a Institut Clinic d'Infeccions i Immunologia, IDIBAPS, Facultat de Medicina, Universitat de Barcelona, Villarroel, 170, 08036 Barcelona; b Parke-Davis, S.L., Poligono Industrial Manso Mateu, s/n, 08820 El Prat de Llobregat, Spain
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Abstract |
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Introduction |
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Previously, quinolones had good activity against Acinetobacter strains1114 even compared with expanded-spectrum cephalosporins and aminoglycosides. However, resistance to these antibiotics has rapidly emerged in clinical isolates.1518 Multiply resistant A. baumannii infections are usually treated with imipenem or sulbactam.19 However, recent data show an emergence of resistance even to these antimicrobial agents.20 Colistin is among the few antimicrobial agents that can still be used to treat infections caused by multiresistant strains, but this antibiotic can cause nephrotoxicity, neuromuscular blockade and neurotoxicity.20 Thus, there is a need to find treatment alternatives and the new fluoroquinolones may be a therapeutic option in the treatment of severe infections caused by multidrug-resistant A. baumannii.
New fluoroquinolones are broad-spectrum antibacterial agents that present an enhanced activity in comparison with old quinolones.21 The protein targets for quinolones are type II topoisomerases (DNA gyrase and topoisomerase IV).22 Both are tetrameric enzymes with two A subunits and two B subunits, encoded by the gyrA and gyrB genes, respectively, in the case of DNA gyrase, and by the parC and parE genes in the case of topoisomerase IV.22,23 There is a region in these genes that is known as the quinolone resistance determining region (QRDR), where mutations associated with the acquisition of quinolone resistance have been located.22 The mutations that play the most important role in the acquisition of resistance are located in the QRDR of the gyrA and parC genes.22,24 Mutations affecting the QRDR of the gyrB gene seem to be more frequent in quinolone-resistant strains obtained in vitro25 than in clinical isolates.26,27
Mutations affecting the parE gene are extremely unusual among clinical isolates of Gram-negative microorganisms,28,29 although mutations in in vitro quinolone-resistant strains of Escherichia coli have been described by Breines et al.30
Another mechanism of resistance to quinolones, different from alterations in target proteins, is a decrease in the accumulation of the quinolone, both by decrease in permeability and/or by an increase in the active efflux of the antibiotic. These mechanisms have not been studied in depth in Acinetobacter.24
Reserpine is a well established inhibitor of efflux pumps among Gram-positive microorganisms,31 and has recently shown its ability to inhibit an active efflux system in Bacteroides fragilis.32,33 Moreover, previous results obtained in our laboratory show its potential to act concomitantly with quinolones against other non-fermenting Gram-negative microorganisms such as Stenotrophomonas maltophilia.34
The main aim of the present study was to compare the activity of clinafloxacin, a novel fluoroquinolone, against clinical isolates of A. baumannii with other quinolones both in the presence and absence of reserpine, and to correlate their activity with mechanisms of resistance.
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Materials and methods |
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A total of 42 epidemiologically unrelated isolates of A. baumannii were recovered from different biological samples, mainly respiratory secretions, submitted to the Clinical Laboratory of Microbiology of the Hospital Clinic of Barcelona, Spain and to other hospitals in Spain (La Princesa, Madrid; Doce de Octubre, Madrid; Ramon y Cajal, Madrid; San Pablo, Barcelona; Vall D'Hebron, Barcelona; Virgen del Rocío, Sevilla; Virgen de la Macarena, Sevilla; San Joan, Reus and Virgen del Pino, Canary Islands). Isolates were identified as A. baumannii using standard biochemical procedures following the criteria of Bouvet & Grimont.35
Antimicrobial susceptibility testing
Susceptibility testing was carried out using a broth microdilution assay, in either the absence or the presence of reserpine at a concentration of 25 mg/L, in accordance with the guidelines established by the National Committee for Clinical Laboratory Standards.36 An inoculum of 5 x 10 5 cfu/mL of each isolate was inoculated on to freshly prepared medium containing serial dilutions of ciprofloxacin and moxifloxacin (Bayer, Leverkusen, Germany), nalidixic acid (Sigma, St Louis, MO, USA), levofloxacin (Hoechst Marion Roussell, Romainville, France), sparfloxacin, (Rhone-Poulenc, Vitry, France), trovafloxacin (Pfizer Ltd, Sandwich, UK) and clinafloxacin (Parke-Davis, Ann Arbor, MI, USA). The quality control strains used were: E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212.
The breakpoints proposed by the NCCLS36 were used for ciprofloxacin (S 1 mg/L; R
4 mg/L); levofloxacin (S
2 mg/L; R
8 mg/L); and nalidixic acid (S
16 mg/L; R
32 mg/L), whereas the breakpoints proposed for MENSURA37 (Mesa Española de Normalización de la Sensibilidad y Resistencia a los Antimicrobianos) were used for the quinolones that do not possess a breakpoint established by the NCCLS,36 as was the case for sparfloxacin (S
1 mg/L; R
4 mg/L); clinafloxacin (S
1 mg/L; R
4 mg/L); moxifloxacin (S
2 mg/L; R
4 mg/L); and trovafloxacin (S
1 mg/L; R
4 mg/L).
To evaluate the intrinsic activity of reserpine, the MIC for this agent was determined following the procedure mentioned above.
Amplification and DNA sequencing of the QRDR of the gyrA and parC genes
The PCR amplification of the QRDR of the gyrA and parC genes was carried out using the primers and following the conditions previously described.38,39 PCR was carried out using a DNA thermal cycler 480 (Perkin-Elmer Cetus, Emeryville, CA, USA). Amplified DNA products were resolved by electrophoresis in agarose gels containing 0.5 mg of ethidium bromide per litre. The PCR product was recovered from the agarose gel and purified with the Concert Rapid Purification System according to the manufacturer's instructions (Gibco-BRL, Life Technologies Inc., Gaithersburg, MD, USA). The sample was then directly processed for DNA sequencing using the dRhodamine Terminator Cycle Sequencing kit and was analysed in an automatic DNA sequencer (Abi Prism 377; Perkin-Elmer).
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Results |
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The correlation between the mutations in the gyrA and parC genes and the MIC of ciprofloxacin and clinafloxacin are presented in Table 3. Twenty-two strains had a double mutation, a mutation at the amino acid codon Ser83 in the gyrA gene, which generated a change from Ser to Leu, plus a mutation at the amino acid codon Ser80 of the parC gene, which also produced a substitution to Leu in all of these strains. The range of MICs of ciprofloxacin for these strains was from 32 to 256 mg/L and of clinafloxacin was from 1 to 8 mg/L.
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Discussion |
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The analysis of the mutations in the QRDR of the gyrA and parC genes showed that alterations in Ser83 of GyrA were most frequently found among the strains with a MIC of ciprofloxacin 2 mg/L, whereas a double mutation at the amino acid codon Ser83 of the gyrA gene plus a mutation at the amino acid codon Ser80 of the parC gene was found in strains with a MIC of ciprofloxacin
32 mg/L. These amino acid substitutions were also identified in our previous studies38,39 and by other authors.40 The substitution at amino acid Ser83 of GyrA to Leu was found in strains with a range of MICs of clinafloxacin from 0.12 to 4 mg/L, although the strain having a MIC of 4 mg/L may possess other mechanisms of resistance to clinafloxacin, which may result in this level of MIC. The strains with a double mutation, one in the gyrA gene and another in the parC gene, showed a range of MICs of clinafloxacin from 1 to 8 mg/L. Therefore, clinafloxacin may retain potentially useful clinical activity against strains carrying a single mutation in the gyrA gene, and a second mutation is required for the microorganism to express higher levels of resistance.
All the strains of A. baumannii analysed in this study were able to grow in the presence of 256 mg/L reserpine. Therefore, the effect observed on the MIC of quinolones in the presence of 25 mg/L of this compound was not due to the antibacterial activity of the reserpine.
The effect of reserpine on the MICs of different quinolones is heterogeneous, affecting from only one quinolone (ciprofloxacin or clinafloxacin) to several quinolones at the same time. It is worth mentioning that the majority of reserpine inhibition is observed in clinical isolates having a MIC of ciprofloxacin equal to or lower than 4 mg/L. Therefore, in a strain with a MIC of ciprofloxacin within the range 0.254 mg/L, a decrease in the MIC due to reserpine can clearly be observed. This effect is more difficult to appreciate in a strain already having a MIC of 128 mg/L, since it is not sufficient to decrease the MIC below the previous dilution (64 mg/L). However, the effect of reserpine was also detected among some strains with high levels of resistance to quinolones.
The exact mechanisms of action of reserpine in A. baumannii remain unknown. It could be possible that reserpine affects some hypothetical efflux pumps, as is well established among Gram-positive microorganisms.31 Previous reports have suggested the ability of reserpine to inhibit an efflux pump of Bacteroides fragilis32,33 and Stenotrophomonas maltophilia.34 However, no direct evidence is available to attribute, unequivocally, the effect of reserpine over A. baumannii to reserpine-inhibited efflux pumps. Therefore, further studies to elucidate this question will be carried out.
In summary, clinafloxacin shows a good activity against A. baumannii, being a potential alternative to conventional treatments. Moreover, in combination with reserpine its activity is clearly enhanced, thus further investigations are required to establish the mechanisms of action of reserpine in A. baumannii.
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Acknowledgements |
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Notes |
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References |
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Received 25 June 2001; returned 16 November 2001; accepted 26 November 2001