Molecular Diagnostics and Typing Unit, Department of Microbiology, University Hospital, Queens Medical Centre, Nottingham NG7 2UH, UK
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Abstract |
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Methods: MICs of ciprofloxacin and moxifloxacin were determined by Etest. PCR analysis was used to detect chromosomal mutations in the gyrA and parC genes. Isolates resistant to ciprofloxacin and susceptible to moxifloxacin were examined for the ability to generate spontaneous moxifloxacin-resistant isolates.
Results: Of 226 isolates, 49.1% were resistant to ciprofloxacin and 39.4% were moxifloxacin-resistant according to BSAC criteria. Approximately 20% of isolates resistant to ciprofloxacin remained susceptible to moxifloxacin. A GyrA mutation at Ser-83 was found in all ciprofloxacin-resistant isolates. Single mutations in both the gyrA and parC genes at codons Ser-83 and Ser-80, respectively, were found in ciprofloxacin- and moxifloxacin-resistant isolates. Isolates that were ciprofloxacin-resistant but moxifloxacin-susceptible generated spontaneous moxifloxacin-resistant mutants when grown on medium containing up to 8x their initial MIC. However, these mutants were not stable and none displayed high-level moxifloxacin resistance.
Conclusions: Moxifloxacin retained in vitro activity against some ciprofloxacin-resistant clinical A. baumannii isolates. Mutations in both gyrA and parC were necessary for resistance to moxifloxacin in most isolates of A. baumannii.
Keywords: Acinetobacter, fluoroquinolones, gyrA, parC, resistant
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Introduction |
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Newer fluoroquinolones, such as moxifloxacin, may have increased activity against A. baumannii in vitro in comparison with older agents such as ciprofloxacin.6,7 The present study compared the in vitro activity of these two compounds against A. baumannii isolates obtained from hospitals throughout the UK. The mechanism(s) of resistance to moxifloxacin were analysed and compared with those reported previously,3,4,6,7 and the frequency at which moxifloxacin-susceptible A. baumannii isolates yielded resistant mutants was assessed.
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Materials and methods |
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Two hundred and twenty-six clinical isolates of A. baumannii sensu stricto were obtained from 44 hospitals throughout the UK and were identified to the genomic species level by tDNA and AFLP fingerprinting.8 Isolates included in the study were a combination of outbreak-related and sporadic isolates belonging to the 35 RAPD genotypes and 15 outliers identified previously.8 Susceptibilities against ciprofloxacin and moxifloxacin were initially established using the British Society for Antimicrobial Chemotherapy (BSAC) standardized disc susceptibility testing method.9 MICs of ciprofloxacin-resistant and moxifloxacin-resistant isolates were determined with Etest strips (AB Biodisk, Solna, Sweden). Breakpoints used were those recommended for Acinetobacter spp. and Enterobacteriaceae by the BSAC.9,10 Escherichia coli (strain ATCC 25922) was used as the control strain for disc susceptibility testing and MICs as recommended by the BSAC.9
DNA extraction, amplification and digestion of the QRDR of gyrA and parC genes
Isolates resistant to ciprofloxacin and/or moxifloxacin were examined for mutations in the gyrA and parC genes at codons Ser-83 and Ser-80, respectively, as described previously.3,4
Selection of spontaneous single-step mutants
A. baumannii isolates resistant to ciprofloxacin but moxifloxacin-susceptible were examined for the generation of spontaneous single-step mutations to moxifloxacin resistance. Ten-fold dilutions of an overnight broth culture of each isolate (107108 cfu/mL) were spread (100 µL portions) on to unsupplemented IsoSensitest agar plates (90 mm diameter), and IsoSensitest agar plates containing moxifloxacin at 1, 2, 4, 8 and 16x MIC for each respective isolate. Plates were incubated aerobically at 37°C for 48 h. The mutation frequency was calculated as the number of resistant colonies divided by the total viable count. Resistant colonies obtained at the highest antibiotic concentration for each respective isolate were screened for high-level moxifloxacin resistance by re-streaking on to IsoSensitest agar plates containing moxifloxacin 8 mg/L. MICs for colonies growing on moxifloxacin 8 mg/L were determined with Etest strips. Resistant colonies were also examined for mutations in the gyrA and parC genes.3,4 The stability of resistance was investigated by subculturing a single resistant colony on to IsoSensitest agar lacking moxifloxacin, followed by the subsequent determination of moxifloxacin MICs with Etest strips.
Effect of gyrA and parC mutations on growth in the presence of moxifloxacin
Three isolates, one (A32) lacking mutations in either gyrA or parC, one (A1069) with a mutation in gyrA, and one (A2180) with mutations in both gyrA and parC, were used to study the effect of gyrA and parC mutations on the growth of A. baumannii isolates in the presence of increasing concentrations of moxifloxacin. Overnight broth cultures of each isolate (107108 cfu/mL) were spread (100 µL portions of 10-fold dilutions of the culture) on to IsoSensitest agar plates (90 mm diameter) containing moxifloxacin at increasing concentrations (0128 mg/L). Following incubation at 37°C for 48 h, colony counts (cfu/mL) were plotted against the moxifloxacin concentration in the agar.
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Results and discussion |
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Of 89 isolates resistant to ciprofloxacin and moxifloxacin, 87 had a mutation in the parC gene at codon Ser-80.4 Two isolates lacked this mutation, despite being resistant to ciprofloxacin and moxifloxacin. These isolates may possess an alternative mutation in a neighbouring codon of parC, or could have a mutation in parE.6,7 Alternatively, these isolates could have altered permeability to fluoroquinolones or an efflux-pump mechanism.2,5,6 Nevertheless, the results from this study suggest that mutations in both gyrA and parC genes are normally required for significant moxifloxacin resistance to develop in A. baumannii. Figure 1(b) illustrates the effect of different combinations of gyrA and parC mutations on the growth of A. baumannii isolates in the presence of increasing concentrations of moxifloxacin.
Twenty-two isolates that were ciprofloxacin resistant but moxifloxacin susceptible did not have the Ser-80 mutation in ParC. The results of spontaneous single-step mutation studies for each of these 22 isolates are displayed in Table 1. Only one isolate (A1066) did not generate spontaneous mutants at a detectable frequency following selection at moxifloxacin concentrations above the initial MIC (Table 1). Ten isolates generated mutants at up to twice the initial MIC, a further eight isolates produced mutants at four times the initial MIC and the remaining three isolates generated mutants at eight times the initial MIC (Table 1). However, when 73 single-step mutants that grew at the highest antibiotic concentration for each respective isolate were examined for high-level moxifloxacin resistance, none had an MIC of >8 mg/L. Five of the 73 single-step mutants examined had a moxifloxacin MIC of 8 mg/L. The remainder had a moxifloxacin MIC of 6 mg/L. None of the five mutants with a moxifloxacin MIC of 8 mg/L had the mutation at Ser-80 of ParC. Resistance to moxifloxacin appeared to be unstable in these mutants as all five demonstrated a moxifloxacin MIC of <1.5 mg/L following subculture on moxifloxacin-free agar.
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Acknowledgements |
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Footnotes |
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References |
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4 . Vila, J., Ruiz, J., Goni, P. et al. (1997). Quinolone-resistance mutations in the topoisomerase IV parC gene of Acinetobacter baumannii. Journal of Antimicrobial Chemotherapy 39, 75762.[Abstract]
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