1 Institute of Environmental Medicine and Hospital of Epidemiology, University Hospital of Freiburg, Hugstetter Strasse 55, D-79106 Freiburg; 2 Freiburg Centre for Data Analysis and Modelling, University of Freiburg and Institute of Medical Biometry and Medical Informatics, University Hospital of Freiburg, Freiburg, Germany
Received 22 July 2002; returned 8 October 2002; revised 17 October 2002; accepted 22 October 2002
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Abstract |
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Keywords: fluoroquinolone resistance, gyrA, Legionella pneumophila
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Introduction |
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Induction of resistance upon exposure to quinolones has been described for many bacterial species. In contrast to legionellae, most nosocomial pathogens are transmitted horizontally between persons, which facilitates a stepwise accumulation of mutations and increases the MIC to clinical ineffectiveness. However, pathogens can become resistant during the course of a patients therapy. The likelihood of this occurring varies between antibiotics.3 According to anecdotal reports, subjects with legionnaires disease may respond only after a change in antimicrobial therapy.4,5 One possible explanation for this phenomenon could be the loss of susceptibility during antimicrobial treatment. However, susceptibility data were not presented in these case reports.
The objectives of this study were: (i) to determine how effective new fluoroquinolones are at inducing a stable increase in MICsthis was conducted by serial passages on media containing subinhibitory concentrations of the antibiotic; (ii) to demonstrate whether there are differences in the number of passages required for each fluoroquinolone; and (iii) to search for resistance mechanisms, i.e. to investigate the quinolone resistance-determining region (QRDR) of the gyrA gene for amino acid changes.
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Materials and methods |
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Trovafloxacin (Pfizer, Karlsruhe, Germany), moxifloxacin and ciprofloxacin (Bayer Vital, Wuppertal, Germany), levofloxacin (Aventis Pharma, Bad Soden, Germany), clinafloxacin (Gödecke Parke-Davis, Freiburg, Germany) and gentamicin (Merck, Darmstadt, Germany) were laboratory reference standard substances. All agents were prepared according to the manufacturers recommendations.
Bacterial strains
Six clinical isolates of L. pneumophila were obtained from bronchoalveolar lavages of patients with legionnaires disease. The isolates were identified by standard microbiological methods and PCR.6 All belonged to serogroup 1, with the exception of strain no. 5, which belonged to serogoup 8. (Serogroup data were kindly provided by Dr H.-G. Meyer, Institute of Medical Microbiology and Hygiene, University of Mainz, Mainz, Germany.) Selected strains represented five different genotypes and were determined by means of arbitrarily primed PCR (AP-PCR data not shown).7 After five passages on buffered charcoal yeast extract -ketoglutarate (BCYE
) agar (Heipha, Heidelberg, Germany), the isolates were stored in aliquots of BYE
brothglycerol (1:1) at 70°C. BYE
broth contained 0.9% (w/v) yeast extract (Difco, Augsburg, Germany) and 10% (v/v) legionella buffered charcoal yeast extract (BCYE) Supplement SR 110A (Oxoid Unipath, Wesel, Germany) adjusted to pH 6.9 at a final volume of 100 mL. For the performance of the experiments, an aliquot was thawed and grown on BCYE
agar in 5% CO2 at 37°C for 4 days.
MIC determinations
Antimicrobial susceptibilities were determined by microdilution susceptibility testing in BYE broth, as described previously.8,9 Each experiment was performed in duplicate. Bacteria were added, at a final concentration of 5 x 104 cfu per 100 µL, to geometric dilution series of the antimicrobial agent. After incubation of the dilution series for 48 h at 37°C, MICs of bacterial growth were read visually.
Serial passages
Passages were performed three times a week by taking an inoculum from the well with the next concentration below the MIC. Wells of the geometric dilution series with the highest antimicrobial concentrations showing growth (i.e. 1/2 x MIC) contained 27 x 106 cfu. Following a 100-fold dilution of these well contents, bacteria were inoculated into a fresh microbroth dilution series. Serial passages were performed until the MIC of a strain had increased at least eight-fold. Subsequently, resistant strains were passaged ten times on antibiotic-free BCYE agar.
The stability of the resistant phenotype, and the cross-resistance between the fluoroquinolones, were then determined by microdilution susceptibility testing.
Typing by AP-PCR
Three to five colonies from a freshly grown culture were resuspended in 100 µL of TE (10 mM TrisHCl pH 8.0, 0.1 mM EDTA) in a microfuge tube and incubated for 15 min at 95°C. After chilling on ice, bacterial debris was pelleted by centrifugation at 15 000g for 20 s. The supernatant was transferred to a fresh microfuge tube.
The AP-PCR was performed as described previously.7 The M13 primer (5'-TTATGTAAAACGACGGCCAGT-3') used for typing was fluorescently labelled with Cy-5 during manufacture (Amersham Pharmacia Biotech, Germany). PCR was performed in a total volume of 25 µL, with 1 µL of the bacterial lysate being added to a PCR mix comprising 10 mM TrisHCl pH 9.0, 3 mM MgCl2, 50 mM KCl, 0.2 mM dNTPs (Amersham Pharmacia Biotech), 1.25 U Taq DNA polymerase (Boehringer Mannheim). The PCR comprised 45 cycles of 60 s at 94°C, 60 s at 36°C, 120 s at 72°C and a final extension step at 72°C for 2 min. PCR products were detected by analysis of a 1.2 µL portion on an ALF Express DNA Sequencer (Pharmacia) as described previously.10 Fingerprints of the parental and mutant strains were analysed visually in the range 1201000 nucleotides.
Sequencing of the gyrA gene
PCR analysis was performed on crude DNA, prepared as described above. Amplification was achieved with consensus primers for the Legionella gyrA gene p39 [5'-GATGG(C/T)- TTAAAGCC(C/T)GT(G/T)CAT] and p179 rev [5'-CGCCAT(A/C)CCTACAGC(G/T)AT(A/C)CC-3'], which comprised codons 39179, according to the Escherichia coli numbering.11 PCR fragments were isolated by use of High Pure PCR Product Purification Kit (Roche Diagnostics GmbH, Mannheim, Germany). DNA sequencing reactions were performed at MWG (MWG Biotech; Ebersberg, Germany) in forward and reverse directions, using the primers p39 and p179rev. Both strands were sequenced as a cross-check.
Statistical analysis
For analysis of the fluoroquinolones and their different propensities to cause resistance, the outcome number of passages was considered. The number of passages are ordinal data. Since all pairwise comparisons of the five different types of compounds were of interest, the multiple two-way rank procedure by Wilcoxon and Wilcox was used.12 The two-way analysis takes into account that bacteria from the same six strains were studied for each of the five antimicrobials. Comparisons were two-sided and at the 5% level.
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Results |
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After 10 antibiotic-free passages, MICs for the mutant strains remained stably increased by at least two doubling dilutions. Clear cross-resistance between all five antimicrobials was observed.
It was confirmed that all resistant strains were derived from their parental strains. Typing the strains by means of AP-PCR revealed five different genotypes. Strains 4 and 6 belonged to the same genotype, as confirmed by macrorestriction analysis (data not shown). The DNA fingerprints of all mutant strains were indistinguishable from their parental strains.
In a tentative explanation of the resistance mechanisms, the DNA sequences of the gyrA gene were investigated.13 Sequencing of the QRDR disclosed non-silent nucleotide changes in five of six, and half of the strains subcultured in the presence of ciprofloxacin and moxifloxacin, respectively (Table 2). All changes occurred at codon position 83, which uniformly coded for threonine in all six parent strains. Four of the 12 investigated mutant strains did not disclose any gyrA changes.
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Discussion |
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Fluoroquinolones are another promising class of antimicrobial for treatment of pneumonia. Since their introduction, they have been increasingly discussed as an alternative in the therapy of legionnaires disease. These drugs are known to induce drug resistance, which spreads through the community, mainly by horizontal transfer. In the presence of rifampicin and ciprofloxacin, mutation frequencies for Legionella have been reported in the range of 14 x 108 to 108>107.1
The present study compares the propensity of ciprofloxacin and other new fluoroquinolones to induce resistance, and gives a tentative explanation about the mechanism.
Even when a smaller number of bacterial counts are subcultured, employing the microbroth dilution technique, fluoroquinolones are able rapidly to induce a stable, decreased susceptibility within fewer than 10 passages. Since every 100 µL subculture at 1/2 x MIC ended in 27 x 106 cfu, in total <108 cfu had been exposed to subinhibitory concentrations; two orders of magnitude less than can be expected in human infections.1820
In contrast to results in vitro, animal experiments showed no evidence of rifampicin-induced resistance to L. pneumophila;21 however, it should be acknowledged that the bacterial burden can vary in vivo.
The clinical importance of these study findings might be questioned, since legionellae are not transmitted horizontally. Therefore, clinical isolates with diminished susceptibility imply development of resistance de novo. However, case reports in the literature about initial treatment failures show that final cures were achieved after changing the antimicrobial class in the treatment regimen.4,5,22,23 Unfortunately, few of the reports indicated the susceptibility of the strains before and after the initial treatment regimen.22,23 Although this has not been documented for legionnaires disease, that pathogens can become resistant during the course of a patients antimicrobial therapy is unequivocal.3
The five quinolones investigated differed with respect to their propensity to induce resistance in a dichotomous manner. For ciprofloxacin, levofloxacin and clinafloxacin this potential was markedly higher than for moxifloxacin and trovafloxacin. Presumably, the replacement of the (C-8) carbon with nitrogen (trovafloxacin) or the addition of a methoxy substituent to C-8 (moxifloxacin) decreases the probability of emerging resistances.24 The low propensity of moxifloxacin to induce resistant mutants has been well documented for other species.2,25
There are limited published data on the mechanisms of antimicrobial resistance in Legionella.2628 Most investigations address the intracellular localization of replicating strains. Furthermore, antimicrobial susceptibility itself seems to depend on the state of growth.27,28 In this study, the QRDR of the gyrA gene was investigated in the search for primary fluoroquinolone targets and resistance mechanisms in L. pneumophila. For this purpose, mutants were chosen from antimicrobial groups with varying activity, which had been rendered resistant in the presence of ciprofloxacin and moxifloxacin. In numerous Gram-negative bacteria, gyrase A has been demonstrated as the primary target structure of most fluoroquinolones.29 To our knowledge, this is the first study to describe amino acid changes at gyrA codon position 83 of L. pneumophila, as found in other fluoroquinolone-resistant species (for review see ref. 30). This residue is assumed to be involved in the drug binding.31 Mutants such as Thr83Ile have been found in Pseudomonas aeruginosa, Klebsiella oxytoca, Citrobacter freundii and Enterobacter aerogenes.29 There is little doubt that the increased resistance against all five fluoroquinolones is caused by these gyrA mutants, even if a reversion of the resistant phenotype by complementation with the wild-type gene has not been performed. But the fact that four of the 12 mutants, appearing in the presence of ciprofloxacin and moxifloxacin, did not display changes in the QRDR of gyrA is highly indicative of additional target structures and resistance mechanisms in Legionella, such as topoisomerase IV, or multi-drug efflux pumps. Detailed investigation of these mechanisms would have been beyond the scope of this study.
In conclusion, this work confirms that multiple sub-MIC passages of L. pneumophila can produce quinolone antimicrobial resistant mutants. However, the potency to evoke such changes seems to differ markedly in different fluoroquinolones. This is the first study to demonstrate that reduced fluoroquinolone susceptibility in L. pneumophila can be associated with gyrA mutations.
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Acknowledgements |
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Footnotes |
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References |
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