Comparison of efficacy of ciprofloxacin and doxycycline against experimental melioidosis and glanders

P. Russell*, S. M. Eley, J. Ellis, M. Green, D. L. Bell, D. J. Kenny and R. W. Titball

CBD Porton Down, Salisbury, Wiltshire SP4 0JQ, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Melioidosis and glanders are caused by the closely related species Burkholderia pseudomallei and Burkholderia mallei, respectively. Whereas melioidosis is a significant cause of morbidity in south-east Asia, glanders is extremely rare. The efficacies of ciprofloxacin and doxycycline were assessed against a strain of B. pseudomallei and a strain of B. mallei which were susceptible to both antimicrobials in vitro. Porton outbred mice and Syrian hamsters were given 40 mg/kg of either doxycycline or ciprofloxacin twice daily by sc injection according to one of three regimens: dosing starting 48 h before challenge and continuing for 5 days postchallenge; 5 days' therapy starting immediately after challenge; 5 days' therapy starting 24 h after challenge. Mice were challenged ip with B. pseudomallei 4845 and hamsters were challenged ip with B. mallei 23344. Antimicrobial efficacy was determined by the shift in the median lethal dose (MLD). Ciprofloxacin prophylaxis and immediate therapy both raised the MLD of B. pseudomallei to 4 x 106 cfu from 19 cfu in untreated animals, but therapeutic ciprofloxacin only raised the MLD to 180 cfu. The results for doxycycline were similar. Ciprofloxacin prophylaxis raised the MLD of B. mallei 23344 to 4.6 x 105 cfu compared with 4 cfu in untreated controls. Immediate therapy raised the MLD to 7.0 x 104 cfu and therapy raised the MLD to 1.6 x 103 cfu. All regimens of doxycycline protected hamsters against challenges of up to 2 x 107 cfu. Despite using a susceptible strain of B. pseudomallei, neither antimicrobial was effective when used therapeutically. The timely administration of either antimicrobial, however, was effective in preventing symptomatic infection. Doxycycline was the superior of the two antimicrobials against experimental glanders although relapse did occur in treated animals approximately 4–5 weeks after challenge.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Melioidosis and glanders are caused by the closely related species Burkholderia pseudomallei and Burkholderia mallei. Whereas melioidosis is an important cause of mortality and morbidity in south-east Asia, northern Australia and the Indian sub-continent, glanders is extremely rare and is generally confined to equine disease in some parts of the Middle East, Asia and South America.1,2

As a consequence, the antimicrobial susceptibilities of B. pseudomallei have been scrutinized35 and the antimicrobial therapy of melioidosis is well established.5,6 The use of fluoroquinolones for treating melioidosis has generally been precluded because of high in vitro MICs for some strains of B. pseudomallei, which exceed levels that can be achieved in serum.4,5 Ciprofloxacin, either alone or in combination, has been used in the treatment of melioidosis despite this contra-indication7,8 on the basis that ciprofloxacin can penetrate phagocytic cells where B. pseudomallei resides, to achieve concentrations 4–12 times the extracellular concentration.9,10 Thus, with serum levels of 2–3 mg/L achievable by standard oral dosing, theoretically, intracellular concentrations of up to 20 mg/L should be attained.11 Furthermore, serum levels of 9 mg/L can be achieved by iv infusion, albeit for short periods.11 A second consideration is that, perhaps fluoroquinolones may be useful for immediate therapy or as prophylaxis for individuals known to have been exposed, or at high risk of exposure, to melioidosis, particularly as there is no current means of immunoprophylaxis.

The scarcity of glanders in the latter half of this century means there is scanty knowledge of the antimicrobial susceptibility of B. mallei, particularly to modern antimicrobials, with the few references in recent microbiology literature principally from Russian sources.1218 Likewise, the efficacy in vivo of modern antimicrobials is not well known. Recent studies suggest that the susceptibility of B. mallei to ciprofloxacin is similar to that of B. pseudomallei in vitro.19 Additionally, B. pseudomallei and B. mallei are very similar with respect to their antigenicity, biochemistry and, very likely, in their pathogenicity, particularly with respect to intracellular survival.20 The arguments promoting the use of ciprofloxacin for melioidosis would therefore apply to glanders but, at the same time, its poor performance in clinical studies should also be considered.

Doxycycline is used alone in the treatment of localized melioidosis, and in combination with other antimicrobials5 for systemic disease, thus it may have some utility as a prophylactic or immediate treatment for melioidosis. Additionally, like ciprofloxacin it can penetrate intracellularly and is effective against a wide variety of intracellular pathogens.21 In previous experiments doxycycline showed good in vitro activity against B. mallei.19

The efficacies of ciprofloxacin and doxycycline prophylaxis and immediate therapy were measured against experimental melioidosis in a murine model and experimental glanders in a hamster model.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Challenge strains, storage and culture

Both organisms are classified by the Advisory Committee on Dangerous Pathogens (ACDP) as category 3 pathogens, thus all bacteriological procedures were carried out in Class 3 safety cabinets complying with BS5726.

B. pseudomallei NCTC 4845 and B. mallei ATCC 23344 were used as the challenge strains for melioidosis and glanders, respectively. Both species were stored at –80°C on a ‘Protect’ bead storage system (TSC Ltd, Heywood, Lancashire, UK) until required. Challenge cultures and counts were grown on nutrient agar or nutrient broth and in vitro antimicrobial susceptibility studies were carried out using Mueller–Hinton broth.

Antimicrobial preparation

For the in vitro experiments, doxycycline (Sigma, Poole, UK) and ciprofloxacin (Bayer, Newbury, UK) powders were dissolved in de-ionized water to give stock solutions of 10 mg/L. Solutions for administration were prepared freshly each day by dissolving doxycycline powder in phosphate-buffered saline and Ciproxin tablets (Bayer) in de-ionized water then filter sterilizing the solutions.

In vitro antimicrobial susceptibility testing

Bacteria were recovered by placing five to six ‘Protect’ beads into Mueller–Hinton broth (Oxoid, Basingstoke, UK), and incubating the cultures statically at 37°C for 24 h for B. pseudomallei and 48 h for B. mallei. A microtitre plate dilution method in accordance with NCCLS guidelines was used.22 Briefly, 96-well microtitre plates containing each antimicrobial ranging in dilution from 0.063 mg/L to 64 mg/L were prepared in advance and stored at –20°C. An inoculum of approximately 5 x 105 cfu/mL (determined using McFarland standard 5) in 100 µL was made from overnight or 48 h cultures in Mueller–Hinton broth and was added to all wells. Plates were incubated at 37°C for 18–20 h. Because of its slow-growing nature it was necessary to incubate B. mallei plates for 36 h before reading the MIC.

Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 (NCIMB, Aberdeen, UK) were used as quality control standards.

Animal models

All animal studies were carried out in accordance with the Scientific Procedures Act (Animals) 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures, 1989.

Female Porton outbred mice (bred in-house) and Syrian hamsters Lakeview strain (Charles River, Margate, UK) were used for experimental melioidosis and glanders, respectively. Mice were maintained in cages of five animals, and hamsters kept in pairs within a rigid-wall half-suit isolator complying to BS5726. They were subjected to a 12 h light–12 h dark cycle and had free access to food and water. Both mice and hamsters were given commercial rodent diet, the hamsters were additionally given supplements of sunflower seeds once weekly.

In previous studies, the nitrile rubber gloves worn with the half-suit have provided adequate protection against accidental bites from mice during husbandry and procedures. Although the Lakeview strain of Syrian hamster is noted for its docility, industrial puncture-resistant Kevlar gloves were worn over the nitrile rubber gloves as a precaution when handling infected hamsters.

Antimicrobial administration

Both antimicrobials were used at a dose of 40 mg/kg body weight, given twice daily at 12 h intervals by sc injection in 0.1 mL in mice and in 0.2 mL in hamsters according to one of three regimens: a prophylaxis regimen, where antimicrobials were initiated 48 h before challenge and continued for 5 days following challenge; an immediate or suppressive therapy with antimicrobials administered immediately after challenge and continued for 5 days; and a therapeutic regimen consisting of a 5 day course of antimicrobials starting 24 h after challenge.

Two further groups of mice challenged with B. pseudomallei were given the prophylaxis regimen as described except that the antibiotics were continued for 10 days after challenge.

Animal challenge and virulence estimation

The virulence of each pathogen was determined by measuring the median lethal dose (MLD), defined as the dose required to kill 50% of a small population of animals (25–30) calculated according to the method of Reed & Muench.23 Antimicrobial efficacy was measured by comparing the MLD of treated animals with the MLD in untreated controls.

Challenge cultures of B. pseudomallei and B. mallei were prepared by recovering the appropriate ‘Protect’ beads into nutrient broth and incubating for 24 h and 48 h, respectively.

Mice, in groups of five, were challenged with 0.1 mL of serial dilutions of the B. pseudomallei suspension by ip injection and observed for 35 days after challenge. Hamsters, in groups of four, were challenged with 0.2 mL of serial dilutions of B. mallei suspension by the ip route and were observed for 23 days after challenge. Eight hamsters given immediate treatment with doxycycline and eight hamsters receiving the doxycycline 24 h therapy regimen were kept for 5 weeks post-challenge for further studies. In both animal models, the prophylaxis groups were challenged approximately midway between antimicrobial doses.

Once the signs and symptoms of each of the infections had been characterized, humane end-points were observed wherever practicable. Autopsies were carried out in the isolator. Organs were bisected and smeared on to nutrient agar which was incubated for 24 h or 48 h at 37°C for B. pseudomallei and B. mallei, respectively. Identification of colonies growing on the plates were confirmed by polymerase chain reaction (PCR).

PCR

Single colonies were picked into 200 µL deionized water and boiled for 5 min. DNA was amplified using oligonucleotide primers complementary to the 16S rRNA-encoding genes of B. pseudomallei and B. mallei. PCR amplification was carried out by adding 18 µL of the following mix: 1 x PCR buffer (Boehringer–Mannheim, Mannheim, Germany); 0.2 mM PCR nucleotide mix (Boehringer–Mannheim), 180 ng of both 3' and 5' oligonucleotide primers, 5% (v/v) dimethylsulphoxide (DMSO) and 0.5 U Taq polymerase (Boehringer–Mannheim) to 2 µL of the colony suspension. Amplification was carried out in a Perkin-Elmer thermal cycler (Perkin–Elmer Cetus, Warrington, UK) under the following conditions: 30 cycles of 95°C for 30 s, 50°C for 1 min and 72°C for 1 min followed by 10 min extension time at 72°C. The PCR products were analysed by agarose gel electrophoresis and the product band visualized by ethidium bromide staining on a 2% TAE (40 mM Tris-acetate, 1 mM EDTA) gel.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In vitro susceptibility

Both B. pseudomallei 4845 and B. mallei 23344 were susceptible or moderately susceptible to ciprofloxacin and doxycycline in vitro, with B. pseudomallei inhibited by 2.0 mg/L ciprofloxacin and 1.0 mg/L doxycycline, and B. mallei inhibited by 1.0 mg/L ciprofloxacin and 0.25 mg/L doxycycline.

In vivo susceptibility of B. pseudomallei

Control mice were challenged intraperitoneally with a series of log dilutions ranging from 0.3 cfu to 2.9 x 103 cfu B. pseudomallei, which resulted in a disseminated and rapidly fatal infection. Animals became sick within 24–48 h post-challenge indicated by non-specific symptoms such as piloerection, with animals given the higher concentrations of B. pseudomallei succumbing within 96 h post-challenge. Autopsy revealed very few gross changes although bacteria could be isolated from liver, spleen, lungs and blood. In animals succumbing later there was marked splenomegaly and, occasionally, hepatomegaly. Numerous small abscesses developed in the spleen and pancreas, and less frequently in the liver. The MLD fell to 111 cfu within 7 days following challenge, decreasing further over the course of 3 weeks to a final MLD of 20 cfu (Figures 1 and 2GoGo). Autopsy of surviving animals revealed no gross changes and the organs were free of B. pseudomallei.



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Figure 1. Survival curve of animals challenged with B. pseudomallei NCTC 4845 by ip injection and treated with 40 mg/kg bd ciprofloxacin given subcutaneously: (i) •, 48 h prophylaxis and continued for 5 days post-challenge; (ii) {circ}, 48 h prophylaxis and continued for 10 days post-challenge; (iii) {blacktriangleup}, 5 day therapy starting immediately after challenge; (iv) {diamondsuit}, 5 day therapy starting 24 h after challenge; (v) {blacksquare}, untreated controls.

 


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Figure 2. Survival curve of animals challenged with B. pseudomallei NCTC 4845 by ip injection and treated with 40 mg/kg bd doxycycline given subcutaneously: (i) •, 48 h prophylaxis and continued for 5 days post-challenge; (ii) {circ}, 48 h prophylaxis and continued for 10 days post-challenge; (iii) {blacktriangleup}, 5 day therapy starting immediately after challenge; (iv) {diamondsuit}, 5 day therapy starting 24 h after challenge; (v) {blacksquare}, untreated controls.

 
Treated mice were challenged with a series of log dilutions ranging from 180 cfu to 1.8 x 107 cfu B. pseudomallei. There was little difference between the efficacy of the two antimicrobials. Prophylactic or immediate use of either agent protected against 1 x 107 cfu bacteria whilst the antimicrobial was administered. A few animals relapsed over the course of 5 weeks, the most dramatic occurring in the doxycycline prophylaxis group, between 11 and 21 days post-challenge. At the end of the experiment, however, the MLDs were substantially higher than the controls, varying between 1.4 x 106 cfu and 4.6 x 106 cfu (Figures 1 and 2GoGo). Autopsy of survivors revealed splenomegaly in a minority of animals although not to the degree observed in the controls, and abscesses were present in some cases. B. pseudomallei could be isolated from the organs of some animals including those with no apparent gross changes.

The therapeutic use of either antimicrobial delayed the course of infection, but over 5 weeks provided minimal protection with the final MLD for ciprofloxacin of 180 cfu and for doxycycline of 640 cfu (Figures 1 and 2GoGo).

In vivo susceptibility of B. mallei

Hamsters were challenged intraperitoneally with a series of log dilutions ranging from 0.3 cfu to 2.9 x 107 cfu B. mallei. The infection followed a very rapid course characterized by distinctive symptoms. Within 24–48 h the animals became subdued, and less active. The initial sign of disease was the production of a watery discharge from the eyes which turned purulent. Later signs included swelling of the limbs and palpable crepitus from the pleura. All fatalities/humane end-points occurred within 7 days following infection (Figure 3Go). Autopsy revealed few changes, with splenomegaly being the dominant feature, occasionally with abscesses occurring in both the spleen and the pancreas.



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Figure 3. Survival curve of animals challenged with B. mallei ATCC 23344 by ip injection and treated with 40 mg/kg bd ciprofloxacin given subcutaneously: (i) •, 48 h prophylaxis and continued for 5 days post-challenge; (ii) {blacktriangleup}, 5 day therapy starting immediately after challenge; (iii) {diamondsuit}, 5 day therapy starting 24 h after challenge; (iv) {blacksquare}, untreated controls.

 
Prophylactic ciprofloxacin and immediate ciprofloxacin treatment protected animals against challenges of up to 2.9 x 107 cfu for 18 days followed by a sudden relapse resulting in the MLDs falling to 4.6 x 105 cfu and 7 x 104 cfu, respectively, at the end of the experiment (Figure 3Go). Autopsy of the fatalities revealed an enlarged spleen containing one or two large abscesses. Autopsy of surviving animals generally revealed splenomegaly with an occasional abscess from which B. mallei could be isolated.

When ciprofloxacin therapy was started, eight animals given the highest challenges presented with signs of the disease. Two animals deteriorated despite treatment, three animals remained stable with no further deterioration but no marked improvement and three animals improved. With the exception of the two early cases, animals survived challenges of up to 2.9 x 107 cfu for 11 days. Relapses occurred between 11 and 18 days initially in those animals that had presented with symptomatic disease. Relapses in the lower challenge groups occurred from 18 days until the end of the experiment and the final MLD at 23 days post-challenge was 1.6 x 103 cfu (Figure 3Go). Autopsy findings of fatalities and surviving animals were identical to those of the other regimens.

All regimens of doxycycline protected hamsters against challenges of up to 2.9 x 107 cfu with no relapses observed during the 23 day experimental period, including the improvement of eight symptomatic hamsters in the therapeutic group. Autopsy of survivors revealed splenomegaly in some animals although no B. mallei were recovered.

Relapse did occur, however, in the animals that were retained, with disease reappearing in four of the eight hamsters given immediate therapy and one of the animals receiving the therapeutic regimen 28–31 days after challenge. Autopsies revealed numerous abscesses in the spleen, liver and pancreas from which B. mallei was isolated.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Both B. pseudomallei and B. mallei given intraperitoneally caused a rapidly invasive and lethal infection in their respective animal models reproducing the fulminant systemic infection similar to that occurring in human melioidosis and glanders. However, melioidosis and glanders have other clinical presentations, including chronic abscessic infection or a quiescent infection that may take several years to become symptomatic.2,24,25 The animal models reported here can be manipulated to reproduce chronic infections. In experimental melioidosis, a chronic abscessic infection in mice can result in some animals following a subcutaneous challenge or by using different murine strains such as C57B6 inbreds or Swiss–Webster outbreds (unpublished data). B. mallei infection in Balb/C, C57B6 and Swiss–Webster outbred mice leads to a chronic, non-lethal disease and chronic, fatal disease with a high MLD in Porton outbred mice (unpublished data). In these experiments the acute model was of interest, arguably representing the ‘worse case' infection, which if prevented by ciprofloxacin or doxycycline may also inhibit the establishment of chronic disease. Furthermore, many cases of melioidosis present as acute disease although it may be unclear whether the infection is recent or represents an acute ‘crisis' in patients who may have harboured subclinical infections over a long period of time.

An interesting feature of disease in the animal models was the involvement of the pancreas as a site of abscesses, particularly noticeable in the hamster model. Both B. pseudomallei and B. cepacia have been reported to bind with insulin26 which may indicate an ability to bind to insulin receptors, which would account for pancreatic involvement. Furthermore diabetes mellitus is a common predisposing factor in human disease, although it is not clear in some cases of chronic disease whether B. pseudomallei causes the diabetes, or underlying diabetes results in symptomatic disease. Pancreatic disease in human melioidosis, however, is rare5 and the significance in glanders is not known.

Ciprofloxacin and doxycycline have been reported to be inferior to other antimicrobials alone, or in combination, in the treatment of melioidosis in humans and are associated with high relapse rates.7,8 The clinical findings were mirrored by the results observed in the therapy groups in these studies even when the experimental strain was susceptible or moderately susceptible to each antimicrobial and serum and tissue levels of the antimicrobial exceeded the in vitro MIC. The MIC of ciprofloxacin against B. pseudomallei 4845 was typical of 50% of the strains of B. pseudomallei tested in a wider study carried out in our laboratory19 but low compared with other studies.3,4 The MIC of doxycycline was the highest measured for all of the strains of B. pseudomallei tested in the laboratory19 but again low when compared with other studies.5 Prophylactic and immediate post-exposure use of ciprofloxacin or doxycycline provided good protection, although the opportunity for prophylaxis or immediate therapy in clinical cases would be confined to situations such as laboratory accidents or precautionary immediate therapy following traumatic injury in endemic areas. The experiments showed that the ‘window of opportunity’, i.e. the time between exposure to the pathogen and successful prevention of infection with antimicrobials was less than 24 h following challenge.

Some relapses did occur with the prophylactic and immediate therapy groups, which could be attributed to a number of factors; principally, that the duration of the antimicrobial administration was short, although clinically the regimen would be extended should prophylaxis be used. The comparative pharmacokinetics of each antimicrobial relative to the in vitro MIC, and the ability of the bacteria to reside in privileged intracellular sites which may be inaccessible to the antimicrobials would also be important.

Extending the administration of doxycycline to 10 days post-challenge prevented relapse (fatalities still occurred during the administration period), but extending the ciprofloxacin administration increased the number of relapses. The pharmacokinetics of doxycycline were superior with respect to eradicating B. pseudomallei compared with ciprofloxacin. Doxycycline reached a peak serum concentration of 3.7 mg/L, and serum concentration exceeded the MIC for 24 h after dosing. The peak splenic concentration was 4.1 mg/kg with levels exceeding 1 mg/kg for 9 h. The peak serum concentration of ciprofloxacin was 2.9 mg/L with the MIC exceeded for only 1 h after dosing, and peak spleen concentration was 10.3 mg/kg remaining above 2 mg/kg for 3 h (unpublished data). Despite favourable pharmacokinetics, B. pseudomallei was recovered from surviving animals from all groups, including those given the antimicrobials for 10 days. The potential for relapse posed by these surviving bacteria is difficult to predict from these experiments.

Similarities in the pharmacokinetics of the two antimicrobials in hamsters accounted for the differences between the efficacies of doxycycline and ciprofloxacin against B. mallei. The strain used was sensitive to both antimicrobials, the MIC for ciprofloxacin was typical of 50% of strains tested, although the MIC for doxycycline was one of the lowest. Peak serum concentrations of ciprofloxacin in the hamster were 2.3 mg/L with the MIC exceeded for only 1 h. Splenic concentrations reached a maximum of 20 mg/kg and remained greater than 1 mg/kg for at least 12 h. Serum concentrations of doxycycline reached 2.6 mg/L and remained above 0.25 mg/L for at least 12 h. Splenic concentrations reached 8.7 mg/kg and remained above 0.25 mg/kg for at least 12 h (unpublished data). As glanders is such a rare disease the pathogenic mechanisms and virulence factors of B. mallei have not been subject to the same degree of scrutiny as those of B. pseudomallei. The similarities between the two organisms, however, are likely to extend to intracellular penetration and survival which would account for relapse. Given these similarities, the clinical experience in the treatment of melioidosis serves as a warning to potential antimicrobial regimens against glanders, notably that despite favourable pharmacokinetics and apparent susceptibility, recurrent infection may still occur.

In these experiments, both antimicrobials demonstrated some utility for melioidosis prophylaxis, but were unsuitable for therapeutic use, reflecting clinical findings. Potentially doxycycline could be used for the prevention and treatment of glanders although the problems encountered in treating melioidosis may also apply to glanders.


    Acknowledgments
 
The abstract was first read at the International Congress on Melioidosis held in Bangkok, Thailand, 22–25 November 1998.


    Notes
 
* Corresponding author. Tel: +44-1980-613-438; Fax: +44-1980-613-097; E-mail: drprussell{at}hotmail.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 7 September 1999; returned 10 November 1999; revised 3 December 1999; accepted 14 January 2000