In vitro antibacterial activity of fluoroquinolones against Porphyromonas gingivalis strains

Sigrun Eick*, Andrea Schmitt, Svea Sachse, Karl-Hermann Schmidt and Wolfgang Pfister

Institute of Medical Microbiology, University Hospital, Semmelweisstrasse 4, D-07740 Jena, Germany

Received 6 April 2004; returned 29 April 2004; revised 1 June 2004; accepted 2 June 2004


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Acknowledgements
 References
 
Objectives: The objective of the study was to evaluate the in vitro activity of ciprofloxacin, gatifloxacin and moxifloxacin against 16 Porphyromonas gingivalis strains.

Methods: MICs of the quinolones were established by Etest and the agar dilution technique. Experiments focused on determination of the spontaneous mutation rate and the induction of resistant strains, using 0.25-fold MIC of antibiotic. Fragments of gyrA and gyrB as well as of parC were sequenced.

Results: Moxifloxacin and gatifloxacin had very low MIC values. Subinhibitory concentrations of the fluoroquinolones rapidly induced mutations. The spontaneous mutation rate was strain- and quinolone-dependent; the lowest rate was encountered after moxifloxacin. The predicted serum concentrations of the quinolones were bactericidal to wild-type strains, but 100 mg/L of each tested quinolone was insufficient to kill a mutant exhibiting moderate resistance. Often the mutants exhibited high resistance to ≥32 mg/L. All these mutants bore a Ser-83->Phe substitution in GyrA.

Conclusions: DNA gyrase is the primary target of fluoroquinolones in P. gingivalis. In terms of the achievable level in the gingival fluid and the MICs, moxifloxacin might prevent the development of resistance and may be an alternative in the antibiotic treatment of P. gingivalis-associated periodontitis.

Keywords: P. gingivalis , periodontitis , GyrA , quinolones


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Acknowledgements
 References
 
Porphyromonas gingivalis, an anaerobic Gram-negative bacterium, is implicated in the pathogenesis of periodontitis.1 Antibiotic treatment is an established recourse in severe cases of periodontitis associated with this species. Commonly used antibiotics are metronidazole, doxycycline and clindamycin.2 Since the newly developed fluoroquinolones gatifloxacin and moxifloxacin show enhanced activity against anaerobes,3 they might be alternative compounds.

The purpose of this study was to gain information about the in vitro activity of two newer quinolones, gatifloxacin and moxifloxacin, in comparison with an older one, ciprofloxacin, against P. gingivalis strains, focusing on induction and selection of resistant strains and the localization of the target of resistance.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial strains and antibiotics

This study included the reference strain P. gingivalis ATCC 33277 and clinical isolates obtained from patients with severe chronic periodontitis.

The following quinolones were tested: ciprofloxacin (Bayer Vital GmbH, Leverkusen, Germany), gatifloxacin (Grünenthal, Stolberg, Germany) and moxifloxacin (Bayer Vital).

Induction and selection of mutants

The Etest (AB BioDisk, Solna, Sweden) was used to determine the MIC. The bacterial strains were cultured on agar plates containing 0.25-fold MIC of antibiotic for up to 50 passages. After 10 passages, the MICs were determined again.

For the determination of the spontaneous mutation rate we used Wilkins–Chalgren agar plates enriched with two-, four-, and eight-fold MIC of each quinolone (determined by the agar dilution technique), respectively. An inoculum of 109 bacteria in total was applied to the plates. After an incubation time of 5 days, the number of cfu was counted.

The susceptibility of each obtained mutant to all three quinolones was determined by Etest. The mutants were frozen and transferred onto Schaedler agar plates without any antibiotic for three passages, after which the MICs were determined again.

Killing of bacteria

Before testing, individual strains were incubated in parallel overnight (18 h) and for 4 days to obtain bacteria in logarithmic and stationary growth phases, respectively. In these assays, the strains ATCC 33277, the clinical isolate J426-1 and its mutant J426-1R2 were tested. The bacteria were placed in tubes containing Wilkins–Chalgren broth enriched with 10% sheep blood and supplemented with antibiotic at 0.25-fold MIC, MIC, two-, four- and eight-fold MIC (determined by the agar dilution technique), as well as the possible serum concentration after usual dosage (ciprofloxacin 2 mg/L, gatifloxacin 3 mg/L, moxifloxacin 2.5 mg/L)3 and 100 mg/L as a concentration achievable in the periodontal pocket if an antibiotic were locally applied. After incubation times of 6 and 24 h, the efficacy of the fluoroquinolone was assessed by determination of viable counts.

To detect any efflux effect, reserpine (10 mg/L) was added to tubes containing 0.25-fold MIC of antibiotic.4 We tested ATCC 33277 and J426-1 strains, including all available mutants. The numbers of cfu were determined after an incubation time of 6 h.

Sequencing of QRDRs of gyrA, gyrB and parC

The sequences of gyrA and gyrB as well as of subunit A of topoisomerase IV (parC) of P. gingivalis were obtained from the database htpp://www.ncbi.nlm.nih.gov (accession numbers: AB055973, AB048190, NC 002950). These sequences were screened for similarities with different QRDRs available in the same database. Similarities were found between the QRDR of Escherichia coli (accession number: AY323806) and regions of gyrA and parC, as well as the QRDR of Haemophilus influenzae (accession number: AJ508044) and a region of gyrB. Primers according to the appropriate regions of gyrA, gyrB and parC had been selected by the program DNASIS: gyrAfwd, 5'-TGATCGTCTCCAGAGCTTTG-3'; gyrArv, 5'-CCTTATCTATGTCCTGAAGC-3'; gyrBfwd, 5'-TGCGAACTCTTCCTTGTCGA-3'; gyrBrv, 5'-TACCATCGGCATAACGATCG-3'; parCfwd, 5'-CCGGATATAGAGTCATCTGT-3'; parCrv, 5'-GAGTTTGGCCTCGATGTAAC-3'.

Using a thermal cycler, amplifications were carried out in 25 µL volumes, each containing 0.5 pmol primer, 0.2 mM deoxynucleoside triphosphates, 1 x reaction buffer with 2.5 mM MgCl2, 1 U of native Taq polymerase (MBI Fermentas) and 2.5 µL of template DNA. The PCR fragments were purified with the Invisorb Gel DNA extraction kit (Invitek, Berlin, Germany). Sequencing was performed using the Big Dye Terminator Sequencing Kit 2.0 and the Genetic Analyser ABI Prism 310 sequence analyser (Applied Biosystems).


    Results and discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Acknowledgements
 References
 
MIC values of the fluoroquinolones against Porphyromonas gingivalis strains and development of resistance

The newer quinolones gatifloxacin (MICs in the range 0.006–0.023 mg/L) and moxifloxacin (MICs in the range 0.006–0.032 mg/L) were more active against P. gingivalis than ciprofloxacin (MICs in the range 0.064–0.25 mg/L). In general, these MIC values determined by Etest were lower in comparison with those found by agar dilution, supporting results of other investigators.5

Exposure of bacteria to low concentrations of fluoroquinolones promoted induction of resistant mutants, at the first determination after 10 passages (nine times after ciprofloxacin, six times after gatifloxacin and five times after moxifloxacin). Except for one strain exposed to ciprofloxacin, rising MICs were found during passages in all other cases. Very often MICs exceeded 32 mg/L (nine times after ciprofloxacin, 13 times after gatifloxacin and seven times after moxifloxacin).

Testing of frequency of spontaneous resistance at two-, four- and eight-fold MIC for strains of P. gingivalis revealed strain-dependent differences. After exposure to ciprofloxacin, the highest mutation rate—up to 10–6 after the two-fold MIC and 1.8 x 10–8 after the eight-fold MIC—was found, followed by gatifloxacin (up to 2 x 10–6 after the two- and 10–8 after the eight-fold MIC) and moxifloxacin (up to 5 x 10–8 after the two- and 1.2 x 10–8 after the eight-fold MIC).

Consequently, especially after a normal dosage of moxifloxacin, the level of the antibiotic should exceed a concentration preventing mutations. Moxifloxacin at subtherapeutic doses may prevent Staphylococcus aureus from developing resistance, whereas gatifloxacin, levofloxacin and ciprofloxacin provided similar effects only at doses that exceeded their usual clinical doses.6

In all obtained mutant cases, rising MIC values to all three quinolones were found. Examples of some strains are shown in Table 1. High resistance (≥32 mg/L) was stable. In contrast, any moderate resistance (e.g. J426-1R2, D5-2-2R2) was not stable and was lost stepwise without antibiotic pressure.


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Table 1. Alterations of amino acid position 83 of GyrA and MICs of fluoroquinolones for bacteria

 
Killing of bacteria and efflux

A difference in the killing activity of gatifloxacin and moxifloxacin was not found for bacteria after pre-incubation times of 18 h and 4 days. The killing efficacy of ciprofloxacin was about one log step lower for the bacteria in the stationary growth phase. The killing experiments underlined fluoroquinolone activity against non-dividing bacteria.7 The three tested quinolones were bactericidal to the two wild-type strains at the concentration corresponding to the serum concentration after 24 h. However, a complete bactericidal effect on a strain exhibiting moderate resistance was not observed, even at a concentration of 100 mg/L, indicating that tested quinolones are completely ineffective. The results for the J426–1 strain and the mutant J426–1R2 in the logarithmic growth phase are presented in Figure 1.



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Figure 1. Bactericidal effect of the fluoroquinolones ciprofloxacin, gatifloxacin and moxifloxacin on P. gingivalis J426–1, including the mutant J426–1R2, after a pre-incubation time of 18 h (MICs determined by agar dilution).

 
An efflux effect could not be demonstrated with any of the variants of ATCC 33277 and J426-1 strains (data not shown). Recently, a resistance-nodulation-cell division family xenobiotic efflux pump was described in a P. gingivalis ATCC 33277 mutant resistant to several drugs.8

Genetic analysis of selected mutants

Table 1 shows the results of the sequence analysis of the QRDR of gyrA. All P. gingivalis mutants with an MIC ≥32 mg/L of a fluoroquinolone underwent Ser-83 ->Phe substitution, but we did not detect any amino acid substitution in gyrB and parC. A strain-dependent diversity at different codon positions was observed, but without any exchange at the amino acid level, in GyrA (Glu-72 and Glu-124), GyrB (Gly-331, Arg-340, Pro-347, Thr-374, Lys-398 and Cys-458) and ParC (Glu-22, Val-29, Ala-33, Ile-55, Glu-57, Gly-66 and Met-78). Position 83 of GyrA is assumed to be similar to other bacteria. Thus, isolates of E. coli strains with ciprofloxacin MICs of 0.125 mg/L had a Ser-83 substitution in GyrA; otherwise strains with MICs ≥8 mg/L had three or four amino acid substitutions in GyrA and ParC, showing a strong correlation between a stepwise accumulation of mutations in gyrA and parC.9 Streptococcus pneumoniae strains with a substitution at Ser-81->Tyr in GyrA developed a resistance only to 2 mg/L.4 In our Ser-83->Phe mutants, often the MICs rose in one step from very low values to high resistance. It can be concluded that gyrase is the primary target of all fluoroquinolones in P. gingivalis.

Resistance to fluoroquinolones might also be associated with a reduction in porins and reduced bacterial accumulation of the drug.10 If these phenomena play a role in the strains with the moderate resistance, this needs further investigation as well as involvement of efflux in development of resistance.

Bearing in mind the achievable level in the gingival fluid and the MICs, a short-term application of moxifloxacin in adequate dosage might prevent the development of resistance and may be an alternative in the antibiotic treatment of P. gingivalis-associated periodontitis.


    Acknowledgements
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Acknowledgements
 References
 
We are grateful to B. Sigusch and B. Noack for sampling subgingival plaque from periodontitis patients.


    Footnotes
 
* Corresponding author. Tel: +49-3641-933587; Fax: +49-3641-933474; Email: sigrun.eick{at}med.uni-jena.de


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Lamont, R. J. & Jenkinson, H. F. (1998). Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis. Review. Microbiology and Molecular Biology Reviews 62, 1244–63.[Abstract/Free Full Text]

2 . Walker, C. & Karpinia, K. (2002). Rationale for use of antibiotics in periodontics. Journal of Periodontology 73, 1267–72.[ISI][Medline]

3 . Dalhoff, A. & Schmitz, F. J. (2003). In vitro antibacterial activity and pharmacodynamics of new quinolones. Review. European Journal of Clinical Microbiology & Infectious Diseases 22, 203–21.[ISI][Medline]

4 . Pestova, E., Millichap, J. J., Noskin, G. A. et al. (2000). Intracellular targets of moxifloxacin: a comparison with other fluoroquinolones. Journal of Antimicrobial Chemotherapy 45, 583–90.[Abstract/Free Full Text]

5 . Andrews, J. M. & Wise, R. (2000). Comparison of the Etest with a conventional agar dilution method evaluating the in vitro activity of moxifloxacin. Journal of Antimicrobial Chemotherapy 45, 257–8.[Free Full Text]

6 . Firsov, A. A., Vostrov, S. N., Lubenko, I. Y. et al. (2003). In vitro pharmacodynamic evaluation of the mutant selection window hypothesis using four fluoroquinolones against Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 47, 1604–13.[Abstract/Free Full Text]

7 . Gradelski, E., Kolek, B., Bonner, D. et al. (2002). Bactericidal mechanism of gatifloxacin compared with other quinolones. Journal of Antimicrobial Chemotherapy 49, 185–8.[Abstract/Free Full Text]

8 . Ikeda, T. & Yoshimura, F. (2002). A resistance-nodulation-cell division family xenobiotic efflux pump in an obligate anaerobe, Porphyromonas gingivalis. Antimicrobial Agents and Chemotherapy 46, 3257–60.[Abstract/Free Full Text]

9 . Quiang, Y. Z., Qin, T., Fu, W. et al. (2002). Use of a rapid mismatch PCR method to detect gyrA and parC mutations in ciprofloxacin–resistant clinical isolates of Escherichia coli. Journal of Antimicrobial Chemotherapy 49, 549–52.[Abstract/Free Full Text]

10 . Hooper, D. C. (2001). Emerging mechanisms of fluoroquinolone resistance. Emerging Infectious Diseases 7, 337–41.[ISI][Medline]





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