The antibacterial efficacy of trovafloxacin against an experimental infection with Listeria monocytogenes in hydrocortisone-treated mice

Marc L. van Ogtrop{dagger}

Department of Medical Microbiology, Leiden University Medical Centre, PO Box 9600, 2300 RC Leiden, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The efficacy of trovafloxacin in treating Listeria monocytogenes infections in glucocorticosteroid-treated mice was compared with the efficacy of amoxycillin. Swiss mice were treated with daily injections of 2.5 mg hydrocortisone sc and then infected iv with 1 x 10 7cfu of L. monocytogenes. Untreated, this level of infection resulted in 100% mortality between day 3 and day 5 after infection.Both sc trovafloxacin and amoxycillin were effective in reducing the number of viable L. monocytogenes in the liver and spleen. Although the MIC of amoxycillin for this isolate of L. monocytogeneswas lower than that of trovafloxacin (0.063 mg/L versus 0.5 mg/L, respectively), trovafloxacin was more efficacious in vivo after a single dose in the dose range between 12.5 and 100 mg/kg than was amoxycillin. After treatment with trovafloxacin at 100 mg/kg bodyweight od for 3 days, a mean log10cfu of 1.58 and 2.52 L. monocytogenescould be recovered from the spleens and livers, respectively, whereas after treatment with amoxycillin at 100 mg/kg bodyweight every 8 h for 3 days, the mean log10cfu values were 2.36 and 2.02, respectively. These differences were statistically not significant. Results of the present study show that the antibacterial efficacy of trovafloxacin against L. monocytogenes in our animal model is equivalent to that of amoxycillin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Listeria monocytogenes is a facultative intracellular bacterial pathogen that causes severe infections, such as septicaemia and meningitis, which can be life-threatening in immunocompromised patients. The patients most often affected by severe listeriosis are neonates, transplant recipients, patients on glucocorticosteroids and patients with lymphomas. 1 L. monocytogenes is widespread in nature, but humans are only sporadically infected. It is estimated that approximately 1850 cases occur yearly in the USA with an overall mortality of 25%. 2,3 Standard therapy for listeriosis consists of ampicillin, amoxycillin or penicillin, sometimes combined with an aminoglycoside. There are few well-documented alternatives for the treatment of listeriosis. Co-trimoxazole, vancomycin, tetracycline, chloramphenicol and cephalothin have all been suggested as alternatives for amoxycillin, but controlled studies have not been performed. Trovafloxacin is a fluorinated quinolone with in-vitro bactericidal activity against L. monocytogenes; 4 moreover, it easily penetrates phagocytic cells. 5 On theoretical grounds these characteristics make trovafloxacin an interesting alternative therapy for listeriosis.

The aim of the present study was to compare the antimicrobial efficacy of trovafloxacin with that of amoxycillin for the treatment of an experimental infection with L. monocytogenes in mice immunocompromised by treatment with glucocorticosteroids. The design of the study was first to determine the dosage interval for the treatment of this experimental infection using a maximally effective dose of amoxycillin or trovafloxacin, then to determine the maximally effective dose of either drug after a single dose, and finally to determine the antibacterial efficacy of both antimicrobial drugs by giving the maximally effective doses at the determined dosage intervals for a period of 3 days.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotics

Trovafloxacin powder (79.8% potency) was obtained from Pfizer (Groton, CT, USA). Stock solutions were prepared in distilled water and used within 1 h. Amoxycillin (85%) was obtained from SmithKline Beecham (Brockham Park, UK). Stock solutions were prepared in distilled water and used within 1 h.

Animals

Female specific pathogen free Swiss mice (IFFA Credo, l'Arbresle, France) were used throughout the study. The animals were housed in groups of 10 in polycarbonate cages on sterile sawdust; they received acidified tap water and non-sterilized food pellets (type AM-II, Hope Farms, Woerden, The Netherlands) ad libitum. This study was approved by the Committee on Animal Experiments of the State University of Leiden.

Bacteria

An overnight culture of L. monocytogenes (isolate EGD) in tryptone soya broth (TSB) (Oxoid Ltd, Basingstoke, UK) was stored in small aliquots at –70°C. The virulence of this isolate was maintained by repeated passage through mice. Before each experiment, aliquots were rapidly thawed in a water bath at 37°C. The MIC of amoxycillin for this isolate was 0.063 mg/L, and that of trovafloxacin was 0.5 mg/L. The MICs were determined by the agar dilution method on Mueller–Hinton agar (Oxoid), using an inoculum of 10 3–10 4 cfu/spot.

In-vitro growth experiments

A 1:2000 dilution of an overnight culture of L. monocytogenes in TSB was incubated in a shaking water bath at 37°C for 1 h and then distributed in 20 mL aliquots over 50 mL flasks that contained various concentrations of the antimicrobial drugs. Samples were taken at 1 h intervals over a period of 6 h. After appropriate dilutions in phosphate-buffered saline (PBS) plating on blood agar and incubation overnight at 37°C, the cfu were counted. To prevent carry-over of the antimicrobial agents, samples expected to have low counts of viable L. monocytogenes were washed once with ice-cold PBS. The washing procedure was performed as follows: a 200 µL sample was diluted with 1800 µL PBS and centrifuged at 2000g for 10 min at 4°C; the upper nine-tenths of the volume were then removed with a pipette. Recovery of the bacteria with this procedure was 99.8% (S.D. 17.7%).

Effect of amoxycillin and trovafloxacin on the growth of L. monocytogenes in vivo

The experimental infection model has been validated previously. 6 In short, mice were treated od with 2.5 mg hydrocortisone sodium succinate (Upjohn, Ede, The Netherlands) subcutaneously in the nuchal region. One day after the start of hydrocortisone treatment the mice received an iv injection of 1 x 10 7 cfu L. monocytogenes. Twenty-four hours after the injection of L. monocytogenes the antimicrobial treatment was started. The dose range studied for amoxycillin as well as trovafloxacin was 12.5–100 mg/kg. The dosage interval in these studies was either 8 or 24 h. The outgrowth of L. monocytogenes in the livers and spleens of hydrocortisone-treated mice was determined by quantitative culturing.

At specific intervals after the start of infection the mice were killed by cervical dislocation and the livers and spleens were excised, weighed and homogenized in a tissue homogenizer (Ystral, type X-1020, International Laboratorium Apparate GmbH, Dottingen, Germany). For counting of the cfu in the homogenate, samples were processed as described for the in-vitro experiments. The lower limit of detection for this assay was approximately 5 cfu/organ; when no bacteria were recovered from the organs, the number of cfu was arbitrarily set at unity for further calculations.

Pharmacokinetics

After a single dose of trovafloxacin 100 mg/kg the pharmacokinetics in plasma were determined in a group of 20 uninfected mice. At consecutive time points between 15 min and 24 h after administration of the antimicrobial agent some of the mice were killed by exposure to 100% CO2. Blood samples were taken by cardiac puncture with heparinized syringes and centrifuged at 1500g for 10 min at room temperature and the plasma was removed; the drug concentration was measured as described below.

Pharmacokinetic analysis was performed by the Scientist program (MicroMath, Salt Lake City, UT, USA). The data were fitted to a one-compartment model.

Pharmacokinetic experiments with amoxycillin were not performed, because there were previous published data from our institute.7

Drug assays

Trovafloxacin concentrations were determined by means of an agar diffusion method in a microbiological assay with an Escherichia coli isolate as test organism and Nutrient agar (pH 7.4) (Oxoid) as medium. Appropriate two-fold dilutions of the samples were prepared with pooled murine plasma. Standards were prepared in the same way. The detection limit of the assay was 0.1 mg/L.

Statistical analysis

The results of assessment of the quantitative cultures are given as the mean log cfu per organ. Sigmastat software (Jandel, San Rafael, CA, USA) was used for making statistical calculations. One-way analysis of variance was used for statistical comparisons between multiple groups, with the Tukey test used as a follow-up test. A P-level of 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In-vitro growth experiments

The exponential growth rate for L. monocytogenes in TSB, as determined by linear regression analysis, was 0.28/h, which corresponds to a doubling time of 1.07 h. Amoxycillin showed antibacterial activity at concentrations above 0.032 mg/L. The maximum antibacterial effect of amoxycillin on L. monocytogenes was demonstrated at concentrations of 0.063 mg/L and higher, apparently without concentration-dependent activity. At concentrations above 0.063 mg/L the effect was bacteriostatic in the first 6 h of the experiment (Figure 1). After this period some extent of bactericidal activity occurred in the first 24 h of exposure to amoxycillin (results not shown).



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Figure 1. (In-vitro growth of L. monocytogenes in the presence of various concentrations of (a) trovafloxacin and (b) amoxycillin.)

 
Trovafloxacin showed antibacterial activity at concentrations above 0.125 mg/L. Concentrations of trovafloxacin above 0.5 mg/L were bactericidal, apparently with concentration-dependent killing (Figure 1).

Effect of a single dose of amoxycillin or trovafloxacin on the growth of L. monocytogenes in vivo

For both amoxycillin and trovafloxacin, initial experiments were carried out using a dosage of 100 mg/kg. This dosage was chosen, because for trovafloxacin the limit of solubility in water was approximately 10 mg/mL. After administration of a single sc dose of amoxycillin 100 mg/kg the number of L. monocytogenes in the liver and spleen initially declined, but regrowth of L. monocytogenes occurred after more than 8 h (Figure 2). For a single sc dose of trovafloxacin 100 mg/kg bactericidal activity was shown for a period of 24 h (Figure 2). Therefore an 8 h dosage interval was considered to be the optimum interval for amoxycillin, whereas this period was 24 h for trovafloxacin.



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Figure 2. (Effect of a single sc dose of amoxycillin 100 mg/kg ({blacktriangleup}), trovafloxacin 100 mg/kg (•) or water ({blacksquare}) on the number of cfu of L. monocytogenes in the livers and spleens of mice treated with hydrocortisone. The mice were infected by iv injection of 1 x 107 cfu L. monocytogenes 24 h before administration of the antimicrobial agent. The mice were killed at various time points after administration of the antimicrobial agent. Each symbol in the figure represents the mean log cfu for four mice. The error bars represent the standard deviation.)

 
To determine the dose–effect relationship for amoxycillin and trovafloxacin, the drugs were administered sc to hydrocortisone-treated mice 24 h after an iv injection of 1 x 10 7 cfu of L. monocytogenes. The mice were killed 24 h after administration of the antimicrobial agent. Treatment with amoxycillin caused a significant decrease in the number of L. monocytogenes in the livers and spleens, with an apparent dose-dependent effect in the dose range between 12.5 and 100 mg/kg (Figure 3). The mean log number of L. monocytogenes in the livers of mice treated with amoxycillin 100 mg/kg 24 h after administration of the antimicrobial agent was 5.18 (S.D. 0.57) compared with 6.97 (S.D. 0.57) for control mice; for the spleen it was 6.41 (S.D. 0.35) compared with 7.66 (S.D. 0.48). For amoxycillin-treated mice, the liver and spleen counts of mice treated with doses of 25 mg/kg or higher were significantly different from those of control mice (P< 0.05). The same was true for the difference in liver counts between mice treated with amoxycillin 12.5 mg/kg and 100 mg/kg, and for the spleen counts between mice treated with amoxycillin 12.5 mg/kg and 50 mg/kg.



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Figure 3. (Effect of various sc doses of amoxycillin ({blacktriangleup}) or trovafloxacin (•) on the number of cfu of L. monocytogenes in the livers and spleens of mice treated with hydrocortisone. The mice were infected by iv injection of 1 x 107 cfu L. monocytogenes 24 h before administration of the antimicrobial agent. The mice were killed 24 h after administration of the antimicrobial agent. Each symbol in the figure represents the mean log cfu for six mice. The error bars represent the standard deviation.)

 
Treatment with trovafloxacin 12.5–100 mg/kg also had an apparent dose-dependent effect (Figure 3). Twenty-four hours after the administration of trovafloxacin 100 mg/kg the mean log number of L. monocytogenes in the livers was 4.54 (S.D. 0.61) compared with 6.97 (S.D. 0.57) for the control mice; for the spleens it was 5.05 (S.D. 0.87) compared with 7.66 (S.D. 0.48).

For trovafloxacin-treated mice, the liver and spleen counts of mice treated with doses of 25 mg/kg or higher were significantly different from those of control mice (P< 0.05). The same was true for the difference in liver and spleen counts between mice treated with trovafloxacin 12.5 mg/kg and 50 or 100 mg/kg.

Comparing equivalent doses between amoxycillin- and trovafloxacin-treated mice, there was no statistically significant difference for the liver counts, but the difference was statistically significant for the spleen counts at doses of 25, 50 and 100 mg/kg (P< 0.05).

Effect of 3 days of amoxycillin or trovafloxacin therapy on the growth of L. monocytogenes in vivo

Treatment with the antibiotics for a prolonged period led to a large reduction in the number of L. monocytogenes in the livers and spleens. Treatment with amoxycillin 100 mg/kg tds resulted in a significant decrease (P< 0.001) in the number of L. monocytogenes after 1 day of treatment (Figure 4). Thereafter the numbers of L. monocytogenes declined to a mean log cfu of 2.02 (S.D. 0.70) in the livers and 2.36 (S.D. 1.18) in the spleens after 3 days. Treatment with trovafloxacin 100 mg/kg od also led to a significant decrease (P< 0.001) in the number of L. monocytogenes in the livers and spleens. After 3 days of treatment the mean log number of L. monocytogenes from the livers was 2.52 (S.D. 0.74), and that from the spleens was 1.58 (S.D. 1.09). The difference in numbers between amoxycillin- and trovafloxacin-treated mice at day 3 was not statistically significant for the spleens (P= 0.22) or for the livers (P= 0.26).



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Figure 4. (Effect of sc amoxycillin 100 mg/kg ({blacktriangleup}) every 8 h or trovafloxacin 100 mg/kg (•) every 24 h on the number of cfu of L. monocytogenes in the livers and spleens of mice treated with hydrocortisone. The mice were infected by iv injection of 1 x 107 cfu L. monocytogenes 24 h before the first dose of the antimicrobial agent. {blacksquare}, Control mice. Each symbol in the figure represents the mean log cfu for six mice. The error bars represent the standard deviation.)

 
Pharmacokinetic studies

The pharmacokinetic data on an sc dose of trovafloxacin 100 mg/kg in the plasma of mice are shown in Figure 5. Trovafloxacin reached a peak concentration of 4.5 mg/L in plasma at 4 h after administration. The apparent elimination half-life was 4.2 h. The AUC0–24 hwas 72 mg h/L.



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Figure 5. (Pharmacokinetics of a single sc dose of trovafloxacin 100 mg/kg in plasma. Each point in the curve represents the measured value from a single mouse.)

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study shows that amoxycillin and trovafloxacin exhibit antibacterial activity in an experimental Listeria spp. infection in mice immunocompromised by treatment with glucocorticosteroids, and that the antibacterial efficacy of trovafloxacin did not differ significantly from that of amoxycillin. The antimicrobial efficacy of trovafloxacin can be explained by the finding that trovafloxacin was bactericidal against this isolate of L. monocytogenes in vitro, and that trovafloxacin has a long plasma half-life. Amoxycillin, on the other hand, has a half-life of approximately 0.40 h in Swiss mice, with a peak concentration of 11.9 mg/L after an sc dosage of 10 mg/kg. 7 Other researchers have found a relatively lower Cmax of 67.0 mg/L at a dosage of 100 mg/kg, but with a comparable elimination half-life of 0.33 h. 8 The pharmacokinetic data explain why for amoxycillin three times daily dosing was optimal, whereas for trovafloxacin od sufficed.

Using the pharmacokinetic data, we can calculate that a single dose of trovafloxacin 100 mg/kg reaches an AUC/MIC ratio of 142. This pharmacokinetic/pharmacodynamic parameter should be considered adequate for treatment of infections, as for quinolones an AUC/MIC ratio of >125 is generally correlated with clinically successful treatment of infections. 9 It can also be calculated that amoxycillin 100 mg/kg tds leads to 54% of the time above the MIC. This is also more than the required 40% of the time above the MIC that is needed for clinical success. 10 Therefore, both antibiotics were administered in doses that reached efficacious concentrations, provided that the data that have been generated for mainly extracellular bacteria also apply for L. monocytogenes.

The value of quinolones in treating listeriosis is questionable. In a previous study comparing ciprofloxacin and ampicillin, ampicillin showed superior efficacy compared with ciprofloxacin, 6 but other researchers found that newer quinolones showed some promise in treating experimental listeriosis. 10,11,12

The pharmacokinetics of trovafloxacin in mice are remarkably similar to those in humans. The plasma elimination half-life in humans is 7.8 h, 13 and the maximum concentration after an oral dose of 200 mg is 2.9 mg/L. In humans, approximately 5% of unchanged trovafloxacin is excreted in the urine. The urinary clearance of trovafloxacin in mice is not known, but the relatively long elimination half-life in mice points to a lack of renal elimination. Considering the similarity of the pharmacokinetics of trovafloxacin in mice and humans, murine infection models are well suited to study the antimicrobial efficacy of this drug. This is in strong contrast to most ß-lactam antibiotics, which have a much shorter half-life in mice.

Despite the different in-vitro behaviour of amoxycillin and trovafloxacin, the antibacterial efficacy in our model of listeriosis was similar. It is especially noteworthy that trovafloxacin was bactericidal, whereas amoxycillin exhibited only delayed bactericidal activity. Furthermore, trovafloxacin accumulates well intracellularly 5 and should therefore theoretically be more efficacious against facultative intracellular bacteria than ß-lactam antibiotics, which accumulate more slowly and to a lesser extent. 14 The results of the present study do not lend support to the theory that rapid intracellular accumulation of antibiotics contributes significantly to the antibacterial efficacy against infections by facultative intracellular bacteria.


    Acknowledgments
 
This work was supported by a grant from Pfizer Pharmaceuticals, The Netherlands. The author thanks W. A. Craig for a critical reading of the manuscript. Part of this work was presented at the Thirty-Seventh International Conference on Antimicrobial Agents and Chemotherapy, Toronto, 1997.


    Notes
 
{dagger} Tel: +31-71-526-3585. Fax: +31-71-524-8148 Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Lorber, B. (1997). Listeriosis. Clinical Infectious Diseases 24, 1–11.[ISI][Medline]

2 . Ciesielski, C. A., Hightower, A. W., Parsons, S. K. & Broome, C. V. (1988). Listeriosis in the United States: 1980–1982. Archives of Internal Medicine 148, 1416–9.[Abstract]

3 . Gelling, B. G., Broome, C. V., Bibb, W. F., Weaver, R. E., Gaventa, S. & Mascola, L. (1991). The epidemiology of listeriosis in the United States—1986. Listeriosis Study Group. American Journal of Epidemiology 133, 392–401.[Abstract]

4 . Neu, H. C. & Chin, N.-X. (1994). In vitro activity of the new fluoroquinolone CP-99,219. Antimicrobial Agents and Chemotherapy 38, 2615–22.[Abstract]

5 . Edelstein, P. H., Edelstein, M. A., Ren, J., Polzer, R. & Gladue, R. P. (1996). Activity of trovafloxacin (CP-99,219) against Legionella isolates: in vitro activity, intracellular accumulation and killing in macrophages, and pharmacokinetics and treatment of guinea pigs with L. pneumophila pneumonia. Antimicrobial Agents and Chemotherapy 40,314 –9.[Abstract]

6 . Van Ogtrop M. L., Mattie, H., Sekh, B. R., van Strijen, E. & van Furth, R. (1992). Comparison of the antibacterial efficacies of ampicillin and ciprofloxacin against experimental infections with Listeria monocytogenes in hydrocortisone-treated mice. Antimicrobial Agents and Chemotherapy 36, 2375–80.[Abstract]

7 . Mattie, H. & van der Voet, G. B. (1981). The relative potency of amoxycillin and ampicillin in vitro and in vivo. Scandinavian Journal of Infectious Diseases 13, 291–6.[ISI][Medline]

8 . Darras-Joly, C., Bédos, J.-P., Sauve, C., Moine, P., Vallée, E., Carbon, C. et al. (1996). Synergy between amoxicillin and gentamicin in combination against a highly penicillin-resistant and -tolerant strain of Streptococcus pneumoniae in a mouse pneumonia model. Antimicrobial Agents and Chemotherapy 40, 2147–51.[Abstract]

9 . Craig, W. A. (1998). Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clinical Infectious Diseases 26, 1–12.[ISI][Medline]

10 . Hof, H. (1990). Treatment of experimental listeriosis by CI 934, a new quinolone. Journal of Antimicrobial Chemotherapy 25, 121–6.[Abstract]

11 . Nichterlein, T., Kretschmar, M., Budeanu, C., Bauer, J., Linss, W. & Hof, H. (1994). Bay Y 3118, a new quinolone derivative, rapidly eradicates Listeria monocytogenes from infected mice and L929 cells. Antimicrobial Agents and Chemotherapy 38, 1501–6.[Abstract]

12 . Michelet, C., Avril, J. L., Arvieux, C., Jacquelinet, C., Vu, N. & Cartier, F. (1997). Comparative activities of new fluoroquinolones, alone or in combination with amoxicillin, trimethoprim–sulfamethoxazole, or rifampin, against intracellular Listeria monocytogenes. Antimicrobial Agents and Chemotherapy 41, 60–5.[Abstract]

13 . Wise, R., Mortiboy, D., Child, J. & Andrews, J. M. (1996). Pharmacokinetics and penetration into inflammatory fluid of trovafloxacin (CP-99,219). Antimicrobial Agents and Chemotherapy 40, 47–9.[Abstract]

14 . Gerding, D. N., Hughes, C. E., Bamberger, D. M., Foxworth, J. & Larson, T. A. (1996). Extravascular antimicrobial distribution and the respective blood concentrations in humans. In Antibiotics in Laboratory Medicine, 4th edn (Lorian, V., Ed.), pp. 835–99. Williams and Wilkins, Baltimore, MD.

Received 30 October 1998; returned 10 February 1999; revised 5 March 1999; accepted 12 April 1999





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