 2 Clinical Microbiology, Health Sciences Centre, MS673-820 Sherbrook Street, Winnipeg, Manitoba, R3A 1R9, Canada
Received 21 June 2005; returned 25 July 2005; revised 5 August 2005; accepted 12 August 2005
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Abstract |
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Methods: Quinolone resistance determining regions of parC and gyrA were sequenced for 111 clinical isolates collected from 1995 to 1997 and 665 isolates collected in 2003. Efflux was assessed using a reserpine agar dilution method.
Results: No isolates exhibited efflux. No significant increase in isolates harbouring amino acid substitutions was observed over time (0.9% in 19951997 to 2.1% in 2003, P = 0.32). However, the proportion of isolates with a ciprofloxacin MIC = 2 mg/L and a levofloxacin MIC = 1 mg/L versus ciprofloxacin MIC = 1 mg/L and a levofloxacin MIC = 1 mg/L increased over time (3.6% to 6.5%, P = 0.0021).
Conclusions: No increase in prevalence of first-step parC mutations was observed among all fluoroquinolone-susceptible clinical isolates of S. pneumoniae with levofloxacin MICs of 1 mg/L after the introduction of the respiratory fluoroquinolones; however, fluoroquinolones appear to be selecting for isolates with elevated ciprofloxacin MICs.
Keywords: levofloxacin , mutations , efflux
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Introduction |
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The fluoroquinolones act in S. pneumoniae by targeting the type II topoisomerases: topoisomerase IV and DNA gyrase.26 Resistance to fluoroquinolones arises by stepwise accumulation of spontaneous point mutations in the quinolone-resistance determining regions (QRDR) of these target enzymes that result in decreased binding affinity of the drug.5,7,8 Topoisomerase IV is composed of two ParC subunits and two ParE subunits. Likewise, DNA gyrase is composed of two GyrA subunits and two GyrB subunits. A primary mutation in parC resulting in an amino acid substitution leads to low-level fluoroquinolone resistance (ciprofloxacin MICs of 4 or 8 mg/L). Upon acquisition of this first mutation, secondary mutations in gyrA may develop resulting in high-level resistance (ciprofloxacin MICs of 16 mg/L).2,58 These secondary gyrA mutations have been shown to arise at a significantly higher rate than the primary parC mutations.7 Resistance may also be attributed to efflux and mutations in parE and gyrB.5 Fluoroquinolone efflux in S. pneumoniae is thought to be mediated by a reserpine-sensitive pump driven by the proton motive force.5,9
Despite the low level of fluoroquinolone resistance in Canada, cases of respiratory fluoroquinolone treatment failure due to isolates possessing first-step parC mutations have been documented.7,10 Although the number of treatment failures is small compared with the hundreds of millions of patients treated with these agents, these failures have emphasized that current susceptibility testing methods are often unable to identify isolates with first-step mutations.7,10 The major concern is that the use of the respiratory fluoroquinolones will create a pool of susceptible isolates that contain unidentified first-step mutations (e.g. parC) and these isolates will readily acquire a second mutation upon treatment with a fluoroquinolone resulting in high-level fluoroquinolone resistance.3,7
Since most research to date has focused on the prevalence of parC mutations in clinical isolates with a levofloxacin MIC of 2 mg/L and reports agree that mutations in these isolates are very common,3,7,8 we focused on clinical isolates with a levofloxacin MIC of 1 mg/L. To assess whether the introduction of respiratory fluoroquinolones has had an impact on fluoroquinolone-susceptible isolates in Canada, we set out to determine the prevalence of resistance-associated mechanisms, including mutations in the QRDRs of parC and gyrA, as well as efflux, among 776 fluoroquinolone-susceptible S. pneumoniae isolates (levofloxacin MIC = 1 mg/L, ciprofloxacin MIC 2 mg/L). This study compares the prevalence of these mechanisms among Canadian respiratory isolates collected before the introduction of any respiratory fluoroquinolone in Canada (19951997, n = 111), and isolates collected well after extensive respiratory fluoroquinolone use in Canada (2003, n = 665) (data presented at the American Society for Microbiology 2005 General Meeting in Atlanta, GA, poster A-008).
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Materials and methods |
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All S. pneumoniae clinical isolates investigated in this study were collected as part of an ongoing national respiratory organism surveillance programme (CROSS: Canadian Respiratory Organism Susceptibility Study).11 The isolates were obtained from 25 medical centres in nine of the 10 Canadian provinces. Isolates were identified using conventional methodology and were considered to be significant respiratory pathogens based on each laboratory's existing protocol. MICs were determined using the National Committee for Clinical Laboratory Standards (NCCLS) microbroth dilution technique after the isolates were sub-cultured twice from frozen stock.12 Broth microtitre plates were made in-house with antimicrobials obtained as laboratory grade powders from their respective manufacturers, stock solutions prepared, and dilutions made as described by NCCLS.12 Antimicrobials tested included ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin and moxifloxacin. The broth microtitre plates were inoculated with a final bacterial concentration of 5 x 105 cfu/mL in MuellerHinton broth supplemented with 35% lysed horse blood and incubated for 2224 h at 35°C in ambient air.
Mutation analysis
Sequencing of the QRDRs of parC and gyrA was performed for all isolates. Primers previously described by Morrissey and George4 were used to generate PCR products of the QRDRs of gyrA and parC, which were then sequenced in the forward and reverse directions. An ABI PRISM Big Dye Terminator kit and ABI PRISM 3100 Genetic Analyzer (PE Applied Biosystems, Mississauga, Ontario, Canada) were used to conduct the sequencing.
Efflux determination
The prevalence of efflux was determined using a random sample consisting of 100 isolates from 1995 to 1997 and 300 isolates from 2003. The presence of efflux was assessed by ciprofloxacin agar dilution MICs on MuellerHinton agar plates supplemented with 5% sheep blood in the presence and absence of reserpine (10 mg/L).2 Isolates for which the ciprofloxacin MIC was reduced fourfold or greater in the presence of reserpine were considered positive for reserpine-sensitive efflux.2
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Results and conclusions |
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In further investigations, our isolates were divided into groups based on ciprofloxacin MIC to assess whether a trend was evolving amongst isolates with a ciprofloxacin MIC of 2 mg/L and a levofloxacin MIC of 1 mg/L in comparison with isolates with a ciprofloxacin MIC of 1 mg/L and a levofloxacin MIC of 1 mg/L. The percentage of isolates harbouring resistance-associated mutations from the cohort with a ciprofloxacin MIC of 2 mg/L increased from 2.6% (19951997) to 10.8% (2003) over the 7 year period (P = 0.12). Additionally, we observed a significant increase over time in the prevalence of isolates that had a ciprofloxacin MIC of 2 mg/L and a levofloxacin MIC of 1 mg/L from 3.9% to 6.5% (P = 0.0021). Although the overall prevalence of mutations among clinical isolates with a levofloxacin MIC of 1 mg/L remains stable, these data suggest that fluoroquinolones appear to be selecting for isolates with elevated ciprofloxacin MICs. A trend towards increased prevalence of mutations among isolates with a ciprofloxacin MIC of 2 mg/L and a levofloxacin MIC of 1 mg/L is probably emerging. This would suggest that there are discrepancies based on ciprofloxacin MIC, and predicting the presence of first-step mutations, and thus the possibility of treatment failure due to emergence of resistance, should be based on the combined ciprofloxacin and levofloxacin MICs as opposed to using a single marker such as levofloxacin alone.
A recent review by Fuller and Low10 suggests that patient risk factors may also aid in identifying infections that have a high risk of fluoroquinolone treatment failure. Such factors include residence in a long-term care facility, hospitalization and a history of fluoroquinolone use.
Recent clinical failures have highlighted the difficulty in detecting first-step mutations with the phenotypic method based on MICs using current levofloxacin breakpoints.7,10 Reducing fluoroquinolone breakpoints, using ciprofloxacin MIC as well as levofloxacin MIC, and identifying patient risk factors may all act to reduce the risk of fluoroquinolone clinical failure due to first-step mutations in S. pneumoniae.
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Acknowledgements |
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References |
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