A comparison of antibiotic regimens in the treatment of acute melioidosis in a mouse model

Glen C. Ulett1, Robert Hirst1, Bruce Bowden2, Kellie Powell1 and Robert Norton3,*

Departments of 1 Microbiology and Immunology and 2 Chemistry, James Cook University; 3 Department of Clinical Microbiology, QHPS, Townsville Hospital, Townsville, Queensland 4814, Australia

Received 26 February 2002; returned 28 June 2002; revised 5 August 2002; accepted 12 September 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Melioidosis is caused by the Gram-negative bacillus Burkholderia pseudomallei. Most clinical reports of disease are from south-east Asia and northern Australia. The organism is intrinsically resistant to most commonly available antibiotics. Standard therapy includes ceftazidime either alone or in combination with co-trimoxazole. The clinical advantage in adding co-trimoxazole has never been determined; nor has the activity of newer, fourth-generation cephalosporins, such as cefepime, been studied in the treatment of this condition. BALB/c mice have been shown to represent an animal model of melioidosis. This animal model was used in this study to compare the efficacy of ceftazidime and cefepime alone or with co-trimoxazole, in the therapy of melioidosis. Antibiotic levels in the mice were determined by HPLC, and dosing was modified to keep plasma antibiotic levels at or above the MIC for the organism–antibiotic combination for a significant part of a 12 h period. Bacterial load, as determined by splenic counts, showed that ceftazidime in combination with co-trimoxazole was the most effective therapeutic option. The animal model described in this study can be used as a preliminary evaluation of therapeutic options for melioidosis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Melioidosis is caused by the Gram-negative soil saprophyte Burkholderia pseudomallei, which is endemic in tropical and subtropical regions of south-east Asia and northern Australia.1 Regional endemic foci of this disease are largely confined to areas between latitudes 20°S and 20°N, although the organism has been isolated in subtropical south-western Australia.2 The organism is intrinsically resistant to a number of antibiotics, and therapeutic trials of potentially useful agents are limited by the relatively few cases encountered. Three general categories of clinical presentation of infection are recognized: acute, subacute and chronic.3,4 Acute melioidosis commonly presents as a fulminant septicaemia, often resulting in death within a few days of exposure.4 Mortality rates for the acute septicaemic form of melioidosis remain high, at 39%, in north-eastern Thailand.5 This is often despite intensive antibiotic therapy.

A BALB/c and C57BL/6 mouse model has been characterized to overcome this limitation and has been used in numerous studies to investigate the pathogenesis of infection in vivo.69 BALB/c mice are susceptible to low inocula, developing a septicaemic disease with disseminated abscesses of visceral organs, not unlike that seen in human infection.9

Clinical problems relating to therapy of melioidosis include the following: (i) the lack of coverage afforded by the broad-spectrum empirical therapy commonly chosen for acute, severe, community-acquired pneumonia in endemic areas during the wet season; (ii) the desire for equally effective, but less costly, therapeutic choices; and (iii) whether combination therapy is superior to monotherapy.

In an attempt to address these questions, BALB/c mice were used in a previous study6 as a model of acute infection, to assess the efficacy of selected antibiotic regimens. Disease in BALB/c mice involves a rapidly increasing bacteraemia, resulting in host death 96 h after infection. C57BL/6 mice are relatively resistant to infection.9 This model has been used for the comparison of ceftazidime and cefpirome in combination with co-trimoxazole (trimethoprim with sulfamethoxazole) for the treatment of melioidosis.6 However, the study was limited because single-therapy antibiotic regimens were not included for direct comparison with combination regimens. In addition, there were no data on mouse plasma antibiotic levels. Despite these limitations, this study showed that the BALB/c mouse model of melioidosis might be used to compare antibiotic regimens in the therapy of melioidosis.

The aim of this study was to determine whether the use of ceftazidime or cefepime alone is of equivalent efficacy compared with when used in combination with co-trimoxazole for treatment of acute murine melioidosis. This has clinical implications in that combination therapy with co-trimoxazole has not been trialled. The argument against using co-trimoxazole routinely is its adverse event profile.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria and infection of mice

The strain of B. pseudomallei used in this study was NCTC 13178. The identity of the isolate was confirmed by colony morphology on Ashdown agar and API 20NE (bioMérieux, La Balme, France). B. pseudomallei was grown in brain–heart infusion broth (Oxoid, Basingstoke, UK) at 37°C for 18 h and stored in 1 mL aliquots at –80°C in 20% glycerol. When required, an aliquot was thawed at 37°C and subcultured on to Ashdown agar. After 24–48 h incubation, 3–5 colonies were used to prepare a single cell suspension in sterile PBS equivalent to 0.5 McFarland standard (optical density of 0.18 at 650 nm; Multiskan EX355 version 1.0, Labsystems, Finland). Appropriate dilutions were prepared in PBS to achieve a concentration of 2.5 x 102 cfu/mL. Inbred BALB/c mice (8–16 weeks) were administered 50 cfu intravenously in 200 µL of PBS. Colony count checks were performed by a standard method after 48 h incubation at 37°C. The MICs of ceftazidime, co-trimoxazole and cefepime for the isolate of B. pseudomallei were 1.25 mg/L, 5 mg/L trimethoprim– 25 mg/L sulfamethoxazole and 5 mg/L, respectively, by the Etest (AB Biodisk, Dalvägen, Sweden).

Antibiotic therapy

This study had animal ethics committee approval from James Cook University, Townsville, Queensland, Australia.

At 48 h post-infection, mice were divided into groups of 10 and commenced antibiotic therapy programmes. Therapy programmes were designated 1–5, where programme 1 was ceftazidime (Glaxo, Australia), 2 was cefepime (Bristol Myers Squibb, Noble Park, Australia), 3 was co-trimoxazole (David-Bull Laboratories, Australia), 4 was ceftazidime with co-trimoxazole and 5 was cefepime with co-trimoxazole. Antibiotics were reconstituted in sterile distilled water as per the manufacturer’s instructions. An additional group of mice was infected and received subsequent injections of PBS only, to represent infected untreated controls. Working solutions of antibiotics were stored in 1 mL syringes at –80°C until use. Antibiotics were administered intraperitoneally twice daily at twice the recommended dose rate for co-trimoxazole, and 10 times the recommended dose rate for ceftazidime and cefepime. These dose rates were calculated based on plasma levels of antibiotics that were determined over a 12 h period by high performance liquid chromatography (HPLC) (see below). At 3 and 10 days after commencement of treatment, five mice per group were euthanized and the bacterial load in the spleen was determined, as described previously.9 Control mice were not assessed beyond day 3, due to progressive illness. Remaining mice were euthanized according to National Health and Medical Research Council guidelines. Briefly, spleens were homogenized in 10 mL of PBS using a Stomacher (Townson and Mercer; Altrincham, Cheshire, UK). Serial dilutions were prepared in PBS and these were dispensed on to Ashdown agar (Micro Diagnostics, Australia) to determine viable B. pseudomallei counts. These were expressed as cfu per mL of splenic homogenate. Clinical parameters such as temperature and weight were not recorded, as bacterial counts in spleen provide a reproducible measure of disease severity in this model.9

Development of standard curves for antibiotic levels in mouse plasma

Antibiotic levels in the mice were assayed using HPLC. Standard curves were drawn up for ceftazidime, cefepime and co-trimoxazole. These were based on HPLC-determined levels of the antibiotics in mouse plasma. Dosages were adjusted to maintain plasma levels above the MIC. Based on these standard curves, antibiotic levels in BALB/c mice, over time, were determined.

Sulfamethoxazole and trimethoprim (co-trimoxazole). The concentrated injection BP solution, which contained 400 mg sulfamethoxazole and 80 mg trimethoprim in 5 mL, was diluted to a concentration of 1562 mg/L sulfamethoxazole and 312.4 mg/L trimethoprim. A series of concentrations was then obtained by two-fold sequential dilutions of this solution with distilled water. Each dilution was analysed twice by HPLC, using a diode array detector. Sulfamethoxazole eluted at 7.7 min with 30% acetonitrile in 10 mM NaH2PO4 buffer, and the areas of sulfamethoxazole peaks were determined at 270 nm. Separate analyses were carried out for trimethoprim, which eluted at 9.1 min with 10% acetonitrile in 10 mM NaH2PO4 buffer, and peak areas were determined at 210 nm.

Ceftazidime and cefepime. Ceftazidime and cefepime were both dissolved in distilled water to a concentration of 1562 mg/L. Concentrations down to 6 mg/L were then obtained by two-fold sequential dilutions of this solution with distilled water. Each dilution was analysed twice by HPLC using a diode array detector. Ceftazidime eluted at 9.5 min with 6% acetonitrile in 10 mM NaH2PO4 buffer, and the areas of ceftazidime peaks were determined at 255 nm. Cefepime eluted at 6.6 min with 4% acetonitrile in 10 mM NaH2PO4 buffer, and the areas of cefepime peaks were determined at 235 nm.

Instrumentation and sample treatment

Standards and samples were analysed by HPLC using a GBC LC1150 pump operating at a flow rate of 2 mL/min for sulfamethoxazole, 1.5 mL/min for trimethoprim and ceftazidime, and 1.2 mL/min for cefepime using a Phenomenex Lunar 5 µm phenylhexyl reverse-phase column (250 x 4.6 mm) fitted with a guard column, and a GBC L5100 diode array detector. An ICI Instruments AS2000 automatic injector was used to inject 10 µL samples. The solvent was acetonitrile/10 mM sodium dihydrogen phosphate (AB Biodisk) buffer in the ratio indicated for each antibiotic. Standards and plasma samples were filtered through 13 mm Millex 0.22 µm filters prior to injection.

Determination of appropriate dosing schedules

Using HPLC-derived mouse plasma antibiotic levels, an appropriate dosing schedule was obtained that maintained antibiotic levels at or above the MIC (1.25, 5/25 and 5 mg/L of ceftazidime, co-trimoxazole and cefepime, respectively) for the isolate of B. pseudomallei used, for a significant part of a 12 h period.

Statistical analysis

This was performed using two independent samples with the Mann–Whitney U-test (SPSS software version 9.0.0). P values of <0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mouse plasma antibiotic levels

Following optimization of the HPLC antibiotic assay system, standard curves for ceftazidime, co-trimoxazole and cefepime were constructed. These were used to assay BALB/c mouse plasma levels of these antibiotics following intraperitoneal administration (Figure 1). At the dosages used, all antibiotics maintained a level at or above the MIC for a significant part of a 12 h period. Higher dose rates were investigated to maintain plasma levels at or above the MIC for the duration of the dosing schedule without observable effect.



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Figure 1. BALB/c mouse plasma levels of ceftazidime, cefepime and sulfamethoxazole (in co-trimoxazole) taken up to 12 h post-dose. Dose rates are shown in the key as multiples of the recommended dose rates for the treatment of acute human melioidosis (HD). Data points represent means ± S.E.M. of data averaged from two HPLC determinations of plasma levels in three treated mice.

 
Splenic bacterial loads at days 3 and 10

The splenic bacterial loads obtained from the different treatment groups are expressed as a logarithmic value in Figures 2 and 3.



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Figure 2. Comparative splenic bacterial loads at day 3 in different BALB/c mouse treatment groups. Error bars represent ± 1 S.E.M.

 


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Figure 3. Comparative splenic bacterial loads at day 10 in different BALB/c mouse treatment groups (PBS-treated mice did not survive beyond day 3). Error bars represent ± 1 S.E.M.

 
Although the use of either ceftazidime or co-trimoxazole alone provided significantly better clearance of B. pseudomallei from the spleen of infected mice at day 10 compared with cefepime-treated mice (P = 0.007 and 0.008, respectively), these single-therapy protocols were inferior compared with the combination of ceftazidime and co-trimoxazole.

Statistical analysis

A summary of the statistical analysis of this study is given in Table 1.


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Table 1.  Comparison of single- and combination-therapy programmes involving co-trimoxazole, ceftazidime and cefepime in the treatment of B. pseudomallei infection in BALB/c mice
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Melioidosis is an important cause of morbidity and mortality in northern Australia and south-east Asia. The organism is intrinsically resistant to a variety of commonly available antibiotics and this can present the treating clinician with few therapeutic options. Furthermore, in vitro susceptibility testing of this organism may not correlate with the likelihood of clinical cure. The relative rarity of this condition, both within Australia and worldwide, makes the setting up of clinical trials difficult. This disease therefore lends itself to the development of suitable animal model studies in which therapeutic regimens can be assessed prior to Phase I clinical trials.

Ceftazidime is considered standard therapy for treatment of acute septicaemic melioidosis. There are no published studies that have compared directly the efficacy of ceftazidime alone with that of co-trimoxazole and ceftazidime for the treatment of this condition. The current study was undertaken to address this question and to investigate the efficacy of cefepime, a recently described fourth-generation cephalosporin. A similar fourth-generation cephalosporin, cefpirome, has been shown previously to have activity against B. pseudomallei in this model.6 Determination of appropriate antibiotic dosing schedules for use in BALB/c mice is an important step in the validation of the animal model of melioidosis previously described for evaluation of novel treatment strategies.6 The importance of maintaining antibiotic levels above the MIC for the organism is particularly acute for the ß-lactam antibiotics.10 In this study, this was achieved through a combination of high antibiotic dosages (up to 10 times the equivalent human dose) and a 12-hourly administration. The control group that was used for toxicity testing did not show any evidence of toxicity at these dosages. This has also been demonstrated in a previous study where there was no evidence of antibiotic toxicity in BALB/c mice at dosages 10 times the human dose rate.6

This study has shown that co-trimoxazole enhances the efficacy of ceftazidime (P = 0.047) when administered in combination therapy for the treatment of melioidosis in this model. This conclusion is based on the significantly lower bacterial numbers in spleen following several days of combination treatment, compared with single-drug therapy. While this measure may not allow direct comparison with other ‘clinical’ measures in patients, such as weight and temperature, we believe that splenic bacterial load does provide important information as to the clinical state of mice in the model used here.6,9

Ceftazidime and co-trimoxazole combination therapy was the only treatment regimen to result in an inability to recover viable B. pseudomallei from the spleen of infected mice. Viable B. pseudomallei was consistently recovered from spleens of infected mice that were given alternative antibiotic regimens in this study. This was particularly apparent in mice that were administered cefepime, where the bacterial load in spleen was as high as 107 cfu/mL after 10 days of therapy.

No animal study can replace controlled human clinical trials. However, the BALB/c mouse represents a unique model for experiments designed to test novel therapeutic strategies in melioidosis. Preliminary data derived from studies such as the one presented here provide useful information on which to base a clinical trial.


    Acknowledgements
 
We would like to thank Bristol Myers Squibb (Australia) for their support of this study.


    Footnotes
 
* Corresponding author. Tel: +61-7-47961111; Fax: +61-7-47962415; E-mail: Robert_Norton{at}health.qld.gov.au Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Dance, D. A. B. (1991). Melioidosis: the tip of the iceberg? Clinical Microbiology Reviews 4, 52–60.[ISI][Medline]

2 . Golledge, C. L., Chin, W. S., Tribe, A. E., Condon, R. J. & Ashdown, L. R. (1992). A case of human melioidosis originating in south-west Western Australia. Medical Journal of Australia 157, 332–4.[ISI][Medline]

3 . Hirst, R. G., Indriana, J. & Cocciolone, R. A. (1992). An introduction to melioidosis. Innate resistance and acquired immunity to Pseudomonas pseudomallei. Australian Biologist 5, 203–13.

4 . Smith, C. J., Allen, J. C., Noor Embi, M., Othman, O., Razak, N. & Ismail, G. (1987). Human melioidosis: an emerging medical problem. MIRCEN Journal 3, 343–66.

5 . Chaowagul, W., White, N. J., Dance, D. A., Wattanagoon, Y., Naigowit, P., Davis, T. M. et al. (1989). Melioidosis: a major cause of community acquired septicemia in northeastern Thailand. Journal of Infectious Diseases 159, 890–9.[ISI][Medline]

6 . Ulett, G. C., Norton, R. & Hirst, R. G. (1999). Combination antimicrobial therapy of acute B. pseudomallei infection in a mouse model. Pathology 31, 264–7.[CrossRef][ISI][Medline]

7 . Barnes, J. L., Ulett, G. C., Ketheesan, N., Clair, T., Summers, P. M. & Hirst, R. G. (2001). Induction of multiple chemokine and colony-stimulating factor genes in experimental Burkholderia pseudomallei infection. Immunology and Cell Biology 79, 490–501.[CrossRef][ISI][Medline]

8 . Ulett, G. C., Ketheesan, N. & Hirst, R. G. (2000). Cytokine gene expression in innately susceptible BALB/c mice and relatively resistant C57BL/6 mice during infection with virulent Burkholderia pseudomallei. Infection and Immunity 68, 2034–42.[Abstract/Free Full Text]

9 . Leakey, A. K., Ulett, G. C. & Hirst, R. G. (1998). BALB/c and C57BL/6 mice infected with virulent Burkholderia pseudomallei provide contrasting animal models for the acute and chronic forms of human melioidosis. Microbial Pathogenesis 24, 269–75.[CrossRef][ISI][Medline]

10 . Cars, O. (1997). Efficacy of beta-lactam antibiotics: integration of pharmacokinetics and pharmacodynamics. Diagnostic Microbiology and Infectious Disease 27, 29–33.[CrossRef][ISI][Medline]