Evaluation of azithromycin, trovafloxacin and grepafloxacin as prophylaxis for experimental murine melioidosis Dermot J. Kenny, Armine M. Sefton, Timothy J.G. Brooks, Thomas R. Laws, Andrew J.H. Simpson, Helen S. Atkins
To cite this version:
Dermot J. Kenny, Armine M. Sefton, Timothy J.G. Brooks, Thomas R. Laws, Andrew J.H. Simpson, et al.. Evaluation of azithromycin, trovafloxacin and grepafloxacin as prophylaxis for experimental murine melioidosis. International Journal of Antimicrobial Agents, Elsevier, 2010, 36 (1), pp.87. 10.1016/j.ijantimicag.2010.03.019. hal-00594507
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Title: Evaluation of azithromycin, trovafloxacin and grepafloxacin as prophylaxis for experimental murine melioidosis
Authors: Dermot J. Kenny, Armine M. Sefton, Timothy J.G. Brooks, Thomas R. Laws, Andrew J.H. Simpson, Helen S. Atkins
PII: S0924-8579(10)00152-4 DOI: doi:10.1016/j.ijantimicag.2010.03.019 Reference: ANTAGE 3292
To appear in: International Journal of Antimicrobial Agents
Received date: 17-11-2009 Revised date: 9-3-2010 Accepted date: 11-3-2010
Please cite this article as: Kenny DJ, Sefton AM, Brooks TJG, Laws TR, Simpson AJH, Atkins HS, Evaluation of azithromycin, trovafloxacin and grepafloxacin as prophylaxis for experimental murine melioidosis, International Journal of Antimicrobial Agents (2008), doi:10.1016/j.ijantimicag.2010.03.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Evaluation of azithromycin, trovafloxacin and grepafloxacin as prophylaxis for experimental murine melioidosis
Dermot J. Kenny a, Armine M. Sefton b, Timothy J.G. Brooks a,1, Thomas R.
Laws a, Andrew J.H. Simpson a, Helen S. Atkins a,*
a Department of Biomedical Sciences, Defence Science and Technology
Laboratory, Porton Down, Salisbury SP4 0JQ, UK b Centre for Infectious Disease, Barts and the London, Queen Mary’s School of Medicine and Dentistry, Whitechapel, London E1 2AD, UK
ARTICLE INFO
Article history:
Received 17 November 2009
Accepted 11 March 2010
Keywords:
Burkholderia
Macrolide FluoroquinoloneAccepted Manuscript Mouse
* Corresponding author. Tel.: +44 1980 614 755; fax: +44 1980 614 307.
E-mail address: [email protected] (H.S. Atkins).
1 Page 1 of 14 1 Present address: Health Protection Agency Centre for Emergency
Preparedness & Response, Porton Down, Salisbury SP4 0JG, UK.
Crown Copyright 2010. Published with the permission of the Defence
Science and Technology Laboratory on behalf of the Controller of HMSO.
Accepted Manuscript
2 Page 2 of 14 ABSTRACT
The efficacies of the azalide azithromycin and the fluoroquinolones trovafloxacin and grepafloxacin for pre- and post-exposure prophylaxis of infection with high or low challenge doses of Burkholderia pseudomallei strain
576 were assessed in an experimental mouse model. Trovafloxacin and grepafloxacin afforded significant levels of protection, whereas azithromycin was ineffective and potentially detrimental. Overall, the data suggest that some fluoroquinolones may have potential utility in prophylaxis of melioidosis and suggest that azithromycin would not be effective in prophylaxis of B. pseudomallei infection.
Accepted Manuscript
3 Page 3 of 14 1. Introduction
Burkholderia pseudomallei is the causative agent of melioidosis, a disease that is endemic in Southeast Asia, Northern Australia, and parts of Africa and
South and Central America. Whilst generally considered a relatively rare disease, melioidosis is being diagnosed with increasing frequency, probably as a result of greater awareness and increased global travel. Burkholderia pseudomallei is an incidental pathogen in those who come into contact with it in the environment. Melioidosis can present acutely as septicaemia or pulmonary infection, or as a chronic disease that is difficult to treat with relapses over many years.
Currently there is no licensed vaccine for melioidosis. Septicaemia is usually rapidly fatal unless promptly treated, which reduces mortality to ca. 50%. The therapeutic regimen of choice is either ceftazidime or a carbapenem given parentally for 10–14 days, followed by oral treatment with co-trimoxazole, either alone or in combination with doxycycline [1]. However, the intrinsic resistance of B. pseudomallei towards many antibiotics means that it is important to find novel effective therapeutics.
BurkholderiaAccepted pseudomallei is known to cause Manuscript latent infection that is believed to be associated with the ability of the pathogen to evade host immune responses within host tissues. Moreover, the pathogen can survive within macrophages. Thus, drugs that are active intracellularly are more likely to eradicate infection and prevent relapses of melioidosis. The azalide azithromycin concentrates inside macrophages at high levels and is effective 4 Page 4 of 14 as treatment for intracellular pathogens such as Salmonella spp. even when the minimum inhibitory concentrations (MICs) are relatively high [2].
Additionally, the fluoroquinolones are active against Burkholderia spp. in vitro
[3] and show good intracellular penetration, suggesting that they may also be effective against the pathogen. Clinical experience with the early fluoroquinolone ciprofloxacin was disappointing and previous work performed in our laboratory suggested that it was less effective against B. pseudomallei than doxycycline [4]. However, the pharmacokinetic profiles of the newer fluoroquinolones, trovafloxacin and grepafloxacin, are improved.
Unfortunately, grepafloxacin has been withdrawn from the market, whilst use of trovafloxacin has been limited due to safety concerns [5]. However, prior to their withdrawal, we initiated studies to evaluate the potential utility of trovafloxacin and grepafloxacin, as well as azithromycin, as prophylaxis or therapy against experimental melioidosis.
2. Materials and methods
2.1. Bacteria
Ten B. pseudomallei strains stored on Protect beads (TSC Ltd., Heywood, UK) at –80AcceptedC in the Defence Science and ManuscriptTechnology Laboratory (Salisbury, UK) culture collection were used in a standard Clinical and Laboratory
Standards Institute microdilution method to determine MICs of the antibiotics.
Bacteria were cultured in 10 mL of nutrient broth (Oxoid, Basingstoke, UK) at
37 C overnight. Burkholderia pseudomallei strain 576 was used for challenge studies.
5 Page 5 of 14 2.2. Antibiotics
Antibiotic solutions were prepared freshly each day. Azithromycin (Pfizer,
Sandwich, UK) was prepared by dissolving in a minimal volume of 98% ethanol (Sigma, Poole, UK) and subsequently dissolving in 0.5% methylcellulose (Sigma) in distilled water to achieve a dose of 40 mg/kg in a volume of 50 L per mouse as previously described [6,7]. Trovafloxacin
(Pfizer) and grepafloxacin (GlaxoSmithKline, Stevenage, UK) were dissolved in 0.5% methylcellulose in distilled water to achieve a dose of 40 mg/kg in a volume of 20 L per mouse as previously described [7].
To determine the efficacy of the antibiotics against B. pseudomallei in vivo, groups of 24 mice were orally administered once daily with 50 L of azithromycin solution, 20 L of trovafloxacin solution or 20 L of grepafloxacin solution. Further groups of five to six control mice were administered 0.5% methylcellulose diluent for 7 days. Two different prophylaxis regimens were studied. Antibiotic treatment was initiated either at 1 h prior to challenge (as pre-exposure prophylaxis) and continued for 7 days, or at 6 h post exposure and continued for 14 days (post-exposure prophylaxis). Accepted Manuscript
2.3. Animals
Antibiotic efficacy was evaluated in female 6–7-week-old BALB/c mice
(obtained from Charles River Laboratories, UK). All experimental work was conducted in flexible film isolators according to a UK Home Office licence. All
6 Page 6 of 14 animal studies were carried out in accordance with the Animals Scientific
Procedures Act 1986 and the Codes of Practice for the Housing and Care of
Animals used in Scientific Procedures 1989. Mice were challenged with either a low dose [10–4 dilution of a log-phase culture, ca. 104 colony-forming units
(CFU)/mL] or a high dose (10–1 dilution of a log-phase culture, ca. 107
CFU/mL) of B. pseudomallei strain 576 by subcutaneous injection (100 L) and antibiotic prophylaxis was evaluated using the dosing regimens described above. Following the last antibiotic treatment, surviving mice were observed for a further 47 days for relapse. At this point the experiment was terminated.
A single experiment at each challenge dose was performed in this study.
2.4. Analysis
Kaplan–Meier plots were generated using Microsoft Excel (Microsoft Corp.,
Redmond, WA). Survival was compared using Mantel–Haenszel log-rank tests. Bonferroni correction was used where multiple comparisons were made.
3. Results and discussion Trovafloxacin,Accepted grepafloxacin and azithromycin Manuscript each showed activity against B. pseudomallei in vitro. MIC ranges for the B. pseudomallei strains were 1
g/mL to >64 g/mL for azithromycin (reported previously [8]), 0.5–8 g/mL for trovafloxacin and 1–8 g/mL for grepafloxacin. These are consistent with previously reported fluoroquinolone MIC results [3].
7 Page 7 of 14 Two experiments were performed to evaluate pre-exposure and post- exposure prophylaxis of B. pseudomallei infection with the antibiotics.
Trovafloxacin, administered either 1 h prior to exposure or 6 h after exposure to a low B. pseudomallei challenge dose, enabled 100% survival compared with 50% of controls (P < 0.001) (Fig. 1A). Impressively, pre- or post-exposure prophylaxis with trovafloxacin following a high-dose B. pseudomallei challenge also provided a high and statistically significant enhancement of survival (P < 0.001) (Fig. 1B), although the ability of this antibiotic to eradicate infection completely has not been determined in this study. Grepafloxacin prophylaxis was also highly effective against a low challenge dose of B. pseudomallei (P < 0.05 both for pre- and post-challenge treatment groups).
Grepafloxacin was less effective against a high challenge, where it provided excellent protection when used as a post-exposure therapeutic (P < 0.001) but did not significantly protect when used as a pre-treatment (P > 0.05). This is a potentially interesting observation and may suggest that delaying some antibiotic treatments may actually enhance bacterial clearance. Further study would be required to determine the significance of this finding.
In comparison with the two other therapies, azithromycin afforded no protection against B. pseudomallei in these experiments (P > 0.05 in all combinationsAccepted of data) (Fig. 1). In fact, these Manuscript data suggest that azithromycin may even be detrimental and exacerbate disease, as survival was lower than controls in both treatment groups, although further experiments would be needed to consolidate this observation. If so, it is possible that this effect may be due to the immunomodulatory role attributed to this azalide [9,10].
8 Page 8 of 14 Azithromycin has been used for eradication therapy in melioidosis in a single clinical trial, in combination with ciprofloxacin. Interestingly, treatment failure and relapse were both significantly higher with this regimen than with a combination of co-trimoxazole and doxycycline [11].
We have previously found the fluoroquinolones ciprofloxacin, moxifloxacin and gatifloxacin to be relatively ineffective as prophylaxis for B. pseudomallei infection [4,12]. However, these data indicate that trovafloxacin in particular has potential utility for therapy of melioidosis. Although this particular antibiotic may be inappropriate for use owing to safety concerns, this does suggest that further assessment of other new fluoroquinolones is still indicated.
Acknowledgments
The authors gratefully acknowledge technical assistance provided by Sarah
Woodger and the animal technicians at the Defence Science and Technology
Laboratory (Salisbury, UK).
Funding This workAccepted was funded by the UK Ministry ofManuscript Defence.
Conflicts of interest
None declared.
Ethical approval
9 Page 9 of 14 All experimental work was conducted according to a UK Home Office licence.
All animal studies were carried out in accordance with the Animals Scientific
Procedures Act 1986 and the Codes of Practice for the Housing and Care of
Animals used in Scientific Procedures 1989.
Accepted Manuscript
10 Page 10 of 14 References
[1] Cheng AC, McBryde ES, Wuthiekanun V, Chierakul W, Amornchai P, Day
NPJ, et al. Dosing regimens of cotrimoxazole (trimethoprim–
sulfamethoxazole) for melioidosis. Antimicrob Agents Chemother
2009;53:4193–9.
[2] Metchock B. In vitro activity of azithromycin compared with other
macrolides and oral antibiotics against Salmonella typhi. J Antimicrob
Chemother 1990;24:29–31.
[3] Ho PL, Cheung TKM, Kinoshita R, Tse CWS, Yuen KY, Chau PY. Activity
of five fluoroquinolones against 71 isolates of Burkholderia pseudomallei.
J Antimicrob Chemother 2002;49:1042–4.
[4] Russell P, Eley SM, Ellis J, Green M, Bell DL, Kenny DJ, et al.
Comparison of efficacy of ciprofloxacin and doxycycline against
experimental melioidosis and glanders. J Antimicrob Chemother
2000;45:813–8.
[5] Mandell L, Tillotson G. Safety of fluoroquinolones: an update. Can J Infect
Dis 2002;13:54–61.
[6] Atkins HS, Spencer S, Brew SD, Jenner DC, Sefton AM, MacMillan AP, et
al. Evaluation of azithromycin, trovafloxacin and grepafloxacin as prophylaxisAccepted against experimental murine Manuscript Brucella melitensis infection. Int J Antimicrob Agents 2010 Forthcoming.
doi:10.1016/j.ijantimicag.2009.10.003
[7] Kenny DJ, Sefton AM, Steward J, Brooks TJG, Simpson AJH, Atkins HS.
Efficacy of the quinolones trovafloxacin and grepafloxacin for therapy of
11 Page 11 of 14 experimental tularaemia and plague. Int J Antimicrob Agents
2009;34:502–3.
[8] Kenny DJ, Russell P, Rogers D, Eley SM, Titball RW. In vitro
susceptibilities of Burkholderia mallei in comparison to those of other
pathogenic Burkholderia spp. Antimicrob Agents Chemother
1999;43:2773–5.
[9] Fernandez AD, Elmore MK, Metzger DW. Azithromycin modulates murine
immune responses to pneumococcal conjugate vaccine and inhibits nasal
clearance of bacteria. J Infect Dis 2004;190:1762–6.
[10] Tsai WC, Rodriguez ML, Young KS, Deng JC, Thannickal VJ, Tateda
K, et al. Azithromycin blocks neutrophil recruitment in Pseudomonas
endobronchial infection. Am J Respir Crit Care Med 2004;170:1331–9.
[11] Chetchotisakd P, Chaowagul W, Mootsikapun P, Budhsarawong D,
Thinkamrop B. Maintenance therapy of melioidosis with ciprofloxacin plus
azithromycin compared with cotrimoxazole plus doxycycline. Am J Trop
Med Hyg 2001:64:24–7.
[12] Steward J, Piercy T, Lever MS, Nelson M, Simpson AJH, Brooks TJG.
Comparison of gatifloxacin, moxifloxacin and ciprofloxacin for treatment of
experimental Burkholderia pseudomallei infection. J Antimicrob ChemotherAccepted 2005;55:523–7. Manuscript
12 Page 12 of 14 Fig. 1. Protective efficacy of azithromycin, grepafloxacin and trovafloxacin against Burkholderia pseudomallei infection in a mouse model. Groups of 24
BALB/c mice were challenged with (A) low or (B) high challenge doses of B. pseudomallei 576 and were treated orally with azithromycin (■, ), grepafloxacin (▲, ) or trovafloxacin (, ○). Antibiotics were administered as pre-exposure prophylaxis (starting at 1 h prior to challenge, once daily for 7 days; solid symbols) or as post-exposure prophylaxis (starting at 6 h post- challenge, once daily for 14 days; open symbols). Groups of 5–6 control animals () were treated with antibiotic diluent for 7 days.
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